Interaction between the Rous sarcoma virus transforming protein and two cellular phosphoproteins: analysis of the turnover and distribution of this complex.

The transforming protein of Rous sarcoma virus (RSV), pp60src, was previously shown to associate with two cellular proteins of Mr 90,000 and 50,000 in RSV-transformed chicken cells. In this report, we demonstrate that this interaction is specific for a discrete population of pp60src molecules. Newly synthesized pp60src was found to preferentially associate with pp90 and pp50 to form a short-lived complex. The half-life of this complex varied from 9 to 15 min in cells transformed by nondefective strains of RSV. This interaction between pp60src, pp50, and pp90 took place in a soluble fraction of the cell, and the complex-bound pp60src molecules were not phosphorylated on tyrosine. These results suggest that pp90 and pp50 may be involved in the processing of pp60src molecules before the association of pp60src with the plasma membrane. The kinetics of dissociation of this complex were shown to be altered in cells infected with viruses containing a temperature-sensitive defect in the src gene. When cells infected with these viruses were grown at the nonpermissive temperature, more than 90% of the pp60src molecules were associated with pp90 and pp50, and little or no dissociation was observed in a 3-h chase period. These results suggest that mutations in the src gene which affect the transforming activity of pp60src also affect the stability of the interaction of pp60src with pp90 and pp50.     

Association of pp60src and src protein kinase activity with the plasma membrane of nonpermissive and permissive avian sarcoma virus-infected cells.

The intracellular localization of pp60src and src protein kinase activity in avian sarcoma virus (ASV)-infected chicken embryo fibroblasts and transformed and morphologically reverted field vole cells was examined by subcellular fractionation procedures. Fractionation by differential centrifugation of Dounce-homogenized cellular extracts prepared from vole cells showed that 83 to 91% of pp60src sedimented with particulate subcellular components from both transformed and revertant vole cells. A slightly lesser amount (60 to 70%) of pp60src was found associated with the particulate fraction from ASV-infected chicken embryo fibroblasts. The distribution of src protein kinase activity in the cytosol and particulate cell fractions was identical to that of pp60src, indicating no detectable differences in the activity of cytosol- and particulate-associated pp60src. When subcellular components of the cell were fractionated by discontinuous sucrose gradient centrifugation, similar amounts of both pp60src and src protein kinase activity cosedimented with the plasma membrane fractions from both transformed and revertant vole cells, as well as from ASV-infected chicken embryo fibroblasts. src protein kinase activity associated with plasma membrane fractions prepared from vole cells and ASV-infected chicken embryo fibroblasts was resistant to extraction with high salt concentrations, but partial elution was achieved with nonionic detergent. Thus, in both transformed and morphologically reverted vole cells, pp60src is intimately associated with the plasma membrane. Since transforming virus can be rescued from revertant vole cells by fusion to chicken embryo fibroblasts, revertant vole cell pp60src is capable of inducing morphological transformation. Thus, although the data presented herein suggest that transformation requires the association of pp60src with the plasma membrane, the binding of pp60src to the plasma membrane per se is insufficient to induce morphological transformation and requires the additional interaction with a specific target membrane protein which appears to be defective in revertant vole cells.     

Isolation and partial characterization of a monoclonal antibody to the Rous sarcoma virus transforming protein pp60src.

Transformation of cells by Rous sarcoma virus is mediated by the product of the viral src gene, pp60src. A hybridoma cell line producing an immunoglobulin G3 antibody to pp60src was isolated after lymph node cells from immune mice were fused with mouse myeloma cells (P3-NS1-1). Mice were immunized with p60src purified from Escherichia coli cells expressing the src gene product. The monoclonal antibody immunoprecipitated pp60src from Rous sarcoma virus-transformed cells and recognized an antigenic determinant located in the amino-terminal third of the pp60src protein.     

Loss of tumorigenicity correlates with a reduction in pp60src kinase activity in a revertant subclone of avian sarcoma virus-infected field vole cells

We have recently isolated an interesting revertant subclone (revertant 866-4) of ESV-infected field vole cells that is indistinguishable from uninfected vole cells with respect to its lack of transformed cell properties. These revertants are not only normal morphologically, but they do not grow in soft agar and are nontumorigenic in athymic nude mice. Despite this lack of transformed cell properties, we have found that this cell line still contains pp60src at concentrations (0.30 microgram/mg cell protein) similar to those (0.13-0.42 microgram/mg cell protein) found in transformed and morphologically reverted, but tumorigenic vole cells (partial revertants). However, the most interesting aspect of this newly isolated subclone is the marked reduction in its pp60src kinase activity (2--3%) when compared with the specific activity of pp60src immunoprecipitated from transformed and partially revertant vole cell lines. Since the reduction in pp60src kinase activity strongly correlates with the loss of tumorigenicity in this particular revertant cell line, these data support the contention that this enzymatic activity is a crucial factor in the tumorigenic conversion of cells by avian sarcoma virus. Proteolytic peptide analysis of the structure of pp60src from revertant 866-4 indicates that it is similar to pp60src obtained from avian sarcoma virus-transformed chick embryo fibroblasts. Moreover, the reduction in kinase activity does not appear to be due to a lack of phosphorylation of the tyrosine residue in pp60src. Thus neither an obvious structural alteration nor a reduction in phosphorylation of pp60src appears responsible for the reduced kinase activity observed, suggesting that some as of yet undetermined feature of pp60src can influence the pp60src phosphorylating event.     

Half-life of the Rous sarcoma virus transforming protein pp60src and its associated kinase activity.

The half-life of metabolically labeled pp60src of the Prague A strain of Rous sarcoma virus and of several transformation-defective, temperature-sensitive mutants was investigated by pulse-labeling infected cells with [35S]methionine, chasing for different times, and immunoprecipitating pp60src with tumor-bearing rabbit serum. These experiments showed that pp60src has a short half-life of approximately 60 min under normal physiological conditions and that the mutant pp60src proteins have similar half-lives to the wild type, irrespective of whether the cells are kept at the nonpermissive (42 degrees C) or permissive (35 degrees C) temperature. The half-life of the pp60src -associated kinase activity was determined by monitoring its decay by the immunoglobulin G heavy chain assay after the cells had been treated with several inhibitors of protein synthesis. In these experiments the kinase half-life was much longer than expected from the half-life of pp60src. The apparent contradiction between the half-lives of the kinase activity and the [35S]methionine-labeled pp60src protein could be resolved by the observation that treatment of cells with inhibitors of protein synthesis stabilized pp60src, resulting in a greatly extended half-life. Inhibitors of protein synthesis also extended the half-life of the gag precursor polypeptide, Pr76, suggesting that a host factor(s) may be required for the efficient intracellular processing of this polypeptide to the gag proteins.     

Isolation of monoclonal antibodies that recognize the transforming proteins of avian sarcoma viruses.

Thirteen clones of hybrid cells which synthesize antibodies directed against the Rous sarcoma virus (RSV) transforming protein, pp60src, were isolated. Mouse myeloma cells were fused with spleen cells from mice that had been immunized with purified pp60src from bacterial recombinants which direct the synthesis of the RSV src gene. The hybridomas which survived the selection medium were screened by immunoprecipitation of pp60src from 32P-labeled lysates of RSV-transformed cells. Monoclonal antibodies produced by subclones derived from 13 hybridomas recognized pp60src encoded by the Schmidt-Ruppin and Prague strains of RSV and the cellular homolog of pp60src. Antibody from clone 261 had a high affinity for the viral yes gene product, and antibodies from clones 443 and 463 recognized the transforming proteins encoded by viruses containing the related transforming genes fps and ros. Several other clones had a low affinity for the viral yes, fps, and ros gene products which could be detected by in vitro phosphorylation of the transforming proteins after immunoprecipitation with the monoclonal antibody. All of the monoclonal antibodies allowed phosphorylation of pp60src and casein in an immune complex-bound reaction.     

Demonstration of avian sarcoma virus-coded pp60src in vivo and the anti-pp60src immune response in chickens.

The avian sarcoma virus (ASV)-coded transforming protein pp60src was originally detected in vitro in ASV-transformed avian and mammalian cells in experiments involving mammalian antisera to ASV-induced tumors. It is demonstrated here that pp60src is also expressed in vivo in ASV tumors of chickens. Furthermore, the existence of the endogenous pp60src in all chicken cells does not impair the immune response to exogenous pp60src in the chicken. Whereas chicken antibodies can bind to pp60src, they do not serve as substrates for the protein kinase activity of this transforming protein.     

Immune response to the src gene product in mice bearing tumors induced by injection of avian sarcoma virus-transformed mouse cells.

A single subcutaneous injection of 10(7) live cells of the highly tumorigenic avian sarcoma virus (Schmidt-Ruppin strain, subgroup D)-transformed BALB/c line into BALB/c mice resulted in the production of an antiserum specific for the avian sarcoma virus gene product pp60src. All sera taken from mice 3 weeks after injection of tumor cells contained antibodies to pp60src. Immunoprecipitation experiments showed that all sera precipitated pp60src from Schmidt-Ruppin-infected chicken cells, but only a portion of these sera precipitated pp60src from chicken cells infected with other strains of avian sarcoma virus, i.e., Prague and Bratislava-77. Analysis of the cross-reactivity patterns of these antisera demonstrated a minimum of three to four antigenic determinants on pp60src. The findings reported here should facilitate the production of monoclonal antibodies to pp60src, which in turn will provide highly specific probes for further investigations into the structure and function of this protein.     

Early activation of endogenous pp60src kinase activity during neuronal differentiation of cultured human neuroblastoma cells.

The proto-oncogene product pp60c-src is a tyrosine-specific kinase with a still unresolved cellular function. High levels of pp60c-src in neurons and the existence of a neuronal pp60c-src variant, pp60c-srcN, suggest participation in the progress or maintenance of the differentiated phenotype of neurons. We have previously reported that phorbol esters, e.g., 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulate human SH-SY5Y neuroblastoma cells to neuronal differentiation, as monitored by morphological, biochemical, and functional differentiation markers. In this report, we describe activation of the pp60src (pp60c-src and pp60c-srcN) kinase activity observed at 6 h after induction of SH-SY5Y cells with TPA. This phenomenon coincides in time with neurite outgrowth, formation of growth cone-like structures, and an increase of GAP43 mRNA expression, which are the earliest indications of neuronal differentiation in these cells. The highest specific src kinase activity (a three- to fourfold increase 4 days after induction) was noted in cells treated with 16 nM TPA; this concentration is optimal for development of the TPA-induced neuronal phenotype. During differentiation, there was no alteration in the 1:1 ratio of pp60c-src to pp60c-srcN found in untreated SH-SY5Y cells. V8 protease and trypsin phosphopeptide mapping of pp60src from in vivo 32P-labeled cells showed that the overall phosphorylation of pp60src was higher in differentiated than in untreated cells, mainly because of an intense serine 12 phosphorylation. Tyrosine 416 phosphorylation was not detectable in either cell type, and no change during differentiation in tyrosine 527 phosphorylation was observed.     

Towards a molecular description of cell transformation by avian sarcoma virus

The avian sarcoma virus transforming gene product has been identified and partially purified from extracts of transformed cells. It is a phosphoprotein with a relative molecular mass of 60 000 (pp60src) with two major sites of phosphorylation. pp60src appears to be a cyclic-AMP-independent protein kinase as judged by protein phosphorylation with partly purified fractions. The specificity of the phosphorylation observed was judged by inhibition with anti-pp60src IgG but not by normal IgG and by the fact that the protein kinase activity isolated from ts transformation-mutant infected cells was more thermolabile than that from wild-type transformed cells, thus showing more directly the origin of the enzymic activity. A cellular protein substrate of pp60src has been identified as a 34 000 molecular mass protein. These data together suggest that protein phosphorylation by pp60src may be a function of the molecule that plays a major role in transformation.     

Forms of pp60v-src isolated from Rous sarcoma virus-transformed cells.

It has previously been shown that an electrophoretic variant form of the Rous sarcoma virus transforming protein, pp60v-src, exists in src-transformed cells. This variant, which was readily observed in vanadate-treated cells, was characterized as possessing extensive amino-terminal domain phosphotyrosine modification. Its appearance was further correlated with increased src-specific protein kinase activity. In this study, we used a src-specific monoclonal antibody (MAb) to resolve immunologic forms of pp60v-src. The MAb was able to distinguish between two populations of typical lower-band pp60v-src and was unreactive with the electrophoretic variant upper-band pp60v-src species. Using serial immunoprecipitations, we resolved four populations of pp60v-src: src protein either immunoreactive or unreactive with the MAb from both untreated and vanadate-treated transformed cells. The pp60v-src in each fraction displayed a distinct phosphoamino acid composition and tryptic phosphopeptide profile. However, analysis of their tyrosyl kinase specific activities showed that the immunologically resolved populations of pp60v-src from a given culture did not differ. Both pp60v-src fractions from vanadate-treated cells exhibited similar kinase specific activities, which were greatly enhanced over those of enzyme preparations from untreated cells. Since the MAb-reactive pp60v-src fraction from vanadate-treated cells lacked the electrophoretic variant upper-band pp60v-src species yet still possessed enhanced enzymatic specific activity, the initially stated correlation between the appearance of the electrophoretic variant src form and increased src kinase activity breaks down. These results suggest that yet to be defined modifications of the src protein may be involved in its functional regulation.     

Isolation and characteristics of antibodies against synthetic fragments of oncoproteins. II. Antiserum against the transforming protein pp60src of Rous sarcoma virus

To generate the antibodies to the transforming protein of Rous sarcoma virus (RSV) pp60src, rabbits were immunized with the peptide, corresponding to 415-421 sequence of pp60src. These antibodies immunoprecipitate pp60src in RSV-transformed chicken and mammalian cells, and also some proteins (45, 85 and 120 kDa), which could be autophosphorylated in vitro. It was shown that 415-421 sequence of pp60src is not recognized by the antibodies to pp60src from RSV-induced tumour bearing rabbits (TBR serum). In contrast to TBR serum, antibodies, generated against synthetic peptide, corresponding 415-421 sequence of pp60src couldn't be phosphorylated in vitro, when [gamma-32P]ATP is added to the immune complex. The antipeptide antibodies, bound to pp60src did not block phosphorylation of TBR immunoglobulins, added to this immune complex. Hence, 415-421 sequence of pp60src RSV containing the major tyrosine phosphorylation site does not take part in the kinase reaction in vitro.     

Stable association of pp60src and pp59fyn with the focal adhesion-associated protein tyrosine kinase, pp125FAK.

Changes in cellular growth and dramatic alterations in cell morphology and adhesion are common features of cells transformed by oncogenic protein tyrosine kinases, such as pp60src and other members of the Src family. In this report, we present evidence for the stable association of two Src family kinases (pp60src and pp59fyn) with tyrosine-phosphorylated forms of a focal adhesion-associated protein tyrosine kinase, pp125FAK. In Src-transformed chicken embryo cells, most of the pp125FAK was stably complexed with activated pp60src (e.g., pp60(527F). The stable association of pp125FAK with pp60(527F) in vivo required the structural integrity of the Src SH2 domain. The association of pp60(527F) and pp125FAK could be reconstituted in vitro by incubation of normal cell extracts with glutathione S-transferase fusion proteins containing SH2 or SH3/SH2 domains of pp60src. Furthermore, the association of isolated SH2 or SH3/SH2 domains with in vitro 32P-labeled pp125FAK protected the major site of pp125FAK autophosphorylation from digestion with a tyrosine phosphatase, indicating that the autophosphorylation site of pp125FAK participates in binding with Src. Immunoprecipitation of Src family kinases from extracts of normal chicken embryo cells revealed stable complexes of pp59fyn and tyrosine-phosphorylated pp125FAK. These data provide evidence for a direct interaction between two cytoplasmic nonreceptor protein tyrosine kinases and suggest that Src may contribute to changes in pp125FAK regulation in transformed cells. Furthermore, pp125FAK may directly participate in the targeting of pp59fyn or possibly other Src family kinases to focal adhesions in normal cells.     

Detection of the Viral Sarcoma Gene Product in Cells Infected with Various Strains of Avian Sarcoma Virus and of a Related Protein in Uninfected Chicken Cells

Genetic analyses have defined a single gene (src) as that portion of the avian sarcoma virus (ASV) genome which encodes the protein directly responsible for ASV-induced neoplastic transformation. We have recently identified the polypeptide product of the src gene of the Schmidt-Ruppin (SR) strain of ASV, a 60,000-dalton phosphoprotein designated pp60(src), and have further determined that pp60(src) acts as a protein kinase. Essential to the identification and characterization of the pp60(src) protein of SR-ASV was the use of serum (TBR serum) from rabbits bearing SR-ASV-induced tumors. TBR serum was, however, strain specific, recognizing pp60(src) from SR-ASV-transformed cells only. We report here that sera from marmosets bearing tumors induced by the Bryan or SR strains of ASV (TBM sera) contain antibody which precipitates the transforming gene product from cells transformed by the SR, Bryan, Prague, or Bratislava strains of ASV. In contrast, rabbits bearing tumors induced by either the Bratislava or Bryan strains of ASV, or hamsters with SR-ASV-induced tumors did not produce antibody to pp60(src) from any strain of ASV. The 60,000-dalton polypeptides immunoprecipitated with TBM serum from cells transformed by each of the above virus strains are phosphoproteins. One-dimensional peptide mapping by limited proteolysis revealed that the pp60(src) proteins are structurally very similar, but not identical. Furthermore, all of the viral pp60(src) proteins have an associated phosphotransferase activity. In addition to detecting the viral src proteins, TBM serum was able to immunoprecipitate an antigenically related protein from normal uninfected avian cells.     

Evidence that the Rous sarcoma virus transforming gene product is associated with glycerol kinase activity

This communication provides biochemical, immunological, and genetic evidence that pp60src, the Rous sarcoma virus transforming gene product, is associated with glycerol kinase activity. Our investigations demonstrated that the compound phosphorylated by pp60src or by glycerol kinase (EC 2.7.1.30) from Candida mycoderma share the same electrophoretic and chromatographic mobilities. The glycerol kinase and protein kinase activities of pp60src were inhibited similarly by preincubation with immune IgG. Both activities were reduced 6-9-fold in pp60src preparations derived by immunoaffinity chromatography from cells which were infected with NY68, a temperature-sensitive transformation mutant of Rous sarcoma virus. The thermolability at 41 degrees C of the glycerol kinase activity of pp60src from the mutant virus-infected cells was greater (t/2 = 1.3 min) than the same activity in pp60src preparations from wild type virus-infected cells (t/2 = 4.8 min).     

Phosphorylation of the solubilized insulin receptor by the gene product of the Rous sarcoma virus, pp60src

Both the insulin receptor and the gene product of the Rous sarcoma virus, pp60src, are protein kinases which phosphorylate themselves and other proteins on tyrosine residues. Addition of the solubilized insulin receptor to purified pp60src increased the phosphorylation of the beta-subunit of the insulin receptor. Phosphorylation of the insulin receptor by pp60src occurred both in the absence and presence of insulin but did not alter the insulin dose response for autophosphorylation of the receptor. Increasing concentrations of pp60src increased the phosphorylation of the receptor and at high concentrations equaled the maximal effect produced by insulin. Our observations suggest a possible mechanism by which the metabolically regulated insulin receptor tyrosine kinase could be altered by other tyrosine kinases such as that associated with pp60src. Further studies will be required to determine if the insulin receptor is phosphorylated by pp60src in Rous sarcoma virus-infected cells.     

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.     

Isolation of proteins with kinase activity and related to pp60 src from human cells

A protein kinase activity (PK) was associated with immunoprecipitates between polypeptides of human lymphoblastoid cells of malignant origin (Raji cell line) or of their normal counterparts ( Priess cell line) and antibodies directed against avian pp60 src or against the carboxyterminal hexapeptide of pp60 src. Therefore, these human cells and Rous Sarcoma Virus (RSV) transformed avian cells share antigenic determinants of pp60 src and, in particular, its carboxyterminal sequence, as well as one of its functions, a protein kinase activity. The protein kinase from Raji cells phosphorylated predominantly tyrosine residues, that from Priess cells threonine residues.     

Stable association of activated pp60src with two tyrosine-phosphorylated cellular proteins.

We have identified two phosphotyrosine-containing cellular proteins with relative molecular masses of 130,000 (pp130) and 110,000 (pp110) daltons in chicken embryo cells that coimmunoprecipitated with pp60v-src and activated forms of chicken pp60c-src (pp60(527)F). Most if not all of the tyrosine-phosphorylated forms of pp130 and pp110 could be immunoprecipitated from lysates with any of several src protein-specific monoclonal antibodies directed against at least three spatially distinct epitopes. Consequently, of the more than 15 prominent phosphoproteins detected on immunoblots with phosphotyrosine-specific antibodies, pp130 and pp110 were selectively removed by src protein-specific immunoprecipitation, and their presence in the immunoprecipitates appears to have been due to a direct interaction with activated src proteins. src protein variants that induce different morphological phenotypes were altered in their ability to form detergent-stable complexes with pp130 and pp110 or with pp110 alone. Mutant src proteins, defective for myristylation, showed increased tyrosine phosphorylation of and association with pp110. Expression of src variants with mutations in the A box (pp60dl92/527F) or B box (pp60dl155/527F) of the src homology region induced differences in phosphorylation of pp130 and pp110, as well as changes in their association with variant src proteins. Sequences within the B-box region appeared to be necessary for stable complex formation with pp130 and pp110 and may be involved in the interaction of activated src proteins with cellular substrates.     

Evidence that the transforming gene of avian sarcoma virus encodes a protein kinase associated with a phosphoprotein.

Avian sarcoma virus (ASV) induces sarcomas in animals and transforms fibroblasts to a neoplastic state in cell culture. A single viral gene (src) is responsible for both the induction and maintenance of neoplastic transformation. Recent work has identified a protein with a molecular weight of 60,000 daltons that is apparently encoded in src and may be the effector molecule for the gene (Brugge and Erikson, 1977; Purchio et al, 1978). The putative product of src can be immunoprecipitated by antisera obtained from rabbits bearing tumors induced by ASV. We have used this approach to isolate the protein to characterize further its genetic origins and possible function. Our rabbit tumor antisera precipitated a protein with a molecular weight of 60,000 daltons; according to serological, biochemical and genetic criteria, this protein is encoded in src. We found that this protein is phosphorylated and therefore denoted it pp60. Phosphorylation of pp60 could be accomplished in vitro with extracts of ASV-infected cells. A temperature-sensitive conditional mutation in src had no demonstrable effect on either the production or stability of pp60 in the infected cell, but phosphorylation of the protein was temperature-sensitive. Since the mutant src is not expressed at the restrictive temperature, our findings raise the possibility that phosphorylation of pp60 is required for its function as the putative effector of src. Immunoprecipitates prepared with extracts of ASV-infected cells and the rabbit tumor antisera contained a protein kinase activity that catalyzed phosphorylation of the heavy chains of immunoglobulin molecules, using either ATP or GTP as phosphate donor. The kinase activity immunoprecipitated in parallel with pp60 was obtained only from cells that contained a functioning product of src and could not be precipitated with antisera directed against structural proteins of ASV. A temperature-sensitive conditional mutation in src caused the kinase activity to be thermally inactivated in vitro far more rapidly than the activity from cells infected with wild-type virus. We conclude that both the protein kinase and pp60 are encoded in src, and that the enzymatic activity may be an intrinsic property of pp60. Phosphorylation of pp60 in cellular extracts was inhibited by calcium ion, whereas the immunoprecipitable kinase activity was not, suggesting that the kinase responsible for pp60 phosphorylation may be distinct from that encoded in src. Collett and Erikson (1978) have also identified a protein kinase activity associated with pp60. These findings raise the possibility that phosphorylation of specific cellular targets might account for transformation of the host cell by src.