Protein Of Rous Sarcoma Virus Research Articles

Phosphorylation and Protein Synthetic Events in Xenopus laevis Oocytes Microinjected with pp60v-src

Microinjection of purified pp60v-src, the transforming protein of Rous sarcoma virus, into Xenopus laevis oocytes accelerated the rate of progesterone- or insulin-induced meiotic maturation. This acceleration was abolished by incubating the oocytes with cycloheximide or puromycin during a 2-h interval between pp60v-src microinjection and progesterone addition. In contrast, exposure to actinomycin D did not alter the acceleration of maturation by microinjected pp60v-src. Associated with progesterone treatment and pp60v-src microinjection were a number of qualitative changes in phosphoproteins; a few of these changes are common to both stimuli. These results indicate that the action of pp60v-src in oocytes involves both phosphorylation and protein synthetic events that affect oocyte maturation.

Protein kinase C phosphorylates pp60 src at a novel site

The transforming protein of Rous sarcoma virus (pp60 v- src ) and its normal cellular homolog (pp60 c- src ) are demonstrated to be phosphorylated at serine 12 in vivo under certain conditions. We propose that protein kinase C is responsible for this modification based on the following evidence. First, the tumor promoters, 12-O-tetradecanoylphorbol-13-acetate and teleocidin, and synthetic diacylglycerol, known activators of protein kinase C in vivo, cause nearly complete phosphorylation of pp60 src at serine 12. Second, among five purified serine/threonine-specific protein kinases tested, only protein kinase C phosphorylates pp60 c- src and pp60 v- src in vitro at serine 12. Third, purified protein kinase C phosphorylates a synthetic peptide corresponding to the N-terminal 20 amino acids of pp60 c- src at serine 12. The physiological significance of this novel phosphorylation is discussed.

Neurones express high levels of a structurally modified, activated form of pp60c-src

Neural tissues contain high levels of the cellular homologue of the transforming protein of Rous sarcoma virus (RSV), but neither the specific cell types expressing high levels of c-src, nor the function of the cellular src (c-src) protein has been determined. Using primary culture methods, we have found that pure neurones and astrocytes derived from the rat central nervous system (CNS) contain 15- to 20-times higher levels of the c-src protein than fibroblasts. However, the specific activity of the c-src protein from the neuronal cultures is 6- to 12-times higher than that from the astrocyte cultures. In addition, the c-src protein expressed in neuronal cultures contains a structural alteration within the amino-terminal region of the molecule that causes a shift in the mobility of the c-src protein on the SDS-polyacrylamide gels. These results indicate that a structurally distinct form of the cellular src protein that possesses an activated tyrosylkinase activity is expressed at very high levels in post-mitotic CNS neurones.

Amino terminal myristylation of the protein kinase p60src, a retroviral transforming protein.

The transforming protein of Rous sarcoma virus, p60src, was shown to be acylated at its amino terminus with the long-chain fatty acid myristic acid by isolation of a tryptic peptide with the following structure: myristylglycylserylseryllysine. The occurrence of this unusual posttranslational modification in the cyclic adenosine monophosphate-dependent protein kinase and in several transforming protein kinases of mammalian retroviruses suggests that myristylation of the amino terminal glycyl residue may be critical for the function of certain proteins related to cell transformation and growth control.

Myristic acid, a rare fatty acid, is the lipid attached to the transforming protein of Rous sarcoma virus and its cellular homolog.

The lipid bound to p60src, the transforming protein of Rous sarcoma virus, has been identified by gas and thin-layer chromatography as the 14-carbon saturated fatty acid, myristic acid. The protein can be labeled biosynthetically with either [3H]myristic acid or [3H]palmitic acid. Incorporation of [3H]myristic acid was noticeably greater than incorporation of [3H]palmitic acid. All of the [3H]myristic acid-derived label in p60src was present as myristic acid. In contrast, none of the radioactivity derived from [3H]palmitic acid was recovered as palmitic acid. Instead, all 3H incorporated into p60src from [3H]palmitic acid arose by metabolism to myristic acid. The cellular tyrosine kinase, p60c-src also contains myristic acid. By comparison of the extent of myristylation of p60v-src with that of the Moloney murine leukemia virus structural protein precursor, Pr65gag, we estimate that greater than 80% of the molecules of p60v-src contain one molecule of this fatty acid. Myristylation is a rare form of protein modification. p60v-src contains 10 to 40% of the myristic acid bound to protein in cells transformed by Rous sarcoma virus and is easily identified in total cell lysates when [3H]myristic acid-labeled proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the amount of [3H]myristic acid-labeled p60src in total cell lysates and in immunoprecipitates suggests that immunoprecipitation with rabbit anti-Rous sarcoma virus tumor sera detects ca. 25% of the p60src present in cells.

Myristic acid is attached to the transforming protein of Rous sarcoma virus during or immediately after synthesis and is present in both soluble and membrane-bound forms of the protein.

Myristic acid, a minor component of cellular fatty acids, has been shown previously to be covalently bound to most molecules of p60src, the transforming protein of Rous sarcoma virus. We have now determined at what time during the life cycle of p60src, and where within the cell, this lipid becomes attached to the protein. p60src was found to acquire myristic acid at only one time, during or immediately after its synthesis. p60src is known to be synthesized on free polysomes and appears at the cytoplasmic face of the plasma membrane after a lag of 10 min. The addition of myristic acid to p60src therefore precedes the binding of the protein to the plasma membrane. The lipid attached to p60src is a permanent, metabolically stable part of the protein; we found no evidence for turnover of the myristyl moiety. However, we did find myristate attached to various soluble forms of p60src and to a large number of cytosolic cellular proteins as well. This demonstrates that the attachment of myristic acid to a protein is not in itself sufficient to convert a soluble protein into a membrane-bound protein.

Myristic acid is attached to the transforming protein of Rous sarcoma virus during or immediately after synthesis and is present in both soluble and membrane-bound forms of the protein.

Myristic acid, a minor component of cellular fatty acids, has been shown previously to be covalently bound to most molecules of p60src, the transforming protein of Rous sarcoma virus. We have now determined at what time during the life cycle of p60src, and where within the cell, this lipid becomes attached to the protein. p60src was found to acquire myristic acid at only one time, during or immediately after its synthesis. p60src is known to be synthesized on free polysomes and appears at the cytoplasmic face of the plasma membrane after a lag of 10 min. The addition of myristic acid to p60src therefore precedes the binding of the protein to the plasma membrane. The lipid attached to p60src is a permanent, metabolically stable part of the protein; we found no evidence for turnover of the myristyl moiety. However, we did find myristate attached to various soluble forms of p60src and to a large number of cytosolic cellular proteins as well. This demonstrates that the attachment of myristic acid to a protein is not in itself sufficient to convert a soluble protein into a membrane-bound protein.

Antibodies to two defined regions of the transforming protein pp60src interact specifically with the epidermal growth factor receptor kinase system.

Antibodies generated against two synthetic peptides corresponding to two defined regions on the transforming protein of Rous sarcoma virus, pp60src, interact specifically with the epidermal growth factor (EGF)-receptor kinase. An antibody directed against a synthetic peptide corresponding to the major phosphorylation site of pp60src interacts specifically with EGF receptor and immunoprecipitates a functional EGF-receptor kinase. The second antibody, which binds close to a region on the src molecule that is required for its kinase activity, also binds to EGF-receptor kinase and prevents the autophosphorylation of the receptor molecules. Neither antibody binds to intact cells, but they do recognize various forms of the solubilized receptor. It is concluded that at least two cytoplasmic domains of the EGF receptor are antigenically and presumably also structurally related to specific domains on pp60src.

Monoclonal antibodies to Rous sarcoma virus pp60src react with enzymatically active cellular pp60src of avian and mammalian origin.

The derivation and characterization of 22 hybridoma clones producing monoclonal antibodies (Mabs) specific for the transforming protein of Rous sarcoma virus, pp60src, are described. All Mabs reacted with pp60v-src encoded by Prague, Schmidt-Ruppin, and Bratislava 77 strains of Rous sarcoma virus. Of these Mabs, 10 efficiently immunoprecipitated pp60c-src from chicken embryo cells. Of these 10 Mabs, 2 (GD11 and EB8) readily detected pp60c-src from a variety of rodent and human cultured cells and from rat brain tissue in an in vitro immune complex kinase assay. Mapping experiments have tentatively localized the determinant(s) recognized by GD11 and EB8 to a region of the src protein bounded by amino acid residues 82 to 169, whereas the remaining Mabs appeared to recognize determinants residing within residues 1 to 82 or 169 to 173. Most of the Mabs complexed denatured pp60v-src in a Western immunoblot, and several were used to localize pp60v-src in Rous sarcoma virus-transformed chicken embryo cells by indirect immunofluorescence microscopy.

Direct evidence that oncogenic tyrosine kinases and cyclic AMP-dependent protein kinase have homologous ATP-binding sites.

p60src, the transforming protein of Rous sarcoma virus (RSV), is a protein kinase that has a strict specificity for tyrosine. The phosphorylation of cellular proteins by p60src (ref. 4) results in transformation. Recently, Barker and Dayhoff discovered that residues 259-485 of p60src have 22% sequence identity with residues 33-258 of the catalytic subunit of cyclic AMP-dependent protein kinase, an enzyme that has a specificity for serine. Because it was necessary to introduce eight gaps to align the two proteins, the question remained as to whether this apparent homology reflected a common evolutionary origin. We demonstrate here that the ATP analogue p-fluorosulphonylbenzoyl 5'-adenosine (FSBA) inactivates the tyrosine protein kinase activity of p60src by reacting with lysine 295. When aligned for maximum sequence identity, lysine 295 of p60src and the lysine in the catalytic subunit which also reacts specifically with FSBA are superimposed precisely. This functional homology is strong evidence that the protein kinases, irrespective of amino acid substrate specificity, comprise a single divergent gene family.

Pp60 c-src kinase is in chick and human embryonic tissues

The normal cellular protein pp60 c-src is a tyrosine-specific protein kinase that is homologous to the transforming protein of Rous sarcoma virus (RSV) but its function is unknown. The expression of pp60 c-src in chick and human embryonic tissues was monitored by the immune complex protein kinase assay, Western transfer analysis, and immunocytochemical staining at the light microscope level. pp60 c-src kinase was expressed in the head and trunk regions of the chick embryo at all stages of development examined; however, expression increased significantly during the major period of organogenesis (Hamburger and Hamilton stages 21 to 32). Western transfer analysis showed that the amount of pp60 c-src protein increased in parallel with the increase in kinase activity. Highest levels of pp60 c-src kinase were present in the neural tube, brain, and heart of the stage 32 chick embryo. Lower levels of activity were found in eye, limb bud, and liver. Immunocytochemical staining of the neural tube region and heart of the chick confirmed the results of biochemical analysis and showed immunoreactive pp60 c-src distributed throughout the neural tube and heart. The distribution of pp60 c-src kinase in human fetal tissues was similar to that in the chick embryo; elevated levels of pp60 c-src kinase were present in cerebral cortex, spinal cord, and heart, but all other tissues examined expressed some pp60 c-src kinase. The results of our studies suggest that pp60 c-src plays a fundamental role in an aspect of cellular metabolism that is particularly important in electrogenic tissues.

The differential expression of the cellular src-gene product pp60 src and its phosphokinase activity in normal chicken cells and tissues

Chick embryos of different ages and adult chickens were examined for the expression of pp60 c-src , the normal cellular homolog of the transforming protein of Rous sarcoma virus. In any brain extract, the pp60 c-src kinase activity was always high, whereas muscle extracts of embryos show an age-dependent decrease in kinase activity. Adult animals show either no or barely measurable activity in muscle tissue. In contrast, liver cell extracts of embryos show an age-dependent increase in pp60 c-src kinase activity, with adult chickens displaying the highest activity, very similar to that found in brain extracts. This demonstrates that increased expression of c-src is not necessarily correlated with cell proliferation, but suggests that, at an early stage of differentiation of mesenchymal cells, the relatively high expression of c-src could be responsible, at least in part, for the control of cell metabolism and proliferation.

Analysis of the catalytic domain of phosphotransferase activity of two avian sarcoma virus-transforming proteins.

Proteolytic digestion of the transforming protein of Rous sarcoma virus (pp60src) with trypsin, chymotrypsin, or thermolysin generated a 29,000-dalton fragment representing the carboxyl half of this molecule. This proteolytic fragment was able to phosphorylate pp60src-specific immunoglobulin as well as exogenous substrates such as angiotensin, casein, and tubulin. When quantitated on a molar basis, the protease-resistant fragment of pp60src had a greater specific activity than the intact enzyme. Digestion of pp90yes, the transforming protein of Y73 sarcoma virus with these proteases yielded a peptide of similar molecular weight which was capable of autophosphorylation as well as the phosphorylation of exogenous substrates. The proteolytic fragments of both pp60src and pp90yes displayed the same strict specificity for phosphorylation of tyrosine as the intact enzymes. These results indicate that the 29,000-dalton carboxyl end of pp60src and pp90yes can function independently as phosphotransferases and indicate that the catalytic domains of these molecules have a conformation which confers protection against limited conditions of proteolysis.

Partial purification and characterization of a 60 000-dalton phosphoprotein from pig heart tissue.

A 60 000-dalton phosphoprotein (pp60) was purified up to 10(4)-fold by a combination of low-ionic-strength extraction, ammonium sulfate fractionation, on-exchange and affinity chromatography, all in detergent-free buffer. Fractionation on omega-aminohexylagarose column shows that pp60 actually consists of two different polypeptides of similar molecular mass (pp60 omega 1 and pp60 omega 2). Partial hydrolysis with proteases of the proteins 32P-labeled in vitro indicates that pp60 omega 1 and pp60 omega 2 are similar but not identical. On the other hand, individual phosphoamino acid analysis reveals that pp60 omega 1 is phosphorylated primarily at serine residues while pp60 omega 1 is phosphorylated almost equally at serine and threonine residues. Partial hydrolysis with proteases has been also used to explore a possible relationship between the pp60's and the transforming protein of Rous sarcoma virus (pp60v-src). Our data suggest that pp60v-src also consists of two different polypeptides chemically homologous to the presently purified pp60's.

It is Rous Sarcoma virus protein P12 and not P19 that binds tightly to Rous Sarcoma virus RNA

The interactions between Rous Sarcoma virus (RSV) RNA and the viral proteins in the virus have been analysed by Sen & Todaro (1977) using ultraviolet light irradiation; they showed that the major protein ultraviolet light cross-linked to the viral RNA was P19 as identified by polyacrylamide gel electrophoresis. We report here that it is not viral protein P19 but P12 that binds tightly to RSV RNA upon ultraviolet light irradiation of the virus. Therefore, the binding sites of the viral protein along RSV RNA that we have characterized previously should be correctly attributed now to P12 and not P19.

Enzymatic characteristics of pp60v-src isolated from vanadium-treated transformed cells.

The transforming protein of Rous sarcoma virus (RSV) typically appears as a single phosphorylated polypeptide designated pp60v-src. pp60v-src possesses a protein kinase activity specific for tyrosine residues on select protein substrates. Treatment of RSV-transformed cells with vanadium ions resulted in the appearance of an electrophoretic variant of pp60v-src and was paralleled by a significant increase in the src kinase specific activity in purified enzyme preparations. Both the normal (standard) src kinase and the src kinase preparations obtained from vanadium-treated cells exhibited similar optimal activity profiles for MgCl2, KCl, and pH. Furthermore, their site specificities of phosphorylation of the substrates casein and vinculin were the same. The reaction kinetic profile of the standard src kinase showed a nonlinear pattern, while the vanadium enzyme exhibited conventional linear Michaelis-Menten kinetics. These results are discussed with respect to the possible functional regulation of pp60v-src activity by a vanadium-sensitive protein phosphatase activity.

Differential inhibition of cellular and viral pp60src kinase by P1,P4-di(adenosine-5')tetraphosphate.

We contrasted the protein kinase activities of pp60v-src, the transforming protein of Rous sarcoma virus, and its normal cellular homolog pp60c-src with respect to inhibition by P1,P4-di(adenosine-5')tetraphosphate by using the immune complex protein kinase assay. The concentration of P1,P4-di(adenosine-5')tetraphosphate required for 50% inhibition of pp60v-src kinase (1 microM) was found to be significantly lower than that required for inhibition of pp60c-src kinase (46 microM). Viral and cellular pp60src kinases differed to a lesser extent with respect to inhibition by adenosine-5'-tetraphosphate, di(guanosine-5')tetraphosphate, and ADP. No significant differences were found in the ATP Km values of pp60v-src (0.108 +/- 0.048 microM) and pp60c-src kinases (0.056 +/- 0.012 microM). These results demonstrate that the protein kinase activities of viral and cellular pp60src are functionally distinguishable, particularly on the basis of enhanced sensitivity of the viral enzyme to inhibition by P1,P4-di(adenosine-5')tetraphosphate. These functional differences are likely to be due to differences in the conformation of the active site and may be important for determining transformation potential.

Differential Inhibition of Cellular and Viral pp60src Kinase by P1,P4-Di(Adenosine-5′)Tetraphosphate

We contrasted the protein kinase activities of pp60v-src, the transforming protein of Rous sarcoma virus, and its normal cellular homolog pp60c-src with respect to inhibition by P1,P4-di(adenosine-5')tetraphosphate by using the immune complex protein kinase assay. The concentration of P1,P4-di(adenosine-5')tetraphosphate required for 50% inhibition of pp60v-src kinase (1 microM) was found to be significantly lower than that required for inhibition of pp60c-src kinase (46 microM). Viral and cellular pp60src kinases differed to a lesser extent with respect to inhibition by adenosine-5'-tetraphosphate, di(guanosine-5')tetraphosphate, and ADP. No significant differences were found in the ATP Km values of pp60v-src (0.108 +/- 0.048 microM) and pp60c-src kinases (0.056 +/- 0.012 microM). These results demonstrate that the protein kinase activities of viral and cellular pp60src are functionally distinguishable, particularly on the basis of enhanced sensitivity of the viral enzyme to inhibition by P1,P4-di(adenosine-5')tetraphosphate. These functional differences are likely to be due to differences in the conformation of the active site and may be important for determining transformation potential.

Antibodies to a defined region of pp60src neutralize the tyrosine-specific kinase activity.

Site-specific antibodies to pp60src, the transforming protein of Rous sarcoma virus (RSV), have been prepared by immunizing rabbits with a chemically synthesized pentadecapeptide corresponding to residues 498-512 (Cys-Trp-Arg-Lys-Asp-Pro-Glu-Glu-Arg-Pro-Thr-Phe-Lys-Tyr-Leu) as deduced from the nucleotide sequence of the Prague C src gene. Antibodies specific for the synthetic peptide were purified from immune sera by affinity chromatography on peptide-bound Sepharose and characterized by a number of immunocytochemical techniques. Immunoprecipitation and Western blot analyses of normal and RSV-transformed cell lines revealed that this peptide antibody identified the authentic viral src gene product. This finding was further supported by indirect immunofluorescence on RSV-transformed rat kidney cells. The anti-peptide antibodies produced dramatic intracellular staining patterns characteristic of the src protein. Although able to immunoprecipitate pp60src, in vitro kinase reactions indicated that, unlike sera from RSV-induced tumor-bearing rabbits, the peptide antibody did not serve as a phosphate acceptor in the immunocomplex. Moreover, immunoprecipitates of pp60src prepared from this site-specific immune reagent were unable to phosphorylate exogenously added casein or the synthetic peptide substrate, Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly. In contrast, pp60src-containing immunoprecipitates made from an anti-peptide serum specific for the COOH-terminal six amino acids (residues 521-526), a region only eight amino acids removed, readily phosphorylated both substrates. This evidence indicates that an antibody directed against residues 498-512 neutralizes the kinase activity of pp60src and suggests that this region may be functionally necessary for the tyrosine-specific kinase activity of this transforming protein.

Transforming activity of polyoma virus middle-T antigen probed by site-directed mutagenesis.

The ability of polyoma virus to transform cells results primarily from the action of one of the virus-coded early proteins, called middle-T antigen. Middle-T has an associated tyrosine-specific protein kinase activity that can be measured in vitro and results in the phosphorylation of middle-T itself. Almost all mutants so far tested that lack the ability to transform cells, also lack associated kinase activity. Attempts to map within middle-T the tyrosine residue(s) that are phosphorylated in vitro suggest that a likely site of phosphorylation is tyrosine 315 (refs 8-10 and unpublished results). The amino acid sequence preceding Tyr 315 includes a tract of six contiguous glutamic acid residues and bears some homology with that preceding the tyrosine phosphorylated in vivo in pp60v-src, the transforming protein of Rous sarcoma virus, and with a region in the polypeptide hormone, gastrin, preceding a tyrosine that is sulphated. Furthermore, although surprisingly large tracts of middle-T may be removed without affecting its transforming activity, mutants that lack the sequences corresponding to amino acids 311-318 inclusive are transformation defective. Because the likely site of phosphorylation, the homology with pp60v-src and gastrin and the sequence apparently required for transformation all overlap, it has generally been accepted that this region of middle-T may form part of an essential region, possibly an active site on the protein. Here we have used techniques of site-directed and site-specific mutagenesis to probe the sequence requirements in more detail. Contrary to expectation, the results obtained strongly suggest that Tyr 315 and conservation of the surrounding amino acid sequence are not essential for transformation.