Tyrosine phosphorylation of a 50K cellular polypeptide associated with the Rous sarcoma virus transforming protein pp60src.

We have examined the phosphorylation of a 50,000-dalton cellular polypeptide associated with the Rous sarcoma virus (FSV) transforming protein pp60-src. It has been shown that pp60src forms a complex with two cellular polypeptides, an 89,000-dalton heat-shock protein (89K) and a 50,000-dalton phosphoprotein (50K). The pp60src-associated protein kinase activity phosphorylates at tyrosine residues, and the 50K polypeptide present in the complex contains phosphotyrosine and phosphoserine. These observations suggest that the 50K polypeptide may be a substrate for the protein kinase activity of pp60src. To examine this possibility, we isolated the 50K polypeptide by two-dimensional polyacrylamide gel electrophoresis from lysates of uninfected or virally infected cells. Tryptic phosphopeptide analysis indicated that the 50K polypeptide isolated by this method was the same polypeptide as that complexed to pp60src. In uninfected cells or cells infected by a transformation-defective mutant, the 50K polypeptide contained phosphoserine but little or no phosphotyrosine. In cells infected by Schmidt-Ruppin or Prague RSV, there was a 40- to 50-fold increase in the quantity of phosphotyrosine in the 50K protein. Thus, the phosphorylation of the 50K polypeptide at tyrosine is dependent on the presence of pp60src. However, the 50K polypeptide isolated from cells infected by temperature-sensitive mutants of RSV was found to be phosphorylated at tyrosine at both permissive and nonpermissive temperatures; this behavior is different from that of other substrates or putative substrates of the pp60src kinase activity. It is possible that the 50K polypeptide is a high-affinity substrate of pp60src.     

Evidence that the phosphorylation of tyrosine is essential for cellular transformation by Rous sarcoma virus

All cells transformed by Rous sarcoma virus contain levels of phosphotyrosine in protein which are 6–10 fold greater than the very low levels present in uninfected cells. The increase is due largely to modification of cellular polypeptides. The abundance of phosphorylated tyrosines in protein in cells infected with tsLA29, a mutant of Rous sarcoma virus which is temperature-sensitive for cellular transformation, increases to 60% of maximum within 60 min of a shift to the permissive temperature and drops to a level close to that in uninfected cells within 60 min of a shift to the restrictive temperature. In light of the fact that pp60^src phosphorylates tyrosine in vitro, these results suggest strongly that the modification of one or more cellular polypeptides by way of pp60^src is critical for cellular transformation by Rous sarcoma virus. There is, however, no increase in the abundance of phosphotyrosine in protein in mouse cells transformed by Kirsten sarcoma virus, Moloney sarcoma virus, or SV40 virus, in chick embryo cells infected with avian myelocytomatosis virus MC29, and in rat and hamster cells transformed by polyoma virus. Thus increased phosphorylation of tyrosine is neither a universal mechanism of transformation nor an inevitable secondary cellular response to transformation.     

Characterization of a normal avian cell protein related to the avian sarcoma virus transforming gene product.

In this paper, we identify and characterize both structurally and functionally a protein from normal uninfected avian cells that is antigenically related to the pp60^src viral protein responsible for transformation by ASV. This protein was detected by immunoprecipitation of radiolabeled normal cell extracts with serum derived from marmosets bearing ASV-induced tumors. The normal avian cell protein, which has been detected in each of the four avian species tested (chicken, duck, quail and pheasant) is a phosphoprotein of 60,000 daltons. This protein is not related to any of the ASV structural proteins; however, its immunoprecipitation is prevented by preadsorption of the antiserum with cell extracts specifically containing pp60^src. Peptide analyses by partial proteolysis using chymotrypsin resulted in a map of the normal cell protein that was very similar to that of pp60^src. When Staphylococcus aureus V8 protease was used, however, one of the major cleavage products of the normal cell protein exhibited an altered migration with respect to the corresponding pp60^src product. Tryptic phosphopeptide analyses demonstrated that phosphorylation of the normal cell protein was also different from that seen in pp60^src. The expression of the normal cell protein did not seem to be affected by cellular growth conditions, maintaining a constant level which was approximately 30–50 fold lower than that of pp60^src in infected cells. The normal cell protein appeared to be functionally dissimilar to pp60^src lacking detectable protein kinase activity in the currently available assay system.     

Highly specific antibody to Rous sarcoma virus src gene product recognizes a novel population of pp60v-src and pp60c-src molecules

Antiserum to the Rous sarcoma virus (RSV)-transforming protein, pp60v-src, was produced in rabbits immunized with p60 expressed in Escherichia coli. alpha p60 serum immunoprecipitated quantitatively more pp60v-src than did tumor-bearing rabbit (TBR) sera. When RSV-transformed cell lysates were preadsorbed with TBR serum, the remaining lysate contained additional pp60v-src, which was recognized only by reimmunoprecipitation with alpha p60 serum and not by TBR serum. In subcellular fractions of RSV-infected chicken embryo fibroblasts (RSV-CEFs) and field vole cells probed with TBR serum, the majority of the pp60v-src was associated with the plasma membrane-enriched P100 fraction. However, alpha p60 serum revealed equal distribution of pp60v-src and its kinase activity between the P1 (nuclear) and P100 fractions. The same results were obtained for pp60c-src in uninfected CEFs. On discontinuous sucrose gradients nearly 50% of the P1-pp60v-src sedimented with nuclei, in fractions where no plasma membrane was detected. Indirect immunofluorescence microscopy of RSV-CEFs with alpha p60 serum revealed a distinct pattern of perinuclear fluorescence, in addition to staining at the cell periphery. Thus the use of a highly specific antibody reveals that enzymatically active pp60v-src and pp60c-src molecules are present in other intracellular structures, probably juxtareticular nuclear membranes, in addition to the plasma membrane in normal, uninfected, and wild-type RSV-infected cells.     

Cytoskeletal association of pp60 : The transforming protein of the rous sarcoma virus

Immunoferritin labelling methods have been employed to examine the distribution of the Rous Sarcoma virus (RSV)-transforming protein pp60^src in the detergent-resistant cytoskeleton of transformed cells. pp60^src was found to be localized on actin microfilaments present in adhesion plaques, at adherens junctions between cells and also in microfilament bundles. This localization is consistent with the hypothesis that some of the morphological effects of transformation result from the interaction in situ of pp60^src with microfilament-bound target proteins.     

Transformation by subgenomic fragments of Rous sarcoma virus DNA

Subgenomic fragments of Rous sarcoma virus (RSV) DNA, generated by Eco RI digestion of DNA of RSV-infected chicken cells, induced transformation of NIH/3T3 mouse cells with efficiencies that were 100–1000 fold lower than the efficiency of transformation by intact RSV DNA. Analysis of the DNAs of NIH cells transformed by Eco RI-digested RSV DNA indicated that these cells contained no more than 2 × 10⁶ daltons of RSV DNA, and did not contain sequences from the 5′ terminus of RSV RNA which are included in the leader sequence of subgenomic src mRNA of RSV-infected cells. The product of the RSV src gene (pp60^src), however, was produced in apparently similar quantities by NIH cells transformed by Eco RI fragments of RSV DNA and by intact RSV DNA. Thus expression of the src gene of RSV in NIH cells transformed by subgenomic fragments of RSV DNA did not require the terminal sequences of the RSV genome, which appear to be involved in synthesis and processing of src mRNA in RSV-infected cells. DNAs of NIH cells transformed by Eco RI-digested RSV DNA were found to induce transformation in secondary transfection assays with efficiencies that were similar to the efficiency of transformation by intact RSV DNA. These results suggest that transformation by subgenomic fragments of RSV DNA may be a consequence of integration of src gene-containing DNA fragments in the vicinity of a promoter site in the recipient cell genome, leading to efficient expression of the RSV src gene.     

Phosphorylations of the subcellular matrix in cells transformed by Rous' sarcoma virus

The transforming protein of Rous' sarcoma virus (RSV) is a phosphoprotein of Mr 60 000 (pp60src) which displays protein kinase activity specific for tyrosine residues; pp60src is associated with the plasma membrane and is recovered in the detergent-insoluble material which represents the subcellular matrix of the cell. After phosphorylation of this material of RSV-transformed cells with [gamma-32P]ATP, five phosphoproteins have been detected which are not seen in normal cells. These proteins (Mr = 135 000, 125 000, 75 000, 70 000, 60 000) contain phosphotyrosine. Their phosphorylation is strongly inhibited by anti-pp60src antibodies. In cells transformed by a temperature-sensitive mutant of RSV, these phosphoproteins, present at the permissive temperature, are no longer detected at the non-permissive temperature. It is concluded that these phosphorylations are mediated by pp60src protein kinase activity. This supports a possible role of the phosphorylation of cytoskeletal proteins in the transformation process.     

Pyruvate kinase type M2 is phosphorylated at tyrosine residues in cells transformed by Rous sarcoma virus

Chicken embryo cells (CECs) contain pyruvate kinase (PK) type M2 (M2-PK). Transformation of CECs by Rous sarcoma virus (RSV) leads to a reduction in the affinity of PK for the substrate phosphoenolpyruvate. In vitro, M2-PK can be phosphorylated at tyrosine residues by pp60v-src, the transforming protein of RSV. To study tyrosine phosphorylation of M2-PK in intact RSV-transformed cells, the protein was immunoprecipitated from 32P-labeled normal and RSV-SR-A-transformed CECs. Phosphoamino acid analysis of immunoprecipitated M2-PK revealed that M2-PK of both normal and transformed CECs contained phosphoserine and small amounts of phosphothreonine. Only M2-PK of transformed CECs contained phosphotyrosine in addition. For enzyme kinetic studies M2-PK was partially purified by chromatography upon DEAE-Sephacel and hydroxyapatite. A decreased affinity for phosphoenolpyruvate was observed 3 h after the onset of transformation using the temperature-sensitive mutant of RSV, ts-NY 68. The kinetic changes were correlated with tyrosine phosphorylation of M2-PK, but there is no direct evidence that they are caused by post-translational modification of the enzyme.     

The use of Rous sarcoma virus transformation mutants with differing tyrosine kinase activities to study the relationships between vinculin phosphorylation, pp60v-src location and adhesion plaque integrity

Tyrosine-specific phosphorylation of cellular proteins has been implicated in the neoplastic transformation of cells by Rous sarcoma virus (RSV). One of the putative substrates for the src gene product (pp60v-src) of RSV is the cytoskeletal protein vinculin, giving rise to the hypothesis that tyrosine-specific phosphorylation of vinculin disrupts adhesion plaque integrity, leading to the characteristic rounded morphology of RSV-transformed cells. We have investigated this hypothesis by analysing the properties of fibroblasts transformed by conditional and non-conditional mutants of RSV which confer different morphologies on infected cells, with respect to formation of microfilament bundles, formation of vinculin-containing adhesion plaques, the deposition of a fibronectin-containing extracellular matrix, the localization of pp60v-src and the tyrosine-specific phosphorylation of vinculin. Cells transformed by the temperature-sensitive (ts) RSV mutant LA32 cultured at 41 degrees C were morphologically normal, and contained prominent microfilament bundles and well-developed adhesion plaques. However, these cells had a fully active pp60v-src kinase, had pp60v-src concentrated in their adhesion plaques and contained vinculin which was heavily phosphorylated on tyrosine residues. Cells transformed by a recovered avian sarcoma virus, rASV 2234.3 exhibited a markedly fusiform morphology with pp60v-src concentrated in well-developed adhesion plaques and an elevation of the phosphotyrosine content of vinculin. Cells transformed by LA32 at restrictive temperature comprise morphologically normal cells, indistinguishable from untransformed CEF, yet which contain tyrosine-phosphorylated vinculin and suggest that neither tyrosine-specific phosphorylation of vinculin nor pp60v-src concentration in adhesion plaques is sufficient for the rounded morphology of RSV-transformed cells.     

Ecto-protein kinase activities in normal and transformed cells

The incubation of intact uninfected and Rous sarcoma virus (RSV)-transformed chicken cells (SR-RSV-A) with micromolar amounts of [gamma-32P]ATP under physiological conditions resulted in the radioactive phosphorylation of a variety of proteins. According to the experimental protocol the detectable phosphorylation was restricted to ATP utilization at the cell surface and was catalyzed by surface located protein kinase (PK). Serine- and to a lesser extent, threonine residues were phosphorylated. With respect to this enzyme the cells under investigation showed upon incubation with phosvitin the release of surface (phosvitin) kinase into the incubation medium. Based on immunochemical analysis and PK-assays using antisera from RSV-tumor bearing rabbits (TBR-serum) the pp60v-src with its associated tyrosine kinase activity was likewise detected in appreciable amounts at the outside of RSV-transformed chicken and mammalian cells. There was no cross reactivity of TBR-serum with phosvitin kinase. Phosvitin was not phosphorylated by the immunoprecipitated pp60v-src. Whereas phosphorylation catalyzed by pp60v-src was blocked with 10 to 20 microM diadenosine 5',5'-P1P4 tetraphosphate (Ap4A) the phosvitin phosphorylation was far less sensitive towards inhibition by Ap4A, similar to the cellular pp60c-src kinase activity in uninfected cells. The functional significance of the PK activities in uninfected and RSV-transformed cells observed at their surface or in cell-free form as well as the nature of their substrates remain to be established.     

Rous sarcoma virus-induced changes in the pattern of phosphorylation of non-histone nuclear proteins

We here studied the protein kinase activity and in vitro phosphorylable sites of non-histone nuclear proteins, 0.4 M NaCl extracts (mostly chromosomal proteins) from chick embryo fibroblasts (CEF), infected or not with a Schmidt Ruppin strain subgroup A of Rous sarcoma virus (RSV).The infection and transformation of chick fibroblasts by RSV induced an increase in kinase activity and endogenous phosphorylation of non-histone chromosomal (NHC) proteins. The stimulation, by a change of medium, of the proliferation of dense cultures of normal chick fibroblasts also induced an increase in the kinase activity and endogenous phosphorylation of NHC proteins.However, two-dimensional gel electrophoresis of the ³²P-phosphorylated proteins showed that stimulation due to a change of medium and that due to the expression of transformation were very different. The stimulation by a change of medium increased to a greater or lesser extent the phosphorylation of the different NHC proteins, with no fundamental variations in the pattern of protein phosphorylation. In contrast, RSV infection induced significant changes in the pattern of protein phosphorylation. One of the most striking feature was the large increase of amount and phosphorylation of high molecular weight (HMW) proteins in particular of phosphoproteins having an evaluated molecular weight (MW) of 78 K and 82 K and pI>8.2.The percent of phosphotyrosine residues in NHC proteins was clearly increased when the proteins were extracted from transformed cells instead of normal cells. But the alkaline treatment of two-dimensional gel electrophoresis indicated that the 80 K phosphoproteins did not contain phosphotyrosine residues, and thus cannot be considered as substrates for pp60^src kinase.     

Detection of phosphotyrosine-containing proteins in polyomavirus middle tumor antigen-transformed cells after treatment with a phosphotyrosine phosphatase inhibitor.

Cells transformed with the middle tumor antigen (mT) of polyomavirus were treated with sodium orthovanadate (Na3VO4), an inhibitor of phosphotyrosine phosphatases, to enhance for the detection of cellular proteins which are phosphorylated on tyrosine. Na3VO4 treatment of mT-transformed rat F1-11 cells resulted in a 16-fold elevation in the level of phosphotyrosine associated with total cellular proteins. Parental F1-11 cells displayed only a twofold increase in phosphotyrosine following Na3VO4 treatment. The abundance of phosphotyrosine in Na3VO4-treated mT-transformed F1-11 cells was twofold higher than in untreated Rous sarcoma virus (RSV)-transformed F1-11 cells and 3.5-fold lower than in Na3VO4-treated RSV-transformed F1-11 cells. Tyrosine phosphorylation of many cellular proteins, including p36, the major substrate of the RSV pp60v-src protein, was detected in Na3VO4-treated mT-transformed F1-11 cells at levels comparable to those observed in RSV-transformed cells. Some of the major protein species recognized by antiphosphotyrosine antibodies in Na3VO4-treated mT-transformed cells displayed electrophoretic mobilities similar to those detected in RSV-transformed F1-11 cells. Tyrosine phosphorylation of p36 was also detected in fibroblasts infected with polyomavirus. There was no detectable difference in the kinase activity of pp60c-src:mT extracted from untreated and Na3VO4-treated mT-transformed cells; however, Na3VO4 treatment of F1-11 and mT-transformed F1-11 cells was shown to inhibit the activity of phosphotyrosine phosphatases in a crude assay of total cellular activity with pp60v-src as the substrate. Thus, Na3VO4 treatment may allow the detection of phosphotyrosine-containing proteins in mT-transformed cells by preventing the turnover of phosphate on substrates phosphorylated by activated cellular protein-tyrosine kinases associated with mT. These results suggest that tyrosine phosphorylation of cellular proteins may be involved in the events that are responsible for mT-induced cellular transformation.     

Changes in amino-terminal sequences of pp60src lead to decreased membrane association and decreased in vivo tumorigenicity

We have suggested previously that the amino-terminal 8 kilodaltons of pp60^src may serve as a structural hydrophobic domain through which pp60^src attaches to plasma membranes. Two isolates of recovered avian sarcoma viruses (rASVs), 1702 and 157, encode pp60^src proteins that have alterations in this amino-terminal region. The rASV 1702 src protein (56 kilodaltons) and the 157 src protein (62.5 kilodaltons) show altered membrane association, and fractionate largely as soluble, cytoplasmic proteins in aqueous buffers, in contrast with the membrane association of more than 80% of the src protein of standard avian sarcoma virus under the identical fractionation procedure. Plasma membranes purified from cells transformed by these rASVs contain less than 10% of the amount of pp60^src found in membranes purified from cells transformed by Rous sarcoma virus or control rASVs. The altered membrane association of these src proteins had little or no effect on the properties of chick embryo fibroblasts transformed in monolayer culture. In contrast, rASV 1702 showed reduced in vivo tumorigenicity compared with Rous sarcoma virus or with other rASVs that encode membrane-associated src proteins. Rous sarcoma virus-induced tumors are malignant, poorly differentiated sarcomas that are lethal to their hosts. rASV 1702 induces a benign, differentiated sarcoma that regresses and is not lethal to its hosts. These data support the role of amino-terminal sequences in the membrane association of pp60^src, and suggest that the amino terminus of pp60^src may have a critical role in the promotion of in vivo tumorigenicity.     

Detection of phosphotyrosine-containing proteins in the detergent-insoluble fraction of RSV-transformed fibroblasts by azobenzene phosphonate antibodies.

The Rous sarcoma virus (RSV) oncogene product pp60src is known to trigger the acquisition of the transformed phenotype by phosphorylating host cell target molecule(s) at tyrosine residues. To identify phosphotyrosine-containing proteins, rabbit antibodies were raised against the synthetic hapten p-azobenzene-phosphonate (ABP) that specifically cross-reacts with phosphorylated tyrosine. By immuno-decoration of proteins extracted from RSV-transformed mouse fibroblasts and transferred to nitrocellulose sheets, phosphoproteins of 130, 70 and 60 kd were identified. These molecules were found to be associated with the cellular fraction insoluble in non-ionic detergent. Moreover, ABP antibodies precipitated detergent-insoluble proteins of 130, 70 and 60 kd, plus two additional components of 85 and 65 kd, that had been phosphorylated in vitro by [gamma-32P]ATP under conditions allowing the kinase reaction catalyzed by pp60src. Phosphoproteins of closely related mol. wts. were immunoprecipitated from RSV-transformed avian fibroblasts. The radioactivity co-migrated with authentic phosphotyrosine in two-dimensional chromatography. The 60-kd protein comigrated with pp60src, while the identity between the 130-kd protein and vinculin was disproved by the lack of cross-reaction with appropriate antisera. In transformed mouse and duck fibroblasts ABP antibodies, employed in indirect immunofluorescence microscopy, stained diffusely the cytoplasm and intensely decorated restricted areas of the ventral cell plasma membrane. These data show that antibodies reacting with phosphotyrosine may be usefully employed in the identification and in the intracellular localization of molecules that are potential targets of the pp60src protein kinase.     

Transformation by Rous sarcoma virus: a cellular substrate for transformation-specific protein phosphorylation contains phosphotyrosine

Transformation of chicken embryo fibroblasts by Rous sarcoma virus (RSV) is caused by a single viral gene, src, which encodes a phosphoprotein, pp60src, with the enzymatic activity of a protein kinase. The relative abundance of a 36,000 molecular weight (36K) phosphorylated polypeptide which can be detected by two-dimensional electrophoresis of 32P-labeled phosphoproteins is greatly increased in RSV-transformed fibroblasts. We have reported previously that phosphorylation of the 36K polypeptide is an early event in the process of transformation and that protein synthesis is not required for its appearance. Here we identify a nonphosphorylated 36K polypeptide, present in both uninfected and transformed cells, which is homologous to the 36K phosphoprotein as judged by limited proteolysis and by tryptic peptide mapping. We conclude that the 36K phosphoprotein is generated by phosphorylation of this 36K polypeptide. It has recently been shown that pp60src phosphorylates tyrosine residues in vitro: phosphotyrosine and also phosphoserine are present in the 36K phosphoprotein isolated from RSV-transformed cells. On the basis of these results we propose that the 36K polypeptide present in chicken fibroblasts is a substrate for the protein kinase activity of pp60src. Phosphorylation of this polypeptide may be important in cellular transformation by Rous sarcoma virus.     

Phosphorylation of connexin43 gap junction protein in uninfected and Rous sarcoma virus-transformed mammalian fibroblasts.

Gap junctions are membrane channels that permit the interchange of ions and other low-molecular-weight molecules between adjacent cells. Rous sarcoma virus (RSV)-induced transformation is marked by an early and profound disruption of gap-junctional communication, suggesting that these membrane structures may serve as sites of pp60v-src action. We have begun an investigation of this possibility by identifying and characterizing putative proteins involved in junctional communication in fibroblasts, the major cell type currently used to study RSV-induced transformation. We found that uninfected mammalian fibroblasts do not appear to contain RNA or protein related to connexin32, the major rat liver gap junction protein. In contrast, vole and mouse fibroblasts contained a homologous 3.0-kilobase RNA similar in size to the heart tissue RNA encoding the gap junction protein, connexin43. Anti-connexin43 peptide antisera specifically reacted with three proteins of approximately 43, 45 and 47 kilodaltons (kDa) from communicating fibroblasts. Gap junctions of heart cells contained predominantly 45- and 47-kDa species similar to those found in fibroblasts. Uninfected fibroblast 45- and 47-kDa proteins were phosphorylated on serine residues. Phosphatase digestions of 45- and 47-kDa proteins and pulse-chase labeling studies indicated that these proteins represented phosphorylated forms of the 43-kDa protein. Phosphorylation of connexin protein appeared to occur shortly after synthesis, followed by an equally rapid dephosphorylation. In comparison with these results, connexin43 protein in RSV-transformed fibroblasts contained both phosphotyrosine and phosphoserine. Thus, the presence of phosphotyrosine in connexin43 correlates with the loss of gap-junctional communication observed in RSV-transformed fibroblasts.     

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

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.     

Elevated phosphatidylinositol kinase activity in Rous sarcoma virus-transformed cells. Lack of evidence for enzyme translocation

Phosphatidylinositol kinase (E.C. 2.7.1.67) activity of rat fibroblasts transformed by Rous sarcoma virus (RSV) was measured and compared with immunoprecipitated protein tyrosine kinase activity associated with pp60v-src. Both enzyme activities were elevated in the particulate fractions from wild-type RSV-transformed cells and cells transformed by a temperature-sensitive mutant of RSV when grown at the permissive temperature. The presence of the non-ionic detergent Nonidet P-40 in the phosphatidylinositol kinase assays stimulated the soluble and particulate forms of the enzyme to different degrees but did not affect the relative differences between transformed and untransformed cells. Our results indicate that phosphatidylinositol kinase activity is a good correlate of RSV transformation and suggest a functional relationship between pp60v-src and phosphatidylinositol kinase.     

In vitro association and phosphorylation of polyoma virus middle T antigen by cellular tyrosyl kinase activity

We have observed increased phosphorylation of tyrosine residues on the polyoma virus middle tumor antigen (MTAg) in in vitro kinase assays of the immune complexes immunoprecipitated from lysates of polyoma virus-infected mouse embryo cells to which increasing amounts of uninfected mouse embryo cell lysate had been added. The components from uninfected mouse cells responsible for increased MTAg phosphorylation were localized by subcellular fractionation to the plasma membrane and found to be sensitive to protease digestion, N-ethylmaleimide, and 5'-p-fluorosulfonylbenzoyladenosine inactivation. The majority of the membrane-associated activity responsible for the increased MTAg phosphorylation in these assays could be cleared from lysates of uninfected mouse cell lysates by centrifugation after reaction with Sepharose-bound monoclonal antibodies which recognize pp60c-src. These results suggest that MTAg can associate with cellular tyrosyl kinases in vitro and be phosphorylated by these enzymes in immune-complex kinase assays. The identity of at least one of these cellular tryosyl kinases which can associate with MTAg in vitro is likely to be pp60c-src.