Oncogene database

55281

TMEM140

transmembrane protein 140;TMEM140;transmembrane protein 140

-

7q33

protein-coding

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.

We have previously shown that ethanol administration results in tyrosine phosphorylation of the 130 kDa protein in rat brain, and identified the protein as Cas, the crk-associated src substrate. In the present study, we demonstrate that Cbl of a 120 kDa protein is also tyrosine-phosphorylated in the cerebellum in response to ethanol administration. We also investigated whether Fyn kinase was involved in ethanol-induced Cbl phosphorylation. Immunoprecipitation experiments showed that the amount of coimmunoprecipitated Fyn kinase with an anti-Cbl antibody increased in extracts from ethanol-administered rats compared to those from saline-administered rats. Exogenous Fyn kinase was shown to phosphorylate on tyrosine residue(s) of Cbl from the cerebellum in vitro. Furthermore, Fyn kinase and Cbl were demonstrated immunohistochemically to be coexpressed in white matter in the cerebellum. These findings indicate that Cbl is tyrosine-phosphorylated in rat cerebellum in response to ethanol administration, and also raise the possibility that Fyn kinase may be involved in the process.

RET gene rearrangements, which generate chimeric RET/PTC oncogenes, are early events in the evolution of thyroid papillary carcinomas. expression of RET/PTC oncogenes promotes neoplastic transformation of cultured thyroid cells and of thyroid glands in transgenic mice. Notwithstanding these oncogenic effects, we have found that the expression of two RET/PTC oncogenes (H4-RET and RFG-RET) induces apoptosis of rat thyroid PC CL 3 cells. Promotion of thyroid cell death depends on the kinase activity of RET/PTC and on the phosphorylation of a tyrosine residue (tyrosine 1062) that maps in the carboxy-terminus of the RET protein. Tyrosine 1062 is essential for RET/PTC-mediated activation of the Ras/ERK pathway. Inhibition of Ras/ERK by a dominant negative Ras or by the MEKI inhibitor, PD98059, obstructed RET/PTC-mediated apoptosis. We also show that signals transmitted by tyrosine 1062 mediate proapoptotic events like Bcl-2 down regulation and Bax upregulation, and that adoptive overexpression of Bcl-2 overcomes RET/PTC-induced apoptosis. Thus, gene rearrangements that generate RET/PTC oncogenes subvert RET function by converting it into a chronically active kinase that is constitutively phosphorylated on tyrosine 1062. In turn, Y1062 phosphorylation transmits not only mitogenic but also proapoptotic signals to thyroid cells.

The pleiotropic proto-oncogene c-kit receptor, implicated in hematopoiesis and melanogenesis, is also known to play an important role in germ cell proliferation and differentiation although the mechanisms for the latter remain unknown. We studied c-kit expression by RT-PCR in various tissues of both fertile and infertile Brown Norway rats. Using different sets of primers, several regions from within the extracellular domain were amplified, cloned, and sequenced. One set of primers, in addition to the expected 352-bp amplicon, revealed a 276-bp transcript, although its biological functions remain unknown. These two transcripts showed varying levels of expression in different tissues of infertile rats against nearly uniform expression in the fertile animals. Significantly, the 352 bp testis transcript showed mutational hotspots from nucleotide 84-266 in the infertile rats. Analysis of testis and brain genomic DNA from these infertile rats showed mutations only in the testis suggesting this to be a postzygotic event. In contrast, no mutation was detected in the genomic DNA of testis and brain of the fertile rats. Protein expression studies showed complete absence of the cytoplasmic kinase domain and soluble c-kit protein in one of the infertile rats. Histological examination of testis of these infertile animals showed stem cell depletion resulting in fewer germ cells. Based on these results, we infer that 352-bp mutant mRNA transcript is implicated in the spermatogenic failure.

Growth hormone releasing hormone (GHRH) signals via G protein-coupled receptors (GHRH-R) to enhance intracellular Galphas/adenylyl cyclase/cAMP signaling, which in turn has positive effects on GH synthesis and release, as well as proliferation of the GH-producing cells of the anterior pituitary gland. Some GH-producing pituitary tumors express a constitutively active mutant form of Galphas (gsp oncogene). It has been reported that these tumors are more responsive to octreotide therapy. In this study we used a rat GH-producing cell line (GH3) stably transfected with the human GHRH-R cDNA (GH3-GHRHR cells) as a model to study the effects of gsp oncogene on somatostatin (SRIH) receptor subtype 1 and 2 (sst1 and sst2) mRNA levels. Transient transfection of gsp oncogene in GH3-GHRHR cells for 48 h increased intracellular cAMP levels and GH release. Phosphodiesterase (PDE) 4, sst1 and sst2 mRNA levels were increased by G protein mutation as assessed by real-time RT-PCR. Increased PDE mRNA levels in gsp-transfected cells may be a compensatory mechanism to the constitutive activation of cAMP-dependent pathway by G protein mutation and is consistent with reports of higher PDE expression in human pituitary tumor that express gsp. Our data suggest that higher expression of sst1 and sst2 mRNA induced by the gsp oncogene may be a mechanism by which gsp-positive tumors show a greater response to SRIH. GH3 cells permanently transfected with GHRH-R can be used for in vitro studies of actions of GHRH.

The c-myc proto-oncogene is rapidly activated by serum and regulates genes involved in metabolism and cell cycle progression. This gene is thereby uniquely poised to coordinate both the metabolic and cell cycle regulatory events required for cell cycle entry. However, this function of Myc has not been evaluated. Using a rat fibroblast model of isogenic cell lines, myc(-/-), myc(+/-), myc(+/+) and myc(-/-) cells with an inducible c-myc transgene (mycER), we show that the Myc protein programs cells to utilize both oxidative phosphorylation and glycolysis to drive cell cycle progression. We demonstrate this coordinate regulation of metabolic networks is essential, as specific inhibitors of these pathways block Myc-induced proliferation. Metabolic events temporally correlated with cell cycle entry include increased oxygen consumption, mitochondrial function, pyruvate and lactate production, and ATP generation. Treatment of normal cells with inhibitors of oxidative phosphorylation recapitulates the myc(-/-) phenotype, resulting in impaired cell cycle entry and reduced metabolism. Combined with a kinetic expression profiling analysis of genes linked to mitochondrial function, our study indicates that Myc s ability to coordinately regulate the mitochondrial metabolic network transcriptome is required for rapid cell cycle entry. This function of Myc may underlie the pervasive presence of Myc in many human cancers.

Cell proliferation requires the coordinated activity of cytosolic and mitochondrial metabolic pathways to provide ATP and building blocks for DNA, RNA and protein synthesis. Many metabolic pathway genes are targets of the c-myc oncogene and cell-cycle regulator. However, the contribution of c-Myc to the activation of cytosolic and mitochondrial metabolic networks during cell-cycle entry is unknown. Here, we report the metabolic fates of [U-(13)C] glucose in serum-stimulated myc(-/-) and myc(+/+) fibroblasts by (13)C isotopomer NMR analysis. We demonstrate that endogenous c-myc increased (13)C labeling of ribose sugars, purines and amino acids, indicating partitioning of glucose carbons into C1/folate and pentose phosphate pathways, and increased tricarboxylic acid cycle turnover at the expense of anaplerotic flux. Myc expression also increased global O-linked N-acetylglucosamine protein modification, and inhibition of hexosamine biosynthesis selectively reduced growth of Myc-expressing cells, suggesting its importance in Myc-induced proliferation. These data reveal a central organizing function for the Myc oncogene in the metabolism of cycling cells. The pervasive deregulation of this oncogene in human cancers may be explained by its function in directing metabolic networks required for cell proliferation.

Although anoxic preconditioning (APC) in the myocardium has been investigated for many years, its physiological mechanism is still not completely understood. Increasing evidence indicates that transiently increased resistance to ischemic damage following APC is dependent on de novo proteins synthesis. However, the key effector pathway(s) associated with APC still remains unclear. The proto-oncogene Pim kinase belongs to a serine/threoine protein kinase family, consists of Pim-1, Pim-2 and Pim-3 and has been implicated in stimulating cell growth and inhibiting cell apoptosis. Therefore we assumed that Pim-3 expression might be aberrantly induced in cardiomyocytes that were subjected to anoxia/reoxygenation (A/R) injury and that Pim-3 might also contribute to cardio-protection after APC. To address this hypothesis, we cloned a Pim-3 expression vector, transfected it into rat cardiomyocytes, and examined Pim-3 expression in rat cardiomyocytes that were subjected to A/R injury. Moreover, we studied the role of three major MAPK pathways, e.g. p38 MAPK, JNK, and ERK1/2, in order to evaluate the molecular mechanism underlying Pim-3 up-regulation and A/R induced cardiomyocyte injury. Our experiments showed that APC induced an up-regulation of Pim-3 and the transfection of Pim-3 gene into the cardiomyocytes attenuated A/R injury. The inhibition of p38 MAPK by SB203580 abolished both the Pim-3 up-regulation and the cardio-protection provided by APC. Overall, these results suggest that APC could act to protect the heart from A/R injury with cooperation from the proto-oncogene Pim-3; in addition, it up-regulates Pim-3 expression through a p38 MAPK signaling pathway.

Osteocalcin (OC) is a major noncollagenous bone matrix protein and an osteoblast marker whose expression is limited to mature osteoblasts during the late differentiation stage. In previous studies we have shown osteosarcomas to lose p53 function with a corresponding loss of osteocalcin gene expression. Introduction of wild type p53 resulted in re expression of the osteocalcin gene. Using gel shift and chromatin immunoprecipitation assays, we have identified a putative p53 binding site within the rat OC promoter region and observed an increase in OC promoter activity when p53 accumulates using a CAT assay. The p53 inducible gene Mdm2 is a well-known downstream regulator of p53 levels. Our results showed a synergistic increase in the OC promoter activity when both p53 and MDM2 were transiently overexpressed. We further demonstrate that p53 is not degraded during overexpression of MDM2 protein. Increased OC expression was observed with concomitantly increased p53, VDR, and MDM2 levels in ROS17/2.8 cells during treatment with differentiation promoting (DP) media, but was significantly decreased when co-treated with DP media and the small molecule inhibitor of MDM2-p53 interaction, Nutlin-3. We have also observed a dramatic increase of the OC promoter activity in the presence of p53 and Mdm2 with inclusion of Cbfa-1 and p300 factors. Our results suggest that under some physiological conditions the oncoprotein MDM2 may cooperate with p53 to regulate the osteocalcin gene during osteoblastic differentiation.

Rcl is a novel N-glycoside hydrolase found in mammals that shows specificity for the hydrolysis of 5 -monophosphate nucleotides. Its role in nucleotide catabolism and the resulting production of 2-deoxyribose 5-phosphate has suggested that it might fuel cancer growth. Its expression is regulated by c-Myc, but its role as an oncoprotein remains to be clarified. In parallel, various nucleosides have been shown to acquire pro-apoptotic properties upon 5 -monophosphorylation in cells. These include triciribine, a tricyclic nucleoside analogue that is currently in clinical trials in combination with a farnesyltransferase inhibitor. Similarly, an N(6)-alkyl-AMP has been shown to be cytotoxic. Interestingly, Rcl has been shown to be inhibited by such compounds in vitro. In order to gain better insight into the precise ligand-recognition determinants, the crystallization of Rcl with these nucleotide analogues was attempted. The first crystal structure of Rcl was solved by molecular replacement using its NMR structure in combination with distantly related crystal structures. The structures of Rcl bound to two other nucleotides were then solved by molecular replacement using the previous crystal structure as a template. The resulting structures, solved at high resolution, led to a clear characterization of the protein-ligand interactions that will guide further rational drug design.

The 14-3-3 family of proteins regulates various signaling pathways involved in cell cycle, apoptosis, stress response, and malignant transformation. We previously demonstrated that the beta isoform of the 14-3-3 protein promotes cell growth and tumorigenicity of rat K2 hepatocellular carcinoma cells. We identified fourteen-three-three beta interactant 1 (FBI1)/Akirin2 as a binding partner of 14-3-3beta and showed that the complex of these proteins promotes tumorigenicity and metastasis of K2 cells. In addition, we demonstrated that FBI1/Akirin2 downregulation shortened the duration of MAPK activity. Because 14-3-3beta and FBI1/Akirin2 overexpression is observed in various cancer cell lines, 14-3-3beta-FBI1/Akirin2 oncogenic function should be elucidated in different types of cancer. In this study, we used LLC1 Lewis lung carcinoma cells as a model. We established FBI1/Akirin2 knockdown cell clones through transfection of an antisense FBI1/Akirin2 expression vector and assessed the capacity for cell growth in vitro and tumorigenicity and metastasis in vivo. FBI1/Akirin2 downregulation decreased anchorage-independent growth, whereas the growth rate in monolayer culture was not affected. Moreover, an in vivo assay in nude mice showed that FBI1/Akirin2 overexpression is required for LLC1 tumor growth and metastasis. These results suggest that FBI1/Akirin2 plays an important role in oncogenesis of LLC1 lung carcinoma cells, and this protein may also serve as an oncogene in other cancers.