Zinc-induced tyrosine phosphorylation of hippocampal p60c-src is catalyzed by another protein tyrosine kinase.

Tyrosine phosphorylation of p60c-src induced by Zn2+ in rat hippocampal membranes is shown to inhibit Src tyrosine kinase activity. Zn2+ catalyzes the phosphorylation of p60c-src in the membranes but does not activate autophosphorylation of p60c-src immunoprecipitated with anti-Src monoclonal antibody. Moreover, the immunoprecipitated Src kinase has no Zn(2+)-induced activity in phosphorylation of exogenous substrate, enolase. Cyanogen bromide cleavage of p60c-src phosphorylated in the presence of Zn2+ yields a 4-kDa phosphopeptide corresponding to phosphorylation of a carboxy-terminal tyrosine residue of Src kinase. In conclusion, hippocampal membranes contain a Zn(2+)-stimulated protein tyrosine kinase capable of regulating the p60c-src activity.     

A protein tyrosine kinase involved in regulation of pp60c-src function

We recently identified a novel protein tyrosine kinase that specifically phosphorylates truncated pp60c-src (Mr = 53,000) at a tyrosine residue(s) distinct from its autophosphorylation site. In this study, we examined whether this enzyme phosphorylates intact pp60c-src (Mr = 60,000) and determined its phosphorylation site. Non-neuronal and neuronal forms of intact pp60c-src were separately purified from the membrane fraction of neonatal rat brain by sequential column chromatographies. The novel kinase phosphorylated tyrosine residues of both forms of intact pp60c-src. The phosphorylation occurred in parallel with autophosphorylation of pp60c-src, and in both forms the final stoichiometry estimated was quite similar to that of autophosphorylation (about 5%). The enzyme also phosphorylated pp60c-src in which the kinase activity had been destroyed by an ATP analogue, p-fluorosulfonylbenzoyl 5'-adenosine. The phosphorylation site of the non-neuronal form was analyzed by sequential peptide mapping with tosylphenylalanyl chloromethyl ketone-treated trypsin and alpha-chymotrypsin. Tryptic digestion of the phosphorylated pp60c-src yielded a unique phosphopeptide that cross-reacted with an antibody specific for the carboxyl-terminal sequence of chicken pp60c-src. Digestion of the phosphopeptide with chymotrypsin yielded a product that comigrated with a synthetic phosphopeptide corresponding to the carboxyl-terminal 15 residues of chicken pp60c-src. These results clearly indicate that the carboxyl-terminal sequence of rat pp60c-src is identical to that of chicken pp60c-src, and a tyrosine residue corresponding to chicken Tyr527 is the phosphorylation site. This phosphorylation resulted in a decrease in the enolase phosphorylating activity of pp60c-src. Kinetic experiments indicated that this decrease in activity was due to a decrease in the Vmax value of pp60c-src. These findings support our previous proposal that the novel tyrosine kinase acts as a specific regulator of pp60c-src in cells.     

Enzymatically inactive p60c-src mutant with altered ATP-binding site is fully phosphorylated in its carboxy-terminal regulatory region

Cellular src protein, p60c-src, is phosphorylated on tyrosine 527 in chicken embryo fibroblasts, and this phosphorylation is implicated in suppressing the protein-tyrosine kinase activity and transforming potential of p60c-src. To determine whether tyrosine 527 phosphorylation is dependent on p60c-src kinase activity, the ATP-binding site of chicken p60c-src was destroyed by substitution of lysine 295 with methionine. The resultant protein, p60c-src(M295), expressed either in chicken cells or in yeast, lacked detectable kinase activity. Nevertheless, tyrosine and serine phosphorylation of p60c-src(M295) overproduced in chicken cells were indistinguishable from that of authentic p60c-src. By contrast, p60c-src(M295) was not phosphorylated on tyrosine in yeast. These results suggest that a protein kinase present in chicken cells but not in yeast phosphorylates tyrosine 527 in trans, and are consistent with the possibility that this kinase is distinct from p60c-src.     

Purification and characterization of a pp60c-src-related tyrosine kinase that effectively phosphorylates a synthetic peptide derived from p34cdc2.

A protein tyrosine kinase has been purified from the particulate fraction of bovine spleen to a specific activity of 0.217 mumol/min/mg at 100 microM ATP and 3 mM [Val5] angiotensin II. Both the angiotensin phosphorylation activity and immunoreactivity towards an antibody preparation raised against a synthetic peptide containing the autophosphorylation site of pp60c-src, Cys-src(403-421), were monitored during the purification. The purified sample displayed three closely spaced protein bands with molecular weights of 50-55 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All bands could be phosphorylated exclusively on tyrosine residues under autophosphorylation conditions. All reacted on immunoblots with an antibody raised against a synthetic peptide corresponding to the consensus autophosphorylation site of members of the pp60c-src family of tyrosine kinases. Tryptic phosphopeptide maps of the three proteins were essentially indistinguishable. The results suggest that the purified enzyme preparation contained mainly three closely related pp60c-src-family protein tyrosine kinases or a pp60src-family protein tyrosine kinase modified posttranslationally to give three closely spaced protein bands on sodium dodecyl sulfate gel. Neither of these proteins appears to be pp60c-src or p56lck. The spleen protein tyrosine kinase was found to phosphorylate a p34cdc2 kinase peptide, Cys-cdc2(8-20), which contained the regulatory tyrosine residue Tyr-15 about 20 times better than [Val5]angiotensin II or Cys-src(403-421) peptide at a peptide substrate concentration of 1 mM. In contrast, epidermal growth factor receptor kinase partially purified from A431 cells did not show preference for Cys-cdc2(8-20) as its substrate. Although Cys-cdc2(8-20) contained two tyrosine residues, only the tyrosine corresponding to Tyr-15 in p34cdc2 was phosphorylated by the spleen tyrosine kinase. The observation suggests that the primary structure surrounding Tyr-15 of p34cdc2 contains substrate structural determinants specific for the spleen tyrosine kinase.     

Investigation of factors that influence phosphorylation of pp60c-src on tyrosine 527.

Phosphorylation at tyrosine 527 of the proto-oncogene product, pp60c-src, has been proposed to decrease the tyrosine kinase activity of the enzyme. We have investigated potential factors that might influence phosphorylation at this site by making mutant variants of the pp60c-src protein. By effectively eliminating the site of N-terminal myristylation, we demonstrated that stable membrane association is not necessary for tyrosine 527 phosphorylation. Furthermore, mutational elimination of the enzymatic activity of this mutant pp60c-src protein did not alter the efficiency of phosphorylation at tyrosine 527. These data are consistent with the proposal that pp60c-src may be phosphorylated at tyrosine 527 by a cellular tyrosine kinase distinct from pp60c-src. In addition, using detergent-permeabilized cells, we established conditions that allow efficient phosphorylation of tyrosine 527 in vitro.     

Association of p60fyn with middle tumor antigen in murine polyomavirus-transformed rat cells.

Rabbit antisera raised against human FYN-specific peptides were used to evaluate the expression of the fyn gene product in normal and murine polyomavirus middle tumor antigen (MTAg)-transformed rat cells. The antisera were capable of detecting p60fyn in both normal and MTAg-transformed cells. Two different antisera directed against unique p60fyn sequences were found to detect p60fyn-MTAg complexes in cell lysates from the MTAg-transformed cells. The MTAg molecules immunoprecipitated by FYN antisera were phosphorylated on tyrosine during immune-complex kinase reactions at sites similar to those found on MTAg in complexes with pp60c-src. Whereas the abundance of p60fyn was estimated to be less in the MTAg-transformed cells than in their normal counterparts, the specific activities of p60fyn molecules in the normal and transformed cells were similar.     

Activation of the pp60c-src kinase by middle T antigen binding or by dephosphorylation.

The transforming protein of polyoma virus, middle T antigen, associates with the protein tyrosine kinase pp60c-src, and analysis of mutants of middle T suggests that this complex plays an important role in transformation by polyoma. It has recently been reported that pp60c-src from polyoma virus-transformed cells has enhanced tyrosine kinase activity in vitro. The data presented here confirm these findings and show that the enhanced kinase activity of pp60c-src is due to an increase in the Vmax of the enzyme. Sucrose density gradient analysis demonstrates that only the form of pp60c-src which is bound to middle T antigen is activated. The difference in enzyme activity between pp60c-src from normal and middle T-transformed cells is more marked when the enzyme is prepared from lysates containing the phosphotyrosine protein phosphatase inhibitor, sodium orthovanadate. pp60c-src from middle T transformed cells is unaffected, but pp60c-src from normal cells has reduced kinase activity if dephosphorylation is prevented. The kinase activity of pp60c-src thus appears to be regulated by its degree of phosphorylation at tyrosine, and data are presented which support this hypothesis. pp60c-src is the first example of a protein tyrosine kinase whose activity is inhibited by phosphorylation at tyrosine. Middle T antigen may increase the kinase activity of pp60c-src by preventing phosphorylation at this regulatory site.     

Low level of cellular protein phosphorylation by nontransforming overproduced p60c-src.

We have previously found that Rous sarcoma virus variants in which the viral src (v-src) gene is replaced by the cellular src (c-src) gene have no transforming activity. In this study, we analyzed the basis for the inability of the p60c-src overproduced by these variants to transform cells. Phosphorylations of tyrosine residues in total cell protein or in cellular 34K protein are known to be markedly enhanced upon infection with wild-type Rous sarcoma virus. We found that these tyrosine phosphorylations were only slightly increased in the c-src-containing virus-infected cells, whereas both levels were significantly increased by infection with wild-type Rous sarcoma virus, or transforming mutant viruses which are derived from c-src-containing viruses by spontaneous mutation. Phosphorylation at tyrosine 416 of p60 itself was also extremely low in overproduced p60c-src and high in p60s of transforming mutant viruses. In immunoprecipitates with monoclonal antibody, the overproduced p60c-src had much lower casein tyrosine kinase activity than did p60v-src. We previously showed that p60 myristylation and plasma membrane localization may be required for cell transformation. p60c-src was similar to transforming p60s in these properties. These results strongly suggest that the low level of tyrosine phosphorylation by overproduced p60c-src accounts for its inability to transform cells.     

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.     

Serine/threonine-specific protein kinase activity associated with viral pp60src protein

Three different types of experiments are presented in this paper, the results of which converge to indicate that the viral src protein associates with and modulates the activity and/or the specificity of a serine/threonine protein kinase. Firstly, a 60-kDa protein from extracts of FR3T3 rat fibroblasts transformed by wild-type Rous sarcoma virus (SRD-FR3T3) is shown to be immunoprecipitated with a monoclonal antibody (mAb) raised against bacterially produced pp60v-src, the mAb327 [Lipsich, L. A., Lewis, A. J. & Brugge, J. S. (1983) J. Virol. 48, 352-360] and to be phosphorylated in vitro at serine/threonine/tyrosine residues, in the ratio 25:53:22. Under the same experimental conditions, the pp60c-src protein immunoprecipitated with mAb327 from extracts of NIH c-src overexpresser cells is phosphorylated exclusively on tyrosine residues. Secondly, the results of immunoprecipitation experiments using a tumor-bearing rabbit (TBR) serum and reported in an earlier work [David-Pfeuty, T. & Hovanessian, A. (1984) Eur. J. Biochem. 140, 325-342], together with those reported here, suggest that the TBR-immunoprecipitated pp60v-src coprecipitates with a cellular protein related to the 60-kDa subunit of the Ca2+/calmodulin protein kinase II from brain. Finally, partially purified preparations of pp60v-src, but not of pp60c-src, are shown to contain a Ca2+/calmodulin-dependent protein kinase activity that phosphorylates a 52-kDa protein substrate.     

Structural and functional modification of pp60c-src associated with polyoma middle tumor antigen from infected or transformed cells.

The polyoma middle tumor antigen (MTAg) associates with the src proto-oncogene product pp60c-src in infected or transformed rodent cells. The tyrosine protein kinase activity of pp60c-src, as measured by in vitro phosphorylation of pp60c-src itself or the exogenous substrate enolase, was increased 10- to 20-fold in cells transformed or infected with transformation-competent polyoma virus compared with controls. pp60c-src associated with MTAg and precipitated with polyoma antitumor serum had a novel site(s) of in vitro tyrosine phosphorylation within its amino-terminal domain. These observations suggest that association of MTAg with pp60c-src alters the accessibility of pp60c-src tyrosine residues for phosphorylation in vitro and increases pp60c-src protein kinase activity. Several transformation-defective mutants of MTAg did not cause amino-terminal tyrosine phosphorylation of pp60c-src in vitro or enhance its protein kinase activity, suggesting that these properties correlate with the transforming ability of MTAg. However, one transformation-defective MTAg mutant, dl1015, did cause amino-terminal tyrosine phosphorylation of pp60c-src in vitro and did enhance its protein kinase activity. This suggests that properties of MTAg, in addition to modifying the structure and function of pp60c-src, may be important for transformation.     

Tyrosine phosphorylation of a c-Src-like protein is increased in membranes of CD4- CD8- T lymphocytes from lpr/lpr mice.

Mice homozygous for the autosomal recessive lpr gene have a disorder that results in autoimmunity and massive accumulation of T lymphocytes lacking CD4 and CD8 surface markers. These abnormal T cells exhibit constitutive tyrosine phosphorylation of a component of the CD3-T-cell receptor complex. We compared membrane tyrosine phosphorylation in lpr/lpr CD4- CD8- T cells and control T cells, lpr membranes exhibited a 7.3-fold increase (n = 16) in tyrosine phosphorylation of a 60-kilodalton protein. The increase was correlated with the Lpr but not the CD4- CD8- phenotype in that p60 phosphorylation was not increased in membranes from normal CD4- CD8- thymocytes. To identify the p60 in lpr cells, we examined the activity of several T-cell tyrosine-specific protein kinases. p56lck phosphorylation was only slightly increased in lpr membranes (2.2-fold; n = 16). Phorbol ester treatment of intact T cells before membrane isolation caused p56lck to migrate as pp60lck; however, pp60lck could be clearly distinguished from the pp60 in lpr cells by two-dimensional gel electrophoresis. The pp60 from lpr cells exhibited several isoforms at pH approximately 6.3 to 6.5. Although on two-dimensional gels pp60c-src had a pI (6.4 to 6.8) within a similar region, p60c-src mRNA, protein, and kinase activities were not increased in lpr cells. In addition, staphylococcal V8 proteolytic cleavage of the lpr pp60 isolated on two-dimensional gels yielded two major fragments, a pattern distinct from that of pp60c-src. However, by using an antiserum against the C-terminal sequence of c-Src and other related kinases, including p59fyn, the pp60 could be immunoprecipitated in greater amounts from lpr than from control T cells. When pp59(fyn) was selectively immunoprecipitated from T-cell membranes with specific antisera, its molecular weight, proteolytic cleavage pattern, and behavior on two-dimensional gels were identical to those of the pp60 from lpr cells. We conclude that p59(fyn) phosphorylation is increased in membranes from lpr/lpr CD4(-) CD8(-) T cells and that the increase is correlated with constitutive tyrosine phosphorylation and perhaps with the expansion of this unusual T-cell population.     

Characterization of purified pp60c-src protein tyrosine kinase from human platelets.

Intact pp60c-src, the cellular homologue of the transforming protein of Rous sarcoma virus, was purified from human platelets. The purified fractions also contained small amounts of a 54-kDa proteolytic degradation product of pp60c-src. We investigated some of the biochemical and kinetic properties of pp60c-src protein tyrosine kinase. Maximum kinase activity occurred at pH 6.5 and required a mixture of 2 mM Mn2+/Mg2+ as divalent cations. The enzyme most strongly phosphorylated casein, followed by enolase and alcohol dehydrogenase. The Km value for ATP was 4 microM for substrate phosphorylation and for autophosphorylation. Using casein, we determined a Vmax for substrate phosphorylation by pp60c-src in the range of 1.9-3.4 nmol.min-1.mg-1. Since the Vmax value for the purified 54-kDa fragment of pp60c-src was also included in this value, we conclude that proteolytic degradation of a 6-kDa fragment from the N-terminus of pp60c-src did not affect its kinase activity. Tryptic phosphopeptide analysis identified Tyr-416 as the major autophosphorylation site. Preincubation of purified pp60c-src with ATP increased the amount of autophosphorylation accompanied by an increase in Vmax, whereas the Km values were not altered. Our data directly demonstrate that autophosphorylation at Tyr-416 exerts, in contrast to phosphorylation at Tyr-527, a positive regulatory effect on the pp60c-src kinase activity.     

Activity of pp60c-src and association of pp60c-src, pp54/58lyn, pp60fyn, and pp72syk with the cytoskeleton in platelets activated by collagen

Collagen stimulation of platelets induced an increase in the specific activity of pp60c-src immunoprecipitated from the Triton-soluble fraction. The earliest time after collagen stimulation that an increase in pp60c-src activity was observed was 30 s. However, the maximum activity of pp60c-src in the Triton-soluble fraction was observed 60 s after collagen stimulation. At this time an approximately twofold increase of pp60c-src activity towards phosphorylation of KVEKIGEGTYGVVKK specific peptide and enolase and a 4.5-fold increase towards phosphorylation of pp60c-src itself was measured. Furthermore, the majority of pp60c-src as well as pp54/58lyn, pp60fyn, and pp72syk were found in the Triton-soluble fraction in resting platelets. Collagen induced, to different extents and velocities, translocation of all of these proteins from the Triton-soluble fraction to the Triton-insoluble, cytoskeleton-rich, platelets fraction. These results provide direct evidence that collagen stimulation of platelets increases the tyrosine kinase activity of pp60c-src and suggest that the platelet cytoskeleton plays an important role in collagen-induced signal transduction by localizing signaling molecules.     

Specific association of the proto-oncogene product pp60c-src with an intracellular organelle, the PC12 synaptic vesicle

The protein product of the proto-oncogene c-src is a membrane-associated tyrosine kinase of unknown function. Identification of pp60c-src target membranes may elucidate the function of the c-src protein. The available evidence indicates that pp60c-src associates with distinct membranes within single cell types and has different distributions in different cell types. Our experiments demonstrate targeting of pp60c-src to an isolatable and biochemically identified membrane fraction in the neuroendocrine cell line PC12. The c-src protein was found to be specifically associated with synaptic vesicles since: (a) the pp60c-src immunofluorescent pattern overlapped with a synaptic vesicle marker, synaptophysin; (b) a significant proportion (44%) of the pp60c-src from PC12 but not fibroblast postnuclear supernatants was recovered in a small vesicle fraction; (c) an anti-synaptophysin cytoplasmic domain antibody immunodepleted all of the pp60c-src vesicles in this fraction, and (d) pp60c-src copurified during a 100-fold purification of PC12 synaptic vesicles. These results suggest a role for the c-src protein in the regulation of synaptic vesicle function.     

Purification and characterization of a possible protooncogene fyn product, p59fyn, from a rat brain particulate fraction.

Four tyrosine-protein kinases that reacted with antibodies specific to p62c-yes, p60c-src, p60c-src+, and p59fyn, respectively, were solubilized from a rat brain particulate fraction and separated by casein-Toyopearl column chromatography. Possible p59fyn, with a pI of 6.5, was purified 490-fold as a single 59-kDa protein band on SDS-PAGE. The purified enzyme contained almost no phosphotyrosine residues but was autophosphorylated with Mg2+. ATP exclusively at tyrosine residues, with a concomitant increase in the kinase activity toward tyrosine-glutamate (1:4) copolymers. The rate of the copolymer phosphorylation was proportional to the square of the enzyme concentration, suggesting activation through intermolecular catalysis. In the presence of Mn2+, however, the reaction showed a first-order dependence on the enzyme concentration.     

Anti-peptide Antibody Identifies a 57 kDa Protein Tyrosine Kinase in the Sea Urchin Egg Cortex

The egg plasma membrane and cortical structures are highly enriched in protein tyrosine kinase activity which is thought to play an important role in the fertilization process. In order to identify the tyrosine protein kinases in the egg cortex, a site directed polyclonal antibody was produced against a peptide duplicating a conserved region of the catalytic domain of the sea urchin c-abl gene product. The region chosen as an antigen had a high degree of homology (57%) to other protein tyrosine kinases. The antibody was found to bind with a high degree of specificity to a 57 kDa protein tyrosine kinase in S. purpuratus eggs. The antibody was capable of immunoprecipitating the enzyme as a 57 kDa phosphoprotein from purified egg cortex fractions solubilized in NP-40. Immunoprecipitation was completely inhibited by prior incubation of the antibody with the synthetic peptide used as an antigen. Binding of the antibody completely inhibited kinase activity. However, the immunoprecipitated kinase activity could be eluted from the Sepharose-coupled antibody and was shown to have catalytic activity towards a tyrosine containing peptide substrate. The enzyme also underwent autophosphorylation on tyrosine in vitro. Ultrastructural localization of the kinase by immuno-electron microscopy revealed that the enzyme was primarily restricted to the egg plasma membrane.     

Phosphoinositide kinase activity and transformation

We have used the DNA tumor virus polyoma as a model system to examine whether the phosphatidylinositol (PI) turnover pathway is a critical target for transforming gene products. Polyoma-infected cells show elevated levels of polyphosphoinositides and polyphosphoinositols, and a PI kinase activity is associated with middle T antigen, a transforming gene product of polyoma virus. In anti-T immunoprecipitates from polyoma-infected or -transformed cells, comparisons of wild-type and polyoma mutants defective for transformation show a strong correlation between middle T-associated PI kinase activity and transforming ability. Middle T has previously been found to associate at the plasma membrane with pp60 c-src and to activate it as a tyrosine kinase. c-src itself does not appear to phosphorylate PI; however, the middle T/pp60 c-src tyrosine kinase activity may be important for activation of PI kinase. Ammonium orthovanadate, a tyrosine phosphatase inhibitor, elevates the middle T/pp60 c-src-associated PI kinase activity. We propose that middle T/pp60 c-src activates a PI kinase and modulates PI turnover in vivo by tyrosine phosphorylation.     

Structural differences between repressed and derepressed forms of p60c-src.

The kinase activity of p60c-src is derepressed by removal of phosphate from Tyr-527, mutation of this residue to Phe, or binding of a carboxy-terminal antibody. We have compared the structures of repressed and active p60c-src, using proteases. All forms of p60c-src are susceptible to proteolysis at the boundary between the amino-terminal region and the kinase domain, but there are several sites elsewhere that are more sensitive to trypsin digestion in repressed than in derepressed forms of p60c-src. The carboxy-terminal tail (containing Tyr-527) is more sensitive to digestion by pronase E and thermolysin when Tyr-527 is not phosphorylated. The kinase domain fragment released with trypsin has kinase activity. Relative to intact p60c-src, the kinase domain fragment shows altered substrate specificity, diminished regulation by the phosphorylated carboxy terminus, and novel phosphorylation sites. The results identify parts of p60c-src that change conformation upon kinase activation and suggest functions for the amino-terminal region.     

Transformation of chicken embryo fibroblasts and tumor induction by the middle T antigen of polyomavirus carried in an avian retroviral vector.

The middle T antigen of polyomavirus transformed primary chicken embryo fibroblasts when expressed from a replication-competent avian retrovirus. This in vitro-constructed retrovirus, SRMT1, is a variant of Rous sarcoma virus that encodes the middle T antigen in place of v-src. Inoculation of SRMT1 into 1-week-old chickens rapidly induced hemangiomas and hemangiosarcomas. As shown with mammalian cells infected with polyomavirus, polyomavirus middle T antigen appears to be associated with p60c-src in chicken cells infected with SRMT1. When lysates of SRMT1-infected cells immunoprecipitated with either a monoclonal antibody against p60src or anti-T serum were assayed in an in vitro kinase reaction, the middle T antigen was heavily phosphorylated. To see whether an excess of p60c-src could alter the extent of phosphorylation of the middle T protein or the process of cell transformation by middle T, cells were doubly infected with SRMT1 and NY501, a virus which overexpresses p60c-src. Doubly infected chicken embryo fibroblasts transformed with the same kinetics and were morphologically indistinguishable from chicken embryo fibroblasts infected with SRMT1 alone. Phosphorylation of the middle T antigen was elevated two- to fivefold relative to cells infected only with SRMT1.