A strain of Fujinami sarcoma virus which is temperature-sensitive in protein phosphorylation and cellular transformation

We have investigated the effect of 12-0-tetradecanoylphorbol 13-acetate (TPA) on the synthesis and modification of polypeptides in normal avian cells and cells infected by wild-type and temperature-sensitive Rous sarcoma virus (RSV). Using two-dimensional gel electrophoresis, we have detected alterations in both the abundance of cellular polypeptides and in their phosphorylation that seem unique to TPA treatment. However, the state of phosphorylation of the major putative substrate for the action of the arc gene-associated protein kinase, the 34-to 36-kilodalton protein, was not altered. Moreover, examination of the phosphorylated amino acid content of total cellular phosphoproteins revealed that the response to TPA was not associated with detectable increases in their phosphotyrosine content. These results make it unlikely that TPA acts by the activation of the phosphorylating activity of the cellular proto-src gene or by the activation ofother cellular phosphotyrosine-specific kinases. We A major achievement oftumor virology in the last few years has been the identification and characterization of the products of viral "oncogenes," which are responsible for the initiation and maintenance ofmalignant transformation (1). In the case ofRous sarcoma virus (RSV) the product of its transforming gene, src, has been shown to be tightly associated with a protein kinase (2, 3) that specifically phosphorylates tyrosine (4). Several putative substrates of this activity have been identified, including the 34-to 36-kilodalton (kDal) protein (5-7) and vinculin (8). However, the link between the function ofthe src gene and the pleiotropic changes in cellular phenotype that are associated with oncogenic transformation by RSV has remained elusive.Uninfected normal vertebrate cells contain loci related to every retrovirus oncogene identified so far (1, 9). In the case of RSV, the cellular sarc (proto-src gene) has been shown to code for a product highly similar to that of the viral src gene (10,-11). Such results have led to proposals that malignant transformation ofcells by retroviruses may be a consequence ofgene dosage: By providing an efficient promoter for transcription, viruses overload cells with gene products that are similar to cellular gene products normally under strict quantitative control (3, 9). These proposals rest on the assumptions that the substrate specificity ofthe viral and cellular gene products are similar and that the products of the proto-oncogenes play an important role in cell differentiation and growth control (9).One way to obtain insights into the possible role that these In this paper we have investigated the effect ofthe TPA treatment of chicken embryonic fibroblasts (CEF) on polypeptide synthesis and protein phosphorylation and compared these changes to those that are characteristic for RSV-induced transformation (5). We also asked whether such effects are accompanied by alterations in two cellular parameters directly related to the action of the src gene-i.e., the alteration of the st...

Section: Methodsmentioning

confidence: 99%

Tumor promoters alter gene expression and protein phosphorylation in avian cells in culture.

Laszlo

Radke

Chin

et al. 1981

“…Transformation by Rous sarcoma virus (RSV) requires the activity of a virally encoded tyrosine-specific protein kinase (4). The viral gene is apparently derived from a normal cellular gene and is present in transformed cells at a level 50-to 100-fold more abundant than in normal cells (5)(6)(7)(8). Tyrosine-specific protein kinase activity is also stimulated after treatment of cells and membranes with several potent polypeptide mitogens, including epidermal growth factor (EGF), insulin, and platelet-derived growth factor (9)(10)(11).…”

mentioning

confidence: 99%

Phorbol esters potentiate tyrosine phosphorylation of epidermal growth factor receptors in A431 membranes by a calcium-independent mechanism.

Moon¹,

Palfrey²,

King³

1984

Incubation of membranes prepared from A431 cells with either epidermal growth factor (EGF) or phorbol 12-myristate 13-acetate (PMA) stimulates the transfer of 32phosphate from [y-32P]ATP into 8-10 membrane proteins. The major phosphorylated protein migrates on NaDodSO4/ polyacrylamide gels with an apparent Mr of 180,000, corresponding to the previously identified EGF receptor. Stimulation of EGF receptor phosphorylation by PMA does not require Ca2+, suggesting-that prior activation of protein kinase C is not a prerequisite for phosphate transfer. PMA-enhanced phosphorylation proceeds at 40C and requires Mn2+, both properties of tyrosine-specific protein kinases. Phospho amino acid analysis of the Mr 180,000 receptor band shows that only tyrosine residues are phosphorylated when A431 membranes are treated with either EGF or PMA. Moreover, proteolysis reveals that these residues are located in the same peptides of the receptor. These results demonstrate that a potent tumorpromoting phorbol ester can mimic a critical early response usually elicited by EGF.Phorbol ester tumor promoters are mitogenic and induce phenotypic changes in mammalian cells that resemble transformation by oncogenic viruses (1-3). The mechanisms that direct tumor promotion, transformation, and growth potentiation are not known; however, recent evidence indicates that protein kinases may be involved in mediation of these effects. Transformation by Rous sarcoma virus (RSV) requires the activity of a virally encoded tyrosine-specific protein kinase (4). The viral gene is apparently derived from a normal cellular gene and is present in transformed cells at a level 50-to 100-fold more abundant than in normal cells (5)(6)(7)(8). Tyrosine-specific protein kinase activity is also stimulated after treatment of cells and membranes with several potent polypeptide mitogens, including epidermal growth factor (EGF), insulin, and platelet-derived growth factor (9-11). In several instances, the major substrate for mitogen-stimulated tyrosine phosphorylation is the receptor specific for that particular mitogen (10-14). For EGF and insulin receptors, the tyrosine-specific protein kinase activity is associated with affinity-purified forms of the receptor and is believed to be an integral part of the receptor itself (14,15). The finding that different activators of cell growth enhance phosphorylation of tyrosine residues has led to the speculation that tyrosine-specific protein kinases may represent cellular control elements that regulate normal and pathologic growth.Receptors for phorbol esters (16)(17)(18) are tightly associated with another protein kinase activity (19,20), protein kinase C. Both phorbol binding (19-21) and kinase activity (22)(23)(24) are dependent on the simultaneous presence of Ca2+ and phospholipid. Protein kinase C is activated by diacylglycerol generated after hormonal stimulation of phosphatidyl inositol breakdown (23, 24). However, tumor-promoting phorbol esters can substitute for diacylglycerol in the activation of protein kina...

“…This protein kinase activity, usually detected as autophosphorylation, has been found to be associated with transforming proteins of Rous sarcoma virus (10,11), Abelson murine leukemia virus (MuLV) (12), Snyder-Theilen strain offeline sarcoma virus (13,14), Y73 avian sarcoma virus (15), and Fuginami avian sarcoma virus (16). First identified in the Rous sarcoma virus (10), the protein kinase activity ofphosphorylated p6{src has been shown to be an intrinsic property of the transforming protein (17,18).…”

mentioning

confidence: 99%

P85 ^gag-mos encoded by ts110 Moloney murine sarcoma virus has an associated protein kinase activity

Kloetzer

Maxwell

Arlinghaus

1983

A protein identified as P859'9-°was shown to be phosphorylated when immunoprecipitates from tsllO Moloney murine sarcoma virus transformed nonproducer cells (clone 6m2) were incubated with [y-32P]ATP. The in vitro-labeled 85,000-dalton phosphoprotein comigrated on NaDodSO4/polyacrylamide gels with authentic phosphorylated P859'9-°. Immunoprecipitates obtained with antisera prepared against Rauscher murine leukemia virus core protein p30 were active in the immune complex kinase assay but anti-murine leukemia virus plO precipitates were not. Previous studies have shown that anti-p30 but not antiplO antisera recognize P859'9-°. The 6m2 clone has been shown to express P85gag-"w8 at 330C but not at 39TC. Anti-p30 immune complexes from 6m2 cells maintained at 390C failed to phosphorylate the 85,000-dalton protein. Furthermore, the in vitro phosphorylated 85,000-dalton protein gave the same pattern of V8 protease-generated cleavage products as in vivo 32P-labeled P85gag-'.8, We conclude from these results that P85gag`m06 is phosphorylated in anti-p30 immune complex kinase reactions. Phosphoamino acid analyses indicated that the in vitro phosphorylated P85gag-tno contained phosphoserine and phosphothreonine. Our findings indicate that incubation of anti-p30 immunoprecipitates at 39°C drastically reduced, in a specific way, the kinase activity associated with P85gag-1l8. This result and other data suggest that the kinase is virus-encoded. Because P85gag-"1w8, but not Pr65gag, is phosphorylated in anti-p30 immunoprecipitates from MuLVMuSV tsllO producer cells, the kinase enzyme is associated with P85gag-' and not gag gene products. A second major polypeptide of the size of P58gag was also phosphorylated in anti-p30 immunoprecipitates from cells maintained at 33°C but not at 39°C. Since 6m2 cells at 39°C contain P58wag, this is also consistent with the kinase activity being associated with P85gag-11W.