Transformation by Rous Sarcoma Virus: Effects of src-gene Expression on the Synthesis and Phosphorylation of Cellular Polypeptides

Radke, Kathryn; Martin, G S

doi:10.1101/sqb.1980.044.01.105

Cited by 50 publications

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“…We observed a 4-to 8-fold increase in the analytic phosphorylation of casein and as much as an 18-fold increase when the 34K protein, a putative in vivo target protein of pp6Ov-sr (8,24,25), was used as substrate. In addition, a protein of 50,000 daltons which copurified with the 34K protein under our conditions served as an excellent substrate for pp6f-esrc kinase activity (Fig.…”

Section: Discussionmentioning

confidence: 84%

Increase in the phosphotransferase specific activity of purified Rous sarcoma virus pp60v-src protein after incubation with ATP plus Mg2+.

Purchio¹,

Wells²,

Collett³

1983

Mol. Cell. Biol.

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pp6O-vsc, the product of the Rous sarcoma virus src gene, was partially purified by immunoaffinity chromatography from extracts of Rous sarcoma virus-transformed field vole cells. Incubation of this preparation with ATP plus Mg2+ and subsequent repurification by chromatography on hexylamine-agarose resulted in a net increase in the specific activity of the src protein kinase. This increase in phosphotransferase activity was detected by using a variety of substrates including casein, tubulin, and a 34,000-dalton protein presumed to be an in vivo target substrate of pp6O-src. In all cases, the phosphorylation was at tyrosine residues, and the kinase activity was inhibited by preincubation of the enzyme with immunoglobulin G prepared from tumor-bearing rabbit sera. The implications of these results for the regulation and control of pp6vOsr,'-associated kinase activity are discussed.The product of the Rous sarcoma virus (RSV) src gene is a 60,000-dalton (60K) phosphoprotein termed pp60vsrc (2, 22). Early experimental results suggested that a tentative function of this transforming protein was that of a protein kinase. This assignment was based on the ability of immune complexes containing pp6Ovsrc to phosphorylate the heavy chain of immunoglobulin G (IgG) molecules directed against the pp60V-sr( protein (5, 16). Subsequent purification of pp60v-sr from cytoplasmic extracts of RSVtransformed cells indicated that pp6Ov-sr either was itself a protein kinase or was tightly associated with a protein kinase (9, 10). More recently, the src gene has been molecularly cloned to expression in Escherichia coli (11,12,18). The polypeptide produced in bacteria appears to possess protein phosphotransferase activity (11, 18), clearly indicating that the RSV-transforming protein pp6Ov-sr is indeed a protein kinase. In all cases studied to date, the pp60v-src protein kinase activity appears to be specific for only tyrosine residues (6, 14, 17).Early experiments with pp6OvsrL indicated that this enzyme was capable of apparent autophosphorylation (9); this was based on the observation that the addition of [-y-32P]ATP to partially purified preparations of pp60v-sr, resulted in the phosphorylation of pp6Ov-src itself, although other workers have been unable to detect this activity (17). Experiments with highly purified preparations of pp6Ov-srC suggest that pp60v-src either does phosphorylate itself or is very tightly associated with a second kinase which is responsible for phosphorylating pp60v-src (21). Analyses of pp6Ov-.r( phosphorylation in vitro indicate that the same major tyrosine residue is phosphorylated on pp6Ov-src in vivo (28).Understanding the regulation of the pp6Ov-sr( kinase activity is an important step in understanding the mechanism of transformation by RSV. In this study, partially purified pp60v5sr( was incubated with ATP plus Mg2+ (ATP/Mg2+) and, after separation from all unlabeled ATP, was analyzed for its kinase activity. We observed a significant increase in the tyrosinespecific protein kinase activity of the enz...

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Section: Discussionmentioning

confidence: 84%

Increase in the phosphotransferase specific activity of purified Rous sarcoma virus pp60v-src protein after incubation with ATP plus Mg2+.

Purchio¹,

Wells²,

Collett³

1983

Mol. Cell. Biol.

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“…The extent of phosphorylation depended on the particular clone of transformed A431 cells, being greater for the clone that had the highest pp60 8" kinase activity when assayed in vitro (Table I). Thus, in human epidermoid cells, as well as a variety of other cells (16)(17)(18)(19)(20), transformation by RSV induces phosphorylation of a 36K cellular protein. The 81K phosphoprotein was not detected in any of the clones of infected A431 cells (Fig .…”

Section: Rapid Communicationsmentioning

confidence: 99%

“…While we find its molecular weight to be 39,000, for clarity we will refer to this protein by its original molecular weight designation, 36K (16)(17)(18)(19)(20)(21). One other protein (81K), whose phosphorylation on serine and tyrosine in A431 cells is EGF dependent (15), appears not to be phosphorylated in RSV-transformed mouse cells (17), but it is quite possible that the human protein is homologous to a mouse protein of different size or isoelectric point.…”

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Similarities and differences between the effects of epidermal growth factor and Rous sarcoma virus.

Cooper¹,

Hunter²

1981

The Journal of Cell Biology

107

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We have derived a line of A431 human tumor cells infected with Rous sarcoma virus (RSV). The infected cells contain the RSV-transforming protein, pp60src, which has characteristic tyrosine specific protein kinase activity. As in other RSV-transformed cells, a 36,000-dalton protein is phosphorylated in RSV-infected A431 cells. Addition of epidermal growth factor (EGF) to the cells induces further phosphorylation of this protein. In contrast, this phosphoprotein is not detected in uninfected A431 cells, except when treated with EGF. Increased phosphorylation of the EGF receptor protein and of an 81,000-dalton cellular protein is dependent upon addition of EGF to the culture fluids, in both control and RSV-infected A431 cells. The results are discussed with reference to the similarities and differences between the tyrosine-specific protein kinases induced by RSV and activated by EGF.

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“…Transformation by Rous sarcoma virus (RSV) causes an eight-to tenfold increase in total cell phosphotyrosine (49) and an increase in the amount of phosphotyrosine found in many cellular proteins (3,36). These include the cytoskeletal protein vinculin (46,48), a 36,000-dalton (36-kDa) membrane-associated protein (pp36) (20,42,43), a 50-kDa cytoplasmic protein (6,7) of unknown function, and three non-rate-determining enzymes involved in the glycolytic pathway (16). The tyrosine phosphorylating activity correlates with transformation in most RSV temperature-sensitive mutants (28,53) and mutant revertants (37), although mutants and revertants exist which have tyrosine phosphorylating activity but which do not induce transformation (37,53).…”

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confidence: 99%

Restriction of the in vitro and in vivo tyrosine protein kinase activities of pp60c-src relative to pp60v-src.

Coussens¹,

Cooper²,

Hunter³

et al. 1985

Mol. Cell. Biol.

115

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The tyrosine protein kinase activities of pp60csrc and pp60v'-were compared. The activities were qualitatively similar in vitro when the src proteins were bound in an immune complex with monoclonal antibody; both proteins utilized either ATP or GTP as phosphate donors, preferred Mn2+ to Mg2+, and had similar exogenous substrate specificities. The specific activity of pp604>snc was about 10-fold lower than that of pp6v.s-r for exogenous substrate phosphorylation but was only 1.1-to 2-fold lower than that of pp60v-sc for autophosphorylation. Six Tyrosine-specific protein kinase activity is rare in normal fibroblasts, and accordingly, the level of phosphotyrosine in these cells is very low, comprising only 0.03% of total phosphoamino acids (49). Transformation by Rous sarcoma virus (RSV) causes an eight-to tenfold increase in total cell phosphotyrosine (49) and an increase in the amount of phosphotyrosine found in many cellular proteins (3, 36). These include the cytoskeletal protein vinculin (46, 48), a 36,000-dalton (36-kDa) membrane-associated protein (pp36) (20,42,43), a 50-kDa cytoplasmic protein (6, 7) of unknown function, and three non-rate-determining enzymes involved in the glycolytic pathway (16). The tyrosine phosphorylating activity correlates with transformation in most RSV temperature-sensitive mutants (28, 53) and mutant revertants (37), although mutants and revertants exist which have tyrosine phosphorylating activity but which do not induce transformation (37, 53). These facts suggest that src tyrosine kinase activity is required but not sufficient for transformation.Immunoaffinity-purified pp60v-src and pp60v-sr bound to some monoclonal antibodies can phosphorylate itself as well as other substrates (11-13, 35, 41). It has a broader substrate range in vitro than in vivo (12, 48) and phosphorylates purified casein, histocompatibility antigens (26), and random tyrosine-containing polypeptides (5). Preparations of pp60v-src also contain enzyme activities that can phosphorylate glycerol (24, 44), diacylglycerol, and phosphoinositides (54). The in vitro protein kinase activity is cyclic AMP independent (21), uses either manganese or magnesium as divalent cation (12,45), and in contrast with most character-* Corresponding author. (25,45). pp6csrc, the normal cellular counterpart of pp6v-src, is also a tyrosine-specific protein kinase in vitro and phosphorylates the immunoglobulin G (IgG) heavy chains of some tumor-bearing rabbit (TBR) antisera (10, 38), but the low level of pp60'csrc in normal cells has prevented a detailed assessment of its in vitro and in vivo substrates. Recent studies with overproduced pp60fcsrc from fibroblasts show that it has lower tyrosine kinase activity than pp6Ov-src when bound by monoclonal antibody (29, 31). pp6Oc-sr binds to polyomavirus middle T antigen, and the phosphorylating activity of the bound pp6Oc-sr population for casein and IgG is stimulated 20-to 50-fold by association with middle T (4; J. Brugge, personal communication). Amino acid sequences inferred from D...

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Transformation by Rous Sarcoma Virus: Effects of src-gene Expression on the Synthesis and Phosphorylation of Cellular Polypeptides

Cited by 50 publications

References 0 publications

Increase in the phosphotransferase specific activity of purified Rous sarcoma virus pp60v-src protein after incubation with ATP plus Mg2+.

Increase in the phosphotransferase specific activity of purified Rous sarcoma virus pp60v-src protein after incubation with ATP plus Mg2+.

Similarities and differences between the effects of epidermal growth factor and Rous sarcoma virus.

Restriction of the in vitro and in vivo tyrosine protein kinase activities of pp60c-src relative to pp60v-src.

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