Oncogene database

1398

CRK

v-crk avian sarcoma virus CT10 oncogene homolog;CRK;v-crk avian sarcoma virus CT10 oncogene homolog

adapter molecule crk|proto-oncogene c-Crk

17p13.3

protein-coding

Human glioma cells frequently overexpress epidermal growth factor receptor (EGFR). We found that the CrkII proto-oncogene product was associated with the EGFR in human glioma cells in the absence of epidermal growth factor (EGF). EGF stimulation of glioma cells induced the phosphorylation of tyrosine 221 of the CrkII protein, which correlates with its dissociation from the EGFR. By contrast, Shc and Grb2 were inducibly associated with the EGFR in response to EGF stimulation of glioma cells. In A431 cells, epidermoid carcinoma cells which overexpress EGFR, CrkII was tyrosine-phosphorylated and associated with the EGFR in an EGF-dependent manner. Therefore, the dissociation of CrkII from the EGFR upon stimulation with EGF appears to be specific to glioma cells. The Cbl oncogene product was also tyrosine-phosphorylated in U87MG glioma cells upon EGF stimulation. However, unlike in other cell lines, CrkII was not inducibly bound to Cbl in U87MG glioma cells. Thus, EGF-dependent binding of CrkII to phosphotyrosine-containing proteins appears to be suppressed in glioma cells. To evaluate the physiological role of dissociation of CrkII from EGFR, we expressed the CrkII-23 mutant in glioma cells. CrkII-23 mutant, which was isolated as a suppressor gene of the EGF-dependent transformation of NRK cells, binds constitutively to EGFR. We found that expression of CrkII-23 inhibited the anchorage-independent growth of the glioma cells in the presence of EGF. Taken together, these data implicate EGF-dependent dissociation of CrkII from EGFR in the oncogenicity of human glioma cells.

Cell transformation is associated with anchorage independent growth and morphological changes characterized by reduced adhesion and spreading. The molecular signals that control these events are poorly understood. The Met receptor tyrosine kinase is deregulated in human tumors and an oncogenic derivative of this receptor transforms cells. In this paper we demonstrate that fibroblasts transformed by the Met oncoprotein display decreased cell spreading consistent with the loss of actin stress fibers and vinculin staining focal adhesions. In contrast to control cells, focal adhesion kinase, p130Cas and paxillin are weakly or not detectably tyrosine phosphorylated in Met transformed cells. Moreover, although paxillin and p130Cas associate with the Crk adapter protein in control cells, they fail to associate with Crk in Met transformed cells, yet these cells are motile and capable of wound closure to the same extent as control cells. In Met transformed cells, Crk predominantly associates with the Cbl and Gab1docking proteins in a tyrosine phosphorylation dependent manner. The coupling of Gab1, but not Cbl, with Crk is retained in cells grown in suspension and enhances JNK activation. We propose that the loss of adhesion dependent signals required for cell cycle progression is compensated through Met induced Gab1/Crk signals.

The C-CRK gene, cellular homolog of the avian v-crk oncogene, encodes two alternatively spliced adaptor signaling proteins, CRKI (28 kDa) and CRKII (40 kDa). Both CRKI and CRKII have been shown to activate kinase signaling and anchorage-independent growth in vitro and CRKI transformed cells readily form tumors in nude mice. Affymetrix oligonucleotide arrays were used to analyse 86 lung adenocarcinomas and 10 uninvolved lung tissues. C-CRK mRNA expression was increased in more advanced (stage III versus stage I), larger (T(2-4) versus T(1)), and poorly differentiated tumors and in tumors from patients demonstrating poor survival (P=0.00034). An overlapping series of 93 lung adenocarcinomas (64 stage I and 29 stage III) and 10 uninvolved lung specimens were measured for quantitative differences in CRKI and CRKII protein levels using 2-D PAGE. CRK protein spots were identified using mass spectrometry and 2-D Western blotting. A significant increase in levels of the CRKI oncoprotein and the phosphorylated isoform of CRKII was observed in tumors (P<0.05). No difference in protein level was evident between stages. Concordant with mRNA expression, CRKI and CRKII were increased in poorly differentiated tumors (P<0.05). CRK immunohistochemical analysis of tumor tissue arrays using the same tumor series also demonstrated increased abundance of nuclear and cytoplasmic CRK in more proliferative tumors (P<0.05). This study provides the first quantitative analysis of discrete CRKI and CRKII protein isoforms in human lung tumors and provides evidence that the C-CRK proto-oncogene may foment a more aggressive phenotype in lung cancers.

An antiserum specific for P47gag-crk, the oncogene product of avian sarcoma virus CT10, was used to investigate possible crk-binding proteins. In in vitro kinase assays, four proteins were phosphorylated in anti-crk immunoprecipitates. Tyrosine, serine, and threonine residues were phosphorylated. A number of tyrosine-phosphorylated proteins were identified in anti-crk immunoprecipitates of 32P-labeled cells, including the three major phosphotyrosine-containing proteins of CT10-infected cells. These three proteins also bound to bacterially synthesized crk protein. These results suggest that the crk transforming protein can stably associate with both endogenous kinases and cellular kinase substrates.

We have constructed a series of mutants with deletion, linker insertion, and point mutations in the v-crk oncogene of avian sarcoma virus CT10. The v-crk gene contains no apparent catalytic domain, but does contain two blocks of homology to putative regulatory domains, termed SH2 and SH3, found in a variety of proteins implicated in signal transduction. Infection with CT10 causes a dramatic increase in the level of tyrosine phosphorylation of several cellular proteins. We found that mutation of either the SH2 or SH3 domain of v-crk reduced or eliminated transforming activity, whereas mutation of regions outside the conserved domains had no effect. Deletion of amino-terminal gag sequences caused a partial loss of transforming activity and a change in subcellular distribution of the crk protein. In ALL cases, there was an absolute correlation between increased cellular phosphotyrosine and transformation.

The oncogene product of the avian sarcoma virus CT10, P47gag-crk, contains the SH2, SH2 , and SH3 domains and binds proteins in a phosphotyrosine (ptyr)-dependent manner. In this study, we have determined the region of P47gag-crk essential for binding to ptyr-containing proteins. Mutant P47gag-crk proteins expressed in Escherichia coli that have the intact SH2 and SH2 regions retained the capacity to bind ptyr-containing proteins obtained from cells transformed by crk and src. The deletion of SH2 resulted in the loss of binding activity. Other mutants that have altered SH2 or SH2 bound few, if any, of the ptyr-containing proteins. Those mutants that bound ptyr-containing proteins associated with tyrosine kinase activity. We also found that polypeptides containing SH2, SH2 , and SH3 of p60v-src and p60c-src associated with ptyr-containing proteins from crk-transformed cells. Thus, the SH2 and SH2 domains of P47gag-crk are responsible for their binding to ptyr-containing proteins.

As a member of adherens junction, p120-catenin (p120ctn) plays a major role in cell adhesions through stabilization of E-cadherin. p120ctn is transcriptionally down-regulated in non-small cell lung cancer (NSCLC), although the molecular mechanisms underlying p120ctn repression are incompletely defined. Here we further investigated transcriptional regulation of p120ctn in NSCLC. We prepared a promoter reporter plasmid construct that contained p120ctn promoter region from position -1082 to +320 relative to transcription start site. Through serial deletion mutation analysis of the p120ctn promoter, we pinpointed cis-acting elements involved in regulation of p120ctn. We identified transcription factor SP1 as a transcriptional repressor of p120ctn that directly binds to segment (-9 to +36) of the p120ctn promoter. SP1 can receive multiple signals from several intracellular signaling pathways. Through examination of SP1 binding partners, we identified proto-oncogene c-Crk to be involved in transcriptional down-regulation of p120ctn. RNAi mediated silencing of CRK in A549, H157 and H358 cells increased p120ctn protein levels. On the other hand, over-expression of CRK-I and CRK-II in NSCLC cells down-regulated p120ctn, an effect that was abrogated by simultaneous silencing of SP1. In summary, our data provide evidence for the role of c-Crk proto-oncogene in transcriptional repression of p120ctn that further clarifies the mechanism by which this biochemical signal promotes metastasis in NSCLC.

Numerous oncogenes have been isolated from acutely transforming retroviruses. To date, the products of these viral oncogenes have been protein kinases, nuclear proteins, growth factors, or GTP-binding proteins. We have cloned the previously uncharacterized avian sarcoma virus CT10 and sequenced its genome. This virus encodes a protein, p47gag-crk, that has blocks of sequence similarity to the amino-terminal, non-catalytic region of the non-receptor class of tyrosine kinases. In addition, the structure of p47gag-crk has striking similarity to a 180-amino acid region of bovine brain phospholipase C. Biochemical data suggest that p47gag-crk activates one or several endogenous tyrosine kinases.

The signaling protein CrkII switches between forms with high or low binding affinity. Both phosphorylation and native-state prolyl isomerization were suggested to regulate the transition between these forms. Here we analyzed how phosphorylation at Tyr222 and Tyr252 and the Pro238Ala substitution affect signal transfer of human and chicken CrkII to a downstream target. Human CrkII is regulated by phosphorylation only, but chicken CrkII is regulated by Pro238 trans-->cis isomerization and by Tyr222 phosphorylation. Surprisingly, they act in an independent fashion. Apparently, the allosteric transition to a low-activity form can be induced by phosphorylation or prolyl isomerization located at distant sites in CrkII.

Avian sarcoma virus 1 (ASV-1) was isolated from a spontaneous sarcoma of an adult chicken. It is a replication-defective virus that induces fibrosarcomas in young chickens and oncogenic transformation in cultured chick embryo fibroblasts. The ASV-1 genome has been cloned in the lambda gt WES.lambda B vector from closed circular viral DNA extracted from infected cells. The nucleotide sequence of the oncogene insert in the ASV-1 genome has been determined. It is virtually identical to the sequence of the oncogene crk recently discovered in the avian sarcoma virus CT10. ASV-1 and CT10 are two independent retrovirus isolates carrying the crk oncogene.

Background and aims. Various morphologies are seen in different salivary gland tumorsor within an individual tumor, and the lesions show divers biological behaviors. Experimental results support the hypothesis that increased CrkII proto-oncogene is associated with cytokine-induced tumor initiation and progression by altering cell motility signaling pathway. The aim of this study was to assess the CrkII expression in common malignant salivary gland tumors and pleomorphic ade-noma. Materials and methods. Immunohistochemical analysis of CrkII expression was performed on paraffin blocks of 64 car-cinomas of salivary glands, 10 pleomorphic adenomas, and 10 normal salivary glands. Biopsies were subjected to immu-nostaining with EnVision detection system using monoclonal anti-CrkII. Evaluation of immunoreactivity of CrkII was based on the immunoreaction intensity and percentage of stained tumor cells which were scored semi-quantitatively on a scale with four grades 0 to 3. Kruskal-wallis test and additional Mann-Whitney statistical test were used for analysis of CrkII expression levels. Results. Increased expression of CrkII was seen (P=0.005) in malignant tumors including: mucoepidermoid carcinoma, adenoid cystic carcinoma, and carcinoma ex pleomorphic adenoma, but CrkII expression in acinic cell carcinoma was weak. CrkII expression in pleomorphic adenoma was weak or negative. A weak staining was sparsely seen in normal acinar serous cell. Conclusion. Increased expression of CrkII and its higher intensity of staining in tumors with more aggressive biologic behavior in carcinomas of salivary gland is consistent with a role for this proto-oncogene in salivary gland tumorigenesis and cancer progression.

The transforming gene of the avian sarcoma virus CT10 encodes a fusion protein (p47gag-crk or v-Crk) containing viral Gag sequences fused to cellular sequences consisting primarily of Src homology regions 2 and 3 (SH2 and SH3 sequences). Here we report a novel function of v-Crk in the mammalian pheochromocytoma cell line, PC12, whereby stable expression of v-Crk induces accelerated differentiation, as assessed by induction of neurites following nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) treatment compared with the effect in native PC12 cells. Surprisingly, however, these cells also develop extensive neurite processes after epidermal growth factor (EGF) stimulation, an event which is not observed in native PC12 cells. Following EGF or NGF stimulation of the v-CrkPC12 cells, the v-Crk protein itself became tyrosine phosphorylated within 1 min. Moreover, in A431 cells or TrkA-PC12 cells, which overexpress EGF receptors and TrkA, respectively, a GST-CrkSH2 fusion protein was indeed capable of binding these receptors in a phosphotyrosine-dependent manner, suggesting that v-Crk can directly couple to receptor tyrosine kinase pathways in PC12 cells. In transformed fibroblasts, v-Crk binds to specific tyrosine-phosphorylated proteins of p130 and paxillin. Both of these proteins are also complexed to v-Crk in PC12 cells, as evidenced by their coprecipitation with v-Crk in detergent lysates, suggesting that common effector pathways may occur in both cell types. However, whereas PC12 cellular differentiation can occur solely by overexpression of the v-Src or oncogenic Ras proteins, that induced by v-Crk requires a growth factor stimulatory signal, possibility in a two-step process.

Products of the crk oncogene are expressed in ALL tissues. Crk proteins are composed exclusively of Src homology 2 (SH2) and Src homology 3 (SH3) domains, and they have been implicated in intracellular signaling. For example, they participate as mediators of Ras activation during nerve growth factor stimulation of PC12 pheochromocytoma cells. We examined the role of Crk proteins during T cell receptor-mediated signaling and observed that Crk proteins specifically interact, via their SH2 domains, with a tyrosine-phosphorylated 116-kDa protein upon T cell activation. p116 may be related to the recently cloned fibroblast p130cas and/or p120-Cbl. In addition, we observed that GST-Crk fusion proteins and Crk-L bind, most likely via their SH3 domain, to C3G, a Ras guanine nucleotide exchange factor. Thus, the interaction of Crk with p116 and C3G strongly implicates Crk as a mediator of T cell receptor signaling, possibly involved in Ras activation.

The transforming gene v-crk found in CT10 and ASV-1 avian sarcoma viruses induces marked phosphorylation of several proteins in cells expressing p47v-crk (v-Crk). In this work, the main tyrosine-phosphorylated proteins in ASV-1-infected chicken cells and v-crk-transfected rat cells were characterized biochemically. Both these proteins have a molecular mass of about 130 kDa and are tightly associated with v-Crk in vivo. Two-dimensional gel electrophoresis revealed that they are both essentially single proteins (p130) with modifications that result in a broad spot in an acidic region. The broad band of semi-purified p130 became sharp at an elevated position in the gel upon treatment with orthovanadate in vivo or with c-Src kinase produced using a baculovirus vector in vitro, whereas it shifted at a lower position upon treatment with alkaline phosphatase in vitro. These results suggest multiple phosphorylation states of p130, which result in a broad band of p130. Two procedures of immunoaffinity purification were used to purify p130 from 3Y1 cells transfected with v-crk. Approximately 30 pmol of purified p130 was obtained in an immobilized form on a filter starting from 3 x 10(10) cells. Peptide mapping of p130 digested in situ by peptidase revealed that the purity and quantity of the final material were enough for peptide sequencing. Several stretches of partial amino acid sequences were determined, and they indicated that p130 is a novel protein.

A genomic DNA fragment, isolated from a human phage library using a chicken crk cDNA probe, was shown to derive from the human CRK locus. We have used fluorescent in situ hybridization (FISH) to map CRK distal in chromosome band 17p13, a region which demonstrates frequent deletion or loss of heterozygosity in a wide range of human cancers.

The cellular homologs of the v-Crk oncogene product are composed exclusively of Src homology region 2 (SH2) and SH3 domains. v-Crk overexpression in fibroblasts causes cell transformation and elevated tyrosine phosphorylation of specific cellular proteins. Among these proteins is a 130-kDa protein, identified as p130cas, that forms a stable complex in vivo with v-Crk. We have explored the role of endogenous Crk proteins in Bcr-Abl-transformed cells. In the K562 human chronic myelogenous leukemia cell line, p130cas is not tyrosine phosphorylated or bound to Crk. Instead, Crk proteins predominantly associate with the tyrosine-phosphorylated proto-oncogene product of Cbl. In vitro analysis showed that this interaction is mediated by the SH2 domain of Crk and can be inhibited with a phosphopeptide containing the Crk-SH2 binding motif. In NIH 3T3 cells transformed by Bcr-Abl, c-Cbl becomes strongly tyrosine phosphorylated and associates with c-Crk. The complex between c-Crk and c-Cbl is also seen upon T-cell receptor cross-linking or with the transforming, tyrosine-phosphorylated c-Cbl. These results indicate that Crk binds to c-Cbl in a tyrosine phosphorylation-dependent manner, suggesting a physiological role for the Crk-c-Cbl complex in Bcr-Abl tyrosine phosphorylation-mediated transformation.

The Crk proto-oncogene product is an SH2 and SH3 domain-containing adaptor protein which we have previously shown to become rapidly tyrosine phosphorylated in response to stimulation with insulin-like growth factor I (IGF-I) in NIH-3T3 cells. In order to further characterize the role of Crk in the IGF-I signaling pathway, NIH-3T3 and 293 cells were stably transfected with an expression vector containing the Crk cDNA. The various resultant 3T3-Crk clones expressed Crk at approximately 2-15-fold higher levels than parental 3T3 cells. In 3T3-Crk cells, Crk immunoreactivity was detected in insulin receptor substrate-1 (IRS-1) immunoprecipitates. Stimulation with IGF-I resulted in a dissociation of Crk protein from IRS-1. In contrast, the association of the related adaptor protein Grb2 with IRS-1 was enhanced by IGF-I stimulation. Similar results were obtained in stably transfected 293-Crk cells, which express both IRS-1 and the IRS-1-related signaling protein 4PS. In these cells, IRS-1 and 4PS both associated with Crk, and this association was also decreased by IGF-I treatment, whereas the association of Grb2 with IRS-1 and 4PS was enhanced by IGF-I. Overexpression of Crk also enhanced IGF-I-induced mitogenesis of NIH-3T3 cells, as measured by [3H]thymidine incorporation. The levels of IGF-I-induced mitogenesis were proportional to the level of Crk expression. These results suggest that Crk is a positive effector of IGF-I signaling, and may mediate its effects via interaction with IRS-1 and/or 4PS.

v-Crk, identified as an oncogene product of the CT10 retrovirus, became the first example of an adaptor protein. It consists mostly of the Src homology 2 (SH2) and Src homology 3 (SH3) domains. Two of the three major proteins bound to Crk SH2 have been identified as paxillin and p130Cas. Both paxillin and p130Cas are phosphorylated upon stimulation by integrin, suggesting that Crk transduces signals from integrin. The cloning of the complementary DNA of two major proteins bound to Crk SH3 was recently completed. Both cDNAs encoded novel proteins: C3G, a guanine nucleotide exchange protein for Rap1, and DOCK180, an SH3-containing protein of unknown function. The SH3 domain of Crk also binds to Sos, Abl, and Eps15. The variety of the proteins bound to Crk SH3 implies that Crk provides a set of effector proteins that are triggered together. Alternatively, other domains of the SH3-binding proteins enable Crk to specifically activate each of the SH3-binding proteins according to the particular form of stimulation.

Human embryonic kidney 293 cells and 293 cells overexpressing different amounts of the adaptor protein Crk-II (ranging from 3- to 10-fold higher levels than the parental cell line) were examined for their ability to undergo apoptosis when maintained in control and serum-free (SF) medium. Parental 293 cells undergo apoptosis only when deprived of serum for prolonged periods of time (24-48 h). On the other hand, 293 cells overexpressing different levels of Crk-II present detectable levels of apoptosis as measured by DNA fragmentation when grown in control medium, with a marked increase when they are deprived of serum for 12-48 h. To determine the pathways involved in Crk-II-induced apoptosis, Crk-II overexpressing cells were transiently transfected with a dominant-negative Ras construct (N17-Ras). Compared to cells transfected with control vectors, the cells overexpressing N17-Ras presented lower levels of apoptosis when maintained in SF-medium. On the other hand, transient transfection of a dominant-negative Raf-1 construct (K375W-Raf-1) did not decrease apoptosis; slightly increasing DNA fragmentation levels were seen. Similar results were obtained when the cells were incubated in the presence of a MEK1 inhibitor. The results presented here suggest that overexpression of Crk-II induces apoptosis via a Ras-dependent, Raf-1/MEK1/ERK-independent pathway.

The Crk proto-oncogene product is an SH2 and SH3 domain-containing adaptor protein. We have previously demonstrated that Crk-II becomes rapidly tyrosine-phosphorylated in response to stimulation with insulin-like growth factor I (IGF-I) and might be involved in the IGF-I receptor signalling pathway. To determine whether this involvement includes the direct interaction of Crk-II with the cytoplasmic region of the receptor, studies were performed in vitro with glutathione S-transferase (GST) fusion proteins containing various domains of Crk-II. The kinase assay in vitro showed that activated IGF-I receptors efficiently phosphorylated the GST-Crk-II fusion protein. This phosphorylation was dependent on the presence of the SH2 domain and Tyr-221 located in the spacer region between the two SH3 domains. mutation of Tyr-221 not only prevented phosphorylation of GST-Crk in vitro, but also significantly increased the ability of GST-Crk proteins to co-precipitate activated IGF-I receptors from total cell lysates. Additional binding experiments in vitro showed that Crk-II might interact with the phosphorylated IGF-I receptor through its SH2 domain. To elucidate which region of the IGF-I receptor interacts with Crk-II, a peptide association assay was used in vitro. Different domains of the IGF-I receptor were expressed as (His)6-tagged fusion peptides, phosphorylated with activated wheat germ agglutinin-purified IGF-I receptors and tested for association with GST-Crk-II fusion proteins. Using wild-type as well as mutated peptides, we showed that the SH2 domain of Crk-II preferentially binds the peptide encoding the juxtamembrane region of the IGF-I receptor. Phosphorylation of Tyr-950 and Tyr-943 of the receptor is important for this interaction. These findings allow us to propose a model of direct interaction of Crk-II and the IGF-I receptor in vivo. On activation of the IGF-I receptor, Crk-II binds to phosphorylated tyrosine residues, especially in the juxtamembrane region. As a result of this binding, the IGF-I receptor kinase phosphorylates Tyr-221 of Crk-II, resulting in a change in intramolecular folding and binding of the SH2 domain to the phosphorylated Tyr-221, which causes rapid disassociation of the Crk-II-IGF-I receptor complex.

The closely related proto-oncogene proteins CrkII and CrkL consist of one SH2 and two SH3 domains and share 60% overall homology with the highest identity within their functional domains. In this study we show that CrkL and CrkII may play overlapping but different roles in insulin-like growth factor (IGF)-I receptor-mediated signal transduction. While both proteins are substrates involved in IGF-I receptor signaling, they apparently demonstrate important different properties and different biological responses. Evidence supporting this hypothesis includes (a) the oncogenic potential of CrkL versus the absence of this potential in CrkII overexpressing cell lines, (b) the inhibition of IGF-I-dependent cell cycle progression by overexpression of CrkII, and (c) the differential regulation of the phosphorylation status of selective proteins in CrkII and CrkL overexpressing cell lines. In addition we demonstrate the specific association of CrkL and CrkII with the newly characterized IRS-4 protein, again in a differential manner. Whereas CrkL strongly interacts with IRS-4 via its SH2 and N-terminal SH3 domains, CrkII interacts only via its SH2 domain, possibly explaining the unstable nature of IRS-4-CrkII association. The results obtained allow us to propose a unique mechanism of CrkL and CrkII tyrosine phosphorylation in response to IGF-I stimulation. Thus these highly homologous proteins apparently possess structural features that allow for the differential association of each protein with different effector molecules, thereby activating different signaling pathways and resulting in unique biological roles of these proteins.

v-Crk is a member of the family of adaptor-type signaling molecules that consist mostly of SH2 and SH3 domains. The cellular homologs of v-Crk includes CrkI, CrkII, and CrkL; these have been isolated from species ranging from lower vertebrates to man. Crk-family proteins are involved in a variety of signaling cascades such as those of growth factor receptor, integrin, T-cell receptor, B-cell antigen receptor, and cytokines. It has been postulated that the primary function of Crk is to recruit cytoplasmic proteins in the vicinity of tyrosine kinases through SH2-phosphotyrosine interaction. Thus, the output from Crk depends on the SH3-binding proteins, which include the C3G guanine nucleotide exchange protein for Rap1, Abl tyrosine kinase, DOCK180, and the Sos guanine nucleotide exchange protein for Ras. The variety of the Crk-binding proteins indicate the pleiotropic function of Crk.