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.     

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.     

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.     

The 34 kd pp60src substrate is located at the inner face of the plasma membrane

The subcellular localization of the 34 kd protein substrate of the pp60src kinase was investigated by immunofluorescence microscopy and subcellular fractionation. When permeabilized fibroblasts were stained with a monoclonal anti-34 kd protein antibody, a diffuse reticular pattern was observed. The 34 kd protein was not exposed on the outside surface of the cell. Double immunofluorescence staining experiments established that the 34 kd protein distribution was similar to that of the membrane-associated protein alpha-spectrin. The 34 kd protein was found in cell sections to be concentrated along the cell edges. Taken together, these results suggested that the 34 kd pp60src substrate was associated with the inside surface of the plasma membrane. This conclusion was supported by subcellular fractionation experiments in which the 34 kd protein was observed to fractionate with the plasma membrane. These localization studies support further the hypothesis that many of the primary effects of the pp60src kinase occur at the plasma membrane.     

Role of the mitogenic property and kinase activity of p60src in tumor formation by Rous sarcoma virus

Expression of the src gene of Rous sarcoma virus in chicken embryo neuroretinal cells results in morphological transformation and sustained proliferation of this normally resting cell population. PA101 and PA104 are two mutants of Rous sarcoma virus which induce neuroretinal cell proliferation in the absence of morphological transformation. Their mitogenic property is temperature sensitive, and they both encode p60src proteins with low kinase activity. To study the role of the mitogenic function and protein kinase activity of p60src in tumorigenesis, we investigated the oncogenicity of PA101 and PA104. Both mutants were less tumorigenic than wild-type virus when injected into chicks. Tumorigenicity was further assayed by inoculating infected chicken embryo fibroblasts and neuroretinal cells onto the chorioallantoid membrane of embryonated duck eggs. This system provides a nonpermissive and immunodeficient environment for xenogenic cell grafting and allows the study of cell tumorigenicity within a temperature range of 37 to 39.5 degrees C. Chicken embryo fibroblasts and neuroretinal cells infected with PA101 were as tumorigenic as wild type-infected cells at 37 degrees C, but tumor development was significantly reduced at 39.5 degrees C. In contrast, both cell types infected with PA104 displayed sharply reduced tumorigenicity. Cell cultures derived from PA101 tumors induced on the chorioallantoid membrane were similar to the corresponding cells maintained in vitro in terms of morphology, production of plasminogen activator, relative amounts of phosphotyrosine in total cellular proteins, and phosphorylation of 34,000-molecular-weight protein. These results indicate that the expression of the mitogenic function of src does not account per se for cell tumorigenicity and that tumor formation is compatible with low levels of p60src protein kinase activity.     

Regulation of cellular morphology by the Rous sarcoma virus src gene: analysis of fusiform mutants.

We have been interested in how Rous sarcoma virus (RSV) influences transformed cell morphology and compared the molecular properties of chicken embryo cells (CEC) infected with mutants of RSV that induce the fusiform transformed cell morphology with those of CEC infected by wild-type RSV, which induces the more normal round transformed cell morphology. We looked for properties shared by all fusiform mutant-infected cells, because these may be responsible for maintaining the fusiform morphology. Five different fusiform mutants, two wild-type RSVs, and one wild-type back revertant of a fusiform mutant were studied. In the fusiform mutant-infected cells, the localization and myristylation of pp60src were determined and the extent of expression of the extracellular matrix protein fibronectin was examined at both the mRNA and protein levels. The phosphorylation of vinculin on tyrosine also was examined in the same CEC. Within all fusiform mutant-transformed CEC, pp60src was dramatically absent from the adhesion plaque sites normally seen in cells transformed with wild-type RSV, and these transformed CEC all expressed more fibronectin mRNA and protein in the extracellular matrix than did the wild-type RSV-transformed CEC. The absence of pp60src from the adhesion plaques was not due to lack of myristylation of the src protein, and tyrosine phosphorylation of vinculin was not related to fibronectin expression. These results suggest that the inverse relationship between pp60src in the adhesion plaques and fibronectin expression in the extracellular matrix may be interconnected phenomena and could be related to the maintenance of the fusiform transformed morphology.     

Tyrosyl kinases acquired from anchorage-independent cells by a membrane-enveloped virus

Tyrosyl kinase activity in vesicular stomatitis virus (VSV) acquired from host cells that differ in morphology was investigated. VSV grown in baby hamster kidney (BHK) cells with rounded morphology and a high efficiency of colony formation in soft agar (Rous sarcoma virus [RSV]- transformed and suspension BHK cells) was compared with VSV grown in BHK cells with a flattened morphology and lower efficiency of colony formation in soft agar (RSV-infected revertant and control BHK cells). Tyrosyl kinase activity measured with the substrates angiotensin II peptide or casein was found at 7-10-fold higher levels in virus released from the anchorage-independent BHK cells. Most of the VSV- associated tyrosyl kinases acquired from the RSV-transformed BHK cells reacted with antiserum to pp60src, whereas the activity acquired from the suspension BHK cells was unaffected by anti-src serum. The overall levels of tyrosyl kinase in subcellular fractions of the host BHK cells were also measured. Like the VSV released from them, the RSV- transformed cell extracts contained high levels. The suspension cells, however, contained the same low levels of tyrosyl kinase as was found in the control BHK cell extracts. Therefore, tyrosyl kinase was concentrated and acquired by VSV from the anchorage-independent suspension BHK cells. VSV-associated protein kinases acquired from other cell types followed a similar pattern. Tyrosyl kinase levels were high in VSV released from suspension cultures (Chinese hamster ovary and HeLa) and from virally transformed cells (Kirsten murine sarcoma virus-transformed rat kidney cells) and low in VSV released from an anchorage-dependent primary cell culture (chick embryo fibroblasts).     

Phosphatidylinositol kinase activities in normal and Rous sarcoma virus-transformed cells.

The phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and diacylglycerol kinase activities in the plasma membrane-rich fraction of chicken embryo fibroblasts infected with a temperature-sensitive mutant of Rous sarcoma virus increased when the cells were shifted from the nonpermissive temperature, 41 degrees C, to the permissive temperature, 35 degrees C. Temperature shift from 35 to 41 degrees C decreased the lipid kinase activities in the membrane vesicles. These changes accompanied the changes observed in pp60v-src protein kinase activity. Thermal inactivation at 41 degrees C did not appreciably reduce PI and PIP kinase activities in membrane vesicles prepared from uninfected or Rous sarcoma virus-transformed cells, whereas pp60v-src protein kinase activity in the membrane vesicles was rapidly inactivated under the same conditions. These data suggest that pp60v-src may indirectly enhance PI and PIP phosphorylation but not directly contribute to this pathway.     

Transformation by the src oncogene alters glucose transport into rat and chicken cells by different mechanisms.

Transformation of both rat and chicken fibroblasts by the src oncogene leads to a four- to fivefold increase in the rate of glucose transport and in the level of the glucose transporter protein. We have previously shown that, with chicken embryo fibroblasts, transformation leads to a reduction in the rate of degradation of the transporter, with little or no increase in the rate of its biosynthesis. We now show that, with the rat-1 cell line, the opposite result was obtained. src-induced transformation led to an increase in transporter biosynthesis, with little effect on turnover. A src-induced increase in transporter mRNA entirely accounted for the increase in biosynthesis of the protein. By contrast, in chicken embryo fibroblasts, the level of transporter mRNA was low and was not induced to rise by src transformation. Thus, src induced an increase in the level of the glucose transport protein by fundamentally different mechanisms in chicken embryo fibroblasts and rat-1 cells. To test whether this difference was due to rat-1 cells being an immortalized cell line, we measured transporter mRNA levels in primary fibroblast cultures from rat embryos and in parallel cultures transformed by src. Transporter mRNA was inducible by src in these cells. Thus, the difference in mRNA inducibility between chicken and rat cells is not due to immortalization.     

Requirement of phosphatidylinositol-3 kinase modification for its association with p60src.

When purified p60v-src was mixed with lysates of chicken embryo fibroblasts and immunoprecipitated with anti-Src antibody, phosphatidylinositol (PI)-3 kinase activity was found to be present in the Src protein immunoprecipitates. The level of bound PI-3 kinase activity was 5 to 10 times higher in lysates obtained from cells transformed by the src, fps, or yes oncogene than in lysates of uninfected cells. This increase in associated PI-3 kinase activity appears to be due to increased binding of this enzyme to p60v-src. This change most likely resulted from tyrosine phosphorylation of PI-3 kinase or an associated protein, since the PI-3 kinase activity that can bind to p60v-src was depleted by antiphosphotyrosine antibody. Binding of PI-3 kinase did not require either p60src protein kinase activity or autophosphorylation of p60v-src tyrosine residues. Furthermore, binding was markedly decreased by deletions in the N-terminal SH2 region but unchanged by deletion of the C-terminal half of p60v-src containing the catalytic domain. Taking these data together, it appears that PI-3 kinase or its associated protein is phosphorylated on tyrosine and that the phosphorylated form can bind to the N-terminal half of p60v-src, which contains the SH2 domain.     

Cyclic AMP treatment of Rous sarcoma virus-transformed Chinese hamster ovary cells increases phosphorylation of pp60src and increases pp60src kinase activity

Treatment of growing Rous sarcoma virus-transformed Chinese hamster ovary cells with the cyclic AMP analog 8-bromo-cyclic adenosine 3',5'-monophosphate (8-bromo-cyclic AMP) stimulates the incorporation of 32Pi into the viral transforming protein pp60src. Based on one-dimensional and two-dimensional peptide analysis and phosphoamino acid analysis, the increase is on a single phosphoserine residue at the NH2 terminus of the protein. The phosphate incorporation increases during the first 4 h of treatment. The pp60src kinase activity in extracts of cells treated with 8-bromo-cyclic AMP was stimulated about 2- to 3-fold. This stimulation of kinase activity increased during the first 3 h of treatment with 1 mM 8-bromo-cAMP and the activity was increased in both the soluble and particulate fraction of the cells. These results suggest that cyclic AMP can modulate the activity of pp60src in transformed cells.     

Protein kinase C in fibroblasts. Characteristics of its intracellular location during growth and after exposure to phorbol esters and other mitogens

Using an N-bromosuccinimide cleavage fragment of histone H1 as a relatively specific substrate for protein kinase C, we evaluated the partitioning of this kinase activity between soluble and particulate cellular fractions in 3T3-L1 fibroblasts. In confluent, serum-deprived cells, protein kinase C activity was approximately equally divided between soluble and detergent-extractable particulate fractions; both rapidly growing and transformed cells appeared to contain higher levels of particulate enzyme activity. Soluble protein kinase C activity and immunoreactivity decreased to virtually undetectable levels after exposure of the cells to phorbol 12-myristate 13-acetate (PMA), associated with a commensurate increase in particulate kinase activity and immunoreactivity. In intact cells, PMA appeared to cause a shift of immunoreactive protein kinase C from the cytosol to the perinuclear region, as assessed by immunofluorescent microscopy; however; subcellular fractionation revealed that PMA caused increases in the protein kinase C activity associated primarily with non-nuclear membranes. Exposure of the cells to sn-1,2-dioctanoylglycerol resulted in a modest and transient membrane association of protein kinase C, whereas platelet-derived growth factor, fibroblast growth factor, and bombesin caused no detectable increases in the membrane association of the kinase. Activation of protein kinase C by growth factors in fibroblasts may occur without the gross disturbances in intracellular kinase location which occur in response to phorbol esters.     

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.     

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.     

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.     

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.     

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.     

Analysis of pp60c-src protein kinase activity in hamster embryo cells transformed by simian virus 40, human adenoviruses, and bovine papillomavirus 1.

We have examined the effect of DNA tumor virus transformation of primary hamster embryo cells on the tyrosyl kinase activity of pp60c-src. Our present study demonstrates that some clones of hamster embryo cells transformed by simian virus 40, adenovirus type 2, adenovirus type 12, or bovine papillomavirus 1 can possess elevated pp60c-src kinase activity when compared with normal hamster embryo cells. However, other clones of hamster embryo cells transformed by these same viruses were found to have normal levels of pp60c-src kinase activity. In those clones of transformed cells where pp60c-src kinase activity was elevated, the increased levels of kinase activity were the result of an apparent increase in the specific activity of the pp60c-src phosphotransferase rather than an increase in the amount of the src gene product. Additionally, pp60c-src was not found to be physically associated with tumor antigens known to be encoded by these viruses. These results indicate that elevated levels of pp60c-src kinase activity can be found in hamster embryo cells transformed by several different DNA tumor viruses and suggest that the molecular mechanism by which pp60c-src kinase activity is elevated may differ from that previously observed in polyomavirus-transformed cells. These results also imply that elevation of pp60c-src kinase activity is not required for the transformation of hamster cells by these viruses.     

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.     

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.