Oncogene database

6714

SRC

SRC proto-oncogene, non-receptor tyrosine kinase;SRC;SRC proto-oncogene, non-receptor tyrosine kinase

proto-oncogene c-Src|proto-oncogene tyrosine-protein kinase Src|protooncogene SRC, Rous sarcoma|tyrosine kinase pp60c-src|tyrosine-protein kinase SRC-1|v-src avian sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog

20q12-q13

protein-coding

Polymoma virus encodes a potent oncogene, the middle T-antigen (MT), that induces cell transformation by copying the actions of tyrosine kinase associated growth factor receptors. A crucial component of MT transformation is its ability to bind and stimulate the activity of src-family kinases. However, the mechanism by which this is achieved remains unclear. Tyrosine phosphorylation of MT by src-kinases then provides binding sites for SH2 and PTB domain containing molecules in a paradigm of receptor action. We present evidence here that the MT/src complex contains equi-molar amounts of PP2A, and that phosphatase activity may be required for the interaction of MT with both PP2A and the src-family. PP2A, then, is a necessary component of the MT-src complex. We also show that two motifs in the 185 to 210 region of MT, each consisting of a basic area followed by a serine or threonine, are essential for interaction with src-kinases, but not PP2A. The spacing between the serine or threonine and the basic sequence also appears to be important. Substituting a cysteine residue in place of Thr203 in MT has no affect on the binding of pp60c-src, showing that these sites interact with src-kinases by a novel mechanism that does not require phosphorylation.

The Src homology 3 (SH3) domain, originally identified in v-Crk, plays an important role in signal transduction. The comparative study with c-src has revealed that v-src oncogene of Schmidt-Ruppin strain of Rous sarcoma virus has three point mutations in its SH3 domain and one in the upstream of SH3. To assess the role of these mutations, each of the single mutations was introduced into c-Src by oligonucleotide-directed mutagenesis and its effect on cell transformation was examined. While variant Src proteins that carry each one of single mutations could not transform cells, double mutation at positions 95 and 117 converted c-Src to be oncogenic and active in kinase. An additional mutation at position 124 together with one at 95 and 117 further activated Src kinase. By use of GST-fusion forms of v-Src SH3 and c-Src SH3, we found that these mutations in SH3 suppressed the binding of SH3 with c-Src protein, possibly with a linker region, while v-SrcSH3 retained the ability to bind a subset of cellular protein to the level similar to those of c-SrcSH3. Taken together, our results suggest that point mutations accumulated in SH3 region can activate, in concert, Src kinase by relaxing the interaction between SH3 and the linker region and subsequently convert Src to be oncogenic.

Signal transducers and activators of transcription (STATs) are transcription factors that mediate normal biologic responses to cytokines and growth factors. However, abnormal activation of certain STAT family members, including Stat3, is increasingly associated with oncogenesis. In fibroblasts expressing the Src oncoprotein, activation of Stat3 induces specific gene expression and is required for cell transformation. Although the Src tyrosine kinase induces constitutive Stat3 phosphorylation on tyrosine, activation of Stat3-mediated gene regulation requires both tyrosine and serine phosphorylation of Stat3. We investigated the signaling pathways underlying the constitutive Stat3 activation in Src oncogenesis. expression of Ras or Rac1 dominant negative protein blocks Stat3-mediated gene regulation induced by Src in a manner consistent with dependence on p38 and c-Jun N-terminal kinase (JNK). Both of these serine/threonine kinases and Stat3 serine phosphorylation are constitutively induced in Src-transformed fibroblasts. Furthermore, inhibition of p38 and JNK activities suppresses constitutive Stat3 serine phosphorylation and Stat3-mediated gene regulation. In vitro kinase assays with purified full-length Stat3 as the substrate show that both JNK and p38 can phosphorylate Stat3 on serine. Moreover, inhibition of p38 activity and thus of Stat3 serine phosphorylation results in suppression of transformation by v-Src but not v-Ras, consistent with a requirement for Stat3 serine phosphorylation in Src transformation. Our results demonstrate that Ras- and Rac1-mediated p38 and JNK signals are required for Stat3 transcriptional activity induced by the Src oncoprotein. These findings delineate a network of tyrosine and serine/threonine kinase signaling pathways that converge on Stat3 in the context of oncogenesis.

We investigated the effect of cell transformation by v-src on the expression and tyrosine phosphorylation of SHPS-1, a putative docking protein for SHP-1 and SHP-2. We found that transformation by v-src virtually inhibited the SHPS-1 expression at mRNA level. While nontransforming Src kinases including c-Src, nonmyristoylated forms of v-Src had no inhibitory effect on SHPS-1 expression, transforming Src kinases including wild-type v-Src and chimeric mutant of c-Src bearing v-Src SH3 substantially suppressed the SHPS-1 expression. In cells expressing temperature sensitive mutant of v-Src, suppression of the SHPS-1 expression was temperature-dependent. In contrast, tyrosine phosphorylation of SHPS-1 was rather activated in cells expressing c-Src or nonmyristoylated forms of v-Src. SHPS-1 expression in SR3Y1 was restored by treatment with herbimycin A, a potent inhibitor of tyrosine kinase, or by the expression of dominant negative form of Ras. Contrary, active form of Mekl markedly suppressed SHPS-1 expression. Finally, overexpression of SHPS-1 in SR3Y1 led to the drastic reduction of anchorage independent growth of the cells. Taken together, our results suggest that the suppression of SHPS-1 expression is a pivotal event for cell transformation by v-src, and the Ras-MAP kinase cascade plays a critical role in the suppression.

Binding constants for triplex formation between purine-rich oligonucleotides and a pyrimidine.purine tract of the human c-src proto-oncogene were measured by fluorescence polarization in the presence of polyamines, Na(+) and K(+). In both the hexamine and tetramine series, the longer polyamines had the larger binding constants for triplex formation at low concentrations of polyamine. At higher concentrations ALL values tended to plateau in the 10(9)/M range. In contrast to previous reports, K(+) did not inhibit triplex formation and at 150 mM the binding constants were again in the 10(9)/M range for both an 11mer and 22mer oligonucleotide. At 150 mM K(+) the addition of polyamines did not lead to any significant increase in the binding constants. It was determined that the lack of inhibition by K(+) was due to the low concentration (1 nM) of purine oligonucleotide required for the fluorescence polarization technique. At higher concentrations (1 microM) self-association of the oligonucleotide was observed. These results suggest that in vivo, at least for the c-src promoter, the inhibition of triplex formation by K(+) may not be detrimental. However, it may be difficult to achieve binding constants above approximately 10(9)/M even in the presence of polycations.

Tyrosine kinase oncoproteins cause simultaneous activation of multiple intracellular signaling pathways. However, the precise mechanisms by which individual pathways induce oncogenesis are not well understood. We have investigated the roles of individual signaling pathways in v-Src-dependent cell growth and survival by inhibiting one particular pathway. v-Src induced constitutive activation of signal transducers and activators of transcription 3 (STAT3), phosphatidylinositol 3-kinase, and Ras in murine Ba/F3 cells and led to factor-independent proliferation. Dominant-negative mutants of STAT3 (STAT3D) and phosphatidylinositol 3-kinase (Deltap85) inhibited v-Src-dependent growth by approximately 60 and approximately 40%, respectively. Moreover, dominant-negative Ras (N17) induced severe apoptosis, which was accompanied by down-regulation of Bcl-2 and activation of caspase-3. Although cells overexpressing Bcl-2 or caspase-3 inhibitors remained viable even when N17 was expressed, the growth was reduced by approximately 85%. During N17- and STAT3D-induced growth suppression, expression of cyclin D2, cyclin D3, c-myc, and c-fos was suppressed by N17, whereas that of cyclin D2, cyclin E, and c-myc was suppressed by STAT3D. Thus, v-Src-activated Ras and STAT3 are involved in distinct but partly overlapping transcriptional regulation of cell cycle regulatory molecules. These results suggest that the full oncogenic activity of v-Src requires simultaneous activation of multiple signalings, in which Ras is particularly required for survival.

A number of oncogenes alter the regulation of the cell cycle and cell death, contributing to the altered growth of tumours. expression of the v-Src oncoprotein in Rat-1 fibroblasts prevented cell cycle exit in response to growth factor withdrawal. Here we investigated whether survival of v-Src transformed cells in low serum is dependent on v-Src activity. We used a temperature sensitive v-Src to study the effect inactivating v-Src on transformed cells growing under low serum conditions. We found when we switched off v-Src the cells died by apoptosis characterised by activation of caspases and the stress-activated kinases, JNK (Jun N-terminal kinase) and p38 MAP (mitogen activated protein) kinase. We were able to prevent cell death by addition of serum or overexpression of Bcl-2. Thus v-Src transformed Rat-1 cells can be protected from apoptosis by serum, v-Src, or Bcl-2. We investigated how v-Src protects from apoptosis under these conditions. Amongst other effects, v-Src activates two kinases which have been shown to protect cells from apoptosis, phosphatidylinositol 3-kinase (PI3-K) and extracellular signal-regulated kinase (ERK1/2). We found that switching off v-Src led to a decrease in the activity of both PI3-K and ERK1/2, however, we found that adding a specific inhibitor of PI3-K (LY294002) to v-Src transformed Rat-1 cells grown in low serum induced apoptosis while a specific ERK kinase (MEK1) inhibitor (PD98059) had no effect. This suggests that v-Src protects from apoptosis under low serum conditions by activating PI3-K.

CMS/CD2AP is a cytoplasmic protein critical for the integrity of the kidney glomerular filtration and the T cell function. CMS contains domains and motifs characteristic for protein-protein interactions, and it is involved in the regulation of the actin cytoskeleton. We report here that the individual SH3 domains of CMS bind to phosphotyrosine proteins of approximately 80, 90, and 180 kDa in cell lysates stimulated with epidermal growth factor. The second SH3 domain of CMS bound specifically to a tyrosine-phosphorylated protein of 120 kDa, which we identified as the proto-oncoprotein c-Cbl. The c-Cbl-binding site for CMS mapped to the carboxyl terminus of c-Cbl and is different from the proline-rich region known to bind SH3-containing proteins. CMS binding to c-Cbl was markedly attenuated in a tyrosine phosphorylation-defective c-Cbl mutant indicating that this interaction is dependent on the tyrosine phosphorylation of CMS. It also implies that CMS interacts with c-Cbl in an inducible fashion upon stimulation of a variety of cell-surface receptors. Immunofluorescence analysis revealed that both proteins colocalize at lamellipodia and leading edges of cells, and we propose that the interaction of CMS with c-Cbl offers a mechanism by which c-Cbl associates and regulates the actin cytoskeleton.

While the activated viral Src oncoprotein, v-Src, induces uncontrolled cell growth, the mechanisms underlying cell cycle deregulation by v-Src have not been fully defined. Previous studies demonstrated that v-Src induces constitutively active STAT3 signaling that is required for cell transformation and recent data have implicated STAT3 in the transcriptional control of critical cell cycle regulators. Here we show in mouse fibroblasts stably transformed by v-Src that mRNA and protein levels of p21 (WAF1/CIP1), cyclin D1, and cyclin E are elevated. Using reporter constructs in transient-transfection assays, the cyclin D1 and p21 promoters were both found to be transcriptionaly induced by v-Src in a STAT3-dependent manner. The kinase activities of cyclin D/CDK4, 6 and cyclin E/CDK2 complexes were only slightly elevated, consistent with the findings that coordinate increases in p21, cyclin D1 and cyclin E resulted in an increase in cyclin/CDK/p21 complexes. Similar results were obtained in NIH3T3 and BALB/c 3T3 cells stably transformed by v-Src, indicating that these regulatory events associated with STAT3 signaling represent common mechanisms independent of cell line or clonal variation. These findings suggest that STAT3 has an essential role in the regulation of critical cell cycle components in v-Src transformed mouse fibroblasts.

We have isolated a novel isoform of phospholipase A(2). This enzyme was designated srPLA(2) because it was discovered while analyzing the proteins interacting with different forms of the v-Src oncoproteins isolated from Rous sarcoma virus-transformed hamster cells. It contains ALL the functional regions of the PLA(2) group IIA proteins but differs at its C-terminal end where there is an additional stretch of 8 amino acids. The SrPLA(2) isoform was detected as a 17-kDa precursor in cells and as a mature 14-kDa form secreted in culture medium. A direct interaction of the 17-kDa precursor with the Src protein was observed in lysates of transformed cells. Both the 17- and 14-kDa forms were found to be phosphorylated on tyrosine. To our knowledge, this is the first report of a PLA(2) group II protein that is tyrosine phosphorylated. We surmise that srPLA(2) interacts with the Src protein at the cell membrane during the process of its maturation.

Oncogenesis associated with human T-cell leukemia virus (HTLV) infection is directly linked to the virally encoded transcription factor Tax. To activate HTLV-1 transcription Tax interacts with the cellular protein CREB and the pleiotropic coactivators CBP and p300. While extensively studied, the molecular mechanisms of Tax transcription function and coactivator utilization are not fully understood. Previous studies have focused on Tax binding to the KIX domain of CBP, as this was believed to be the key step in recruiting the coactivator to the HTLV-1 promoter. In this study, we identify a carboxy-terminal region of CBP (and p300) that strongly interacts with Tax and mediates Tax transcription function. Through deletion mutagenesis, we identify amino acids 2003 to 2212 of CBP, which we call carboxy-terminal region 2 (CR2), as the minimal region for Tax interaction. Interestingly, this domain corresponds to the steroid receptor coactivator 1 (SRC-1)-interacting domain of CBP. We show that a double point mutant targeted to one of the putative alpha-helical motifs in this domain significantly compromises the interaction with Tax. We also characterize the region of Tax responsible for interaction with CR2 and show that the previously identified transactivation domain of Tax (amino acids 312 to 319) participates in CR2 binding. This region of Tax corresponds to a consensus amphipathic helix, and single point mutations targeted to amino acids on the face of this helix abolish interaction with CR2 and dramatically reduce Tax transcription function. Finally, we demonstrate that Tax and SRC-1 bind to CR2 in a mutually exclusive fashion. Together, these studies identify a novel Tax-interacting site on CBP/p300 and extend our understanding of the molecular mechanism of Tax transactivation.

DNA vaccination is particularly efficient for induction of cytotoxic T-lymphocyte (CTL) response. In our experiments, we used MHC(B) congenic chicken lines CB and CC (regressors and progressors of v-src-induced tumours, respectively) and a mutated, non-oncogenic v-src gene construct as the DNA vaccine. A high degree of vaccine protection against oncogenic v-src challenge was achieved in the CB line chickens. CTL response was demonstrated in vitro and by adoptive transfer of immune cells to the syngeneic host and to the CC line chickens rendered tolerant to CB cells. In the CC line chickens we observed tumour growth retardation after a low-dose DNA vaccination administered to immature recipients while higher amounts of DNA vaccine in immunocompetent chickens exerted an enhancing effect.

We previously reported the occurrence of novel dinucleotide mutations of the K-RAS gene (KRAS2) in 2% of pancreatic tumors sampled, but it remained unknown whether these were functional mutations that convert the proto-oncogene to an oncogene, or unselected mutations that might inactivate protein function. In the current study, the functionality of these rare mutations was quantitated via a mitogen-activated protein kinase (MAPK) pathway-specific transactivational reporter system. pathway activation by dinucleotide mutant proteins was comparable to that of the common G12V mutant K-Ras protein. Current allele-specific technologies often employed to detect K-RAS mutations in clinical tumor samples produce false results when dinucleotide mutations are present. Therefore, it is advisable to consider dinucleotide KRAS2 mutants in the strategic design of mutational screens used to assay clinical tumor samples. Hum Mutat 18:357, 2001.

One of the prominent effects of the Src kinase is to reduce cell adhesion. The small GTPase, R-Ras, affects cell adhesion by maintaining integrin activity, and the ability of R-Ras to do so can be regulated by phosphorylation of a tyrosine residue located in its effector domain by an Eph receptor kinase (Zou, J. X., Wang, B., Kalo, M. S., Zisch, A. H., Pasquale, E. B., and Ruoslahti, E. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 13813-13818). Here we show that Src regulates cell adhesion through R-Ras and integrins. Reduced substrate attachment of 293T cells transfected with the cDNA for an activated form of Src (v-Src) was accompanied by phosphorylation of endogenous R-Ras. v-Src also phosphorylated R-Ras in vitro. An activated form of Src similar to one that has been found in human cancers, Src527, shared with v-Src the ability to phosphorylate R-Ras. Stronger R-Ras phosphorylation was seen in Madin-Darby canine kidney cells cells transformed with temperature-sensitive v-Src at the permissive temperature than at the non-permissive temperature, and R-Ras and Src co-immunoprecipitated at the permissive temperature. mutation analysis showed that the Src phosphorylation site in R-Ras was tyrosine 66, the position critical to the ability of R-Ras to support integrin activity. Finally, activated R-Ras in which tyrosine 66 is mutated to phenylalanine rendered cells partially resistant to the effects of Src on cell adhesion. Regulation of cell adhesion by Src through R-Ras may be at least partially responsible for the reduced adhesion and the resulting increased invasiveness of Src-transformed cells.

Bcl-3 is a distinctive member of the IkappaB family of NF-kappaB inhibitors because it can function to coactivate transcription. A potential involvement of Bcl-3 in oncogenesis is highlighted by the fact that it was cloned due to its location at a breakpoint junction in some cases of human B-cell chronic lymphocytic leukemia and that it is highly expressed in human breast tumor tissue. To analyze the effects of Bcl-3 dysregulation in breast epithelial cells, we created stable immortalized human breast epithelial cell lines either expressing Bcl-3 or carrying the corresponding vector control plasmid. Analysis of the Bcl-3-expressing cells suggests that these cells have a shortened G(1) phase of the cell cycle as well as a significant increase in hyperphosphorylation of the retinoblastoma protein. Additionally, the cyclin D1 gene was found to be highly expressed in these cells. Upon further analysis, Bcl-3, acting as a coactivator with NF-kappaB p52 homodimers, was demonstrated to directly activate the cyclin D1 promoter through an NF-kappaB binding site. Therefore, our results demonstrate that dysregulated expression of Bcl-3 potentiates the G(1) transition of the cell cycle by stimulating the transcription of the cyclin D1 gene in human breast epithelial cells.

When c-Src and v-Src were immunoprecipitated together from hamster fibroblasts transformed by Rous sarcoma virus containing v-src oncogene, the total Src activity was almost threefold higher compared to c-Src activity in the control cells. The activity of v-Src immunoprecipitated separately, however, accounting for only 40% of the total Src activity, indicating that c-Src is activated upon transformation. An increased activity of Csk was also found in RSV-transformed cells. It decreased upon serum stimulation in parallel with an increase in Src kinase activity. In nontransformed cells, serum stimulation induced an enhanced Csk activity, but no changes in c-Src activity were observed. This may suggest that Csk may have more functions in hamster fibroblasts, in addition to its inhibitory effect on c-Src.

The E5 oncoprotein of bovine papillomavirus type 1 is a Golgi-resident, hydrophobic polypeptide that can transform immortalized fibroblasts by activating endogenous platelet-derived growth factor receptor beta (PDGF-R). However, the existence of E5 mutants that dissociate transformation from PDGF-R activation implies that there are additional mechanism(s) by which E5 can transform cells. We now show that both wt E5, and transforming E5 mutants that are defective for PDGF-R activation, constitutively activate endogenous c-Src in NIH3T3 cell lines to levels normally associated with acute growth factor stimulation. The ubiquitous Src family protein tyrosine kinase (PTK) Fyn is not activated by these E5 constructs, nor are focal adhesion kinase and endogenous receptor PTKs for insulin, epidermal growth factor, basic fibroblast growth factor and insulin-like growth factor. We further demonstrate that transforming activity of the L26A E5 mutant, which is highly defective for PDGF-R activation, depends on its ability to activate Src. L26A E5 does not transform SYF cells that are deficient for Src, Fyn and Yes, unless Src expression is reconstituted, and does not transform NIH3T3 cells in which Src PTK activity is maintained at a basal level by means of kinase-defective K295R Src overexpression.

The cellular homolog of the oncogene v-src, the proto-oncogene c-src, was cloned from rat testis using a high stringency polymerase chain reaction. Rat c-src cDNA shared identity with chicken and mouse, and Rous sarcoma virus c-src and v-src, respectively. Rat c-Src protein was 98% homologous to both human and mouse c-Src. Interestingly, rat Src contained one extra amino acid compared to the mouse protein. As expected, the rat testis Src lacked the six extra residues common to the neuronal Src identified in human and mouse. Reporting of the cDNA sequence for non-neuronal, rat c-src should facilitate experimentation into cell growth and transformation using rat tissues as models of human disease.

The QM gene encodes a 24.5 kDa ribosomal protein L10 known to be highly homologous to a Jun-binding protein (Jif-1), which inhibits the formation of Jun-Jun dimers. Here we have carried out screening with the c-Yes protein and found that a QM homologous protein showed interactions with c-Yes and other Src family members. We have found that two different regions of QM protein were associated with the SH3 domain of c-Yes. The QM protein does not contain canonical SH3 binding motifs or previously reported amino acid fragments showing interaction with SH3 domains. Several c-Yes kinase activity assays indicated that the QM protein reduced c-Yes kinase activity by 70% and that this suppression is related not only to the two SH3 binding regions but also to the C-terminal region of QM. Moreover, our autophosphorylation assays clarified that this regulation resulted from the inhibition of c-Yes autophosphorylation. Immunofluorescence studies showed that the QM proteins and c-Yes are able to interact in various tumor cell lines in vivo. The increases of the c-Yes protein and mRNA levels were detected when the QM was transfected. These results suggest that the QM protein might be a regulator for various signal transduction pathways involving SH3 domain-containing membrane proteins.

Cellular Src and epidermal growth factor receptor (EGFR) collaborate in the progression of certain human malignancies, and their cooverexpression characterizes relatively aggressive animal tumors. Our study addressed the mode of oncogenic cooperation and reports that overexpression of c-Src in model cellular systems results in the accumulation of EGFR at the cell surface. The underlying mechanism involves inhibition of the normal, c-Cbl-regulated process of ligand-induced receptor down-regulation. In response to activation of c-Src, c-Cbl proteins undergo tyrosine phosphorylation that promotes their ubiquitylation and proteasomal destruction. Consequently, ubiquitylation of EGFR by c-Cbl is restrained in Src-transformed cells, and receptor sorting to endocytosis is impaired. In conclusion, by promoting destruction of c-Cbl, c-Src enables EGFR to evade desensitization, which explains Src-EGFR collaboration in oncogenesis.

Our data show that in hamster fibroblasts transformed by Rous sarcoma virus (RSV), the phosphoinositide 3 -kinase (PI-3K)/Akt/glycogen synthase kinase 3 antiapoptotic pathway is upregulated and involved in increased protein synthesis through activation of initiation factor eIF2B. Upon inhibition of PI-3K by wortmannin, phosphorylation of 70-kDa ribosomal protein S6 kinase (p70 S6k) and its physiological substrate, ribosomal protein S6, decreased in the non-transformed cells but not in RSV-transformed cells. Thus PI-3K, which is thought to be involved in regulation of p70 S6k, signals to p70 S6k in normal fibroblasts, but it does not appear to be an upstream effector of p70 S6k in fibroblasts transformed by v-src oncogene, suggesting that changes in the PI-3K signalling pathway upstream of p70 S6k are induced by RSV transformation.

Infection of the IL-3-dependent, myeloid progenitor cell line 32D cl 3 with murine retroviruses that contain either the wild-type or a temperature-sensitive mutant v-src can render these cells growth-factor independent. These cells also became resistant to gamma irradiation administered at the low-dose rate of 0.05 Gy/min, which is used clinically. The v-src-dependent nature of resistance to gamma irradiation was examined by studying four clones of 32D cl 3 cells that had been infected with a retrovirus carrying the tsLA31A mutant of v-src. The tyrosine-specific kinase activity of this mutant is dramatically reduced at the nonpermissive temperature of 39 degrees C. Cells transformed by v-src and grown at either 34 or 39 degrees C, in the presence or absence of IL-3, demonstrated a significantly higher D0 compared to parental cells examined under identical conditions. In addition, expression of v-src abrogated the synergistic killing effect of heat and gamma irradiation. The D0 of parental 32D cl 3 cells kept at 39 degrees C after gamma irradiation was reduced significantly compared to the D0 of these cells kept at 34 degrees C. This contrasts with data from 32D cl 3 cells infected with either the wild-type v-src or the temperature-sensitive mutant, neither exhibited a synergistic effect in the D0 at either 34 or 39 degrees C. Therefore, while continuous expression of a v-src gene product is required for maintenance of the growth-factor-independent state, v-src does not appear to be responsible for the increased gamma-radiation resistance of these cells at low dose rate.

To analyze the signaling pathways utilized in malignant transformation by pp60v-src, we have isolated and characterized src mutants which possess normal levels of protein tyrosine kinase activity but which cause only a partially transformed phenotype. Our hypothesis is that such mutants are partially defective for transformation because they are defective in their ability to activate specific components of the cellular signaling machinery while still activating others. In this communication, we report on the molecular and biochemical characterization of one such mutant, CU12 (D. D. Anderson, R. P. Beckmann, E. H. Harms, K. Nakamura, and M. J. Weber, J. Virol. 37:455-458, 1981). Cells infected with this mutant are capable of anchorage-independent growth, but rather than exhibiting the rounded and refractile morphology characteristic of wild-type-infected cells, they display an extremely elongated, fusiform morphology. The morphological properties of this mutant src could be accounted for entirely by a single mutation in the SH3 domain (lysine 106 to glutamate). Other mutations were constructed in this region by in vitro mutagenesis, both in a v-src and in an activated c-src background, and several of them also induced a fusiform morphology. ALL of the mutations inducing fusiform morphology also resulted in decreased association of pp60src with phosphatidylinositol 3 -kinase activity. In addition, association of pp60src with some tyrosine-phosphorylated proteins was altered. We propose that the SH3 domain participates (along with the SH2 domain) in the interaction of pp60src with cellular signaling proteins, and we speculate that the association with phosphatidylinositol 3 -kinase plays an important role in the regulation of cellular morphology.

Transplantation of spleen cells from primary reconstituted mice expressing the v-src oncogene to secondary and tertiary irradiated recipients resulted in the emergence of erythroid precursors with a transformed phenotype. When cultured in methyl cellulose, these precursors generated colonies of undifferentiated cells that could be expanded into continuously growing factor-dependent cell lines in liquid culture. ALL lines tested had a similar phenotype and expressed the v-src oncogene. In addition they responded to factors that regulate normal erythroid development, namely erythropoietin (Epo), interleukin-3 (IL-3), and mast cell growth factor (MGF), the ligand to the c-kit encoded receptor. When cells from one of the lines were maintained in the absence of factor, a "factor independent" subpopulation emerged that appeared to grow in an autocrine fashion. Conditioned medium from these cells stimulated their own growth as well as the growth of broad spectrum of normal precursors. Studies with neutralizing antibodies indicated that the predominant colony-stimulating factor produced by these cells is IL-3.

Rapid endothelial cell migration and inhibition of thrombosis are critical for the resolution of denudation injuries to the vessel wall. Inhibition of the endothelial cell autocrine angiotensin system, with either the angiotensin-converting enzyme inhibitor lisinopril or the angiotensin II receptor antagonist sar1, ile8-angiotensin II, leads to increased endothelial cell migration and urokinase-like plasminogen activator (u-PA) activity (Bell, L., and J. A. Madri. 1990. Am. J. Pathol. 137:7-12). Inhibition of the autocrine angiotensin system with the converting-enzyme inhibitor or the receptor antagonist also leads to increased expression of the proto-oncogene c-src: pp60c-src mRNA increased 7-11-fold, c-src protein 3-fold, and c-src kinase activity 2-3-fold. Endothelial cell expression of c-src was constitutively elevated after stable infection with a retroviral vector containing the c-src coding sequence. Constitutively increased c-src kinase activity reconstituted the increases in migration and u-PA observed with angiotensin system interruption. Antisera to bovine u-PA blocked the increase in migration associated with increased c-src expression. These data suggest that increases in endothelial cell migration and plasminogen activator after angiotensin system inhibition are at least partially pp60c-src mediated. Elevated c-src expression with angiotensin system inhibition may act to enhance intimal wound closure and to reduce luminal thrombogenicity in vivo.

The protein product of the proto-oncogene c-src is a membrane-associated tyrosine kinase of unknown function. Identification of pp60c-src target membranes may elucidate the function of the c-src protein. The available evidence indicates that pp60c-src associates with distinct membranes within single cell types and has different distributions in different cell types. Our experiments demonstrate targeting of pp60c-src to an isolatable and biochemically identified membrane fraction in the neuroendocrine cell line PC12. The c-src protein was found to be specifically associated with synaptic vesicles since: (a) the pp60c-src immunofluorescent pattern overlapped with a synaptic vesicle marker, synaptophysin; (b) a significant proportion (44%) of the pp60c-src from PC12 but not fibroblast postnuclear supernatants was recovered in a small vesicle fraction; (c) an anti-synaptophysin cytoplasmic domain antibody immunodepleted ALL of the pp60c-src vesicles in this fraction, and (d) pp60c-src copurified during a 100-fold purification of PC12 synaptic vesicles. These results suggest a role for the c-src protein in the regulation of synaptic vesicle function.

Tyrosine phosphorylation is an important regulatory mechanism in response to the action of growth factors and oncogenes. Since many oncogenes code for tyrosine kinases, increased or altered oncogene expression may be reflected in increased tyrosine kinase activity. In a recent study (Hennipman et al., cancer Res., 49: 516-521, 1989), we found that the tyrosine kinase activity of the cytosolic and membrane fractions of malignant human breast tissue was significantly higher compared to the benign or the normal breast tissue. Moreover, the increase in the cytosolic fractions was found to be of prognostic value. In the present study we determined the protein tyrosine kinase (PTK) activity of another 72 breast cancer specimens, and it could be shown again that the PTK activity in ALL 72 of these tumors was elevated compared to normal controls. We characterized these cytosolic PTKs by anion exchange chromatography using fast protein liquid chromatography, and it could be shown that at least two different forms of PTK exist. Using antibodies against a number of known oncogene products, we could determine that at least 70% of the PTK activity in the cytosol originated from the presence of the c-src oncogene product. Both of the PTK activity peaks seen in the fast protein liquid chromatography patterns could be precipitated with the anti-Src antibody. Furthermore, using the MCF-7 breast cancer cell line, it could be shown that the antibody against c-src also precipitated a part of the cytosolic PTK activity. In normal human peripheral lymphocytes, no precipitation of the cytosolic and membrane PTK activity could be achieved using the anti-Src antibody. Inasmuch as the cytosolic PTK activity parallels the malignancy in breast tumors (Hennipman et al., cancer Res., 49: 516-521, 1989), and the majority of this activity is precipitated by anti-Src antibodies, the c-src protooncogene may play a key role in the manifestation of breast cancer.

Growth factor receptor tyrosine kinases can form stable associations with intracellular proteins that contain src homology (SH) 2 domains, including the p85 regulatory subunit of phosphatidylinositol (PI)-3 kinase. The activation of this enzyme by growth factors is evaluated in PC12 pheochromocytoma cells and NIH 3T3 fibroblasts expressing the pp140c-trk nerve growth factor (NGF) receptor (3T3-c-trk). NGF causes the rapid stimulation of PI-3 kinase activity detected in anti-phosphotyrosine, but not in anti-trk, immunoprecipitates. This effect coincides with the tyrosine phosphorylation of two proteins, with molecular masses of of 100 kd and 110 kd, that coimmunoprecipitate with p85. Similar phosphorylation patterns are induced when an immobilized fusion protein containing the amino-terminal SH2 domain of p85 is used to precipitate tyrosine-phosphorylated proteins. Thus, although NGF produces the rapid activation of PI-3 kinase through a mechanism that involves tyrosine phosphorylation, there is no evidence for tyrosine phosphorylation of p85, or for its ligand-dependent association with the NGF receptor. Perhaps another phosphoprotein may link the NGF receptor to this enzyme.

The expression of proteins that play a role in neuronal differentiation was examined in central nervous system (CNS) micromass embryo cell cultures and compared to expression at comparable developmental stages in vivo. The protein product of the src proto-oncogene (pp60c-src) has been postulated to have a specific role in development because, although it is expressed in many tissues, marked increases in amount and activity of pp60c-src occur in neurons at the time of differentiation. Another protein of interest, high molecular weight neurofilament (NF) protein, is found in differentiated neurons. In the present study, changes over time in the expression of these two proteins in vitro and in vivo were examined. In the micromass cell cultures, primary cells from day 12 rat embryo CNS are plated at high density and differentiate into neurons during five days in culture. Tissues from embryos grown in vivo were assessed at 12 and 17 days post-coitum. Proteins were quantified by PAGE separation of equal amounts of total protein followed by transfer to membranes, immunoblotting, and densitometric scanning of blots. Increases in the amount of both proteins with neuronal differentiation was shown. Protein kinase activity of immunoprecipitated pp60c-src also increased in cell cultures and in embryos. Similarity in patterns of expression between in vitro and in vivo tissue samples provides further evidence that the cultures closely simulate in vivo differentiation and are a useful system for examining expression of developmental genes in vitro.

The ability of the focal adhesion kinase (FAK) to integrate signals from extracellular matrix and growth factor receptors requires the integrity of Tyr397, a major autophosphorylation site that mediates the Src homology 2-dependent binding of Src family kinases. However, the precise roles played by FAK in specific Src-induced pathways, especially as they relate to oncogenic transformation, remain unclear. Here, we investigate the role of FAK in v-Src-induced oncogenic transformation by transducing temperature-sensitive v-Src (ts72v-Src) into p53-null FAK+/+ or FAK-/- mouse embryo fibroblasts (MEF). At the permissive temperature (PT), ts72v-Src induced abundant tyrosine phosphorylation, morphological transformation and cytoskeletal rearrangement in FAK-/- MEF, including the restoration of cell polarity, typical focal adhesion complexes, and longitudinal F-actin stress fibers. v-Src rescued the haptotactic, linear directional, and invasive motility defects of FAK-/- cells to levels found in FAK+/+ or FAK+/+-[ts72v-Src] cells, and, in the case of monolayer wound healing motility, there was an enhancement. Src activation failed to increase the high basal tyrosine phosphorylation of the Crk-associated substrate, CAS, found in FAK-/- MEF, indicating that CAS phosphorylation alone is insufficient to induce motility in the absence of FAK- or v-Src-induced cytoskeletal remodeling. Compared with FAK+/+[ts72v-Src] controls, FAK-/-[ts72v-Src] clones exhibited 7-10-fold higher anchorage-independent proliferation that could not be attributed to variations in either v-Src protein level or stability. Re-expression of FAK diminished the colony-forming activities of FAK-/-[ts72v-Src] without altering ts72v-Src expression levels, suggesting that FAK attenuates Src-induced anchorage independence. Our data also indicate that the enhanced Pyk2 level found in FAK-/- MEF plays no role in v-Src-induced anchorage independence. Overall, our data indicate that FAK, although dispensable, attenuates v-Src-induced oncogenic transformation by modulating distinct signaling and cytoskeletal pathways.

B(12) haplotype of the inbred chicken line CB (B12/B12) contains, like the bulk of chicken MHC(B) haplotypes, only a single dominantly expressed class I molecule (B-F). The peptide binding motifs for this major B-F12 molecule in chickens of Rous sarcoma regressor line CB (B12/B12) have been determined. Using stringent and relaxed motifs, several peptides were found in the v-src molecule of the PR-RSV-C, but most of these peptides are identical with that of endogenous c-src. Only the v-src C-tail peptide(517-524) (LPACVLEV) contains critical anchor amino acids (valine at positions 5 and 8) and shows a sequence different from the corresponding c-src peptide. This v-src C-tail peptide up-regulates expression of the B-F12 class I molecule on PBL, as assessed by FACS analysis, and stimulates T cell proliferation in a [3H]thymidine uptake assay. A protective effect of the immune response to LPACVLEV against RSV challenge was demonstrated in CB (B12/B12) chickens immunised with peptides encapsulated in liposomes.

Mushroom bodies (MB) are substructures in the Drosophila brain that are essential for memory. At present, MB anatomy is rather well described when compared to other brain areas, and elucidation of the genetic control of the development and projection patterns of MB neurons will be important to the understanding of their functions. We have performed a gain-of-function screen in order to identify genes that are involved in MB development. We drove expression of genes in MB neurons by crossing 2407 GAL4-driven UY element lines to lines containing an MB GAL4 source and UAS-GFP elements, and looked for defects in the MB structure. We have molecularly identified the genomic regions adjacent to the 26 positive UY insertions and found 18 potential genes that exhibit adult MB gain-of-function phenotypes. The proteins encoded by these candidate genes include, as well as genes with yet unknown function, transcription factors (e.g., tramtrack), nanos RNA-binding protein, microtubule-severing protein, vesicle trafficking proteins, axon guidance receptor, and the Src64 cytoplasmic protein tyrosine kinase. These genes are involved in key features of neuron cell biology. In three cases, tramtrack, nanos, and Src64, we show that the open reading frame located directly downstream of the UY P element is indeed the expressed target gene. Loss-of-function mutations of both ttk and Src64 lead to MB phenotypes proving that these genes are involved in the genetic control of MB development. Moreover, Src64 is shown here to act in a cell-autonomous fashion and is likely to interact with the previously-identified linotte/derailed receptor tyrosine kinase in MB development.

While some low molecular weight GTPases such as Ras and RhoA contribute to malignant transformation, a closely related family member, RhoB, has tumor-suppressive activity, but little is known about its regulation by oncogenes. In this study, we show that H-Ras, N-Ras, K-Ras, EGFR and ErbB2 but not v-Src suppress RhoB promoter transcriptional activity in NIH3T3 cells and human cancer cell lines derived from lung (A-549), pancreatic (Panc-1) and cervical (C33A) tumors. The EGFR and ErbB2 suppression of RhoB promoter activity is mediated by Ras. Furthermore, Ras suppresses basal as well as 5-fluorouracil (5-FU)-induced RhoB promoter activity and RhoB protein levels. Ectopic expression of RhoB, but not the closely related family member RhoA, antagonizes the ability of EGFR, ErbB2, H-Ras, N-Ras and K-Ras but not v-Src to transform NIH3T3 cells. Furthermore, RhoB, but not RhoA, inhibits colony formation and proliferation and induces anoikis in A-549 cells and Ras-transformed NIH3T3 cells. Finally, Ras-mediated resistance to 5-FU-induced apoptosis is reversed by RhoB. These results demonstrate that RhoB expression is negatively regulated by oncogenes that are prevalent in human cancers, and that ectopic expression of RhoB antagonizes the ability of these oncogenes to induce transformation. Taken together the data suggest that certain oncogenes suppress RhoB as one of the critical steps leading to malignant transformation.

A novel compound depudecin inducing the flat phenotype of ras- and src- transformed NIH3T3 cells at a concentration of 1 microgram/ml was isolated from the culture broth of Alternaria brassicicola. Based on its spectroscopic characteristics and X-ray crystallographic analysis of its bis-(1S)-(-)-camphanate, the structure of depudecin was determined to be (2R,3S,4S,5E,7S,8S,9R)-2,9- dihydroxy-3,4;7,8-diepoxy-undeca-5,10-diene.

Transformation of fibroblasts by oncogenic Src causes disruption of actin stress fibers and formation of invasive adhesions called podosomes. Because the small GTPase Rho stimulates stress fiber formation, Rho inactivation by Src has been thought to be necessary for stress fiber disruption. However, we show here that Rho[GTP] levels do not decrease after transformation by activated Src. Inactivation of Rho in Src-transformed fibroblasts by dominant negative RhoA or the Rho-specific inhibitor C3 exoenzyme disrupted podosome structure as judged by localization of podosome components F-actin, cortactin, and Fish. Inhibition of Rho strongly inhibited Src-induced proteolytic degradation of the extracellular matrix. Furthermore, development of an in situ Rho[GTP] affinity assay allowed us to detect endogenous Rho[GTP] at podosomes, where it colocalized with F-actin, cortactin, and Fish. Therefore, Rho is not globally inactivated in Src-transformed fibroblasts, but is necessary for the assembly and function of structures implicated in tumor cell invasion.

The oncogenic potential of the viral tyrosine kinase v-Src is due to its constitutive activity. Unlike the highly homologous cellular c-Src kinase, a C-terminal deletion of the regulatory tail and numerous point mutations make the viral kinase uncontrollable. To determine the basis of these differences, we analysed the structure and stability of v-Src and c-Src in vitro. We show that the stability of v-Src against unfolding and irreversible aggregation is significantly lower than that of c-Src. Furthermore, in v-Src hydrophobic residues are more exposed already in the native state. In consequence, v-Src was found to be inactive close to physiological temperatures. We thus suggest that the ensemble of mutations that transform c-Src into the oncogenic variant cause a concomitant destabilisation of the kinase.

We investigated the ability of overexpression of the c-myc proto-oncogene to potentiate in vitro transformation by model chemical carcinogens. A mouse c-myc gene was introduced to C3H 10T1/2 and Rat 6 embryo fibroblast cell lines via a retroviral vector containing the gene for neomycin resistance. Our present work extends previous findings by showing that individual vectored C3H 10T1/2 clones have enhanced (two-fold to sevenfold) sensitivity to benzo[a]pyrene (BP) and N-methyl-N-nitro-N -nitrosoguanidine (MNNG). Rat 6 clones acquiring the c-myc gene display various degrees of altered morphology. They form orderly but densely packed cells, grow to higher saturation density, and yield microcolonies in soft agar. The degree of altered growth properties is directly correlated with the level of c-myc expression. Transient exposure of c-myc-expressing clones to BP and MNNG induced the formation of distinct, large colonies in soft agar, whereas the untreated cells formed microcolonies and the parental Rat 6 cells remained single cells in soft agar. We also demonstrated that the degree of responsiveness to chemical carcinogens of the clones correlates with their ability to form microcolonies in soft agar. These cells overexpressing c-myc may be used as a model system to study the interaction between oncogenes and chemical carcinogens in the process of multistage carcinogenesis.

Substrate specificities of two protein tyrosine kinases were compared using nine undecapeptides modeled after human gastrin. Using the proto-oncogene product, p56lck, Vmax decreased with the distance between glutamate and tyrosine, whereas for the oncogene product, pp60v-src there was no relation. For pp60v-src there was a precipitous rise in Km, from 2.9 to 20 mM when glutamate was greater than three residues away from tyrosine. For p56lck, Km was a minimum when glutamate occupied either a position 3 residues, or both positions 3 and 4, N-terminal to tyrosine. An important factor in enzyme recognition of these peptides is glutamate three residues N-terminal to tyrosine.

BALB/3T3 cells transfected with plasmids pcDNA3.1-[S-ras(Q(61)K)] of shrimp Penaeus japonicus were applied to reveal a complex of p120-GAP/syndecan-2 being highly expressed upon transformation. Of interest, most of the p120-GAP/syndecan-2 complex was localized at caveolae, a membrane microdomain enriched with caveolin-1. To confirm the molecular interaction between syndecan-2 and p120-GAP, we further purified p120-GAP protein from mouse brains by using an affinity column of HiTrap-RACK1 and expressed mouse RACK1-encoded fusion protein and mouse syndecan-2-encoded fusion protein in bacteria. We report molecular affinities exist between p120-GAP and RACK1, syndecan-2 and RACK1 as well as p120-GAP and syndecan-2. The selective affinity between p120-GAP and syndecan-2 was found to be sufficient to detach RACK1. The p120-GAP/syndecan-2 complex was demonstrated to keep Src tyrosine kinase in an activated form. On the other hand, the syndecan-2/RACK1 complex was found to have Src in an inactivated form. These data indicate that the p120-GAP/syndecan-2 complex at caveolae could provide a docking site for Src to transmit tyrosine signaling, implying that syndecan-2/p120-GAP functions as a tumor promoter upon transformation with oncogenic ras of shrimp P. japonicus.

HiTrap-syndecan-2/p120-GAP and HiTrap-syndecan-2/RACK1 affinity columns were applied to reveal that Src tyrosine kinase was highly expressed in BALB/3T3 cells transfected with plasmids pcDNA3.1-[S-ras(Q(61)K)] of shrimp Penaeus japonicus. Both columns were effective to isolate Src tyrosine kinase. The selective molecular affinity for Src was found to be stronger with HiTrap-syndecan-2/RACK1, as revealed with competitive RACK1 to dislodge Src from HiTrap-syndecan-2/p120-GAP. We thus challenged the syndecan-2/p120-GAP and syndecan-2/RACK1 with GTP-K(B)-Ras(Q(61)K). The reaction between RACK1 and syndecan-2 was sustained in the presence of mutant Ras proteins, but not the reaction between p120-GAP and syndecan-2. In the presence of syndecan-2, GTP-K(B)-Ras(Q(61)K) exhibited sufficient reactivity with p120-GAP to discontinue the reaction between p120-GAP and syndecan-2. But the interference of mutant Ras disappeared when Src tyrosine kinase was introduced to stabilize the syndecan-2/p120-GAP complex. On the other hand, in the absence of syndecan-2, GTP-K(B)-Ras(Q(61)K) was found to react with RACK1. The reaction between GTP-K(B)-Ras(Q(61)K) and RACK1 could provide a mechanism to deprive RACK1 for the organization of syndecan-2/RACK1 complex and to facilitate the formation of syndecan-2/p120-GAP complex, as well as to provide docking sites for Src signaling upon transformation with oncogenic ras.

Many studies have shown the importance of altered cellular proto-oncogene expression in contributing to changes in cell survival, cell transformation, and cell cycle progression. In these experiments we examined the effects of total-body exposure of BCF1 mice to gamma rays (3 Gy) in modulating expression of cellular oncogenes in both gut and liver tissues. We selected specific cellular oncogenes (c-fos, c-myc, c-src, and c-H-ras), based on their normal expression in liver and gut tissues from untreated mice. As early as 5 min following whole-body exposure of BCF1 mice to gamma rays we detected induction of mRNA specific for c-src and c-H-ras in both liver and gut tissues. Accumulation of c-fos-RNA was slightly decreased in gut but was unaffected in liver tissue from irradiated mice relative to untreated controls. Accumulation of c-myc mRNA was unaffected in ALL tissues examined. These experiments document that modulation of cellular proto-oncogene expression can occur as an early event in tissues following irradiation and suggest that this modulation may play a role in radiation-induced cellular changes.

The MUC1 heterodimeric transmembrane glycoprotein is aberrantly overexpressed by most human carcinomas. The MUC1 C-terminal subunit localizes to mitochondria and blocks stress-induced activation of the intrinsic apoptotic pathway. How MUC1 is delivered to mitochondria is not known. The present studies demonstrate that MUC1 forms intracellular complexes with HSP70 and HSP90. We show that the MUC1 cytoplasmic domain binds directly to HSP70 in vitro. By contrast, binding of MUC1 to HSP90 in vitro is induced by c-Src-mediated phosphorylation of the MUC1 cytoplasmic domain. c-Src also increases binding of MUC1 to HSP90 in cells. In concert with these results, we show that heregulin (HRG), a ligand for ErbB receptors, activates c-Src and, in turn, stimulates binding of MUC1 to HSP90. We also show that inhibitors of c-Src or HSP90 block HRG-induced targeting of MUC1 to mitochondria and integration of MUC1 into the mitochondrial outer membrane. These findings indicate that MUC1 is delivered to mitochondria by a mechanism involving activation of the ErbB receptor-->c-Src pathway and transport by the molecular chaperone HSP70/HSP90 complex.

Src, a proto-oncogene, has been strongly implicated in the growth, progression and metastasis of a number of human cancers. Its role in lung cancer is, however, still unknown. In the present study, we assessed the expression of Src in three different human lung adenocarcinoma cell lines (PC-9, PC14PE6, A549), and explored the effect of a novel Src kinase inhibitor, M475271, on the behavior of the cell lines. The three cell lines expressed various levels of auto-phosphorylated Src. While M475271 reduced Src-phosphorylation and invasiveness of ALL three cell lines, it inhibited the proliferation of PC-9 and A549 cells with highly phosphorylated Src, but not PC14PE6 cells. We further examined the effect of M475271 on subcutaneous tumors and lung metastasis caused by PC-9 and/or A549 cells in NK-cell depleted SCID mice. Daily oral treatment with M475271 inhibited the growth of subcutaneous tumors with PC-9 and A549 cells via inhibition of tumor cells proliferation, VEGF production and/or vascularization in the mice in a dose-dependent manner. In the metastasis model with A549 cells, the lung weight in the M475271 (50 mg/kg)-treated group was less than that of the control group, despite no difference in the number of metastatic nodules. Our results suggest that inhibition of tyrosine kinase Src by M475271 could reduce the growth, invasion and VEGF-mediated neovascularization of lung adenocarcinoma cells, resulting in inhibition of growth of subcutaneous tumors and lung metastasis. Therefore, a novel Src tyrosine kinase inhibitor, M475271, might be helpful for controlling the progression of human lung adenocarcinoma.

Rat fibroblast 3Y1 cells transformed by Rous sarcoma virus (RSV) or transfected with the v-src gene showed a highly constitutive v-src gene expression. Simultaneously, marked increases in the cellular level of nerve growth factor (NGF) mRNA and NGF content in the culture medium were observed. The levels of NGF mRNA and NGF secreted into the medium were correlated with the expression level of v-src mRNA gene in both transformants and control 3Y1 cells. These results suggest that v-src gene expression is relevant to regulation of NGF synthesis in rat 3Y1 fibroblasts.

Because functionally significant substrates for the tyrosyl protein kinase activity of pp60v-src are likely to include membrane-associated proteins involved in normal growth control, we have tested the hypothesis that pp60v-src could phosphorylate and alter the signaling activity of transmembrane growth factor receptors. We have found that the epidermal growth factor (EGF) receptor becomes constitutively phosphorylated on tyrosine in cells transformed by the src oncogene and in addition displays elevated levels of phosphoserine and phosphothreonine. High-performance liquid chromatography phosphopeptide mapping revealed two predominant sites of tyrosine phosphorylation, both of which differed from the major sites of receptor autophosphorylation; thus, the src-induced phosphorylation is unlikely to occur via an autocrine mechanism. To determine whether pp60v-src altered the signaling activity of the EGF receptor, we analyzed the tyrosine phosphorylation of phospholipase C-gamma, since phosphorylation of this enzyme occurs in response to activation of the EGF receptor but not in response to pp60v-src alone. We found that in cells coexpressing pp60v-src and the EGF receptor, phospholipase C-gamma was constitutively phosphorylated, a result we interpret as indicating that the signaling activity of the EGF receptor was altered in the src-transformed cells. These findings suggest that pp60v-src-induced alterations in phosphorylation and function of growth regulatory receptors could play an important role in generating the phenotypic changes associated with malignant transformation.

We have investigated the ability of the v-src oncogene to block the differentiation of the murine myeloid progenitor cell line 32D cl3. In response to granulocyte-colony stimulating factor (G-CSF), 32D cl3 cells are induced to differentiate into mature granulocytes (Valtieri et al., 1987). In contrast, no differentiation was observed following G-CSF treatment of 32D cl3 cells infected with a murine retrovirus carrying the wild-type v-src oncogene. Furthermore, cells infected with a v-src temperature-sensitive (ts) mutant did not differentiate at the permissive temperature, however, at the nonpermissive temperature G-CSF induced granulocytic differentiation. Differentiation of 32D cl3 cells infected with ts WP31A (ts LA31A src gene inserted into amphotropic murine leukemia virus 4070A; Anderson et al., 1987) occurred with the same kinetics as uninfected 32D cl3 cells. Temperature-shift experiments indicate that after 72 hours of treatment with G-CSF at the nonpermissive temperature, approximately half of the 32D cl3 cells infected with ts WP31A virus become committed to differentiation. Prior to that time, activation of v-src by shifting the cells to the permissive temperature resulted in the presence of only undifferentiated blast cells after six days in culture. In contrast to normal 32D cl3 cells, cells infected with the wild-type v-src were tumorigenic when injected into nu/nu Swiss mice. Lesions appeared in the spleen, liver, kidney, lungs and lymph nodes following subcutaneous injection. Growth factor-independent cells were recovered from the tumor, spleen, bone marrow and a lymph node of tumor-bearing nude mouse. Analysis of the proviral integration site by inverse polymerase chain reaction (PCR) demonstrated that the tumor cells were of donor cell origin.

To approach the transmembrane signaling pathway in the cell-to-cell adherens junctions (AJ), AJ-specific tyrosine phosphorylation was analyzed. When various types of rat adult tissues were pretreated with sodium orthovanadate, a potent inhibitor of tyrosine phosphatase, immunofluorescence microscopy showed that anti-phosphotyrosine polyclonal antibody specifically stained the undercoat of the cell-to-cell AJ. This indicates that the tyrosine kinase activity is elevated at the undercoat of the cell-to-cell AJ of adult tissues. To identify tyrosine kinases responsible for the high level of tyrosine phosphorylation at AJ, we have performed in vitro phosphorylation experiments with cell-to-cell AJ isolated from rat liver (Tsukita, Sh. and Sa. Tsukita. 1989. J. Cell Biol. 108:31-41) and immunoblotting analyses with specific antibodies for tyrosine kinases. As a result, three proto-oncogenic tyrosine kinases of src family, c-yes, c-src, and lyn kinases, were identified as major tyrosine kinases in the cell-to-cell AJ of hepatocytes. Furthermore, it was immunofluorescently shown that at least two of these kinases, c-yes and c-src kinases, were enriched at the cell-to-cell AJ of various types of cells including hepatocytes. Based on these findings, it is concluded that, in various types of cells, specific proto-oncogenic tyrosine kinases of src-family (c-yes and c-src) are enriched to work as signal mediators in the cell-to-cell AJ where the level of tyrosine phosphorylation is elevated.

To further characterize the gene structure of the proto-oncogene c-src and the mechanism for the genesis of the v-src sequence in Rous sarcoma virus, we have analyzed genomic and cDNA copies of the chicken c-src gene. From a cDNA library of chicken embryo fibroblasts, we isolated and sequenced several overlapping cDNA clones covering the full length of the 4-kb c-src mRNA. The cDNA sequence contains a 1.84-kb sequence downstream from the 1.6-kb pp60c-src coding region. An open reading frame of 217 amino acids, called sdr (src downstream region), was found 105 nucleotides from the termination codon for pp60c-src. Within the 3 noncoding region, a 39-bp sequence corresponding to the 3 end of the RSV v-src was detected 660 bases downstream of the pp60c-src termination codon. The presence of this sequence in the c-src mRNA exon supports a model involving an RNA intermediate during transduction of the c-src sequence. The 5 region of the c-src cDNA was determined by analyzing several cDNA clones generated by conventional cloning methods and by polymerase chain reaction. Sequences of these chicken embryo fibroblast clones plus two c-src cDNA clones isolated from a brain cDNA library show that there is considerable heterogeneity in sequences upstream from the c-src coding sequence. Within this region, which contains at least 300 nucleotides upstream of the translational initiation site in exon 2, there exist at least two exons in each cDNA which fall into five cDNA classes. Four unique 5 exon sequences, designated exons UE1, UE2, UEX, and UEY, were observed. ALL of them are spliced to the previously characterized c-src exons 1 and 2 with the exception of type 2 cDNA. In type 2, the exon 1 is spliced to a novel downstream exon, designated exon 1a, which maps in the region of the c-src DNA defined previously as intron 1. Exon UE1 is rich in G+C content and is mapped at 7.8 kb upstream from exon 1. This exon is also present in the two cDNA clones from the brain cDNA library. Exon UE2 is located at 8.5 kb upstream from exon 1. The precise locations of exons UEX and UEY have not been determined, but both are more than 12 kb upstream from exon 1. The existence and exon arrangements of these 5 cDNAs were further confirmed by RNase protection assays and polymerase chain reactions using specific primers. Our findings indicate that the heterogeneity in the 5 sequences of the c-src mRNAs results from differential splicing and perhaps use of distinct initiation sites. ALL of these RNAs have the potential of coding for pp60c-src, since their 5 exons are ALL eventually joined to exon 2.

Activation of the Ha-ras proto-oncogene, but not the Ki-ras or N-ras genes, has been found in mammary gland carcinomas induced in female rats by a single dose of methylnitrosourea (MNU). Here we show that a 10-kb restriction fragment containing the Ha-ras gene was extensively methylated by MNU in DNA isolated from mammary glands of female rats 4 h after carcinogen treatment. Fragments of similar size containing either the Ki-ras or N-ras genes were methylated less extensively. The extent of methylation of the three ras genes by MNU correlated with their transcriptional activity. These results suggest that the extent of interaction of a carcinogen with an oncogene, which depends on its transcriptional activity, may be a factor in determining whether the gene is mutated during the initiation of carcinogenesis.

The saimiri transforming protein oncogene, called STP-A, of herpesvirus saimiri (HVS) subgroup A is not required for viral replication but is required for lymphoid cell immortalization in culture and lymphoma induction in primates. Here we report that STP-A interacts with cellular tumor necrosis factor receptor-associated factors (TRAF2 and TRAF6) and Src family protein tyrosine kinases (SF-PTKs) in a genetically and functionally separable manner and that each interaction constitutively elicits independent cellular signal transduction. The amino-terminal and central proline-rich motifs of STP-A were responsible for TRAF6 and TRAF2 interactions, respectively, and STP-A and TRAF6 interaction contributed to the majority of NF-kappaB activation, whereas STP-A and TRAF2 interaction played a minor role in NF-kappaB activation. On the other hand, interaction of STP-A with SF-PTKs through its SH2 binding motif effectively elicited AP-1 and NF-AT transcription factor activity. One cellular gene targeted by STP-A is intercellular adhesion molecule 1 (ICAM-1), which participates in a wide range of inflammatory and immune responses. Both TRAF and SF-PTK signal transductions induced by STP-A were required for the marked increase of ICAM-1 expression. These results demonstrate that the viral oncogene STP-A independently targets two vital cellular signaling molecules and that these activities likely contribute to HVS-mediated lymphoid cell immortalization in culture and lymphoma induction in primates.

Repression of the tyrosine kinase activity of the cellular src protein (pp60c-src) depends on the phosphorylation of a tyrosine residue (Tyr-527) near the carboxy terminus. Tyr-527 is located 11 residues C terminal from the genetically defined end of the kinase domain (Leu-516) and is therefore in a negative regulatory region. Because the precise sequence of amino acids surrounding Tyr-527 appears to be unimportant for regulation, we hypothesized that the conformational constraints induced by phosphorylated Tyr-527 may require the correct spacing between the kinase domain (Leu-516) and Tyr-527. In this report, we show that deletions at residue 518 of two, four, or seven amino acids or insertions at this residue of two or four amino acids activated the kinase activity and thus the transforming potential of pp60c-src. As is the case for the prototype transforming variant, pp60527F, activation caused by these deletions or insertions was abolished when Tyr-416 (the autophosphorylation site) was changed to phenylalanine. In comparison with wild-type pp60c-src, the src proteins containing the alterations at residue 518 showed a lower phosphorylation state at Tyr-527 regardless of whether residue 416 was a tyrosine or a phenylalanine. Mechanisms dealing with the importance of spacing between the kinase domain and Tyr-527 are discussed.

We have studied by in situ hybridization the mRNA expression of the pp60c-src proto-oncogene in grafts of foetal striatal neurons implanted into the ibotenic acid lesioned adult rat neostriatum. Animals were studied at 5, 10, 15, 30 days and 4 months after transplantation. Using 35S-labelled specific oligonucleotide probes, high levels of mRNA for pp60c-src+ but not pp60c-src- were found in the normal embryonic striatum and in striatal grafts up to 1 month after implantation; no hybridization signal was obtained in the normal adult striatum or in 4-month old grafts. The data suggest that pp60c-src+ is highly expressed in developing, but not mature, neurons and may play a fundamental role in the maturation and differentiation of grafted embryonic striatal neurons.

Recent work has implicated the activated ras oncogene, whose gene product is a G-protein located in the plasma membrane, as well as the activated raf oncogene, whose gene product is a membrane-associated protein kinase, in contributing to radioresistance. Another transforming oncogene whose gene product is localized to the plasma membrane is v-src. We have examined a rat fibroblast line (RAT-1) infected with an avian sarcoma virus carrying a temperature-sensitive mutation in the v-src tyrosine kinase domain (LA-24). At 40 degrees C, LA-24 cells have a flat morphology and grow as a contact-inhibited monolayer, while at 35 degrees C, LA-24 cells have a transformed morphology, lose contact inhibition, grow in soft agar, and exhibit 3.5-fold higher tyrosine kinase activity. The parental RAT-1 line, not infected by the virus, grows at both temperatures as a contact-inhibited monolayer. This well-characterized system represents a good model for examining the effect of v-src transformation on radiosensitivity. RAT-1 and LA-24 cells grown at 35 and 40 degrees C were irradiated with graded doses of radiation, and clonogenic survival was assayed. For LA-24 cells grown at 35 and 40 degrees C, and for RAT-1 cells grown at 35 and 40 degrees C, calculated D0, n, alpha, and beta values did not differ significantly. To determine whether there might be differences in radiation damage repair capacity too subtle to detect by comparing radiation survival curves, sublethal damage repair capacity was assessed. There was no difference in sublethal damage repair capacity for LA-24 cells grown at 35 or 40 degrees C. Other studies have associated multidrug resistance with radioresistance. We have examined the radiation sensitivity of two colchicine-resistant LA-24 clones with four- to fivefold amplification of the P-glycoprotein gene, which are four-to fivefold more resistant to colchicine than the parental LA-24 line. In these multidrug-resistant clones, v-src activation does appear to increase radiation resistance. This did not appear to be due to alteration in cell cycle kinetics. We conclude that oncogene activation, or even protein kinase activity per se, does not necessarily lead to radiation resistance. Rather, radiation resistance following oncogene activation depends upon the oncogene and cell line studied, and perhaps upon specific protein phosphorylation.

Increased activity of the proto-oncogene c-Src and elevated levels of integrin alpha(v)beta(3) are found in melanomas and multiple carcinomas. Regulation of c-Src involves "priming" through disruption of intramolecular interactions followed by "activation" through phosphorylation in the kinase domain. Interactions with overexpressed receptor tyrosine kinases or mutations in the SRC gene can induce priming of c-Src in cancer. Here, we show that alpha(v)beta(3) promotes activation of primed c-Src, causing enhanced phosphorylation of established Src substrates, survival, proliferation, and tumor growth. The beta(3) cytoplasmic tail is required and sufficient for integrin-mediated stimulation of ALL these events through a mechanism that is independent of beta(3) tyrosine phosphorylation. Instead, experiments using Src variants containing the v-Src Src homology 3 (SH3) domain and using mutant beta(3) subunits indicate that a functional interaction of the beta(3) cytoplasmic tail with the c-Src SH3 domain is required. These findings delineate a novel integrin-controlled oncogenic signaling cascade and suggest that the interaction of alpha(v)beta(3) with c-Src may represent a novel target for therapeutic intervention.

SWAP-70 is a phosphatidylinositol trisphosphate (PtdIns(3,4,5)P(3)) binding protein, which acts in F-actin rearrangement. The role of SWAP-70 in oncogenic transformation of mouse embryo fibroblasts (MEFs) by v-Src was examined by use of MEFs defective in SWAP-70. v-Src morphologically transformed MEFs lacking SWAP-70, but growth of the transformed cells in culture was slower than that of cells supplemented with exogenous SWAP-70. The v-Src-transformed MEFs deficient in SWAP-70 were unable to grow in soft agar while those expressing SWAP70 readily formed colonies, suggesting that SWAP-70 is required for anchorage independent growth of v-Src transformed MEFs. When transplanted in nude mice, tumors formed by the v-Src transformed SWAP-70(-/-) MEFs were smaller than those formed by cells expressing exogenous SWAP-70. These results suggest that SWAP-70 may be required for oncogenic transformation and contributes to cell growth in MEFs transformed by v-Src.

Src-specific activity has been reported to be elevated in a high percentage of colon cancer cell lines and tumors, but the underlying mechanisms are largely unknown. In this study, we report that, in the seven cancer cell lines tested, Src-specific activity was elevated (5.2- to 18.7-fold) relative to normal colon cells (FHC). This activation of Src correlated with reduced phosphorylation at Y530 of Src, whereas there was no significant change in the level of phosphorylation at Y419. The membrane tyrosine phosphatase activity for a Src family-specific phosphopeptide substrate FCP (Fyn COOH-terminal peptide phosphorylated by Csk) was greatly increased in the cancer cells and was attributed to PTP1B in most of the cell lines. Membrane PTP1B protein levels were also greatly increased. Overexpression of PTP1B increased Src specific activity in colon cancer cells by reducing phosphorylation at Y530 of Src. It also increased anchorage-independent cell growth and this increase was blocked by the Src inhibitor PP2 and Src small interfering RNA (siRNA). Down-regulating PTP1B activity by PTP1B inhibitor CinnGEL 2Me or knocking down PTP1B using siRNA also reduced Src kinase activity and colony formation ability of colon cancer cells. PTP1B siRNA reduced tumor growth in nonobese diabetic/severe combined immunodeficient mice. This study suggests that (a) PTP1B can act as an important activator of Src in colon cancer cells via dephosphorylation at Y530 of Src and (b) elevated levels of PTP1B can increase tumorigenicity of colon cancer cells by activating Src.

Tyrosine phosphorylation of proteins was examined in NIH3T3 cells transformed by an oncogenic form of the trk protein. Proteins of 148, 140, 70, and 55 kDa were phosphorylated on tyrosine residues in trk-transformed cells but not control NIH3T3 cells. The 70-kDa protein may represent the trk oncogene protein itself which was shown to be tyrosine-phosphorylated in vivo using trk-specific antiserum. Phospholipase C-gamma 1 (PLC-gamma 1) was also found to be constitutively tyrosine-phosphorylated in trk-transformed cells and the trk protein co-immunoprecipitated with PLC-gamma. The GTPase-activating protein of ras (GAP) and the 62-kDa GAP-associated protein were tyrosine-phosphorylated in trk-transformed cells, and a lesser amount of trk co-immunoprecipitated with GAP relative to with PLC-gamma. The trk oncogene product bound specifically to a bacterially expressed fusion protein containing the src homology domains of PLC-gamma. The data suggest a significant role for PLC-gamma in intracellular signaling by the trk oncogene.

SRC-3/AIB1 is a steroid receptor coactivator with potent growth-promoting activity, and its overexpression is sufficient to induce tumorigenesis. Previous studies indicate that the cellular level of SRC-3 is tightly regulated by both ubiquitin-dependent and ubiquitin-independent proteasomal degradation pathways. Atypical protein kinase C (aPKC) is frequently overexpressed in cancers. In the present study, we show that aPKC phosphorylates and specifically stabilizes SRC-3 in a selective ER-dependent manner. We further demonstrate that an acidic residue-rich region in SRC-3 is an important determinant for aPKC-mediated phosphorylation and stabilization. The mechanism of the aPKC-mediated stabilization appears due to a decreased interaction between SRC-3 and the C8 subunit of the 20S core proteasome, thus preventing SRC-3 degradation. Our results demonstrate a potent signaling mechanism for regulating SRC-3 levels in cells by coordinate enzymatic inhibition of both ubiquitin-dependent and ubiquitin-independent proteolytic pathways.

The tyrosine kinase c-Src is upregulated in various human cancers irrespective of its negative regulator Csk, but the regulatory mechanisms remain unclear. Here, we show that a lipid raft-anchored Csk adaptor, Cbp/PAG, is directly involved in controlling the oncogenicity of c-Src. Using Csk-deficient cells that can be transformed by c-Src overexpression, we found that Cbp expression is markedly downregulated by c-Src activation and re-expression of Cbp efficiently suppresses c-Src transformation as well as tumorigenesis. Cbp-deficient cells are more susceptible to v-Src transformation than their parental cells. Upon phosphorylation, Cbp specifically binds to activated c-Src and sequesters it in lipid rafts, resulting in an efficient suppression of c-Src function independent of Csk. In some human cancer cells and tumors, Cbp is downregulated and the introduction of Cbp significantly suppresses tumorigenesis. These findings indicate a potential role for Cbp as a suppressor of c-Src-mediated tumor progression.

The present study aimed to investigate the effect of proto-oncogene c-src on the viability of rat spermatogonial stem cells from 9 day-old rat in vitro. MTT method was used to observe the viability of the spermatogonial stem cells treated with antisense c-src oligodeoxynucleotides (ODNs) in vitro; RT-PCR was utilized to observe the expression of c-src mRNA and Western blot was used to observe the protein expressions of pp60c-src and phosphorylated signal transducer and activator of transcription-3 (p-STAT3). Compared with that in control group, the viability of spermatogonial stem cells decreased by 8.1% (P<0.05) and the expression of c-src mRNA decreased significantly after treatment with 10 mumol/L antisense c-src ODNs for 12 h. Compared with that in the control group, the protein expressions of pp60c-src and p-STAT3 decreased by 33.8% and 45.3% (both P<0.01), respectively, in the spermatogonial stem cells after being transfected with antisense c-src ODNs. The results suggest that proto-oncogene c-src regulates the viability of rat spermatogonial stem cells through p-STAT3.

Elevated activity of Src, the first characterized protein-tyrosine kinase, is associated with progression of many human cancers, and Src has attracted interest as a therapeutic target. Src is known to act in various receptor signaling systems to impact cell behavior, yet it remains likely that the spectrum of Src protein substrates relevant to cancer is incompletely understood. To better understand the cellular impact of deregulated Src kinase activity, we extensively applied a mass spectrometry shotgun phosphotyrosine (pTyr) proteomics strategy to obtain global pTyr profiles of Src-transformed mouse fibroblasts as well as their nontransformed counterparts. A total of 867 peptides representing 563 distinct pTyr sites on 374 different proteins were identified from the Src-transformed cells, while 514 peptides representing 275 pTyr sites on 167 proteins were identified from nontransformed cells. Distinct characteristics of the two profiles were revealed by spectral counting, indicative of pTyr site relative abundance, and by complementary quantitative analysis using stable isotope labeling with amino acids in cell culture (SILAC). While both pTyr profiles are replete with sites on signaling and adhesion/cytoskeletal regulatory proteins, the Src-transformed profile is more diverse with enrichment in sites on metabolic enzymes and RNA and protein synthesis and processing machinery. Forty-three pTyr sites (32 proteins) are predicted as major biologically relevant Src targets on the basis of frequent identification in both cell populations. This select group, of particular interest as diagnostic biomarkers, includes well-established Src sites on signaling/adhesion/cytoskeletal proteins, but also uncharacterized sites of potential relevance to the transformed cell phenotype.

Sphingosine kinase 1 (SPHK1) is overexpressed in solid tumors and leukemia. However, the mechanism of SPHK1 overexpression by oncogenes has not been defined. We found that v-Src-transformed NIH3T3 cells showed a high SPHK1 mRNA, SPHK1 protein and SPHK enzyme activity. siRNA of SPHK1 inhibited the growth of v-Src-NIH3T3, suggesting the involvement of SPHK1 in v-Src-induced oncogenesis. v-Src-NIH3T3 showed activations of protein kinase C-alpha, signal transducers and activators of transcription 3 and c-Jun NH(2)-terminal kinase. Their inhibition suppressed SPHK1 expression in v-Src-NIH3T3, whereas their overexpression increased SPHK1 mRNA in NIH3T3. Unexpectedly, the nuclear run-on assay and the promoter analysis using 5 -promoter region of mouse SPHK1 did not show any significant difference between mock- and v-Src-NIH3T3. Furthermore, the half-life of SPHK1 mRNA in mock-NIH3T3 was nearly 15 min, whereas that of v-Src-NIH3T3 was much longer. Examination of two AU-rich region-binding proteins, AUF1 and HuR, that regulate mRNA decay reciprocally, showed decreased total AUF1 protein associated with increased tyrosine-phosphorylated form and increased serine-phosphorylated HuR protein in v-Src-NIH3T3. Modulation of AUF1 and HuR by their overexpression or siRNA revealed that SPHK1 mRNA in v-Src- and mock-NIH3T3 was regulated reciprocally by these factors. Our results showed, for the first time, a novel mechanism of v-Src-induced SPHK1 overexpression.

The stimulation of quiescent murine fibroblasts by growth factors and by phorbol esters results in a rapid and transient transcriptional activation of a large group of so-called immediate early genes. Several such genes were found to be induced in chicken embryo fibroblasts following activation of a temperature sensitive (ts) Rous sarcoma virus v-src mutant following temperature shift (Simmons et al., 1989). In contrast, the classical immediate early genes c-myc, c-fos and c-jun were essentially uninducible upon activation of a ts v-src mutant in rat-1 fibroblasts (Welham et al., 1990). We have cloned 9 cDNAs of genes that are rapidly and transiently inducible in rat fibroblasts by ts v-src mutants, and by a ts Fujinami sarcoma virus v-fps mutant. Six of these cDNAs are derived from the known immediate early genes NGFI-A, KC, c-fos, tissue factor, PC4 and ornithine decarboxylase; the other three cDNAs have not been described before. These 9 genes showed individual profiles of inducibility by fetal calf serum, epidermal growth factor (EGF) and by phorbol esters. Their response to the retroviral oncogenic protein-tyrosine kinases correlated best with the one to EGF, suggesting a common pathway of signal transduction. c-fos did not respond strongly to this pathway but was well induced by fetal calf serum. NGFI-A, however, was induced to a similar extent by ALL activators tested. Furthermore, we demonstrated that the induction of several of these genes by the retroviral oncogenic protein-tyrosine kinases is rapid, direct and occurs at the transcriptional level.

Prostate cancer is the most frequently diagnosed cancer in American men. We have previously demonstrated that Src mediates androgen-independent proliferation in prostate cancer. We sought to investigate the Src-mediated oncogenic pathways and tumor biology using AZD0530, a novel Src family kinase/Abl dual-kinase inhibitor that is entering phase II clinical trials. We show that while both Src and Abl are expressed in ALL prostate cancer cell lines, Src but not Abl is activated in the prostate. Furthermore, Src activation is inhibited by AZD0530 in a rapid and dose-dependent manner. We show that Src mediates cell proliferation in DU145 and PC3 cells at the G1 phase of cell cycle. Src inhibition resulted in decreased binding of beta-catenin to the promoters of G1 phase cell cycle regulators cyclin D1 and c-Myc. C-Myc may also be regulated at the protein level by extracellular signal-regulated kinase 1/2 and GSK3beta. Cell motility factors focal adhesion kinase, p130CAS and paxillin activation in DU145 and PC3 cells were also inhibited. Administration of AZD0530 in mice reduced orthotopic DU145 xenograft growth by 45%. We have further delineated the Src-mediated oncogenic growth and migration pathways in prostate cancer and established mechanistic rationale for Src inhibition as novel therapy in the treatment of prostate cancer.

Rat-1 fibroblasts transformed with the v-src oncogene show a 6-fold increase in the apparent amount of an inositol polyphosphate which has a high performance liquid chromatography (HPLC) elution characteristic of the D/L-myo-inositol 1,4,5,6-tetrakisphosphate enantiomeric pair (Johnson, R.M., Wasilenko, W.J., Mattingly, R.R., Weber, M.J., and Garrison, J.C. (1989) Science 246, 121-124). By chemical and enzymatic analysis, the structure of this compound produced in both normal and v-src-transformed rat-1 fibroblasts has been determined to be principally D-myoinositol 1,4,5,6-tetrakisphosphate (D-Ins(1,4,5,6)P4). Chronic stimulation with endothelin-1 in the presence of Li+ significantly increased the amount of D/L-Ins(1,4,5,6)P4 only in the v-src-transformed rat-1 cells, suggesting that production of this compound may be remotely coupled to long term agonist-induced phosphatidylinositol turnover. Further evidence for such a link is provided by the progressive loss of D-Ins(1,4,5,6)P4 from the normal cells deprived of serum stimulation. To define a possible synthetic pathway for D-Ins(1,4,5,6)P4, cytosolic extracts of normal and v-src-transformed cells were incubated with [3H]inositol polyphosphates, and the reaction products were identified by HPLC elution and chemical analysis. Although inositol 1,3,4-trisphosphate 6-kinase activity was prominent in extracts of both normal and transformed cells, only the cytosol from v-src-transformed cells ultimately formed measurable amounts of D-Ins(1,4,5,6)P4 from [3H]inositol 1,3,4-trisphosphate. Approximately 6% of 0.1 microM inositol 1,3,4-trisphosphate was converted to D-Ins(1,4,5,6)P4 during a 2-h incubation at 37 degrees C. Inositol pentakisphosphate was identified as a likely intermediate in this conversion, and extracts of both normal and transformed cells converted [3H]inositol 1,3,4,5,6-pentakisphosphate to D-Ins(1,4,5,6)P4. The synthetic pathway described is consistent with the long term regulation of D/L-Ins(1,4,5,6)P4 levels in rat-1 fibroblasts seen in response to src transformation, serum withdrawal, and chronic endothelin treatment, and identifies several new potential interactions between the pathways of inositol polyphosphate metabolism and those of src transformation.

The increase in glucose transport that occurs when chicken embryo fibroblasts (CEFs) are transformed by src is associated with an increase in the amount of type 1 glucose transporter protein, and we have previously shown that this effect is due to a decrease in the degradation rate of this protein. The rate of CEF type 1 glucose transporter biosynthesis and the level of its mRNA are unaffected by src transformation. To study the molecular basis of this phenomenon, we have been isolating chicken glucose transporter cDNAs by hybridization to a rat type 1 glucose transporter probe at low stringency. Surprisingly, these clones corresponded to a message encoding a protein which has most sequence similarity to the human type 3 glucose transporter and which we refer to as CEF-GT3. CEF-GT3 is clearly distinct from the CEF type 1 transporter that we have previously described. Northern (RNA) analysis of CEF RNA with CEF-GT3 cDNA revealed two messages of 1.7 and 3.3 kb which were both greatly induced by src transformation. When the CEF-GT3 cDNA was expressed in rat fibroblasts, a three-to fourfold enhancement of 2-deoxyglucose uptake was observed, indicating that CEF-GT3 is a functional glucose transporter. Northern analyses using a CEF-GT3 and a rat type 1 probe demonstrated that there is no hybridization between different isoforms but that there is cross-species hybridization between the rat type 1 probe and the chicken homolog. Southern blot analyses confirmed that the chicken genomic type 1 and type 3 transporters are encoded by distinct genes. We conclude that CEFs express two types of transporter, type 1 (which we have previously reported to be regulated posttranslationally by src) and a novel type 3 isoform which, unlike type 1, shows mRNA induction upon src transformation. We conclude that src regulates glucose transport in CEFs simultaneously by two different mechanisms.

A series of recombinant retroviral genomes was generated by cotransformation of NIH 3T3 cells with a mixture of cloned DNAs: a proviral copy of the wild-type Moloney murine leukemia virus, and Moloney-based vectors containing defective copies of the chicken v-src and the murine v-abl oncogenes. Morphologically transformed foci, appearing at low frequencies in these cultures, released high titers of transforming viruses. Analysis of one group of these viruses showed that the genomes were recombinants containing portions of the viral gag gene juxtaposed to the v-src oncogene. Biologically active cloned DNAs of two of these viruses were obtained and mapped in detail. One of these viruses did not cause disease after inoculation into newborn mice, but the other induced rapidly fatal hemangiosarcomas located exclusively in the brain.

Hck is a phagocyte specific proto-oncogene of the Src family expressed as two isoforms, p59Hck and p61Hck. It plays a critical role in Bcr/Abl-chronic myeloid leukaemia and is able to transform fibroblasts in vitro. However, the tumourigenic activity of Hck and the respective oncogenic functions of Hck isoforms have not been examined. Tet-Off fibroblasts expressing constitutively active mutants of p59Hck and p61Hck together or individually were used. In contrast to cells expressing p59Hck(ca) or p61Hck(ca) alone, cells expressing both isoforms were transformed in vitro and induced tumour formation in 90% of nude mice within 2 weeks. This is the first demonstration of (i) the tumourigenic activity of Hck in mice, (ii) the cooperative action of the two Hck isoforms in vitro and in vivo. To our knowledge, this is the first example of a transforming activity split in two requisite isoforms.

As we enter the era of targeted therapy for melanoma, attempts are being made to sub-group tumors on the basis of their driving oncogenic mutations, with the hope of developing truly personalized therapeutic regimens. c-KIT is a receptor tyrosine kinase whose aberrant activation is implicated in the progression of gastrointestinal stromal tumors and some acute myeloid leukemias. The role of c-KIT signaling in melanoma has been controversial; although c-KIT activity is critical to melanocyte development, its expression tends to be lost in most melanomas. Some reports have even shown that the re-expression of c-KIT induces apoptosis in melanoma cell lines. The recent publication of work showing the presence of activating c-KIT mutations in acral and mucosal melanomas, as well as melanomas arising on skin with chronic sun damage, has renewed interest in c-KIT signaling in melanoma. Recent work from our own laboratory has further identified melanomas with constitutive c-KIT signaling activity resulting from c-KIT receptor overexpression. Although the initial clinical trials of the c-KIT inhibitor imatinib mesylate in melanoma were negative, some dramatic responses have been seen in patients with very high c-KIT expression and/or documented activating mutations, fostering the belief that focused studies in patients selected on the basis of c-KIT mutational status will yield more encouraging results. The current review discusses the role of c-KIT signaling in melanoma progression and how this new information can be applied to the targeted therapy of melanoma.

Non-small cell lung cancer (NSCLC)-associated epidermal growth factor receptor (EGFR) mutants are constitutively active and induce ligand-independent transformation in non-malignant cell lines. We investigated the possibility that the ability of mutant EGFRs to transform cells reflects a constitutive cooperativity with Src using a system in which the overexpression of mutant, but not wild-type, EGFR induced anchorage-independent cell growth. Src was constitutively activated and showed enhanced interaction with mutant EGFRs, suggesting that constitutive EGFR-Src cooperativity may contribute to mutant EGFR-mediated oncogenesis. Indeed, the mutant EGFR-mediated cell transformation was inhibited by Src- as well as EGFR-directed inhibitors. Importantly, a tyrosine to phenylalanine mutation of the major Src phosphorylation site on EGFR, Y845, reduced the constitutive phosphorylation of NSCLC-EGFR mutants, as well as that of STAT3, Akt, Erk and Src, and reduced the mutant EGFR-Src association as well as proliferation, migration and anchorage-independent growth. Reduced anchorage-independent growth and migration were also observed when dominant-negative-Src was expressed in mutant EGFR-expressing cells. Overall, our findings show that mutant EGFR-Src interaction and cooperativity play critical roles in constitutive engagement of the downstream signaling pathways that allow NSCLC-associated EGFR mutants to mediate oncogenesis, and support the rationale to target Src-dependent signaling pathways in mutant EGFR-mediated malignancies.

Activating epidermal growth factor receptor (EGFR) mutations are common in many cancers including glioblastoma. However, clinical responses to EGFR inhibitors are infrequent and short-lived. We show that the Src family kinases (SFK) Fyn and Src are effectors of oncogenic EGFR signaling, enhancing invasion and tumor cell survival in vivo. expression of a constitutively active EGFR mutant, EGFRvIII, resulted in activating phosphorylation and physical association with Src and Fyn, promoting tumor growth and motility. Gene silencing of Fyn and Src limited EGFR- and EGFRvIII-dependent tumor cell motility. The SFK inhibitor dasatinib inhibited invasion, promoted tumor regression, and induced apoptosis in vivo, significantly prolonging survival of an orthotopic glioblastoma model expressing endogenous EGFRvIII. Dasatinib enhanced the efficacy of an anti-EGFR monoclonal antibody (mAb 806) in vivo, further limiting tumor growth and extending survival. Examination of a large cohort of clinical samples showed frequent coactivation of EGFR and SFKs in glioblastoma patients. These results establish a mechanism linking EGFR signaling with Fyn and Src activation to promote tumor progression and invasion in vivo and provide rationale for combined anti-EGFR and anti-SFK targeted therapies.

Integrins regulate adhesion-dependent growth, survival and invasion of tumor cells. In particular, expression of integrin alpha(v)beta(3) is associated with progression of a variety of human tumors. Here we reveal a previously undescribed adhesion-independent role for integrin alpha(v)beta(3) in pancreatic cancer and other carcinomas. Specifically, alpha(v)beta(3) expressed in carcinoma cells enhanced anchorage-independent tumor growth in vitro and increased lymph node metastases in vivo. These effects required recruitment of c-Src to the beta(3) integrin cytoplasmic tail, leading to c-Src activation, Crk-associated substrate (CAS) phosphorylation and tumor cell survival that, unexpectedly, was independent of cell adhesion or focal adhesion kinase (FAK) activation. Pharmacological blockade of c-Src kinase activity or decreased expression of endogenous alpha(v)beta(3) integrin or c-Src not only inhibited anchorage-independent growth but also suppressed metastasis in vivo, yet these manipulations did not affect tumor cell migration or invasion. These data define an unexpected role for an integrin as a mediator of anchorage independence, suggesting that an alpha(v)beta(3)-c-Src signaling module may account for the aggressive behavior of integrin alpha(v)beta(3)-expressing tumors in humans.

To understand the normal, physiological role of the c-src proto-oncogene, a null mutation was introduced into the gene by homologous recombination in mouse embryonic stem cells. Two independent targeted clones were used to generate chimeras that transmitted the mutated allele to their offspring. Intercrossing of heterozygotes gave rise to live born homozygotes, but most of these mice died within the first few weeks of birth. Histological and hematological examination of the homozygous mutants did not reveal detectable abnormalities in the brain or platelets, where src is most highly expressed. However, these mutants were deficient in bone remodeling, indicating impaired osteoclast function, and developed osteopetrosis. These results demonstrate that src is not required for general cell viability (possibly because of functional overlap with other tyrosine kinases related to src) and uncover an essential role for src in bone formation.

The Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) plays a pivotal role in growth factor and cytokine signaling. Gain-of-function SHP2 mutations are associated with Noonan syndrome, various kinds of leukemias, and solid tumors. Thus, there is considerable interest in SHP2 as a potential target for anticancer and antileukemia therapy. We report a salicylic acid based combinatorial library approach aimed at binding both active site and unique nearby subpockets for enhanced affinity and selectivity. Screening of the library led to the identification of a SHP2 inhibitor II-B08 (compound 9) with highly efficacious cellular activity. Compound 9 blocks growth factor stimulated ERK1/2 activation and hematopoietic progenitor proliferation, providing supporting evidence that chemical inhibition of SHP2 may be therapeutically useful for anticancer and antileukemia treatment. X-ray crystallographic analysis of the structure of SHP2 in complex with 9 reveals molecular determinants that can be exploited for the acquisition of more potent and selective SHP2 inhibitors.

Two members of the myocardin protein family, myocardin-related transcription factor (MRTF)-A and MRTF-B are co-activators of serum response factor (SRF). We recently reported that MRTF-A/B activates the transcription of several actin cytoskeletal/focal adhesion genes SRF dependently, thereby enhancing the formation of stress fibers and focal adhesions. Here, we showed that the levels of caldesmon and tropomyosin, both SRF/MRTF-regulated actin cytoskeletal proteins, were reduced in rat intestinal epithelial (RIE) cell lines that had been transformed with oncogenic ras (RIE-ras) or src (RIE-src) compared with their parental cell line. These cells exhibited morphological abnormalities associated with a disorganized actin cytoskeleton. The serum-stimulated nuclear translocation of MRTF-A/B was suppressed in the RIE-ras and RIE-src cells. However, the transient expression of constitutively active (CA) MRTF-A or MRTF-B reversed the reduced expression levels of caldesmon and tropomyosin and the associated morphological phenotypes. We isolated stable CA-MRTF-A-expressing cell lines from transfected RIE-ras and RIE-src cells and found that their levels of caldesmon and tropomyosin were close to those of untransformed RIE cells. Their morphologies were also normal, with a flattened cell shape and well-developed stress fibers. The CA-MRTF-A-expressing RIE-ras and RIE-src lines also showed lower invasiveness and anchorage-independent growth than their transformed parental cells, in vitro. In vivo, CA-MRTF-A expression suppressed tumor formation and reduced liver metastases. Therefore, we concluded that MRTF-A/B are potent repressors of cancer progression and metastasis and may be good targets for cancer therapy.

One of the characteristics of gliomas is a decrease in the expression of connexin43, a protein that forms gap junctions. Restoring connexin43 expression in glioma cells reduces their exacerbated rate of cell growth, although it is not yet known how connexin43 modifies the expression of genes involved in cell proliferation. Here, we show that restoring connexin43 to C6 glioma cells impedes their progression from G0/G1 to the S phase of the cell cycle by reducing retinoblastoma phosphorylation and cyclin E expression through the upregulation of p21 and p27. Interestingly, connexin43 diminishes the oncogenic activity of c-Src exhibited by glioma cells. By studying a Tyr247 and Tyr265 mutant connexin43, we show that these residues are required for connexin43 to inhibit c-Src activity and cell proliferation. In conclusion, by acting as a substrate of c-Src, connexin43 reduces its oncogenic activity and decreases the rate of glioma cell proliferation, potentially an early step in the antiproliferative effects of connexin43. Although c-Src is known to phosphorylate connexin43, this study provides the first evidence that connexin43 can also inhibit c-Src activity.

Tyrosine-specific protein kinase activity in neuronal differentiation was studied in a PC12 pheochromocytoma cell line (PC12-B9) produced by stable transfection with an inducible v-src gene encoding an activated tyrosine kinase (pp60v-src) under the transcriptional control of the mouse metallothionine I gene promoter. Induction of pp60v-src expression with Cd2+ and Zn2+ resulted in the reversible differentiation of PC12-B9 cells into neuron-like cells. pp60v-src elicited morphological differentiation with apparent first order kinetics at the same rate as NGF-directed neurite outgrowth in PC12-B9 cells. v-src gene expression enhanced the rate of NGF-directed neurite extension in an additive manner. Induction of pp60v-src alone constitutively increased the levels of phosphotyrosine-modified proteins (130-120, 90, 83, 65, 60/59, 36 kDa) detected by immunoblotting with phosphotyrosine antibodies. NGF treatment of PC12-B9 cells transiently increased the levels of distinct phosphotyrosine-modified proteins (108, 46, 42 kDa), as well as common substrates, including a 59-kDa protein that comigrated with alpha-tubulin. Phosphotyrosine-modified proteins were not synergistically increased in PC12-B9 cells induced for both v-src and NGF. The nonsynergistic effects of v-src gene expression on neurite outgrowth and phosphorylation suggest that pp60v-src induces PC12 cell differentiation by an intracellular signaling pathway that is largely distinct from that induced by NGF.

The proto-oncogene Src tyrosine kinase (Src) is overexpressed in human cancers and is currently a target of anti-invasive therapies. Activation of Src is an essential catalyst of invadopodia production. Invadopodia are cellular structures that mediate extracellular matrix (ECM) proteolysis, allowing invasive cell types to breach confining tissue barriers. Invadopodia assembly and maturation is a multistep process, first requiring the targeting of actin-associated proteins to form pre-invadopodia, which subsequently mature by recruitment and activation of matrix metalloproteases (MMPs) that facilitate ECM degradation. We demonstrate that active, oncogenic Src alleles require the presence of a wild-type counterpart to induce ECM degradation at invadopodia sites. In addition, we identify the phosphorylation of the invadopodia regulatory protein cortactin as an important mediator of invadopodia maturation downstream of wild-type Src. Distinct phosphotyrosine-based protein-binding profiles in cells forming pre-invadopodia and mature invadopodia were identified by SH2-domain array analysis. These results indicate that although elevated Src kinase activity is required to target actin-associated proteins to pre-invadopodia, regulated Src activity is required for invadopodia maturation and matrix degradation activity. Our findings describe a previously unappreciated role for proto-oncogenic Src in enabling the invasive activity of constitutively active Src alleles.

We have previously shown that an intracellular mechanism down regulates epidermal growth factor (EGF) receptor levels in rodent fibroblasts transformed by the src oncogene (W. J. Wasilenko, L. K. Shawver, and M. J. Weber, J. Cell. Physiol. 131:450-457, 1987). We now report that this down regulation is due to an inhibition of EGF receptor biosynthesis. With Rat-1 (R1) cells infected with a temperature-sensitive src mutant, we found that 125I-labeled EGF binding to cells began to decrease soon after the activation of pp60v-src by shift down to the permissive temperature for transformation. This effect of src on EGF receptors was reversible. Pulse-chase studies with [35S]methionine-labeled cells revealed that the tyrosine protein kinase activity of pp60v-src had little if any effect on EGF receptor degradation rate. By contrast, the expression of pp60v-src caused a large reduction in the apparent rate of EGF receptor biosynthesis. Northern (RNA) blot analysis demonstrated that pp60v-src also caused marked reductions in the steady-state level of EGF receptor mRNA. These data indicate that one way the expression of the src oncogene can affect the machinery of growth control is by affecting the expression of specific genes for growth factor receptors.

The products of the viral and cellular src genes, p60v-src and p60c-src, appear to be composed of multiple functional domains. Highly conserved regions called src homology 2 and 3 (SH2 and SH3), comprising amino acid residues 88 to 250, are believed to modulate the protein-tyrosine kinase activity present in the carboxy-terminal halves of the src proteins. To explore the functions of these regions more fully, we have made 34 site-directed mutations in a transformation-competent c-src gene encoding phenylalanine in place of tyrosine 527 (Y527F c-src). Twenty of the new mutations change only one or two amino acids, and the remainder delete small or large portions of the SH2-SH3 region. These mutant alleles have been incorporated into a replication-competent Rous sarcoma virus vector to examine the biochemical and biological properties of the mutant proteins after infection of chicken embryo fibroblasts. Four classes of mutant proteins were observed: class 1, mutants with only slight differences from the parental gene products; class 2, mutant proteins with diminished transforming and specific kinase activities; class 3, mutant proteins with normal or enhanced specific kinase activity but impaired biological activity, often as a consequence of instability; and class 4, mutant proteins with augmented biological and catalytic activities. In general, there was a strong correlation between total kinase activity (or amounts of intracellular phosphotyrosine-containing proteins) and transforming activity. Deletion mutations and some point mutations affecting residues 109 to 156 inhibited kinase and transforming functions, whereas deletions affecting residues 187 to 226 generally had positive effects on one or both of those functions, confirming that SH2-SH3 has complex regulatory properties. Five mutations that augmented the transforming and kinase activities of Y527F c-src [F172P, R175L, delta(198-205), delta(206-226), and delta(176-226)] conferred transformation competence on an otherwise normal c-src gene, indicating that mutations in SH2 (like previously described lesions in SH3, the kinase domain, and a carboxy-terminal inhibitory domain) can activate c-src.

To investigate the importance of a conserved region spanning residues 137 to 241 in the noncatalytic domain of p60c-src (SH2 region), we used oligonucleotide-directed mutagenesis to change residues that are highly conserved in this region. Chicken embryo fibroblasts infected with a p60c-src variant containing arginine instead of tryptophan at residue 148 (W148R) appeared more rounded than cells overexpressing a normal c-src gene, and they formed colonies in soft agar. p60c-src variants containing serine instead of arginine at residue 155 (R155S) or isoleucine instead of glycine at residue 170 (G170I) also appeared transformed and were anchorage independent, but to a lesser extent than W148R. mutation of residue 201 from histidine to leucine (H201L) had no observable effect. The in vitro kinase activity of cells infected with W148R or G170I was elevated twofold. expression of p60W148R (or, to a lesser extent, of p60G170I) increased the number of proteins phosphorylated on tyrosine in infected cells. ALL of the mutants were phosphorylated in vivo on Tyr-527, instead of Tyr-416 as observed for p60v-src. Immunoprecipitated p60W148R and p60G170I were found to be associated with a phosphatidylinositol kinase activity, a factor which appears to be necessary for transformation by tyrosine-specific protein kinases. These results show that a single point mutation in the SH2 region of the cellular src gene can activate its transforming potential. This type of activation is in a new category of alterations at the amino terminus that activate but do not cause a shift in phosphorylation at the carboxy terminus.

Monoclonal antibodies were raised against a synthetic NH2-terminal myristoyl tetrapeptide (N-myristoyl-Gly-Ser-Ser-Lys) which is characteristic of an NH2-terminal portion of pp60src, the transforming protein of src-oncogene. The antibody reacted with the albumin conjugated with both the N-myristoyl and N-lauroyl-tetrapeptides, but concentrations at which 50% of the immunoreaction was inhibited were 5 pmol for the N-myristoyl and 830 pmol for N-lauroyl tetrapeptidyl albumin. On the other hand, N-palmitoyl tetrapeptidyl and underivatized albumin, and Gly-Ser-Ser-Lys-Ser-Lys-Pro-Lys octapeptide had no effects. These results suggest a high affinity of the antibody for an N-myristoyl-Gly-Ser-Ser-Lys moiety. src-Oncogene products in Rous sarcoma virus-transformed cells and human colon carcinoma tumor cells were selectively identified as myristoylated pp60src by immunoprecipitation analyses with the antibody.

Pancreas cancer is one of the most lethal malignancies and is characterized by activating mutations of Kras, present in 95% of patients. More than 60% of pancreatic cancers also display increased c-Src activity, which is associated with poor prognosis. Although loss of tumor suppressor function (for example, p16, p53, Smad4) combined with oncogenic Kras signaling has been shown to accelerate pancreatic duct carcinogenesis, it is unclear whether elevated Src activity contributes to Kras-dependent tumorigenesis or is simply a biomarker of disease progression. Here, we demonstrate that in the context of oncogenic Kras, activation of c-Src through deletion of C-terminal Src kinase (CSK) results in the development of invasive pancreatic ductal adenocarcinoma (PDA) by 5-8 weeks. In contrast, deletion of CSK alone fails to induce neoplasia, while oncogenic Kras expression yields PDA at low frequency after a latency of 12 months. Analysis of cell lines derived from Ras/Src-induced PDA s indicates that oncogenic Ras/Src cooperativity may lead to genomic instability, yet Ras/Src-driven tumor cells remain dependent on Src signaling and as such, Src inhibition suppresses growth of Ras/Src-driven tumors. These findings demonstrate that oncogenic Ras/Src cooperate to accelerate PDA onset and support further studies of Src-directed therapies in pancreatic cancer.

c-Yes, a member of the Src tyrosine kinase family, is found highly activated in colon carcinoma but its importance relative to c-Src has remained unclear. Here we show that, in HT29 colon carcinoma cells, silencing of c-Yes, but not of c-Src, selectively leads to an increase of cell clustering associated with a localisation of beta-catenin at cell membranes and a reduction of expression of beta-catenin target genes. c-Yes silencing induced an increase in apoptosis, inhibition of growth in soft-agar and in mouse xenografts, inhibition of cell migration and loss of the capacity to generate liver metastases in mice. Re-introduction of c-Yes, but not c -Src, restores transforming properties of c-Yes depleted cells. Moreover, we found that c-Yes kinase activity is required for its role in beta-catenin localisation and growth in soft agar, whereas kinase activity is dispensable for its role in cell migration. We conclude that c-Yes regulates specific oncogenic signalling pathways important for colon cancer progression that is not shared with c-Src.

AIM: To investigate the role of insulin receptor substrate 2 (IRS-2) in oncogenic transformation induced by v-src. METHODS: IRS-2 gene was silenced using small interfering RNAs (siRNAs). Nuclear translocation and interaction of IRS-2 with v-src was determined using subcellular fractionation, confocal microscopy, and immunoprecipitation. The activity of the cyclin D1 promoter and r-DNA promoter was measured with a luciferase assay. RESULTS: Depletion of IRS-2 inhibited R-/v-src cell growth and reverse the oncogenic transformation. IRS-2 bound to src via its two PI3-K binding sites, which are critical for activities involved in the transformation. Nuclear IRS-2 occupied the cyclin D1 and rDNA promoters. The combination of IRS-2 and v-src increased the activity of the two promoters, especially the rDNA promoter. CONCLUSION: Depletion of insulin receptor substrate 2 could reverse oncogenic transformation induced by v-src.

When fibroblasts are transformed by the src oncogene, there is a two- to fivefold increase in glucose transport and in the level of immunoprecipitable glucose transporter protein. In chicken embryo fibroblasts (CEFs), this increase is correlated with a comparable reduction in the rate at which the glucose transporter protein is turned over. In contrast, in mammalian fibroblasts glucose transporter biosynthesis is increased by src, but there is little or no change in its turnover. To further understand the action of src on transporter turnover, we investigated whether a mammalian transporter can be stabilized by src in a chicken cell environment. The human type 1 glucose transporter protein (hGT), originally cloned from HepG2 cells, was expressed in CEFs or Rat-1 fibroblasts by using a retroviral vector. In CEFs transformed by a temperature-sensitive src mutant, tsNY68, turnover of hGT was lower at the permissive temperature (36 degrees C) than at the nonpermissive temperature (42 degrees C). When this protein was expressed in CEFs transformed by wild-type src, no difference in turnover was observed at the two temperatures. In the case of Rat-1 cells transformed by the temperature-sensitive src mutant tsLA29, turnover of hGT was the same at the permissive temperature (35 degrees C) as at the nonpermissive temperature (39.5 degrees C). These data demonstrate that a heterologous glucose transporter behaves in the same way in chicken and rat cells as the respective endogenous transporter, i.e., when src is active, the protein is stablilized against turnover in chicken cells but not in rat cells.

Steroid receptor co-activator-3 (SRC-3/AIB1) is an oncogene that is amplified and overexpressed in many human cancers. However, the molecular mechanisms that regulate activated SRC-3 oncoprotein turnover during tumorigenesis remain to be elucidated. Here, we report that speckle-type POZ protein (SPOP), a cullin 3 (CUL3)-based ubiquitin ligase, is responsible for SRC-3 ubiquitination and proteolysis. SPOP interacts directly with an SRC-3 phospho-degron in a phosphorylation-dependent manner. Casein kinase Ivarepsilon phosphorylates the S102 in this degron and promotes SPOP-dependent turnover of SRC-3. Short hairpin RNA knockdown and overexpression experiments substantiated that the SPOP/CUL3/Rbx1 ubiquitin ligase complex promotes SRC-3 turnover. A systematic analysis of the SPOP genomic locus revealed that a high percentage of genomic loss or loss of heterozygosity occurs at this locus in breast cancers. Furthermore, we demonstrate that restoration of SPOP expression inhibited SRC-3-mediated oncogenic signaling and tumorigenesis, thus positioning SPOP as a tumor suppressor.

A Balb/c 3T3 cell-line infected with the temperature-sensitive Rous sarcoma virus LA90 has been used to investigate the mitogenic activity of p60v-src and the possible role of phosphatidylinositol turnover and protein kinase C activation in this process. Activation of the p60v-src tyrosine kinase alone was only weakly mitogenic to serum-deprived cells. However, transient activation of the kinase dramatically increased the mitogenic response to epidermal growth factor. Mitogenicity was not additive with phorbol esters. Down-regulation of protein kinase C by chronic administration of phorbol ester did not block the mitogenic effect of p60v-src. Following short-term activation of p60v-src, analysis of protein phosphorylations by giant two-dimensional electrophoresis revealed only very minor changes in any of the 1000 or so detectable phosphoproteins, whereas the activation of protein kinase C by a sub-optimal dose of phorbol ester significantly increased the phosphorylation of at least 10 proteins. Short-term activation of p60v-src did not cause any detectable increase in phosphatidylinositol turnover, or in diacylglycerol level. These results indicate that p60v-src can act, like platelet-derived growth factor, to induce competence in Balb/c 3T3 fibroblasts. They also suggest that even though p60v-src may activate phosphatidylinositol turnover in some other cell-lines, this effect is probably indirect and is not essential for induction of the transformed phenotype or for p60v-src-dependent mitogenesis.

Many oncogene products have been shown to bear strong homology to or to interact with components of normal cellular signal transduction. We have previously shown that a glycoprotein band of 95 kilodaltons (kDa) becomes tyrosine phosphorylated in chick cells transformed by Rous sarcoma virus and that tyrosine phosphorylation of this protein band correlates tightly with phenotypic transformation in cells infected with a large and diverse panel of src mutants (L. M. Kozma, A. B. Reynolds, and M. J. Weber, Mol. Cell. Biol. 10:837-841, 1990). In this communication, we report that a component of the 95-kDa glycoprotein band is related or identical to the 95-kDa beta subunit of the receptor for insulinlike growth factor I (IGF-I). We found that the beta subunit of the IGF-I receptor comigrated on polyacrylamide gels with a component of the 95-kDa glycoprotein region from src-transformed cells under both reducing and nonreducing gel conditions and had a very similar partial phosphopeptide map. To further test the hypothesis that the beta subunit of the IGF-I receptor becomes tyrosine phosphorylated in cells transformed by pp60src, a human cell line that expressed the IGF-I receptor was transformed by src. Comparison of IGF-I receptors immunoprecipitated from normal and transformed cells revealed that the beta subunit of the IGF-I receptor became constitutively tyrosine phosphorylated in src-transformed cells. Moreover, IGF-I receptor phosphorylation induced by src was synergistic with that induced by the hormone: IGF-I-stimulated autophosphorylation of the receptor was much greater in src-transformed cells than in untransformed HOS cells even at maximal concentrations of IGF-I. This increased responsiveness to IGF-I was not due to increases in receptor number, time course of phosphorylation, or affinity for hormone. Finally, no IGF-I-like activity could be detected in culture supernatants collected from the src-transformed cells, suggesting that the increased receptor phosphorylation observed in the src-transformed cells may be mediated by an intracellular mechanism rather than an external autocrine stimulation. Our data demonstrate that the IGF-I receptor becomes constitutively tyrosine phosphorylated in src-transformed cells. This finding raises the possibility that pp60v-src alters growth regulation at least in part by phosphorylating and activating this growth factor receptor.

1. Recombinant retroviruses were used to introduce a temperature-sensitive v-src gene and oncogenic c-Ha-ras into PC12 cells, and stable cell lines expressing these genes were established. 2. As previously reported, expression of v-src (Alema et al., 1985) or c-Ha-ras (Noda et al., 1985) in PC12 cells results in neurite outgrowth resembling that induced by NGF. We report here that v-src but not oncogenic c-Ha-ras induces a stable morphologic neuronal differentiation similar to treatment with NGF. Oncogenic c-Ha-ras-induced neurite outgrowth is not stable with long-term culture, rather the cells revert to an undifferentiated morphology with altered cell cycle kinetics. 3. The stable neuronal phenotype induced by v-src and NGF is characterized by the functional expression of dihydropyridine-insensitive calcium currents.

Ras genes are the most common targets for somatic gain-of-function mutations in human cancers. In this study, we found a high incidence of correlation between Ras oncogenic mutations and c-Src activation in human cancer cells. We showed that oncogenic Ras induces c-Src activation mainly on the Golgi complex and endoplasmic reticulum. Moreover, we identified p120RasGAP as an effector for oncogenic Ras to activate c-Src. The recruitment of p120RasGAP to the Golgi complex by oncogenic Ras facilitated its interaction with c-Src, thereby leading to c-Src activation, and this p120RasGAP-mediated activation of c-Src was important for tumor invasion induced by oncogenic Ras. Collectively, our findings unveil a relationship between oncogenic Ras, p120RasGAP, and c-Src, suggesting a critical role for c-Src in cancers evoked by oncogenic mutations in Ras genes.

The non-receptor tyrosine kinases of the SRC family (SFK) play important roles in signal transduction induced by a large variety of extracellular stimuli, including growth factors and Integrins. When deregulated, SFKs show oncogenic activity, as originally reported for v-Src, the transforming product of the avian retrovirus RSV, and then, in many human cancers, particularly colorectal cancer (CRC). In CRC, SFK deregulation largely occurs in the absence of mutations of the corresponding genes, but the underlying molecular mechanisms involved are still unclear. In addition to a role in early tumor progression, SFK deregulation may also be important in advanced CRC, as suggested by the association between increased SFK activity and poor clinical outcome. However, SFK contribution to CRC metastasis formation is still poorly documented. Here, we will review recent findings that broaden our understanding of the mechanisms underlying SFK deregulation and signaling in advanced CRC. We will also discuss the implication of these observations for SFK-based therapy in metastatic CRC.

Increased inducible nitric oxide synthase (NOS2) expression in breast tumors is associated with decreased survival of estrogen receptor negative (ER-) breast cancer patients. We recently communicated the preliminary observation that nitric oxide (NO) signaling results in epidermal growth factor receptor (EGFR) tyrosine phosphorylation. To further define the role of NO in the pathogenesis of ER- breast cancer, we examined the mechanism of NO-induced EGFR activation in human ER- breast cancer. NO was found to activate EGFR and Src by a mechanism that includes S-nitrosylation. NO, at physiologically relevant concentrations, induced an EGFR/Src-mediated activation of oncogenic signal transduction pathways (including c-Myc, Akt, and beta-catenin) and the loss of PP2A tumor suppressor activity. In addition, NO signaling increased cellular EMT, expression and activity of COX-2, and chemoresistance to adriamycin and paclitaxel. When connected into a network, these concerted events link NO to the development of a stem cell-like phenotype, resulting in the upregulation of CD44 and STAT3 phosphorylation. Our observations are also consistent with the finding that NOS2 is associated with a basal-like transcription pattern in human breast tumors. These results indicate that the inhibition of NOS2 activity or NO signaling networks may have beneficial effects in treating basal-like breast cancer patients.

BACKGROUND: C-src is an evolutionarily conserved proto-oncogene that regulates cell proliferation, differentiation and apoptosis. In our previous studies, we have reported that another proto-oncogene, c-erbB2, plays an important role in primordial follicle activation and development. We also found that c-src was expressed in mammalian ovaries, but its functions in primordial follicle activation remain unclear. The objective of this study is to investigate the role and mechanism of c-src during the growth of primordial follicles. METHODS: Ovaries from 2-day-old rats were cultured in vitro for 8 days. Three c-src-targeting and one negative control siRNA were designed and used in the present study. PCR, Western blotting and primordial follicle development were assessed for the silencing efficiency of the lentivirus c-src siRNA and its effect on primordial follicle onset. The expression of c-src mRNA and protein in primordial follicle growth were examined using the PCR method and immunohistochemical staining. Furthermore, the MAPK inhibitor PD98059, the PKC inhibitor Calphostin and the PI3K inhibitor LY294002 were used to explore the possible signaling pathways of c-src in primordial folliculogenesis. RESULTS: The results showed that Src protein was distributed in the ooplasmic membrane and the granulosa cell membrane in the primordial follicles, and c-src expression level increased with the growth of primordial follicle. The c-src -targeting lentivirus siRNAs had a silencing effect on c-src mRNA and protein expression. Eight days after transfection of rat ovaries with c-src siRNA, the GFP fluorescence in frozen ovarian sections was clearly discernible under a fluorescence microscope, and its relative expression level was 5-fold higher than that in the control group. Furthermore, the c-src-targeting lentivirus siRNAs lowered its relative expression level 1.96 times. We also found that the development of cultured primordial follicles was completely arrested after c-src siRNA knockdown of c-src expression. Furthermore, our studies demonstrated that folliculogenesis onset was inhibited by Calphostin, PD98059 or LY294002 treatment,but none of them down-regulated c-src expression. In contrast, the expression levels of p-PKC, p-ERK1/2 and p-PI3K in the follicles were clearly decreased by c-src siRNA transfection. Correspondingly, both Calphostin and LY294002 treatment resulted in a decrease in the p-PKC level in follicles, but no change was observed in the PD98059 group. Finally, LY294002 treatment decreased the p-PI3K expression level in the follicles, but no changes were observed in the PD98059 and Calphostin groups. CONCLUSIONS: C-src plays an important role in regulating primordial follicle activation and growth via the PI3K-PKC- ERK1/2 pathway.

The non-receptor tyrosine kinase SRC is frequently deregulated in human colorectal cancer (CRC), and SRC increased activity has been associated with poor clinical outcomes. In nude mice engrafted with human CRC cells, SRC over-expression favors tumor growth and is accompanied by a robust increase in tyrosine phosphorylation in tumor cells. How SRC contributes to this tumorigenic process is largely unknown. We analyzed SRC oncogenic signaling in these tumors by means of a novel quantitative proteomic analysis. This method is based on stable isotope labeling with amino acids of xenograft tumors by the addition of [(13)C(6)]-lysine into mouse food. An incorporation level greater than 88% was obtained in xenograft tumors after 30 days of the heavy lysine diet. Quantitative phosphoproteomic analysis of these tumors allowed the identification of 61 proteins that exhibited a significant increase in tyrosine phosphorylation and/or association with tyrosine phosphorylated proteins upon SRC expression. These mainly included molecules implicated in vesicular trafficking and signaling and RNA binding proteins. Most of these proteins were specific targets of SRC signaling in vivo, as they were not identified by analysis via stable isotope labeling by amino acids in cell culture (SILAC) of the same CRC cells in culture. This suggests that oncogenic signaling induced by SRC in tumors significantly differs from that induced by SRC in cell culture. We next confirmed this notion experimentally with the example of the vesicular trafficking protein and SRC substrate TOM1L1. We found that whereas TOM1L1 depletion only slightly affected SRC-induced proliferation of CRC cells in vitro, it drastically decreased tumor growth in xenografted nude mice. We thus concluded that this vesicular trafficking protein plays an important role in SRC-induced tumor growth. Overall, these data show that SILAC analysis in mouse xenografts is a valuable approach for deciphering tyrosine kinase oncogenic signaling in vivo.

Gap junctions are channels that connect the interiors of neighboring cells and are formed by the connexin (Cx) proteins. A reduction in gap junctional, intercellular communication (GJIC) often correlates with increased growth and neoplastic transformation. Cx43 is a widely expressed connexin which can be phosphorylated by the Src oncoprotein tyrosine kinase on tyr247 and -265, and this reduces communication. However, Src activates multiple signalling pathways such as the Ras/Raf/Erk and PLCgamma/protein kinase C, which can also phosphorylate Cx43 and interrupt communication. In addition, the Src effector Cas, which has an adaptor function, binds Cx43 to suppress gap junctional communication. In sharp contrast, activation of a different Src effector, the cytoplasmic transcription factor Signal transducer and activator of transcription-3 (Stat3) is not required for the Src-mediated, GJIC suppression. In fact, Stat3 is actually required for the maintenance of gap junctional communication in normal cells with high GJIC.

BACKGROUND: The Src family tyrosine kinases (SFK) are cellular regulatory proteins that influence cell adhesion, proliferation, invasion and survival during tumor development. Elevated activity of Src was associated with increased cell proliferation and invasivity in human papillomavirus (HPV)-associated malignancies; therefore, transduced human foreskin keratinocytes (HFK) were used to investigate whether SFK activation is a downstream effect of papillomaviral oncoproteins. Activation of ubiquitously expressed SFKs, namely Src, Yes and Fyn, was investigated in both proliferating and differentiating keratinocytes. RESULTS: In proliferating keratinocytes, Src, Yes and Fyn mRNA levels were not affected by HPV 16 E6 or E7 oncoproteins, while at the protein level as detected by western blot, the presence of both E6 and E7 resulted in substantial increase in Src and Yes expression, but did not alter the high constitutive level of Fyn. Phospo-kinase array revealed that ALL ubiquitously expressed SFKs are activated by phosphorylation in the presence of HPV 16 E7 oncoprotein. Keratinocyte differentiation led to increased Yes mRNA and protein levels in ALL transduced cell lines, while it did not influence the Src transcription but resulted in elevated Src protein level in HPV16 E7 expressing lines. CONCLUSIONS: This study revealed that HPV 16 oncoproteins upregulate Src family kinases Src and Yes via posttranscriptional mechanisms. A further effect of HPV 16 E7 oncoprotein is to enhance the activating phosphorylation of SFKs expressed in keratinocytes.

Phosphatase of Regenerating Liver (PRL) family members have emerged as molecular markers that significantly correlate to the ability of many cancers to metastasize. However, contradictory cellular responses to PRL expression have been reported, including the inhibition of cell cycle progression. An obvious culprit for the discrepancy is the use of dozens of different cell lines, including many isolated from tumors or cultured cells selected for immortalization which may have missing or mutated modulators of PRL function. We created transgenic Drosophila to study the effects of PRL overexpression in a genetically controlled, organismal model. Our data support the paradigm that the normal cellular response to high levels of PRL is growth suppression and furthermore, that PRL can counter oncogenic activity of Src. The ability of PRL to inhibit growth under normal conditions is dependent on a CAAX motif that is required to localize PRL to the apical edge of the lateral membrane. However, PRL lacking the CAAX motif can still associate indiscriminately with the plasma membrane and retains its ability to inhibit Src function. We propose that PRL binds to other membrane-localized proteins that are effectors of Src or to Src itself. This first examination of PRL in a model organism demonstrates that PRL performs as a tumor suppressor and underscores the necessity of identifying the conditions that enable it to transform into an oncogene in cancer.

The Src family kinases c-Src, and its downstream effectors, the Rho family of small GTPases RhoA and Jun N-terminal kinase (JNK) have a significant role in tumorigenesis. In this report, using the Drosophila wing disc epithelium as a model system, we demonstrate that the actin-Capping Protein (CP) alphabeta heterodimer, which regulates actin filament (F-actin) polymerization, limits Src-induced apoptosis or tissue overgrowth by restricting JNK activation. We show that overexpressing Src64B drives JNK-independent loss of epithelial integrity and JNK-dependent apoptosis via Btk29A, p120ctn and Rho1. However, when cells are kept alive with the Caspase inhibitor P35, JNK acts as a potent inducer of proliferation via activation of the Yorkie oncogene. Reducing CP levels direct apoptosis of overgrowing Src64B-overexpressing tissues. Conversely, overexpressing capping protein inhibits Src64B and Rho1, but not Rac1-induced JNK signaling. CP requires the actin-binding domain of the alpha-subunit to limit Src64B-induced apoptosis, arguing that the control of F-actin mediates this effect. In turn, JNK directs F-actin accumulation. Moreover, overexpressing capping protein also prevents apoptosis induced by ectopic JNK expression. Our data are consistent with a model in which the control of F-actin by CP limits Src-induced apoptosis or tissue overgrowth by acting downstream of Btk29A, p120ctn and Rho1, but upstream of JNK. In turn, JNK may counteract the effect of CP on F-actin, providing a positive feedback, which amplifies JNK activation. We propose that cytoskeletal changes triggered by misregulation of F-actin modulators may have a significant role in Src-mediated malignant phenotypes during the early stages of cellular transformation.

Analysis of the Polyp Prevention Trial showed an association between an isorhamnetin-rich diet and a reduced risk of advanced adenoma recurrence; however, the mechanism behind the chemoprotective effects of isorhamnetin remains unclear. Here, we show that isorhamnetin prevents colorectal tumorigenesis of FVB/N mice treated with the chemical carcinogen azoxymethane and subsequently exposed to colonic irritant dextran sodium sulfate (DSS). Dietary isorhamnetin decreased mortality, tumor number, and tumor burden by 62%, 35%, and 59%, respectively. MRI, histopathology, and immunohistochemical analysis revealed that dietary isorhamnetin resolved the DSS-induced inflammatory response faster than the control diet. Isorhamnetin inhibited AOM/DSS-induced oncogenic c-Src activation and beta-catenin nuclear translocation, while promoting the expression of C-terminal Src kinase (CSK), a negative regulator of Src family of tyrosine kinases. Similarly, in HT-29 colon cancer cells, isorhamnetin inhibited oncogenic Src activity and beta-catenin nuclear translocation by inducing expression of csk, as verified by RNA interference knockdown of csk. Our observations suggest the chemoprotective effects of isorhamnetin in colon cancer are linked to its anti-inflammatory activities and its inhibition of oncogenic Src activity and consequential loss of nuclear beta-catenin, activities that are dependent on CSK expression.

The diagnosis of chronic obstructive pulmonary disease (COPD) confers a 2-fold increased lung cancer risk even after adjusting for cigarette smoking, suggesting that common pathways are operative in both diseases. Although the role of the tyrosine kinase c-Src is established in lung cancer, less is known about its impact in other lung diseases, such as COPD. This study examined whether c-Src activation by cigarette smoke contributes to the pathogenesis of COPD. Cigarette smoke increased c-Src activity in human small airway epithelial (SAE) cells from healthy donors and in the lungs of exposed mice. Similarly, higher c-Src activation was measured in SAE cells from patients with COPD compared with healthy control subjects. In SAE cells, c-Src silencing or chemical inhibition prevented epidermal growth factor (EGF) receptor signaling in response to cigarette smoke but not EGF stimulation. Further studies showed that cigarette smoke acted through protein kinase C alpha to trigger c-Src to phosphorylate EGF receptor and thereby to induce mitogen-activated protein kinase responses in these cells. To further investigate the role of c-Src, A/J mice were orally administered the specific Src inhibitor AZD-0530 while they were exposed to cigarette smoke for 2 months. AZD-0530 treatment blocked c-Src activation, decreased macrophage influx, and prevented airspace enlargement in the lungs of cigarette smoke-exposed mice. Moreover, inhibiting Src deterred the cigarette smoke-mediated induction of matrix metalloproteinase-9 and -12 in alveolar macrophages and lung expression of cathepsin K, IL-17, TNF-alpha, MCP-1, and KC, ALL key factors in the pathogenesis of COPD. These results indicate that activation of the proto-oncogene c-Src by cigarette smoke promotes processes linked to the development of COPD.

A fundamental property of tumor cells is to defy anoikis, cell death caused by a lack of cell-matrix interaction, and grow in an anchorage-independent manner. How tumor cells organize signaling molecules at the plasma membrane to sustain oncogenic signals in the absence of cell-matrix interactions remains poorly understood. Here, we describe a role for phosphatidylinositol 4-phosphate 5-kinase (PIPK) Igammai2 in controlling anchorage-independent growth of tumor cells in coordination with the proto-oncogene Src. PIPKIgammai2 regulated Src activation downstream of growth factor receptors and integrins. PIPKIgammai2 directly interacted with the C-terminal tail of Src and regulated its subcellular localization in concert with talin, a cytoskeletal protein targeted to focal adhesions. Co-expression of PIPKIgammai2 and Src synergistically induced the anchorage-independent growth of nonmalignant cells. This study uncovers a novel mechanism where a phosphoinositide-synthesizing enzyme, PIPKIgammai2, functions with the proto-oncogene Src, to regulate oncogenic signaling.

The advent of recombinant DNA technology has led to the identification in the DNA of normal animal cells of over 30 proto-oncogenes that are homologous to retroviral transforming genes. One of these encodes a protein kinase (pp60c-src) of unknown function, that is preferentially synthesized in brain and neural retina. Here the expression of pp60c-src in the peripheral nervous system was examined in sensory neurons from chick dorsal root ganglia with antisera raised against the transforming protein of Rous sarcoma virus (pp60v-src) expressed in Escherichia coli carrying the cloned v-src gene. This antiserum recognizes pp60c-src specifically in normal chicken cells. Western immunoblotting showed that dorsal root ganglia of stage 30 (day 6.5) chick embryos contained elevated levels of pp60c-src. Immunoperoxidase staining of neuron-enriched cultures prepared from chick dorsal root ganglia showed pp60c-src immunoreactivity in cells with neuronal morphology; flat, fibroblastic cells contained no detectable immunoreactivity. Indirect double immunofluorescence with pp60src antibodies and monoclonal antibodies against the 200-kD subunit of neurofilament protein confirmed that the cells expressing pp60c-src were neurons. Ninety-six percent of the neurofilament-positive cells were immunoreactive with pp60src antibodies, and conversely, ALL pp60c-src-positive cells were immunoreactive with neurofilament antibodies. pp60c-src immunofluorescence appeared to be distributed over the cell body, processes, and growth cones. These results clearly demonstrate that pp60c-src is a product of neurons and is expressed in sensory neurons in culture.

BALB-/MK-2 mouse epidermal keratinocytes required epidermal growth factor for proliferation and terminally differentiated in response to high Ca2+ concentration. Infection with retroviruses containing transforming genes of the src and ras oncogene families led to rapid loss of epidermal growth factor dependence, in some cases, accompanied by alterations in cellular morphology. The virus-altered cells continued to proliferate in the presence of high levels of extracellular calcium but exhibited alterations in normal keratinocyte terminal differentiation that appear to be specific to the particular oncogene. These alterations bore similarities to abnormalities in differentiation observed in naturally occurring squamous epithelial malignancies.

The Src-like-adaptor protein (SLAP) is an adaptor protein sharing considerable structural homology with Src. SLAP is expressed in a variety of cells and regulates receptor tyrosine kinase signaling by direct association. In this report, we show that SLAP associates with both wild-type and oncogenic c-Kit (c-Kit-D816V). The association involves the SLAP SH2 domain and receptor phosphotyrosine residues different from those mediating Src interaction. Association of SLAP triggers c-Kit ubiquitylation which, in turn, is followed by receptor degradation. Although SLAP depletion potentiates c-Kit downstream signaling by stabilizing the receptor, it remains non-functional in c-Kit-D816V signaling. Ligand-stimulated c-Kit or c-Kit-D816V did not alter membrane localization of SLAP. Interestingly oncogenic c-Kit-D816V, but not wild-type c-Kit, phosphorylates SLAP on residues Y120, Y258 and Y273. Physical interaction between c-Kit-D816V and SLAP is mandatory for the phosphorylation to take place. Although tyrosine-phosphorylated SLAP does not affect c-Kit-D816V signaling, mutation of these tyrosine sites to phenylalanine can restore SLAP activity. Taken together the data demonstrate that SLAP negatively regulates wild-type c-Kit signaling, but not its oncogenic counterpart, indicating a possible mechanism by which the oncogenic c-Kit bypasses the normal cellular negative feedback control.

We studied the expression of a molecularly cloned human c-src gene, c-src-1, localized on chromosome 20, whose coding region consists of 11 exons and spans a 19.5-kilobase (kb) distance. Using a replication-competent retroviral vector derived from molecularly cloned Rous sarcoma virus DNA (pSRA-2), we obtained two constructs: one (pSR-CS) carrying the unmodified human c-src coding sequence and another (pSR-CVS) with a chimeric gene formed between the human c-src gene and the carboxy-terminal 12-amino acid v-src-specific coding sequence. From chicken embryo fibroblasts transfected with these DNA constructs, infectious viruses designated as WO CS and WO CVS, respectively, were recovered. WO CS virus did not cause cell transformation, whereas WO CVS induced cell transformation. Analyses of the proviral DNAs indicated that ALL introns were spliced out such that the 19-kb inserts were converted to 1.7-kb cDNA forms. Analyses of src proteins in infected cells, using monoclonal antibody MAb327 against v-src protein, showed the following results. The CVS and CS src proteins were about 60 and 61 kilodaltons in size, respectively; the specific protein kinase activity assayed in vitro of the CVS src protein was about 20-fold higher than that of the CS src protein and comparable to that of the v-src protein; the transforming CVS src protein reacted to an antibody against a v-src-specific peptide, whereas the CS src protein did not. These results indicate that the human c-src gene has a potential transforming ability and suggest that the v-src-specific sequence played an important role in the generation of Rous sarcoma virus.

The rate of glucose transport in cultured fibroblasts is regulated to a number of physiological variables, including malignant transformation by src, glucose starvation, and stimulation with mitogens. Much of this transport regulation can be accounted for by variations in the amount of transporter protein in the cells. To determine the mechanisms by which levels of the transporter are regulated, we measured the rates of synthesis and degradation of the transporter by pulse-chase experiments and immunoprecipitation of the transporter. We found that transformation by the src oncogene results in a large decrease in the rate at which the transporter protein is degraded but that it does not appreciably increase the rate of transporter biosynthesis. On the other hand, glucose starvation and mitogen stimulation increase the rate of transporter biosynthesis, although a role for control of degradation is possible in these circumstances also. Variations in the rate of glucose transport or the amount of the transporter are not associated with phosphorylation of the transporter protein.

The chicken cellular proto-oncogene c-src was cotransfected into normal Rat-1 cells with the mouse dhfr gene. Selection for amplification of dhfr sequences resulted in co-amplification of the chicken c-src gene. Cell clones expressing varying levels of c-src associated kinase activity were isolated, none of these had a transformed morphology. In contrast, expression of v-src in Rat-1 cells resulted in morphological transformation and the ability to grow in soft agar in an anchorage independent way.

LMO2 was discovered via chromosomal translocations in T-cell leukaemia and shown normally to be essential for haematopoiesis. LMO2 is made up of two LIM only domains (thus it is a LIM-only protein) and forms a bridge in a multi-protein complex. We have studied the mechanism of formation of this complex using a single domain antibody fragment that inhibits LMO2 by sequestering it in a non-functional form. The crystal structure of LMO2 with this antibody fragment has been solved revealing a conformational difference in the positioning and angle between the two LIM domains compared with its normal binding. This contortion occurs by bending at a central helical region of LMO2. This is a unique mechanism for inhibiting an intracellular protein function and the structural contusion implies a model in which newly synthesized, intrinsically disordered LMO2 binds to a partner protein nucleating further interactions and suggests approaches for therapeutic targeting of LMO2.

Chicken c-src sequences have been transduced by avian leukosis viruses (ALV) and by partial src-deletion (td) mutants of Rous sarcoma virus in several independent events. Analyses of the recombination junctions in the genomes of src-containing viruses and the c-src DNA have shed light on the mechanism of transduction, which involves at least two steps of recombination. The initial recombination between a viral genome and the 5 region of c-src appears to occur at the DNA level. This step does not require extensive homology and can be mediated by stretches of sequences with only partial homology. The 5 recombination junction can also be formed by splicing between viral and c-src sequences. The second recombination is presumed to occur between the transducing ALV or td viral RNA and the viral-c-src hybrid RNA molecule generated from the initial recombination. This step involving recombination at the 3 ends of those molecules restores the 3 viral sequences essential for replication to the viral-c-src hybrid molecule. High frequency of c-src transduction by partial td mutants suggests that the second recombination is greatly enhanced when there is sequence homology between the transducing virus and the 3 region of c-src. Incorporation of the c-src sequences into an ALV genome results in greatly elevated expression of the gene. However, increased expression of c-src alone is insufficient to activate its transforming potential. Structural changes in c-src are necessary to convert it into a transforming gene. The changes can be as small as single nucleotide changes resulting in single amino aid substitutions at certain positions. mutations can occur rapidly during viral replication after c-src is incorporated into the viral genome. Therefore, it is most likely that transduction of c-src by ALV is followed by subsequent mutation and selection for the sarcomagenic virus. In the case of transduction by td viruses that retain certain src sequences, joining of these sequences with the transduced c-src apparently is sufficient to activate its transforming potential.

1. oncogenes appear to play an important role in the physiology of transformed as well as of normal cells. 2. The src oncogene is by far the most investigated oncogene in birds and mammals with respect to the biochemical characteristics of its tyrosine kinase activity, although the specific function of this enzymatic activity still remains to be uncovered. 3. Systematic studies on the src related kinase activity in lower animals are lacking. 4. To contribute to a better understanding of the function of the c-src gene, we performed a comparative study on lower chordates. 5. We were able to demonstrate the presence of c-src related sequences in Acrania, Cyclostomata, cartilagenous and bony fish. 6. By performing the pp60src-specific immune complex assay we detected a tyrosine specific kinase activity, that shows the same biochemical properties as the pp60c-src from higher vertebrates. 7. The level of kinase activity is regulated in an organ specific manner. 8. In lymphocystis tumors of flat-fish and in stomatopapilloma of freshwater eels a considerable amount of pp60c-src kinase activity was found, which, however, never exceeded the levels found in the normal brain.

B cell activation is associated with a marked transient rise in expression of the c-myc proto-onco-gene. A unique opportunity to examine the effects of constitutive c-myc expression upon B cell function is provided by transgenic mice in which the c-myc oncogene is regulated by the enhancer (E mu) from the immunoglobulin locus (E mu-myc mice). We have examined the immunologic competence of B cells from E mu-myc mice both in vitro and in vivo. Upon stimulation, many E mu-myc B cells can proliferate to form clones most of which contain antibody-forming cells. However, the frequency of responsive B cells from E mu-myc donors is only about 30% that of B cells from normal littermates. Thus, enforced myc expression is not sufficient to block the differentiation of ALL B cells, but a much larger fraction of the immunoglobulin-bearing cells from E mu-myc mice are incompetent. Upon immunization, E mu-myc mice mounted specific antibody responses, although some responses were delayed. Isotype switching can occur, since we observed hemolytic plaques of both IgM and IgG type and detected specific antibody of both classes in the serum. Moreover, the serum from nonimmunized E mu-myc mice contained normal levels of both IgM and IgG. Thus constitutive expression of the c-myc gene appears to retard B cell differentiation, but does not grossly impair immunologic function in the intact animal.

Antibodies to phosphotyrosine were used to identify proteins phosphorylated on tyrosine in chicken embryo fibroblasts transformed by the oncogenes, v-src, v-yes, v-fps, v-ros, or v-erb-B. These antibodies allowed detection of a minimum of 50, 44, and 47 bands in immunoblots of cellular lysates from fibroblasts transformed by v-src, v-yes, or v-fps respectively. Eight of these bands were also detected in fibroblasts transformed by v-ros and v-erb-B, suggesting that the cellular transformation induced by v-ros, v-erb-B, v-src, v-yes, and v-fps may be achieved by the phosphorylation of some of the same proteins. The viruses SD10 and SD11 are point mutants of the Prague-C strain of Rous sarcoma virus that encode non-myristylated p60v-src. They do not induce the transformation of chicken fibroblasts. Approximately thirty bands were detected by antibodies to phosphotyrosine in fibroblasts transformed by wild-type Prague-RSV-C, only four of which were not substrates nonmyristylated mutant p60v-src. This suggests that the phosphorylation of a large subset of the substrates of p60v-src is not sufficient for transformation.

expression of the c-src locus in skeletal muscle of the chicken was found to be different from other tissues. A 4 kilobases (kb) cellular src (c-src) mRNA, which most likely codes for pp60c-src, is expressed in various chicken tissues examined, and most of its approximately 2.2 kb non-coding sequences are present at the 3 end of the RNA molecule. Whereas the 4 kb RNA is also expressed in embryonic chicken muscle, it disappears shortly before hatching and is replaced by a smaller-sized class of c-src mRNAs of 2.8 to 3.3 kb. The transition of expression from the 4 kb to the 2.8-3.3 kb RNA in skeletal muscle occurs shortly before hatching of the chicken and persists into adulthood. The two classes of the c-src mRNA share sequences both within the 5 coding and the 3 non-coding regions of pp60c-src, however, the 2.8 to 3.3 kb RNA lacks most of the kinase domain, but it contains upstream c-src sequences missing in the 4 kb RNA. The two classes of c-src mRNA most likely are generated by alternative splicing, and the smaller c-src RNA most likely codes for a product other than tyrosine protein kinase.

We found local sequence-similarity between the non-catalytic region of the Rous sarcoma virus oncogene product, p60v-src, and the core region of cytoskeletal keratin through an extensive similarity search of segments of proteins. The segments showing similarity in p60v-src were in the region that is important for morphological transformation, and corresponded to segments with unique structural features predicted for intermediate filament proteins. We suggest that cellular components related with intermediate filament proteins or the sequence shared by the two proteins may be involved in the regulation of the kinase activity or substrate specificity of p60v-src.

We used myristylated and nonmyristylated c-src-based variants and phosphotyrosine-specific antibodies to reevaluate the role of tyrosine phosphorylation in cellular transformation by pp60src. Prior methods used to detect tyrosine-phosphorylated proteins failed to discriminate predicted differences in tyrosine phosphorylation which are clearly observed with phosphotyrosine-specific antibodies and Western blotting (immunoblotting). Here we report the observation of a 120,000-Mr protein whose phosphorylation on tyrosine correlates with the induction of morphological transformation. p120 was not observed in cells overexpressing the regulated, nononcogenic pp60c-src, whereas phosphorylation of p120 was greatly enhanced in cells expressing activated, oncogenic pp60527F. Furthermore, phosphorylation of p120 was not induced by expression of the activated but nonmyristylated src variant pp602A/527F, which is transformation defective. p120 partitioned preferentially with cellular membranes, consistent with the observation that transforming src proteins are membrane associated. Although a number of additional putative substrates were identified and partially characterized with respect to intracellular localization, tyrosine phosphorylation of these proteins was not tightly linked to transformation.

The observation of a slower migrating form of pp60c-src in neural tissue of chicken and mouse has recently been shown to be due to an alternative transcript form of the c-src gene (Martinez et al.: Science 237:411-415, 1987; Levy et al.: Mol Cell Biol 7:4142-4145, 1987). An insertion of 18 basepairs between exons 3 and 4, presumed to be due to alternative splicing of a mini-exon, gives rise to six amino acid residues not found in the non-neuronal (termed fibroblastic) form of pp60c-src. We have addressed the question of the evolutionary origin of the c-src neuronal insert and its functional significance regarding neural-specific expression of the c-src gene. To this end we have investigated whether the c-src gene of a lower vertebrate (the teleost fish Xiphophorus) gives rise to a neural-specific transcript in an analogous manner. We could show that the fish c-src gene does encode for a "fibroblastic" and a "neuronal" form of transcript and that the neuronal transcript does indeed arise by way of alternative splicing of a mini-exon. The mini-exon is also 18 basepairs long and we could demonstrate directly that this exon lies within the intron separating exons 3 and 4. For comparative purposes we have examined whether the fish c-yes gene, the member of the src gene family most closely related to c-src, also encodes a neural tissue-specific transcript. No evidence for a second transcript form in brain was obtained.(ABSTRACT TRUNCATED AT 250 WORDS)FAU - Raulf, F

In situ hybridization of the pHul-c-src probe to metaphase cells from three normal donors and two leukemic patients showed significant labeling in the proximal region of the long arm of chromosome 20q, with modal peaks of grains consistently at band 20q11.2. A secondary peak of grains was detected in the region 20q13.2-qter, the localization of SRC suggested by previous in situ studies. The exact localization of SRC is important for understanding the del(20q) chromosomal abnormality in myeloid neoplasias. Chromosome in situ hybridization and genomic studies showed loss of one allele of SRC in two patients with the deletion (20q). These results differ from previously published findings and suggest heterogeneity of the breakpoint at 20q11.2 in interstitial deletions of 20q, which characterize myeloid disorders.

Oncogene-mediated cellular transformation is a multistep process involving activation of growth-promoting pathways as well as inactivation of tumor suppressors. We recently found that ISGylation of the p53 tumor suppressor is an important novel mechanism to control its stability. Here we identified that Isg15-dependent regulation of p53 can be enhanced by different oncogenes. We further show that the Src-mediated phosphorylation of p53 on Tyr126 and Tyr220 has a positive effect on p53 ISGylation by enhancing Herc5 binding. In turn, deletion of Isg15 results in accumulation and activation of native p53 in transformed cells thus increasing its anti-cancer activity and suppressing tumorigenesis in mice. We propose that Isg15-dependent degradation of p53 is an alternative pathway for oncogenes to regulate p53 activity, and thus is an attractive pathway for development of new anti-cancer drugs.

There is an established role for the endocytic pathway in regulation of epidermal growth factor receptor (EGFR) signaling to downstream effectors. However, because ligand-mediated EGFR endocytosis utilizes multiple "moving parts," dissecting the spatial versus temporal contributions has been challenging. Blocking ALL endocytic trafficking can have unintended effects on other receptors as well as give rise to compensatory mechanisms, both of which impact interpretation of EGFR signaling. To overcome these limitations, we used epidermal growth factor (EGF) conjugated to polystyrene beads (EGF beads). EGF beads simultaneously activate the EGFR while blocking its endocytosis and allow analysis of EGFR signaling from the plasma membrane. Human telomerase immortalized corneal epithelial (hTCEpi) cells were used to model normal epithelial cell biology. In hTCEpi cells, both cell surface and intracellular EGFRs exhibited dose-dependent increases in effector activity after 15 min of ligand stimulation, but only the serine phosphorylation of signal transducer and activator of transcription 3 (STAT3) was statistically significant when accounting for receptor phosphorylation. However, over time with physiological levels of receptor phosphorylation, cell surface receptors produced either enhanced or sustained mitogen-activated protein kinase kinase (MEK), Casitas B-lineage lymphoma (c-Cbl), and the pro-oncogene Src activity. These increases in effector communication by cell surface receptors resulted in an increase in EGFR ubiquitylation with sustained ligand incubation. Together, these data indicate that spatial regulation of EGFR signaling may be an important regulatory mechanism in receptor down-regulation.

The cytolytic effect of the autonomous parvovirus minute virus of mice, prototype strain (MVMp), was studied in cultures of ts 339/NRK rat cells that display a temperature-sensitive transformed phenotype as a result of their transformation with a Rous sarcoma virus strain matured in the v-src oncogene. A shift from restrictive (39.5 degrees C) to permissive (34.5 degrees C) temperature was associated with a marked sensitization of these cells to killing by MVMp. In contrast, ts 339/NRK cell derivatives supertransformed with a wild-type src oncogene were sensitive to MVMp at both temperatures, suggesting that the expression of a functional oncogene product may determine, at least in part, the extent of the parvoviral cytopathic effect. Although ts 339/NRK cells were quite resistant to parvoviral attack at 39.5 degrees C, they were similarly proficient in MVMp uptake, viral DNA and protein synthesis, and infectious particle production at both permissive and restrictive temperatures. Consistently, electron microscopic examination of infected ts 339/NRK cultures incubated at 39.5 degrees C revealed the presence, in the majority of the cells, of numerous full and empty virions that were predominantly located in autophagic-type vacuoles. Thus, in this system, the reversion of transformed and MVMp-sensitive phenotypes appears to correlate with the setting up of a noncytocidal mode of parvovirus production. These results raise the possibility that the physiological state of host cells may affect their susceptibility to parvoviruses by modulating not only their capacity for virus replication but also cellular processes controlling the cytopathic effect of viral products.

Dysregulation of the von Hippel-Lindau/hypoxia-inducible transcription factor (HIF) signaling pathway promotes clear cell renal cell carcinoma (ccRCC) progression and metastasis. The protein kinase GAS6/AXL signaling pathway has recently been implicated as an essential mediator of metastasis and receptor tyrosine kinase crosstalk in cancer. Here we establish a molecular link between HIF stabilization and induction of AXL receptor expression in metastatic ccRCC. We found that HIF-1 and HIF-2 directly activate the expression of AXL by binding to the hypoxia-response element in the AXL proximal promoter. Importantly, genetic and therapeutic inactivation of AXL signaling in metastatic ccRCC cells reversed the invasive and metastatic phenotype in vivo. Furthermore, we define a pathway by which GAS6/AXL signaling uses lateral activation of the met proto-oncogene (MET) through SRC proto-oncogene nonreceptor tyrosine kinase to maximize cellular invasion. Clinically, AXL expression in primary tumors of ccRCC patients correlates with aggressive tumor behavior and patient lethality. These findings provide an alternative model for SRC and MET activation by growth arrest-specific 6 in ccRCC and identify AXL as a therapeutic target driving the aggressive phenotype in renal clear cell carcinoma.

One of the main targets of pp60v-src tyrosine kinase is a 34 to 39-kilodalton protein of chicken embryo fibroblasts called p36 or calpactin I. We have previously reported an association of the cytoplasmic fraction of p36 (10-20% of the total cellular p36) with three chicken polypeptides named p32, p48, and p54. We have now raised and affinity-purified antibodies against each of these proteins. This has allowed their identification: p32 is lactate dehydrogenase, p48 is enolase, and p54 is phosphoglucose isomerase. An association between p36 and two other known substrates of pp60v-src, the glycolytic enzymes enolase and lactate dehydrogenase, suggests a cellular organization of the various targets of the oncogene tyrosine kinases. Furthermore, a possible relationship between p36 and glycolysis is questioned.

Src family kinases (SFK) integrate signal transduction for multiple receptors, regulating cellular proliferation, invasion, and metastasis in human cancer. Although Src is rarely mutated in human prostate cancer, SFK activity is increased in the majority of human prostate cancers. To determine the molecular mechanisms governing prostate cancer bone metastasis, FVB murine prostate epithelium was transduced with oncogenic v-Src. The prostate cancer cell lines metastasized in FVB mice to brain and bone. Gene expression profiling of the tumors identified activation of a CCR5 signaling module when the prostate epithelial cell lines were grown in vivo versus tissue cultures. The whole body, bone, and brain metastatic prostate cancer burden was reduced by oral CCR5 antagonist. Clinical trials of CCR5 inhibitors may warrant consideration in patients with CCR5 activation in their tumors.

We previously demonstrated that sialidase NEU3, a key glycosidase for ganglioside degradation, is up-regulated in various human cancers, leading to increased cell invasion, motility and survival of cancer cells possibly through activation of EGF signaling. Its up-regulation is also important for promotion of the stage of colorectal carcinogenesis in vivo in human NEU3 transgenic mice treated with azoxymethane for the induction of aberrant crypt foci in the colon mucosa, accompanied by enhanced phosphorylation of EGF receptor (EGFR). To address whether the activation of EGF signaling by the sialidase is associated with oncogenic transformation, we here analyzed the effects of overexpression of NEU3 and EGFR in NIH-3T3 cells. When NEU3 was stably transfected with or without EGFR, it was associated with significant increases in clonogenic growth, clonogenicity on soft agar and in vivo tumor growth in nude mice either with or without the receptor overexpression in the presence of EGF, compared with the levels in their vector controls. Despite the fact that the endogenous level of EGFR is known to be extremely low in these cells, NEU3 significantly enhanced the phosphorylation of Akt and ERK, as well as that of the receptor. The NEU3-mediated activation was largely abrogated by the EGFR inhibitor AG1478 or PD153035, but significant clonogenic growth still remained. NEU3 was then found to activate Src kinase, and the clonogenicity was completely suppressed by an Src inhibitor, PP2. The activity-null mutants failed to activate Src and EGFR, indicating that ganglioside modulation by NEU3 may be necessary for the activation. NEU3 and Src were co-immunoprecipitated with EGFR in NEU3- and EGFR- transfected cells. These findings identify NEU3 as an essential participant in tumorigenesis through the EGFR/Src signaling pathway and a potential target for inhibiting EGFR-mediated tumor progression.

The cellular Src (c-Src) tyrosine kinase is upregulated and believed to play a pivotal role in various human cancers. However, the molecular mechanism underlying c-Src-mediated tumour progression remains elusive. Recent studies have revealed that several microRNAs (miRNAs) function as tumour suppressors by regulating the malignant expression of signalling molecules. Aberrant expression of miRNAs is frequently observed in human cancers and should be exploited to seek related molecular targets. In this review, we focus on miRNAs found to be involved in Src signalling in various cancers. We summarize recent findings on Src-related miRNAs, their target genes, mechanisms behind their interplay and their implications for cancer therapeutics.

Hsp90 is a molecular chaperone involved in the activation of numerous client proteins, including many kinases. The most stringent kinase client is the oncogenic kinase v-Src. To elucidate how Hsp90 chaperones kinases, we reconstituted v-Src kinase chaperoning in vitro and show that its activation is ATP-dependent, with the cochaperone Cdc37 increasing the efficiency. Consistent with in vivo results, we find that Hsp90 does not influence the almost identical c-Src kinase. To explain these findings, we designed Src kinase chimeras that gradually transform c-Src into v-Src and show that their Hsp90 dependence correlates with compactness and folding cooperativity. Molecular dynamics simulations and hydrogen/deuterium exchange of Hsp90-dependent Src kinase variants further reveal increased transitions between inactive and active states and exposure of specific kinase regions. Thus, Hsp90 shifts an ensemble of conformations of v-Src toward high activity states that would otherwise be metastable and poorly populated.

Given the side effects of vaccination against infectious laryngotracheitis (ILT), novel strategies for ILT control and therapy are urgently needed. The modulation of host-virus interactions is a promising strategy to combat the virus; however, the interactions between the host and avian ILT herpesvirus (ILTV) are unclear. Using genome-wide transcriptome studies in combination with a bioinformatic analysis, we identified proto-oncogene tyrosine-protein kinase Src (Src) to be an important modulator of ILTV infection. Src controls the virulence of ILTV and is phosphorylated upon ILTV infection. Functional studies revealed that Src prolongs the survival of host cells by increasing the threshold of virus-induced cell death. Therefore, Src is essential for viral replication in vitro and in ovo but is not required for ILTV-induced cell death. Furthermore, our results identify a positive-feedback loop between Src and the tyrosine kinase focal adhesion kinase (FAK), which is necessary for the phosphorylation of either Src or FAK and is required for Src to modulate ILTV infection. To the best of our knowledge, we are the first to identify a key host regulator controlling host-ILTV interactions. We believe that our findings have revealed a new potential therapeutic target for ILT control and therapy. IMPORTANCE: Despite the extensive administration of live attenuated vaccines starting from the mid-20th century and the administration of recombinant vaccines in recent years, infectious laryngotracheitis (ILT) outbreaks due to avian ILT herpesvirus (ILTV) occur worldwide annually. Presently, there are no drugs or control strategies that effectively treat ILT. Targeting of host-virus interactions is considered to be a promising strategy for controlling ILTV infections. However, little is known about the mechanisms governing host-ILTV interactions. The results from our study advance our understanding of host-ILTV interactions on a molecular level and provide experimental evidence that it is possible to control ILT via the manipulation of host-virus interactions.

The Georgiev promotor hypothesis about the cutting of cell genome and the latter being subjected to promotor control and virus regulation was used. On its basis the mechanism of temperature switching off of Src oncogene TS-mutant was investigated. It was shown that the model can correlate with the experiment in suggesting the regulating enzyme sigmoid kinetics and the temperature optimum below 35 degrees C. Hysteretic effect allows one to maintain a "healthy" state throughout the temperature switching off.

The role of tyrosine phosphorylation in the regulation of tyrosine protein kinase activity was investigated using site-directed mutagenesis to alter the structure and environment of the three tyrosine residues present in the C terminus of avian pp60c-src. mutations that change Tyr 527 to Phe or Ser activate in vivo tyrosine protein kinase activity and induce cellular transformation of chicken cells in culture. In contrast, alterations of tyrosine residues present at positions 511 or 519 in c-src do not induce transformation or in vivo tyrosine protein kinase activity. Amber mutations, which alter the structure of the pp60c-src C terminus by inducing premature termination of the c-src protein at either residue 518 or 523 also induce morphological transformation and increase in vivo tyrosine phosphorylation, whereas removal of the last four residues of c-src by chain termination at residue 530 does not alter the kinase activity or the biological activity of the resultant c-src protein. We conclude from these studies that C-terminal alterations which either remove or replace Tyr 527 serve to activate the c-src protein resulting in cellular transformation and increased in vivo tyrosine protein kinase activity.

The transforming protein of Rous sarcoma virus, pp60v-src, is covalently coupled to myristic acid by an amide linkage to glycine 2. Myristylation promotes the association of pp60v-src with cellular membranes, and this subcellular location is essential for transforming activity. The findings presented here, in conjunction with the previous reports of others, imply that the seventh amino acid encoded by v-src might be important in the myristylation reaction. Replacement of lysine 7 by asparagine greatly reduced the myristylation, membrane association, and transforming activity of pp60v-src. In contrast, substitution of arginine at residue 7 had no effect on any of these properties of pp60v-src. Addition of amino acids 1 to 7 encoded by v-src was sufficient to cause myristylation of a src-pyruvate kinase fusion protein. We conclude that the recognition sequence for myristylation of pp60v-src comprises amino acids 1 to 7 and that lysine 7 is a critical component of this sequence.

We previously described a recombinant Moloney murine leukemia virus (Mo-MuLV) carrying the v-src oncogene, Mo-MuLV(src). Mo-MuLV(src) encodes a gag-src fusion protein, transforms cells in culture, and induces fibrosarcomas in vivo. To compare transforming properties of the gag-src fusion protein to pp60src encoded by Rous sarcoma virus, we constructed a new recombinant virus, Mo-MuLV(+ src). Mo-MuLV(+ src) encodes pp60src in the context of Mo-MuLV. Cells transformed by Mo-MuLV(+ src) were round and formed colonies in soft agar, whereas Mo-MuLV(src)-infected cells were fusiform and did not grow in suspension. Thus, the extent of transformation induced by Mo-MuLV(+ src) was greater than that induced by Mo-MuLV(src). Subcutaneous inoculation of either virus into neonatal NIH Swiss mice resulted in fibrosarcomas at the site of injection. Further studies indicated that tumors induced by Mo-MuLV(+ src) grew rapidly but rarely metastasized. In contrast, tumors induced by Mo-MuLV(src) grew somewhat more slowly but metastasized with a high frequency (60%). These viruses may provide a useful model system for tumor metastasis. Another src-containing virus was also studied, MRSV (constructed by Anderson and Scolnick). MRSV also encodes pp60src but in the context of amphotropic MuLV. When injected intravenously into six-week-old mice, MRSV induced splenomegaly and spleen foci but no solid tumors, as reported previously. In contrast, Mo-MuLV(src)-induced fibrosarcomas mostly in the spleen under the same inoculation protocol. These results suggest that the v-src oncogene was the major pathogenic determinant in neonatal mice for ALL three src-containing viruses; however, variations in the nature of the transforming protein modulated the behavior of the induced tumors. In adult mice, greater differences in pathogenicity were observed.

A novel gene was created that linked complementary portions of two different tyrosine kinase oncogenes: v-erB and v-src. The v-erbB/src chimera encoded a glycoprotein exhibiting the subcellular distribution of the v-erbB protein but containing the kinase catalytic domain of the v-src parent. Fibroblasts expressing the v-erbB/src gene product became transformed to an oncogenic state and closely resembled cells expressing the v-erbB parent oncogene. Our results indicated that v-erbB sequences can be functionally replaced by sequences derived from a different oncogene, v-src, and that important determinants of the transformed phenotype appear to be encoded in oncogene sequences distinct from those defining the kinase catalytic domain itself.

Interstrain restriction fragments length polymorphism (RFLP) was detected after Southern blot hybridization of DNA from spontaneously hypertensive rats (SHR) and WKY rats treated with Bam HI restrictase with c-fos probe. The SHR genome is characterized by an additional miner band of 4.0 kilobase. RFLP was also revealed in c-src locus by Eco RI and Hind III restrictases. The major characteristic bands are 1.6 kb (SHR) and 2.4 kb (WKY) after Eco RI restriction and 3.4 kb (SHR) and 4.1 kb (WKY) after Hind III restriction. These RFLP can be used as mendelian traits in the linkage studies of distribution of blood pressure and other quantitative physiological traits in (SHR x WKY) F2 hybrids. The interstrain polymorphism determined in c-fos and c-src can also appear important in the evaluation of their physiological role in the cell.

Interstrain restriction fragment length polymorphism was detected after Southern blot hybridization of spontaneously hypertensive (SHR) and control (WKY) rat DNA digested by Bam HI restrictase with a v-fos probe. The SHR genome was characterized by an additional minor band of 4.0 kb. Other restriction fragment length polymorphism was revealed in the c-src locus by Eco RI, Hind III and Pst I restrictases. The major characteristic bands were 1.6 kb (SHR) and 2.4 kb (WKY) after Eco RI restriction; 3.4 kb (SHR) and 4.1 kb (WKY) after Hind III restriction and 4.0 kb (SHR) and 4.6 kb (WKY) after Pst I restriction. This restriction fragment length polymorphism can be used as Mendelian traits in linkage studies on the distribution of blood pressure and other quantitative physiological traits in (SHR x WKY)F2 hybrids.

The concentration of fructose 2,6-bisphosphate and the activity of 6-phosphofructo-2-kinase are increased after infection of chick-embryo fibroblasts with the Rous sarcoma virus, or with a temperature-sensitive mutant of this virus at the permissive, but not at the non-permissive, temperature. This is observed after transformation by retroviruses carrying either the v-src or v-fps, but not the v-mil and/or v-myc, oncogenes. Comparison of the effects of the Rous sarcoma virus with those of phorbol myristate acetate on fructose 2,6-bisphosphate suggests that both result from the stimulation of a step which is rate-limiting for 6-phosphofructo-2-kinase activation and which is also controlled by protein kinase C.

The ability of cloned Rous sarcoma virus (RSV) DNA encoding the v-src oncogene to neoplastically transform normal, diploid Syrian hamster embryo (SHE) cells was examined. Transfection of RSV DNA into early passage SHE cells resulted in a low but significant number of tumors when treated cells were injected into nude mice. tumors formed with a low frequency (two tumors out of ten sites injected) and only after a long latency period (14 weeks). In contrast to the normal SHE cells, several different carcinogen-induced preneoplastic immortal SHE cell lines were highly susceptible to transformation by the v-src oncogene to the neoplastic phenotype. tumors formed with high efficiency and a short latency period (less than 3 weeks). Further studies were performed to determine the basis for the inefficient transformation of the normal SHE cells. NeoR clones isolated after cotransfection of SHE cells with pSV2-neo and RSV DNAs were neither morphologically altered nor immortal and did not contain detectable levels of the v-src gene product. These results suggest that neoplastic transformation by v-src DNA in the normal cells is initially suppressed. However, cells from a v-src-induced tumor expressed v-src RNA, and antibody to v-src protein precipitated from the tumor cells a 60,000-molecular-weight protein which displayed protein kinase activity. Karyotypic analyses confirmed that the tumor was derived from Syrian hamster cells and suggested that it was clonal in nature. These results indicate that the v-src oncogene was primarily responsible for neoplastic transformation of SHE cells. In contrast to the results with the v-src oncogene, our previous studies showed that v-Ha-ras oncogene alone is unable to induce neoplastic transformation of SHE cells. Furthermore, the v-myc oncogene was able to compliment v-Ha-ras to neoplastically transform SHE cells, while cotransfection with v-src plus v-myc did not increase the incidence of tumors.

PC12 rat phaeochromocytoma cells are a model system that can be used to study both neuronal differentiation and the mechanism of action of nerve growth factor (NGF). PC12 cells respond to NGF protein by shifting from a chromaffin-cell-like phenotype to a neurite-bearing sympathetic neurone-like phenotype. Here we present data on the effect of infection of PC12 cells with retroviruses carrying the src oncogene of Rous sarcoma virus. Previous studies have demonstrated that the expression of src severely affects the synthesis and accumulation of differentiated cell products in a variety of cell types. We show that in the PC12 cell system, expression of v-src appears to have an inductive effect on differentiation that resembles the action of a physiological growth factor.

Density-dependent inhibition of growth has been assumed to be under the control of inhibitory molecules diffusing from dense cell cultures. Growth inhibitory factors have been fractionated or purified from medium conditioned by different cell types. In the present work, it was shown that IDF45 (inhibitory factor diffusing from 3T3 cells) decreased DNA synthesis in chick embryo fibroblasts (CEF) and was an inhibitor of CEF growth; this inhibition was reversible. Since similitudes between oncogene products and growth factors have been observed, it was of interest to compare the inhibitory effect of IDF45 upon the stimulation of DNA synthesis induced either by serum or by pp60-src. CEF infected by Ny68 virus (a mutant of Rous sarcoma virus ts for the expression of transformation) were density-inhibited at 41 degrees C, but were stimulated at this temperature by addition of 1% serum. This stimulation was 94% inhibited by IDF45. The same Ny68-infected cells could also be stimulated by transfer to 37 degrees C, the permissive temperature (in the absence of serum). The stimulation of DNA synthesis by src expression was poorly inhibited by IDF45. From our results, it appears that oncogene expression in CEF induces a loss in their sensitivity to IDF45. This would explain why transformed cells escape DDI of growth.

The effects of src oncogene expression on epidermal growth factor (EGF) receptors have been investigated in mouse 3T3 and rat-1 fibroblasts. Transformation of both cell types with src resulted in marked reductions in cellular EGF receptor levels, as assayed by either 125I-EGF binding or immunoprecipitation of receptor protein from radiolabeled cell lysates. In contrast to cells transformed by other types of retroviral oncogenes, the loss of EGF receptors in the src-transformed cells did not appear to be due to secreted transforming growth factor-alpha (TGF-alpha), since such factors were undetectable in culture fluids from the src-transformed cells. By several criteria of transformation, an EGF-receptorless cell line infected with src was shown to be transformed, suggesting that EGF receptors themselves are not obligatory to the src transformation process. We suggest that pp60src down-modulates EGF receptors by an intracellular mechanism and that the loss of the receptors is symptomatic of more general effects of pp60src on the machinery of growth regulation.

Insulin receptors resemble receptors for certain growth factors (epidermal growth factor, platelet-derived growth factor, and insulin-like growth factor I) in that ALL possess tyrosine-specific protein kinase activity. These cell surface receptors resemble protein kinases encoded by viral oncogenes in that both groups of enzymes phosphorylate proteins on tyrosine. Recently, we reported that there is immunological similarity between the insulin receptor and pp60src [the protein encoded by the src oncogene of Rous sarcoma virus (RSV)]. This is supported by the observation that anti-pp60src antiserum (TBR serum) immunoprecipitated radiolabeled insulin receptors derived from cultured human cells (IM-9 lymphoblasts and U-937 monocytes) and rabbit liver. Moreover, highly purified preparations of src protein inhibit the immunoprecipitation of insulin receptors by TBR serum, and the inhibition is correlated with the src kinase activity present in the preparation used. However, two observations suggested that there were immunological differences between pp60src and mammalian insulin receptors. 1) Even at a relatively high concentration (dilution, 1:10), TBR serum immunoprecipitated a relatively small percentage (approximately 20%) of the labeled insulin receptors. 2) Some lots of TBR serum with a high titer against pp60src failed to immunoprecipitate the insulin receptor. Viral oncogenes are thought to have been derived from proto-oncogenes in the host cell. Therefore, because the chicken is the natural host for RSV, we inquired whether there might be closer homology between pp60src and avian insulin receptors. Surprisingly, under conditions where TBR serum immunoprecipitates human insulin receptors, we could not detect immunoprecipitation of avian insulin receptors from chicken liver, chicken embryo fibroblasts, or turkey erythrocytes. The immunoprecipitation of human insulin receptor is not dependent on the method used for labeling the cells ([125I]insulin cross-linking), inasmuch as the receptor labeled by autophosphorylation with [gamma-32P]ATP could also be immunoprecipitated by TBR serum. These observations suggest that there is structural homology between pp60src and the insulin receptor (most likely the beta-subunit). Nevertheless, it seems unlikely that the insulin receptor gene is the proto-oncogene for the src gene of RSV.

We have isolated cDNA molecules representing the complete coding sequence of a new human gene which is a member of the src family of oncogenes. Nucleotide sequence analysis revealed that this gene, termed slk, encoded a 537-residue protein which was 86% identical to the chicken proto-oncogene product, p60c-src, over a stretch of 191 amino acids at its carboxy terminus. In contrast, only 6% amino acid homology was observed within the amino-terminal 82 amino acid residues of these two proteins. It was possible to activate slk as a transforming gene by substituting approximately two-thirds of the slk coding sequence for an analogous region of the v-fgr onc gene present in Gardner-Rasheed feline sarcoma virus. The resulting hybrid protein molecule expressed in transformed cells demonstrated protein kinase activity with specificity for tyrosine residues.

Both interleukin 2 (IL-2) and epidermal growth factor (EGF) receptors exist in two forms that differ with respect to affinity for their ligand. Only the high-affinity receptors appear to be responsible for the proliferation signal delivered upon binding of the growth factor. Fibroblasts transfected with IL-2 receptor cDNA generate only low-affinity receptors for IL-2, but fusion of membranes from these fibroblasts with T-cell membranes converts some receptors to high affinity, indicating the involvement of a T cell-specific factor in the generation of high-affinity receptors. We have constructed a chimeric cDNA molecule containing the extracellular IL-2-binding domain of the IL-2 receptor cDNA and the transmembrane and intracellular tyrosine kinase domains of the EGF receptor cDNA. When transfected into fibroblasts, this IL-2-EGF receptor cDNA generated high-affinity receptors for IL-2. Moreover, fibroblasts transfected with the chimeric molecule were morphologically transformed and produced rapidly growing tumors in nude mice.

This study examined the possibility of using an immunohistochemical technique to detect the expression of myc and src oncogene proteins (ops) in livers of male Sprague-Dawley rats after treatment with the carcinogen diethylnitrosamine (with or without phenobarbital promotion) or untreated. We found that the majority of nodules and tumors from these livers stained for myc and src ops, indicating that myc and src expression did occur in these structures. These results were expected, since myc and src expression has been previously observed by others using different techniques. However, in our study, myc and src op staining was also noted in normal liver areas from rats in any of the four treatment groups (DENA, DENA + PB, PB alone, or untreated). The staining pattern of normal liver was different for each oncogene probe but was consistent within the four groups. In most cases, oncogene expression of normal liver occurred at sites of abnormal (but non-neoplastic) hepatocytes. The method reported here used both a qualitative technique of op expression analysis and a quantitative method using a Zeiss computer-driven image analysis system.

NIH 3T3 cells were transfected by plasmid containing v-src under control of hormone-regulated LTR MMTV (pMLsrc10). This plasmid caused the foci of morphologically transformed cells. The transformed cells induced rapidly growing tumours in nude mice. In the presence of dexamethasone the efficiency of NIH 3T3 cell transformation increased ten times, while tumourigenicity remained unchanged.

Three cloned murine interleukin 3 (IL-3)-dependent cell lines have been converted to interleukin 2 (IL-2) or granulocyte-macrophage colony-stimulating factor (GM-CSF) growth-dependent states. FD.C/1 32Dcl-23 and GM cells grown and maintained as IL-3-dependent cell lines, and cells grown with GM-CSF have been infected with a murine recombinant retrovirus containing the v-src oncogene, and grown as lymphokine-independent cell lines. There is a significant increase in tyrosine kinase activity in cells which become lymphokine-independent. FD.C/1 and 32Dcl-23 cells maintained as IL-2-dependent cells lines and infected with the same virus did not grow as IL-2-independent cells. The lymphokine-independent cells FD.C/1src, 32Dsrc, and GMsrc ALL expressed high levels of tyrosine kinase activity, ranging from 5- to 20-fold more than levels measured in virus-infected cell lines maintained as IL-2-dependent cells. The exposure of FD.C/1src and 32Dsrc cells to IL-3, and GMsrc cells to IL-3 or GM-CSF, resulted in significant decreases in tyrosine kinase activity. These changes were rapidly reversed by removal of IL-3 or GM-CSF from these cells. However, the synthesis of v-src-specific RNA was not affected by the presence of IL-3 or GM-CSF in these cell lines. The biochemical pathways activated by IL-3 or GM-CSF inhibit the activity of the tyrosine kinase encoded by the v-src oncogene without altering gene transcription.

Transformation of both rat and chicken fibroblasts by the src oncogene leads to a four- to fivefold increase in the rate of glucose transport and in the level of the glucose transporter protein. We have previously shown that, with chicken embryo fibroblasts, transformation leads to a reduction in the rate of degradation of the transporter, with little or no increase in the rate of its biosynthesis. We now show that, with the rat-1 cell line, the opposite result was obtained. src-induced transformation led to an increase in transporter biosynthesis, with little effect on turnover. A src-induced increase in transporter mRNA entirely accounted for the increase in biosynthesis of the protein. By contrast, in chicken embryo fibroblasts, the level of transporter mRNA was low and was not induced to rise by src transformation. Thus, src induced an increase in the level of the glucose transport protein by fundamentally different mechanisms in chicken embryo fibroblasts and rat-1 cells. To test whether this difference was due to rat-1 cells being an immortalized cell line, we measured transporter mRNA levels in primary fibroblast cultures from rat embryos and in parallel cultures transformed by src. Transporter mRNA was inducible by src in these cells. Thus, the difference in mRNA inducibility between chicken and rat cells is not due to immortalization.

The cloned murine interleukin 3 (IL 3)-dependent cell lines FD.C/1, 32Dc1-23, and KP3 can each be switched to interleukin 2 (IL 2)-dependent growth states. Replication-defective retroviral vectors have been used to introduce the v-src oncogene into each of these cell lines maintained in either an IL 3- or an IL 2-dependent growth state. These cell lines maintained in an IL 3-dependent growth state were converted to lymphokine-independent growth after infection with v-src. These same cells maintained in an IL 2-dependent growth state and infected with v-src maintained strict lymphokine dependence for growth. Another cloned murine IL 3-dependent cell line, GM, can be switched to a granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent growth state. GM cells maintained as IL 3- or GM-CSF-dependent cells readily converted to a lymphokine-independent growth state when infected with v-src. These experiments indicate that either there exist differences in the biochemical mechanisms of signal transduction through the IL 3- and IL 2-specific receptors, or developmental processes associated with the switching of cells to an IL 2-dependent growth state influence expression of the v-src gene product. These cell lines offer new ways not only for analyzing biochemical pathways that regulate cell growth, but also for analyzing the control of oncogene expression.

In the haemopoietic system the mature blood cells have only a finite lifetime. For example, a circulating granulocyte in the peripheral blood has an approximate half-life of 7 h (Cartwright, Athens & Wintrobe, 1964; Dancey, Dubelbeiss, Harker & Finch, 1976) whilst the lifetime of an erythrocyte is approximately 120 days (Wickramasinghe & Weatherall, 1982). This constant death of mature functional haemopoietic cells means that new blood cells must replace those that are removed. The process of haemopoiesis provides the mature functional blood cells to replace those lost as a consequence of performing their biological functions (e.g. lymphocytes and macrophages in the immune response) or through apparent old age and breakdown (e.g. erythrocytes that are 110-120 days old). The major questions that we are required to answer about this process are where do ALL these new cells come from? , what regulates their production? and how is this mechanism of control lost in haemopoietic disorders such as leukaemia, hyperproliferative diseases and anaemias? . Recent work in the field of haemopoiesis has given some clues to the answers to the questions, which provide an intriguing insight into not only haemopoiesis itself but the possible lesions associated with the various blood disorders.

C3H/10T1/2-CL8 mouse cells were shown to take up and express a plasmid-cloned drug resistance gene (Ecogpt) after DNA transfection at a frequency (2-6 X 10(-4) which is acceptable for routine recovery of gene-transformed populations. Transfection of 10T1/2 cells with a mutant c-Ha-ras oncogene (pEJ6.6 plasmid) results in neoplastically transformed 10T1/2 cell populations as judged by colony morphology and tumorigenic growth in nude mice. The levels of mutant c-Ha-ras gene integration and expression in the tumorigenic cell populations and 10T1/2 cell controls were determined, and the highest level of mutant ras transcript was seen in the most tumorigenic cell population. A preliminary comparison of 10T1/2 and NIH/3T3 cells showed similar frequencies for pEJ 6.6-induced transformed foci and a similar lack of sensitivity to the transforming effects of a cloned B-lym oncogene. The results identify a genetic event, which has previously been shown to be carcinogen-inducible, that is permissive for neoplastic transformation of the widely used carcinogen-transformable 10T1/2 mouse cell line.

The nucleotide sequence of a PstI fragment prepared from a cloned MH2 virus genome, pMH2-Hd, has been deduced using chemical and enzymatic methods. This fragment, 1862 nucleotides in length, starts with the gag gene, encodes the v-mil sequence and stops within the v-myc gene. This sequence shows that the v-mil gene is fused to the gag gene giving rise to a fused polyprotein of 98 000 daltons: 515 amino acids at the amino terminus would correspond to p10, p19, p27 and part of p12 determinants, 347 amino acids at the carboxy terminus correspond to the v-mil specific sequence. The mil protein shares homology with a number of onc proteins such as src, fes, fms, mos, yes, fps and erbB, as well as with the catalytic chain of the cAMP-dependent protein kinase. This PstI fragment also encodes the beginning of the myc gene which was integrated in MH2 along with the 3 end of the preceding intron placing an acceptor splice site in front of the used open reading frame. As deduced from the sequence, the MH2 myc protein is not identical to the MC29 myc protein. It differs at its amino terminus, which contains little or no gag determinants, depending on the ATG used to initiate translation.

NRK cells infected with a temperature-sensitive, transformation-defective mutant of avian sarcoma virus (ASV), tsLA23, are transformed at 36 degrees C, but at 40 degrees C they behave as nontransformed cells because of the inactivation of the abnormally thermolabile pp60v-src product of the virus transforming src gene. At 40 degrees C, these tsLA23-NRK cells were arrested in G1/G0 by severe serum deprivation. They were induced to enter G1, initiate DNA synthesis 7 or 10 hours later, and then divide as (1) nontransformed cells by adding serum or platelet-derived growth factor (PDGF) at 40 degrees C, or (2) transformed cells by lowering the temperature to a pp60v-src-activating 36 degrees C without adding exogenous growth factor(s). The level of pp60v-src kinase activity rose dramatically in these serum-deprived cells within 30 minutes of lowering the temperature to the permissive 36 degrees C, and it fell just as rapidly when the cells were returned to the restrictive 40 degrees C. As little as a 2-hour exposure to 36 degrees C, with an attendant 2-hour burst of pp60v-src kinase activity, was enough to stimulate serum-deprived tsLA23-NRK cells to transit G1 and initiate DNA replication, but not to divide. Much more prolonged pp60v-src activity was needed for these serum-deprived cells to complete their cycle and divide. The prereplicative development of quiescent tsLA23-NRK cells stimulated by serum or PDGF was accompanied by greatly increased protein synthesis and slightly decreased protein degradation, but the pp60v-src-stimulated cells progressed through G1 and initiated DNA replication without appreciably affecting the protein synthetic machinery of the cell. The cells stimulated by the mitogenic action of pp60v-src, like the cells stimulated by serum, needed to activate early prereplicative genes in order to initiate DNA replication. The needed RNA transcripts induced by serum and pp60v-src were produced with comparable efficiency, although it took longer for pp60v-src-stimulated cells to translate these transcripts and to initiate DNA replication, probably because of their unstimulated protein-synthetic machinery.

Rous sarcoma virus (RSV) is an acutely oncogenic avian retrovirus which induces sarcomas in animals and transforms fibroblasts in cell culture. Genetic analysis indicates that the viral src gene (v-src) mediates neoplastic transformation. The product of v-src is a 60,000 molecular weight (MW) phosphoprotein (pp60v-src) possessing the enzymatic activity of a tyrosine-specific protein kinase. The viral src gene is derived from a cellular gene (c-src) which also encodes a 60,000 MW phosphoprotein (pp60c-src) with tyrosine-specific protein kinase activity. Both birds and mammals are known to possess c-src. Shilo and Weinberg have reported that the genome of the fruit fly, Drosophila melanogaster, contains nucleotide sequences that are homologous to v-src. We report here the molecular cloning and chromosomal mapping of three loci from the Drosophila genome that contain such sequences. We also show that Drosophila contain both phosphotyrosine and a tyrosine-specific protein kinase activity immunoprecipitated by antisera directed against pp60v-src. It should now be possible to identify the precise locus that encodes a src-specific protein kinase in Drosophila, and to explore the role of c-src in the growth and development of D. melanogaster.

The oncogenic pp60v-src product of ASV (avian sarcoma virus) is shown to be a potent endogenous mitogen, which, unlike mitogens such as PDGF (platelet derived growth factor), is able to stimulate host cell proliferation without the help of other growth factors. Thus, NRK rat cells, infected with a temperature-sensitive ASV mutant which produces an abnormally thermolabile pp60v-src, became proliferatively quiescent at a pp60v-src-inactivating 40 degrees C in medium containing either 0.2% calf serum or no serum at all. Adding PDGF stimulated the quiescent tsASV-NRK cells at 40 degrees C to initiate DNA replication in medium containing 0.2% serum, but not in serum-free medium. By contrast, activating internal pp60v-src by dropping the temperature to a permissive 36 degrees C stimulated these quiescent cells to transit G1, initiate DNA replication and to enter mitosis even in serum-free medium. Thus, relative to PDGF, endogenous pp60v-src behaves as a complete mitogen.

Effects of exposure to ethylnitrosourea (ENU) on expression of proteins that play a role in neuronal differentiation were examined in central nervous system (CNS) micromass embryo cell cultures. ENU is a known developmental toxicant which affects neuronal development. The proteins selected were the protein product of the src proto-oncogene (pp60c-src) and high-molecular-weight neurofilament protein (NF). pp60c-src has marked increases in amount and kinase activity in neurons at the time of differentiation and NF is found in differentiated neurons. CNS micromass cultures are primary cells from Day 12 rat embryo midbrains which are plated at high density and differentiate into neurons during 5 days in culture. Proteins were quantitated by polyacrylamide gel electrophoresis separation of equal amounts of total cell protein followed by transfer to membranes, immunoblotting, and densitometric scanning of blots. Dose-dependent decreases in cell growth and differentiation were confirmed using endpoints of cell number, protein content of cultures, neutral red uptake, hematoxylin staining of differentiated cells, and levels of binding of the neurotransmitter [3H]gamma-aminobutyric acid. Concentrations which inhibited response by 50% compared to controls ranged from 232 to 455 microM ENU. Dose-related decreases in amounts of pp60c-src and NF proteins relative to total protein were seen in CNS cultures treated with ENU. Results confirm the usefulness of the micromass cultures in following chemical effects on neuronal differentiation. The effects of ENU on specific proteins associated with neuronal differentiation were shown.

SH2 domains are protein modules specialized in recognizing phosphorylated tyrosine residues and their sequence contexts. The mechanism of phosphotyrosine recognition and context recognition was elucidated in a series of X-ray crystallographic experiments which are discussed below.

Cytokines and growth factors induce tyrosine phosphorylation of signal transducers and activators of transcription (STATs) that directly activate gene expression. Cells stably transformed by the Src oncogene tyrosine kinase were examined for STAT protein activation. Assays of electrophoretic mobility, DNA-binding specificity, and antigenicity indicated that Stat3 or a closely related STAT family member was constitutively activated by the Src oncoprotein. Induction of this DNA-binding activity was accompanied by tyrosine phosphorylation of Stat3 and correlated with Src transformation. These findings demonstrate that Src can activate STAT signaling pathways and raise the possibility that Stat3 contributes to oncogenesis by Src.

The c-src proto-oncogene encodes a M(r) 60,000 phosphoprotein, pp60c-src, with tyrosine-specific protein kinase activity. We have used an immune complex protein kinase assay for pp60c-src to analyze a spectrum of B-cell neoplasms. pp60c-src activity was elevated in ALL five hairy cell leukemia specimens and in a number of the large cell and immunoblastic lymphomas; neoplasms representing later stages in B-cell development. pp60c-src activity was low in neoplastic cells which correspond to early and intermediate stages in B-cell development (acute and chronic lymphatic leukemia, lymphoblastic lymphoma, small lymphocytic lymphoma). The enhanced pp60c-src activity was associated with high levels of pp60c-src protein. However, increased expression of c-src was not associated with amplification or gross structural rearrangement of the c-src gene. This preliminary study demonstrates elevated levels of pp60c-src protein and tyrosine protein kinase activity in neoplasms corresponding to the later stages of B-cell ontogeny.

The roles of activated ras and src oncogene products in the acquisition of fully neoplastic phenotype by human gallbladder adenocarcinoma cells were investigated by co-transfecting non-tumorigenic HAG-I human gallbladder carcinoma cells with the pSV2neo plasmid and a plasmid carrying either activated c-H-ras or v-src oncogene. G418-resistant clones were isolated and assessed for the acquisition of anchorage-independent growth potential. Neither the 10 established clones transfected with pSV2neo alone nor the 17 clones transfected with activated c-H-ras, including 4 clones expressing the mutated p21H-ras protein, could form colonies in soft agar. By contrast, out of 10 clones transfected with v-src, 2 formed colonies in soft agar and produced tumors in athymic nude mice, the resulting progressive neoplasms being poorly differentiated adenocarcinomas. These tumorigenic clones were shown to have v-src DNA and mRNA levels with p60v-src protein, but there were no significant chromosomal alterations following tumorigenic conversion. Moreover, herbimycin A, a selective src-kinase inhibitor, markedly reduced clonogenic growth of these cells in soft agar rather than monolayer growth, suggesting that anchorage-independent growth of the v-src-transformed HAG-I cells might be driven directly by p60v-src kinase activity. Taken together, our data suggest that the fully neoplastic conversion of HAG-I cells depends on src-related tyrosine-kinase activity, but not solely on the function mediated by activated ras, thus providing evidence of an src-related signaling pathway for the acquisition of tumorigenic potential by human gallbladder adenocarcinoma cells.

The epidermal growth factor (EGF)-induced endocytosis of its receptor is an obligatory pathway for the cellular regulation of the EGF-specific receptor (EGF-R). BNER4 is a mouse Balb/3T3 cell line transfected with human EGF-R complementary DNA (cDNA). B4/src-13 and B4/src-24 are BNER4 cells transfected with a viral oncogene v-src. Indirect immunofluorescence study demonstrated that EGF-R was mostly localized at the perinuclear region in BNER4 cells at 60 min after EGF addition, whereas it was diffusely distributed throughout the cytoplasm in its v-src transfectants. Double indirect immunofluorescence study further confirmed that EGF-R was localized in lysosomes in BNER4 and B4/src-13 cells at 60 min after EGF addition. Intracellular distribution of the Golgi apparatus, clathrin-coated vesicles and early endosomes were similar in ALL cell lines. However, the lysosomes detected by anti-lysosomal membrane protein (LGP85) antibodies were diffusely distributed throughout the cytoplasm in the v-src transfectacts. By contrast, in the parental BNER4 cells, the lysosomes were mostly localized in the perinuclear region. The organization of microtubules, but not of actin, was markedly different between BNER4 cells and its v-src transfectants. Nocodazole, which depolymerizes microtubules, altered the distribution of the lysosomes and EGF-R in BNER4 cells. Both intracellular lysosome distribution and microtubule organization in nocodazole-treated BNER4 cells were found to be similar to those in its v-src transfectants without nocodazole treatment. These findings support the notion that changes in lysosome distribution may be correlated with microtubule reorganization by v-src in mouse Balb/3T3 cells.

The widely expressed c-Crk protein, composed of one SH2 and two SH3 domains, lacks an apparent catalytic domain, suggesting that it functions through the formation of specific complexes with other proteins. Bacterially expressed c-Crk formed in vitro highly stable complexes via the first SH3 domain [SH3(N)]. Most prominent were a 185 kDa protein of unknown identity (p185), Sos- immunoreactive bands of 170 kDa (p170) and 145 to 155 kDa bands, corresponding to the recently cloned C3G protein. p170 also bound to Ash/Grb2 and Nck while p185 and C3G bound only to Crk. Additional Crk binding proteins were found in hematopoietic cells, particularly the myeloid-monocytic lineage. The protein binding properties of Crk were subsequently compared to CRKL, the product of a homologous but distinct gene, and found to be very similar. The binding of two guanine nucleotide exchange factors, Sos and C3G, to Crk and CRKL indicates that Ras or related proteins likely play a role in signaling through Crk family proteins.

src and erbB are two tyrosine kinase-encoding oncogenes carried by retroviruses, which have distinct disease specificities. The former induces predominantly sarcomas, and the latter, leukemias. Src and ErbB have similar catalytic domains but have very different regulatory domains. A wealth of information exists concerning how different regulatory domains [Src homology 2 (SH2) and SH3 domains and autophosphorylation sites] control substrate and disease specificities. Whether the catalytic domain helps determine these specificities remains to be explored. Here we show that the Src catalytic domain is enzymatically active when substituted into the ErbB backbone and interacts with the ErbB regulatory domain. This ErbB/Src chimera displays autophosphorylation and substrate phosphorylation patterns different from those of both Src and ErbB. Neither SH2 and SH3 nor autophosphorylation sites are required for the Src catalytic domain to exert its fibroblast transforming ability. Most significantly, the catalytic domain can convert erbB from a leukemogenic oncogene into a sarcomagenic oncogene, suggesting that the leukemogenic determinants in part reside within the ErbB catalytic domain.

In view of the potent mitogenic effect exerted by insulin in human colonic cells, we used Caco-2 cells transfected with an activated (Val12) human Ha-ras gene or the polyoma middle T (PyMT) oncogene, a constitutive activator of pp60c-src tyrosine kinase activity, to investigate the effect of oncogenic p21ras and PyMT/pp60c-src on insulin mitogenic signaling. As compared to vector control Caco-2 cells, both oncogene-transfected cells exhibited: 1) a lost of response to insulin s stimulatory effect on mitogen-activated protein (MAP) kinase activity and cell proliferation, both of which were constitutively increased; 2) a decrease in insulin receptor (IR) affinity and insulin-stimulated exogenous tyrosine kinase activity, which resulted, at least in part, from increased protein kinase C (PKC) activity (4), since both IR alterations were partially corrected by PKC down-regulation; and 3) a decrease in both insulin receptor mRNA level and insulin receptor number, which was independent of PKC since it persisted after PKC down-regulation. In conclusion, oncogenic p21ras and PyMT/pp60c-src abolished insulin mitogenic signaling in Caco-2 cells through mechanisms involving (i) constitutive activation of MAP kinase, and (ii) marked decreases in both insulin receptor function and expression which were mediated by PKC-dependent and PKC-independent pathways respectively. This is the first evidence that, when oncogenically activated, p21ras and pp60c-src not only exert a negative control on insulin receptor function but also repress insulin receptor gene expression in human colonic cells.

The observation that v-src-induced tumors contain tumor cells of differing morphology, notably fibroblastoid or polygonal, raised the question as to whether the tumor cells are also heterogeneous with respect to expression of markers of cellular differentiation. Of the markers tested here, consistent reactivity for tumor tissue was noted only for antibody probes reactive to muscle actin (HHF35, alpha sm-1) or to procollagen type I (SP1. D8); for any given tumor, whether induced by v-src DNA or by Rous sarcoma virus, each of these markers was found only in a subpopulation of tumor cells. The observation of marker heterogeneity in the one v-src DNA-induced tumor examined here that typed as monoclonal suggests that v-src-induced transformation is consonant with a degree of plasticity in the phenotypes of the clonal progeny of a single transformant.

Several genes are induced constitutively in cells transformed by the v-src oncoprotein. This induction is generally dependent on the activation of transcription factors binding to src-responsive elements of the promoter. In previous studies, we showed that the induction of the CEF-4/9E3 cytokine gene by pp60v-src is dependent on the PRDII/kappa B domain of the promoter (Dehbi et al., 1992). In this investigation, we describe the activation of the HIV-1 LTR by pp60v-src and show that a region of 30 bp containing the two NF-kappa B binding sites is critical for activation of the promoter. The induction was dependent on transformation since non-transforming forms of pp60v-src had little or no effect on the promoter. The expression of proviral DNA and the release of p24 antigen were also increased by v-src indicating that viral replication was stimulated in src-transformed cells. The effect of v-src on HIV-1 gene expression occurred in the presence or in the absence of the tat viral trans-activator, in fibroblasts and in Jurkat T lymphocytes. These results indicate that several promoters controlled by PRDII/kappa B may be activated constitutively in v-src transformed cells and suggest that oncogenic tyrosine kinases may play a role in the induction of viruses with a PRDII/kappa B-controlled promoter.

The molecular mechanisms by which oncogenic tyrosine kinases induce cellular transformation are unclear. Herbimycin A, geldanamycin, and certain other benzoquinone ansamycins display an unusual capacity to revert tyrosine kinase-induced oncogenic transformation. As an approach to the study of v-src-mediated transformation, we examined ansamycin action in transformed cells and found that drug-induced reversion could be achieved without direct inhibition of src phosphorylating activity. To identify mechanisms other than kinase inhibition for drug-mediated reversion, we prepared a solid phase-immobilized geldanamycin derivative and affinity precipitated the molecular targets with which the drug interacted. In a range of cell lines, immobilized geldanamycin bound elements of a major class of heat shock protein (HSP90) in a stable and pharmacologically specific manner. Consistent with these binding data, we found that soluble geldanamycin and herbimycin A inhibited specifically the formation of a previously described src-HSP90 heteroprotein complex. A related benzoquinone ansamycin that failed to revert transformed cells did not inhibit the formation of this complex. These results demonstrate that HSP participation in multimolecular complex formation is required for src-mediated transformation and can provide a target for drug modulation.

1. Rat phaeochromocytoma (PC12) cells were used to investigate the expression of Ca2+ channel types during neuronal differentiation. Neuronal differentiation was induced by treatment with nerve growth factor (NGF) or by activation of a temperature-sensitive tyrosine kinase (pp60v-src) in genetically modified PC12 (PC12/v-src) cells. PC12 cells differentiated morphologically in the presence of NGF. When grown at the permissive temperature of 37 degrees C which activates the kinase activity of pp60v-src, PC12/v-src cells differentiated morphologically with the extension of neurites. In contrast, PC12/v-src cells grown at the non-permissive temperature of 40 degrees C continued to divide and were morphologically indistinguishable from control PC12 cells. 2. Whole-cell Ca2+ currents were measured in PC12 cells using Ba2+ as the charge carrier. Ba2+ currents measured at the peak of the current-voltage curve from a holding potential of -80 mV were -0.28 +/- 0.04 nA (mean +/- S.E.M.) in control PC12 cells compared to -1.25 +/- 0.16 nA in NGF-differentiated cells. The current density increased from 9.4 +/- 0.7 pA/pF in control PC12 cells to 22.8 +/- 2.4 pA/pF in NGF-differentiated PC12 cells. Ba2+ currents were -0.24 +/- 0.04 nA in undifferentiated PC12/v-src cells grown at the non-permissive temperature of 40 degrees C compared to -0.95 +/- 0.16 nA in differentiated PC12/v-src cells grown at the permissive temperature of 37 degrees C. The current density increased from 4.5 +/- 0.5 pA/pF in PC12/v-src cells grown at the non-permissive temperature of 40 degrees C to 13.3 +/- 2.4 pA/pF in PC12/v-src cells grown at the permissive temperature of 37 degrees C. 3. The sensitivity of Ba2+ currents to omega-conotoxin GVIA (omega-CgTX) was determined for currents measured at the peak of the current-voltage curve (0 mV in 10 mM Ba2+) from a holding potential of -80 mV. In NGF-differentiated PC12 cells, 10 microM omega-CgTx inhibited 68.1 +/- 3.2% of the total Ba2+ current compared to 35.9 +/- 4.1% in control cells. The density of the omega-CgTX-sensitive current increased from 3.3 +/- 0.4 pA/pF in control cells to 15.7 +/- 2.0 pA/pF in NGF-differentiated cells. In differentiated PC12/v-src cells grown at 37 degrees C, omega-CgTX inhibited 52.2 +/- 4.2% of total Ba2+ current compared to 41.1 +/- 3.8% in PC12/v-src cells grown at 40 degrees C. The density of the omega-CgTX-sensitive current increased from 1.9 +/- 0.3 to 7.4 +/- 2.0 pA/pF with v-src-mediated differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)FAU - Lewis, D L

The c-ret proto-oncogene encodes a receptor tyrosine kinase whose normal function has yet to be determined. To begin to investigate the potential role of this gene in vertebrate development, we have isolated cDNA clones representing the murine c-ret gene, and have analyzed the pattern of expression during mouse embryogenesis, using northern blotting, in situ hybridization to histological sections and whole-mount hybridization histochemistry. c-ret transcripts were detected beginning at day 8.5 of embryogenesis, and were observed in a number of cell lineages in the developing peripheral and central nervous systems, as well as in the excretory system. In the cranial region at day 8.5-9.5, c-ret mRNA was restricted to a population of neural crest cells migrating from rhombomere 4 and forming the anlage of the facioacoustic ganglion, as well as to a closely associated domain of surface ectoderm and pharyngeal endoderm. At later stages (10.5-14.5 days), c-ret mRNA was observed in ALL cranial ganglia. In the peripheral nervous system of the trunk, c-ret was expressed in the autonomic ganglia and in subsets of cells in the dorsal root ganglia. In the enteric nervous system, c-ret was expressed in the presumptive enteric neuroblasts of the vagal crest (day 9.0-11.5), and in the myenteric ganglia of the gut (day 13.5-14.5). c-ret mRNA was observed in several regions of the central nervous system, including the undifferentiated neuroepithelial cells of the ventral neural tube (8.5 days), the motor neurons in the spinal cord and the hindbrain (10.5-14.5 days), the embryonic neuroretina (day 13.5) and the layers of the postnatal retina containing ganglion, amacrine and horizontal cells. Outside the nervous system, c-ret was expressed in the nephric (Wolffian) duct at day 8.5-10.5, the ureteric bud epithelium (but not the surrounding metanephric mesenchyme) at day 11.0-11.5, and the growing tips of the renal collecting ducts (but not the previously formed, subcortical portions of the collecting ducts, or the mesenchyme-derived renal vesicles) at day 13.5-17.5. Our results suggest that the c-ret gene may encode the receptor for a factor involved in the proliferation, migration, differentiation or survival of a variety of neuronal cell lineages, as well as in inductive interactions during organogenesis of the kidney.

Activation of the tyrosine kinase of a temperature sensitive v-Src mutant of Rous sarcoma virus in quiescent Rat-1 cells leads to passage through the cell cycle. This is accompanied by a transient increase of the DNA binding activity of the transcription factor AP-1 which is not sufficient for the v-Src mediated cell cycle traverse. There is another need for v-Src later in the G1 phase of the cycle, and after completion of that event, cells are able to progress through DNA synthesis and division in the absence of either v-Src or other growth factors. When cells are exposed to v-Src activity for periods insufficient for it to behave as a complete mitogen, it can act as either a competence or progression factor in conjunction with appropriate purified growth factors.

The possible influence of expression of v-src oncogene harboring tyrosine-specific protein kinase activity on the physiological state of E. coli cells, carrying plasmid which expresses src oncoprotein was studied. The realization of this activity in the cells brings to the phosphorylation of tyrosine in some additional cellular proteins and, as a consequence, to an increased proliferative activity of cells expressing src oncoprotein.

In order to help clarify the cellular mechanisms that regulate expression of the human c-src proto-oncogene, we have isolated a series of overlapping genomic clones that contain the c-src promoter region, as well as three previously uncharacterized exons. These exons encode the 350-bp 5 untranslated region of the c-src mRNA and span 35 kb of genomic DNA, extending the human c-src locus to approximately 60 kb. Subcloning and sequence analysis of the 5 flanking region of the gene revealed a high GC content and several consensus Sp1 and AP2 binding sites. However, TATA or CAAT boxes were not present, a characteristic shared by other GC-rich promoters. Promoter-CAT constructs demonstrated that the promoter was functional in transfection assays and that its activity was dependent on correct orientation. CAT-promoter deletion constructs were used to define the 5 boundary for maximal promoter activity and to reveal the presence of both positive and negative regulatory elements. S1 analyses of human c-src mRNA from cell lines indicated that multiple transcription start sites were utilized.

We have sought to identify genes whose expression is altered as a consequence of transformation by p60v-src. Using the mRNA differential display method, we have identified the nov proto-oncogene as one gene that is down regulated in chicken embryo fibroblasts (CEFs) transformed by p60v-src. nov transcripts were also found to be present at only very low levels in proliferating CEFs in comparison with quiescent CEFs. Serum stimulation of quiescent CEFs also resulted in a decline in the steady-state level of nov transcripts. Taken together, these findings suggest that the nov gene is expressed only in quiescent fibroblasts and that its down regulation may contribute to cellular transformation by the v-src oncogene. Down regulation of the nov gene appears to occur at both the transcriptional and posttranscriptional levels. Results obtained from experiments with a protein kinase inhibitor suggest that protein kinase C may be a key downstream effector in mediating the down regulation of nov transcripts in response to activation of p60src or serum stimulation. In addition, we found that transcription of an unknown gene is required for the decline in the steady-state level of nov transcripts in response to serum stimulation.

The role of the protein tyrosine kinase pp60c-src in the expression of prostaglandin G/H synthase (PGHS), the key enzyme of prostaglandin synthesis, was investigated in rat renal mesangial cells. Transfection of mesangial cells with the proto-oncogene c-src resulted in nontransformed cells with constitutively enhanced pp60c-src kinase activity. As a control, mesangial cells were transfected with inactive pp60c-src, mutated in position 295 (lysine replaced by methionine). expression of the constitutive isoform PGHS-1 was enhanced in cells overexpressing wild-type c-src compared to cells transfected with the kinase negative c-src mutant. Levels of other constitutively expressed proteins such as GAPDH and beta-actin were also enhanced. PGHS-2 was barely detectable in resting cells but was inducible by PDGF-AB, PDGF-BB, serotonin, FCS, and calcium ionophore A23187. Induction was diminished in pp60c-src kinase-overexpressing cells, independent of the stimulus used, suggesting interference at a late step in the signaling cascade. No induction of PGHS-2 mRNA was observed in mesangial cells transformed by the oncogene v-src. An increase in intracellular calcium levels is an early step in signal transduction by PDGF and serotonin in mesangial cells. c-src kinase overexpression reduced PDGF- and serotonin-mediated changes in Ca2+ signaling, indicating multiple targets of pp60c-src action. Overexpression of pp60c-src in mesangial cells thus affected basal protein expression, reflected by the enhanced PGHS-1 mRNA and protein expression. With regard to induction of PGHS-2, overexpression of pp60c-src reduced induction by stimuli coupled to different types of signaling pathways.

Four different transformed cell lines were isolated as a result of independent infection of primary hamster fibroblasts by Rous sarcoma virus (RSV SR-D stocks). These lines differ by the level of their spontaneous metastatic activity: HET-SR-1, HET-SR-8, and HET-SR-10 cell lines induced 70-200 metastatic nodules in the lung and/or lymph nodes of inoculated animals (high metastatic lines, HM). Metastatic activity was not identified after injection of HET-SR cells (low metastatic line, LM). ALL cell lines contained one copy of integrated and expressed intact RSV provirus. The difference in the amount of v-src protein in cell lines was not correlated with their metastatic potential in vivo. Complete v-srcHM and v-srcLM genes were cloned from corresponding gene libraries and sequenced. In the unique region of both v-src isoforms a GC-rich insert of 60 nucleotides (20 a.a.) was found. The presence of this insert explains the unusual apparent molecular weight of protein encoded by v-srcHM and vsrcLM: 62 kDA. Both genes had 10 identical amino acid changes when compared to the known RSV SR-D v-src sequence. v-srcHM and v-srcLM differ by several amino acid changes. Most of them are localized in the unique domain and the extreme carboxy-terminal region of the of the oncoprotein. Both v-src variants and chimeric v-src with mutually substituted parts were subcloned in a retroviral vector and introduced into avian neuroretina cells. Significant differences in the morphology of transformed neuroretinal cells were associated with the mutations in the carboxy-terminal region of the v-src oncogene. Low metastatic HET-SR cells transfected with v-srcHM and the chimeric gene v-src-LH remarkably increased their metastatic potential. In contrast, this effect was not observed when the same cells were transfected with v-srcLM and the chimeric v-srcHL gene. Specific changes in the distribution of fibronectin matrix typical for high metastatic cells were found in the lines transfected with v-srcHM.

Introduction of v-src or c-src527F, a transforming mutant of the c-src proto-oncogene, into the growth factor-dependent cell line FDCP-1 resulted in growth factor independence. Temperature-shift studies with cells carrying the tsLA29 mutant of v-src demonstrated that growth factor independence was oncogene-dependent; that is, the cells were growth factor-independent at the permissive temperature but became growth factor-dependent at the nonpermissive temperature. Introduction of the c-src proto-oncogene did not result in growth factor independence. The c-src2A,527F mutant, which encodes an activated tyrosine kinase but does not transform fibroblasts due to a mutation in the membrane localization sequence, induced growth factor independence. This suggests that the presence of an activated tyrosine kinase is necessary for this process but that membrane localization is not. Bioassays indicated that conditioned medium from growth factor-independent cells contained a growth factor identified by antibody neutralization studies as granulocyte-macrophage colony-stimulating factor (GM-CSF). Secretion of GM-CSF was confirmed by a quantitative enzyme-linked immunosorbent assay (ELISA) specific for GM-CSF. The presence of GM-CSF mRNA in src-infected FDCP-1 cells was demonstrated by PCR amplification of cDNAs with primers specific for GM-CSF. While GM-CSF mRNA was detected in FDCP/ts29 cells grown at 34 degrees C, it was not observed in cells infected with the tsLA29 mutant grown at the nonpermissive temperature of 39 degrees C. Transfection of v-src-infected FDCP-1 cells with a GM-CSF promoter reporter plasmid revealed src-dependent expression of luciferase; that is, while expression was observed at the permissive temperature, no expression was detected in FDCP/ts29 clone 6 cells grown at the nonpermissive temperature. No expression of the GM-CSF promoter reporter plasmid was observed in uninfected FDCP-1 cells.

Avian sarcoma virus PR2257 contains 952 bp transduced from the left part of the 3 UTR of the chicken c-src oncogene. Deletion mutants were constructed to determine the effect of the 3 UTR on tumorigenicity in vivo and in vitro. In the presence of the 3 UTR, tumours were 3.4 times larger in vivo, and tumorigenicity was increased 2.5-fold in vitro. Several regulatory submotifs were also found within the 3 UTR. Parts of the 3 UTR were cloned into the LTR CAT plasmid and analysed for CAT expression. A 170 bp element was found to be responsible for the enhanced expression of the CAT gene. These results demonstrate the effect of the transduced 3 UTR sequence during long-term interaction between PR2257 virus and the chicken genome, and suggest a novel regulatory mechanism of the src oncogene.

Two major intermediaries in signal transduction pathways are pp60v-sre family tyrosine kinases and heterotrimeric guanine nucleotide-binding proteins. In Rat-1 fibroblasts transformed by the v-src oncogene, endothelin-1 (ET-1)-induced inositol 1,4,5-trisphosphate accumulation is increased 6-fold, without any increases in the numbers of ET-1 receptors or in the response to another agonist, thrombin. This ET-1 hyperresponse can be inhibited by an antibody directed against the carboxyl terminus of the Gq/G11 alpha subunit, suggesting that the Gq/G11 protein couples ET-1 receptors to phospholipase C (PLC). While v-src transformation did not increase the expression of the Gq/G11 alpha subunit, immunoblotting with anti-phosphotyrosine antibodies and phosphoamino acid analysis demonstrated that the Gq/G11 alpha subunit becomes phosphorylated on tyrosine residues in v-src-transformed cells. Moreover, when the Gq/G11 protein was extracted from control and transformed cell lines and reconstituted with exogenous PLC, AIF*4-stimulated Gq/G11 activity was markedly increased in extracts from v-src-transformed cells. Our results demonstrate that the process of v-src transformation can increase the tyrosine phosphorylation state of the Gq/G11 alpha-subunit in intact cells and that the process causes an increase in the Gq/G11 alpha-subunit s ability to stimulate PLC following activation with AIF-4.

Transformation of Rat-1 fibroblasts with the v-src oncogene leads to a 6- to 8-fold enhancement of the activity of the Ins(1,4,5)P3 3-kinase in cytosolic extracts [Johnson, Wasilenko, Mattingly, Weber and Garrison (1989) Science 246, 121-124]. This study confirms these results using another v-src-transformed Rat-1 cell line (B31 cells) and investigates the molecular mechanism by which pp60v-src activates Ins(1,4,5)P3 3-kinase. The mRNA and protein levels for two rat isoforms of Ins(1,4,5)P3 3-kinase were determined in the v-src-transformed cell line. Both the mRNA and protein levels for isoform A were elevated in v-src-transformed Rat-1 cells while those for isoform B were not significantly affected. Moreover, stable expression of either form of Ins(1,4,5)P3 3-kinase in the B31 v-src-transformed Rat-1 cell line did not result in tyrosine phosphorylation of Ins(1,4,5)P3 3-kinase A or B. These results suggest that at least one mechanism by which the v-src oncogene increases the activity of the Ins(1,4,5)P3 3-kinase in the Rat-1 transformed fibroblast is by increasing the level of expression of Ins(1,4,5)P3 3-kinase A.

Integrin-mediated cell adhesion triggers intracellular signaling cascades, including tyrosine phosphorylation of intracellular proteins. We show in this report that p120(cbl) (Cbl), the 120-kDa c-cbl proto-oncogene product, becomes tyrosine-phosphorylated during integrin-mediated macrophage cell adhesion to extracellular matrix substrata and anti-integrin antibodies. This tyrosine phosphorylation does not occur when cells attach to polylysine, to which cells adhere in a nonspecific fashion. It also does not take place when adhesion-induced reorganization of the cytoskeleton is inhibited with cytochalasin D. In contrast to the rapid and transient tyrosine phosphorylation of Cbl by CSF-1 stimulation, tyrosine phosphorylation of Cbl by cell attachment was gradual and persistent. Tyrosine-phosphorylated Cbl was found to form complexes with the SH2 domain-containing signaling proteins Src and phosphatidylinositol 3-kinase; in vitro kinase assays demonstrated that these kinases were active in the Cbl complexes following integrin ligand binding. Furthermore, Cbl was found to translocate to the plasma membrane in response to cell adhesion to fibronectin. These observations suggest that Cbl serves as a docking protein and may transduce signals to downstream signaling pathways following integrin-mediated cell adhesion in macrophages.

The v-src oncogene encodes a nonreceptor tyrosine kinase. When this gene was expressed in the myeloblastic cell line 32Dcl3, it was found to abrogate interleukin-3 (IL-3) dependence of this cell line and to block its ability to terminally differentiate into granulocytes in response to granulocyte colony-stimulating factor (GCSF). In contrast, a highly related tyrosine kinase gene, v-fgr, fails to render this cell line IL-3 independent for growth or to block its ability to undergo terminal differentiation in the presence of GCSF. The active structural domains of v-src that are responsible for the abrogation of IL-3 dependence of myeloid cells and the mechanisms by which v-src transforms these cells are at present unclear. To identify the domains in v-src which are responsible for this activity, we constructed several chimeric recombinants between the v-src and the related Src family member v-fgr by replacing portions of v-src with corresponding domains of v-fgr. These chimeric DNAs were transfected into 32Dcl3 cells and examined for their abilities to render this cell line IL-3 independent. Our results show that only chimeras containing both the SH3 and the SH2 domains of v-src were capable of rendering the 32Dcl3 cell line IL-3 independent. To understand the possible mechanisms underlying the IL-3-independent growth of v-src-transformed 32Dcl3 cells, we examined the phosphorylation status of JAK-1, JAK-2, and JAK-3 kinases in the v-src- and v-fgr-transformed 32Dcl3 cells. Our results show that none of the JAK kinases are constitutively phosphorylated by v-src or v-fgr. We then examined the phosphorylation status of the STAT (signal transducers and activators of transcription) family of transcription factors. Our results show that STAT1, STAT3, and STAT5 exist in a constitutively phosphorylated state in v-src-transformed 32Dcl3 cells, while such constitutive phosphorylation is not seen in v-fgr-transformed cell lines. Our results also show that STAT3 coimmunoprecipitates with v-Src, suggesting that the activation of STAT3 occurs due to direct association with v-Src. However, STAT1 and STAT5, which also exist in a constitutively phosphorylated state in v-src-transformed 32Dcl3 cells, do not coimmunoprecipitate with v-Src, suggesting that these proteins either interact weakly with v-Src or are phosphorylated by a mechanism distinctive from that of STAT3.

The protein encoded by ras and src protooncogenes are frequently activated in a constitutive state in human colorectal cancer. In this study, we investigated the effect of oncogenic p21ras and Py-MT/pp60c-src on the synthesis of syndecan-1, a membrane anchored proteoglycan playing a role in cell-matrix interaction and neoplastic growth control. To this end, we used Caco-2 cells transfected with an activated (Val-12) human Ha-ras gene or the polyoma middle T (Py-MT) oncogene, a constitutive activator of pp60c-src tyrosine kinase activity. As compared to control vector-transfected Caco-2 cells, both oncogene-transfected cells exhibited: (1) a decrease in syndecan-1 specific activity; (2) a decrease in size and sulfation of syndecan-1 ectodomain glycosaminoglycan side chains; and (3) an active heparanase specifically degrading the heparan sulfate chains. In conclusion, the tumorigenic progression induced by oncogenic p21ras or Py-MT/pp60c-src is associated with marked alterations of syndecan-1 at the post-translational level.

The colony stimulating factor 1 (CSF-1) regulates osteoclastogenesis and bone resorption. mutations in the CSF-1 gene cause an osteopetrosis characterized by the absence of osteoclasts. Mature osteoclasts respond to CSF-1 with inhibition of bone resorption and an increment of cell spreading. Herein we demonstrate that CSF-1-induced osteoclast spreading depends on the substrate the osteoclast interacts with and requires integrity of the vitronectin receptor and of the c-src proto-oncogene. Rabbit osteoclasts were allowed to attach to glass, serum, osteopontin, and bone substrates, and were treated with 10 ng/ml human recombinant CSF-1 for 4 h. In osteoclasts plated on glass, the cytokine induced 70% inhibition of bone resorption and 1.8-fold stimulation of cell spreading, without changes in podosome expression and microfilament array. In contrast, CSF-1 induced a 2.5-fold increase of osteoclasts showing filopodia, and a 9.5-fold increase of osteoclasts presenting lamellipodia, indicating that membrane motility was required for cell spreading. Osteoclasts plated on serum substrates showed a 50% reduction of spontaneous spreading. However, in this circumstance, CSF-1 still stimulated an increase of osteoclast area. In osteoclasts cultured on osteopontin substrate or on bone slices, an inhibition of CSF-1-induced osteoclast spreading was observed. To establish involvement of the vitronectin receptor and c-src proto-oncogene, cells were treated with the alpha vbeta3 integrin neutralizing antibody, LM609, or c-src antisense oligonucleotides, which reduced CSF-1-induced osteoclast spreading by 57% and 60%, respectively. The results demonstrate that CSF-1-induced osteoclast spreading requires both the vitronectin receptor and the c-src proto-oncogene and that this action is modulated by the adhesion substrata.

Alterations in cellular signaling underlie the transforming actions of many oncogenes. The vsrc oncogene tyrosine kinase, pp60vsrc, is known to alter multiple signal transduction pathways, including those involving phosphatidylinositol (PI) metabolism. In this study, we investigated the effects of vsrc-transformation on lysophosphatidic acid (LPA) receptor coupling to intracellular free calcium [Ca2+]i and PI turnover in rat-1 fibroblasts. In normal rat-1 cells, LPA rapidly elevated [Ca2+]i (EC50 = 10nM). In contrast, the ability of LPA to mobilize calcium was markedly attenuated in rat-1-vsrc cells. Further study revealed that the LPA-mediated generation of inositol (1,4,5)P3 and other inositol polyphosphates was also markedly attenuated in the vsrc-transformed cells. Although LPA caused a transient reduction in the level of PI(4,5)P2 in normal rat-1 cells, the agonist elevated the level of PI(4,5)P2 in the vsrc-transformed cells. These findings demonstrate that vsrc-transformation alters the coupling of LPA receptors to PI turnover and calcium signaling in rat-1 cells, and point to G protein-coupled receptor systems as targets for modulation by the vsrc kinase.

Oncogenic forms of the Abl and Src tyrosine kinases trigger the destruction of the Abi proteins, a family of Abl-interacting proteins that antagonize the oncogenic potential of Abl after overexpression in fibroblasts. The destruction of the Abi proteins requires tyrosine kinase activity and is dependent on the ubiquitin-proteasome pathway. We show that degradation of the Abi proteins occurs through a Ras-independent pathway. Significantly, expression of the Abi proteins is lost in cell lines and bone marrow cells isolated from patients with aggressive Bcr-Abl-positive leukemias. These findings suggest that loss of Abi proteins may be a component in the progression of Bcr-Abl-positive leukemias and identify a novel pathway linking activated nonreceptor protein tyrosine kinases to the destruction of specific target proteins through the ubiquitin-proteasome pathway.

To determine how an oncogenic tyrosine kinase disturbs cell cycle control we examined expression of cell cycle proteins and growth of fibroblasts reversibly transformed by a temperature sensitive mutant of v-Src (ts LA 29). ts LA 29 Rat-1 cells and normal Rat-1 cells had similar growth rates but the transformed cells traversed the G1 phase of the cell cycle more rapidly and failed to exit cycle efficiently in response to serum starvation and cell confluence. Cyclin D1 and cyclin E levels were not elevated in growing ts LA 29 Rat-1 cells and the abbreviated G1 was further accelerated by overexpression of cyclin E. A fall in cyclin E and cyclin A dependent kinase activities in Rat-1 cells in response to inhibitory growth conditions was abrogated in ts LA 29 Rat-1 cells and correlated with lack of p27 accumulation or cyclin A down regulation, the latter due to sustained cyclin A promoter activity. The expression of p27 mRNA was lower in ts LA 29 Rat-1 cells than Rat-1 cells and was elevated following v-Src inactivation concurrent with an increase in p27 promoter activity and temporary cell cycle exit. The suppression of mRNA or transcription is a novel way an oncoprotein can induce down regulation of p27 and contributes to the G1 shortening and perturbed cell cycle regulation of the v-Src transformed cells.

To determine its role in cell transformations, the v-src oncogene was introduced into the human foetal diploid fibroblasts MRC-5 and into MRC-SV1, a simian virus 40 (SV40)-transformed cell line derived from them. Infected cells were found to contain stably integrated intact proviruses, as determined by Southern blot analysis. Although highly expressed, v-src did not change the morphology or growth patterns of MRC-5 cells and failed to induce foci or alter their saturation densities. However, overexpression of v-src reduced the plating efficiencies of MRC-5 and induced anchorage-independent growth in a low but significant number of cells. Northern blot analysis showed that v-src selectively abolished the expression of decorin, a small dermatan/chondroitin sulphate proteoglycan that interacts with extracellular-matrix components and modulates collagen-fibril formation and the activity of transforming growth factor (TGF) beta1. Addition of herbimycin A, a potent pp60src tyrosine-kinase inhibitor, resulted in the reexpression of decorin in MRC-5 carrying v-src. There were no changes in the expressions of fibronectin, procollagen type I, or tissue plasminogen activator, an activator of extracellular-matrix-degrading enzymes. Moreover, v-src did not alter the expressions of the epidermal-growth-factor receptor or TGFbeta1 or reduce the growth-factor requirements of MRC-5 fibroblasts. MRC-5 and MRC-SV1 expressing v-src remained non-tumourigenic when injected into nude mice. Constitutive expression of v-src did not alter the mRNA levels of c-jun and junB, suggesting that the effects of the oncogene are not mediated by AP-1. Decorin gene expression has been shown previously to be maximal in quiescent cells and virtually absent in transformed ones. Our data indicate that the ability to synthesise decorin can be suppressed in human fibroblasts without their becoming transformed, and that the relations between decorin synthesis and growth controls need further clarification.

The thermodynamic and kinetic parameters of triplex formation between four purine-rich oligonucleotides and a 22 bp pyrimidine. purine tract in the promoter region of the c-src gene were determined by fluorescence polarization studies. Three of these four oligonucleotides were 11 nt in length, corresponding to the left, central or right portion of the tract, while the fourth was a 22mer covering the whole tract. Binding constants ( Ka) were measured as a function of Mg2+ concentration (0-10 mM) and temperature (0-41 degrees C). In 10 mM Mg2+, K a for the left, central and right 11mers were 0.26, 0.75 and 1.4 x 10(8)/M, respectively, while for the 22mer the value was 1.8 x 10(8)/M at 22 degrees C. Under the same conditions, Ka was estimated by an electrophoretic band shift technique. The agreement between the two methods was acceptable for the 22mer but not for the 11mers. Kinetic measurements demonstrated that the rate of dissociation of the 22mer from the triplex was significantly slower than that of the 11mers, providing an explanation for the observed discrepancy. The entropy and enthalpy of triplex formation were calculated from van t Hoff plots. In ALL cases the entropy was favourable, especially for the 22mer and for the 11mer with the lowest guanine content. The enthalpy was unfavourable for the 22mer and most favourable for the 11mer with the highest guanine content. These results provide a thermodynamic explanation for length and sequence effects on the formation of purine.pyrimidine.purine triplexes.

The overexpression and activation of the human c-Src proto-oncogene is closely associated with cancer of the colon and breast. Characterization of the 5 region of the c-Src gene revealed that the promoter is very GC rich, regulated by the Sp family of transcription factors, and contains four perfect homopolypurine/homopolypyrimidine tracts (Pu:Py tracts). These Pu:Py tracts (TC1, TC1.1, TC2, and TC3) are located near or overlap critical Spl binding sites required for full activation of the gene. Triplex-forming oligonucleotides (TFOs) can be targeted to such sequences with high affinity to form intermolecular triple-helical DNA and modulate transcriptional activity. We therefore designed a series of antiparallel purine-based TFOs and measured their ability to form triplexes with the c-Src promoter Pu:Py tracts using comigration, bandshift, and chemical footprint techniques. With one interesting exception, ALL of the TFOs were found to bind with specificity and high affinity (67 nM-28 nM) to their target sequences at physiologic pH. These results indicate that the c-Src gene can successfully form stable triplexes under physiologic conditions and is, therefore, an excellent candidate for triplex-mediated transcriptional downregulation.