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.     

Regulation of pp60c-src expression in rat and mouse fibroblasts by an inducible antisense gene: effects on serum regulation of growth and polyoma virus middle T function.

Expression of antisense c-src RNAs in rat and mouse fibroblasts had a dramatic effect on the function of polyoma virus middle T (mT). Antisense c-src RNA decreased the amount of mT:pp60c-src complexes in de novo virus-infected cells and prevented expression of the transformed phenotype in rat F111 cells. Expression of antisense c-src RNA in infected NIH3T3 cells also reduced the formation of mT:pp60c-src complexes but did not affect the ability of polyoma virus to carry out a productive infection. Further analysis of the effects of antisense c-src RNA in uninfected cells revealed that pp60c-src is required for cell growth. When pp60c-src synthesis was reduced, F111 cells stopped proliferating and showed decreased S6 phosphorylation in response to serum. However, F111 cells expressing reduced pp60c-src could be efficiently transformed by v-rasHa, even in the presence of low serum. Thus, pp60c-src appears to function as a component of a signal transduction pathway which regulates cell proliferation in response to serum.     

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.     

Protein kinase C promotes the phosphorylation of immunoprecipitated middle T antigen from polyoma-transformed cells

Stimulation of protein kinase C in polyoma virus-transformed cells increased the phosphorylation of tyrosine residues of the viral middle T (mT) antigen in mT:pp60c-src complexes precipitated by anti-mT antibodies. This increase might have been due to a stimulation of the complex's pp60c-src tyrosine kinase activity or to an increased ability of the mT protein to be phosphorylated by pp60c-src. These observations suggest that cellular protein kinase C might control the ability of polyoma virus to transform its host cell.     

Phosphorylation of middle T by pp60c-src: a switch for binding of phosphatidylinositol 3-kinase and optimal tumorigenesis

Substitution of phenylalanine for tyrosine 315 of the polyoma virus middle T (mT) protein lowers the incidence and limits the spectrum of tumors induced following inoculation of the virus into newborn mice. This substitution removes the major site of phosphorylation by pp60c-src without altering the ability of mT to associate with or to activate pp60c-src. The mutant mT fails to show binding of a phosphatidylinositol 3-kinase (Ptdlns 3-kinase) activity that is normally present in wild-type mT complexes. Furthermore, an anti-peptide antiserum that specifically recognizes mT lacking phosphate at tyrosine 315 precipitates binary (mT-pp60c-src) but not ternary (mT-pp60c-src-Ptdlns 3-kinase) complexes from wild-type infected cell extracts. Reprecipitation with either anti-pp60c-src or anti-mT serum brings down ternary complexes containing mT phosphorylated on tyrosine 315. Phosphorylation of mT by pp60c-src in vivo is therefore a critical event for binding of Ptdlns 3-kinase and for expression of the full tumorigenic potential of the virus.     

Mutations around the NG59 lesion indicate an active association of polyoma virus middle-T antigen with pp60c-src is required for cell transformation.

The transforming activity of polyoma virus middle-T antigen is believed to be dependent on its ability to form a complex with the cellular tyrosine protein kinase, pp60c-src. This hypothesis is based on observations of mutants of middle-T which demonstrated a correlation between these two activities. To investigate further the significance of pp60c-src association in transformation by middle-T, a series of deletion and point mutants were constructed around the NG59 lesion since this region has been implicated in pp60c-src binding. Analysis of the middle-T variants revealed a complete correlation between the presence of associated activated pp60c-src and the ability to transform. Further, this ability of pp60c-src to associate with middle-T may depend on the presence of a beta-turn between amino acids 177 and 180. The results indicate the NG59 phenotype results from the introduction of an isoleucine residue between amino acids 177 and 178 rather than the transition mutation at 179. The mutant MG1 is a single point mutation (at residue 180) and represents the smallest change in the middle-T which abolishes both the transformating and kinase activity of middle-T. Taken together, the data suggest the region surrounding the NG59 lesion is involved in the formation of an active complex between middle-T and pp60c-src and strongly suggest that this association is an absolute requirement for polyoma virus-induced transformation.     

Protein kinase C stimulation increases the transforming ability of the polyoma virus middle T antigen

Exposure of dexamethasone-treated cells of the mT-1 line of F111 rat cells bearing a dexamethasone-inducible polyoma virus middle T (mT) antigen gene to very low concentrations of the protein kinase C-stimulating phorbol ester TPA increased the association of mT antigen with the cellular pp60c-src tyrosine protein kinase, as indicated by an increased phosphorylation of tyrosine residues of mT in mT:pp60c-src complexes precipitated from extracts of the TPA-treated cells by anti-mT antibodies. This TPA (hence probably protein kinase C)-enhanced association of mT with pp60c-src was accompanied by a large increase in the transforming ability of mT as indicated by a much enhanced ability of TPA-treated mT-1 cells producing submaximal levels of mT to proliferate while suspended in semi-solid medium and to form foci on confluent monolayers of normal F111 cells. NRCC NO: 26558.     

Polyoma virus middle T antigen-pp60c-src complex associates with purified phosphatidylinositol 3-kinase in vitro.

Reconstitution of the polyoma virus middle T antigen (mT)-pp60-src complex and phosphatidylinositol 3-kinase (PtdIns 3-kinase) has been accomplished in vitro with immunopurified baculovirus-expressed mT-pp60c-src and PtdIns 3-kinase purified from rat liver. Both the 110- and 85-kDa subunits of the PtdIns 3-kinase associated with the mT-pp60c-src complex. The association of PtdIns 3-kinase with the mT-pp60c-src complex was dependent on the protein-tyrosine kinase activity of pp60c-src as a kinase-inactive mutant (pp60(295c-src)) still complexed with mT, but the mT-pp60(295c-src)) complex was unable to bind PtdIns 3-kinase. The mT-pp60c-src complex phosphorylated both subunits of PtdIns 3-kinase on tyrosine residues. The immunopurified mT-pp60c-src complex also associated with PtdIns 3-kinase activity from whole cell lysates, and this association was dependent upon the protein-tyrosine kinase activity of pp60c-src. Comparison of 35S-labeled proteins from whole cell lysates which associated with immunopurified mT-pp60c-src and mT-pp60(295c-src) revealed proteins of 110 and 85 kDa as the major peptides dependent on protein-tyrosine kinase activity for association with the complex. In addition, a synthetic phosphopeptide (13-mer) containing sequences conserved between the major tyrosine phosphorylation site of murine polyoma virus mT, hamster polyoma virus mT, and the insulin receptor substrate (IRS-1) specifically blocked the association of the 85- and 110-kDa polypeptides with the mT-pp60c-src complex. The ability to block the association was dependent on the tyrosine phosphorylation of the peptide. Association of PtdIns 3-kinase activity was blocked concurrently. This is the first demonstration that the 110-kDa subunit of PtdIns 3-kinase can associate with mT-pp60c-src. This association in vitro is a step toward understanding protein-protein interactions important in the signal transduction pathway of oncogenic proteins.     

Cell transformation by pp60c-src mutated in the carboxy-terminal regulatory domain

We introduced two mutations into the carboxy-terminal regulatory region of chicken pp60c-src. One, F527, replaces tyrosine 527 with phenylalanine. The other, Am517, produces a truncated pp60c-src protein lacking the 17 carboxy-terminal amino acids. Both mutant proteins were phosphorylated at tyrosine 416 in vivo. The specific activity of the Am517 mutant protein kinase was similar to that of wild-type pp60c-src whereas that of the F527 mutant was 5- to 10-fold higher. Both mutant c-src genes induced focus formation on NIH 3T3 cells, but the foci appeared at lower frequency, and were smaller than foci induced by polyoma middle tumor antigen (mT). The wild-type or F527 pp60c-src formed a complex with mT, whereas the Am517 pp60c-src did not. The results suggest that one, inability to phosphorylate tyrosine 527 increases pp60c-src protein kinase activity and transforming ability; two, transformation by mT involves other events besides lack of phosphorylation at tyrosine 527 of pp60c-src; three, activation of the pp60c-src protein kinase may not be required for transformation by the Am517 mutant; and four, the carboxyl terminus of pp60c-src appears to be required for association with mT.     

Altered sites of tyrosine phosphorylation in pp60c-src associated with polyomavirus middle tumor antigen.

We characterized the tyrosine phosphorylation sites of free pp60c-src and of pp60c-src associated with the polyomavirus middle tumor antigen (mT) in transformed avian and rodent cells. The sites of tyrosine phosphorylation in the two populations of pp60c-src were different, both in vitro and in vivo. Free pp60c-src was phosphorylated in vitro at a single site, tyrosine 416. pp60c-src associated with mT was phosphorylated in vitro on tyrosine 416 and on one or more additional tyrosine residues located in the amino-terminal region of the molecule. Free pp60c-src in polyomavirus mT-transformed cells was phosphorylated in vivo on tyrosine 527. In contrast, pp60c-src associated with mT was phosphorylated in vivo on tyrosine 416 and not detectably on tyrosine 527. Thus, the in vivo phosphorylation sites of pp60c-src associated with mT in transformed cells are identical to those of pp60v-src, the Rous sarcoma virus transforming protein. The results suggest that altered phosphorylation of pp60c-src associated with mT may play a role in the enhancement of the pp60c-src protein kinase activity and in cell transformation by polyomavirus.     

Phosphorylation of polyoma T antigens.

The T antigens of polyoma virus have been examined for phosphorylation in vivo and associated protein kinase activities in vitro. The 100K "large" T antigen is the major phosphoprotein among the T antigen species in vivo as determined by labeling virus-infected cells with 32P-orthophosphate. Hr-t mutants show normal phosphorylation of their 100K T antigens. The wild-type 56K plasma membrane-associated "middle" T antigen is also phosphorylated in the cell, but to a lesser extent than the 100K; this low level phosphorylation is also observed in the presumably altered 56K protein induced by hr-t mutant NG59 and in the 50K truncated "middle" T of hr-t mutant SD15. Addition of dibutyryl cyclic AMP to the medium does not affect labeling of either large or middle T antigens in wild-type- or mutant-infected cells. Thus no differences are observed in T antigen phosphorylation in vivo between wild-type virus and hr-t mutants. Hr-t mutants are defective in a protein kinase activity assayed in vitro by adding gamma-32P-ATP to T antigen immunoprecipitates. In the case of wild-type virus, the 56K protein is the major phosphate acceptor in the in vitro kinase reaction, with a somewhat lower level of phosphorylation observed in the 100K band. Hr-t mutants NG59 and SD15 show no labeling of the altered 56K or 50K, respectively, but do show detectable levels of 32P in the 100K bands. A wild-type virus carrying a small deletion affecting the 100K and 56k bands shows a normal level of kinase activity associated with the truncated T antigens. Ts-a mutants appear to be normal with respect to the middle T antigen-associated kinase. Photoaffinity labeling of infected cell extracts with 8-azido cyclic AMP shows that the two major classes of regulatory subunits of cyclic AMP-dependent protein kinases are present in the immunoprecipitates. Phosphorylation of histone H1 occurs when this substrate is added to immunoprecipitates of either mock-infected or virus-infected cells, again demonstrating the presence of cellular kinases. Further experiments will be required to determine whether the middle T antigen of polyoma virus is itself a protein kinase or simply a substrate for one or more cellular kinases.     

Identification and characterization of p59fyn (a src-like protein tyrosine kinase) in normal and polyoma virus transformed cells.

fyn is a member of the growing family of protein tyrosine kinase genes whose sequences are highly related to that of c-src. We have generated antibodies to peptides corresponding to two different amino-terminal sequences encoded by this gene. Antisera to both peptides recognized a 59 kd protein from human and mouse fibroblasts. p59fyn was phosphorylated in vivo on serine and tyrosine residues and was also myristylated. Furthermore, immune precipitates of p59fyn had tyrosine kinase activity in vitro, as measured by autophosphorylation and by phosphorylation of substrates such as enolase. This kinase activity was shown to be negatively regulated by tyrosine phosphorylation. We have also established that, like pp60c-src and p62c-yes, p59fyn was complexed with middle T antigen, the transforming protein of polyoma virus. However, the tyrosine kinase activity of p59fyn was not elevated in middle T transformed cells. Possible explanations for this are discussed.     

Inositol trisphosphate levels in cells expressing wild-type and mutant polyomavirus middle T antigens: evidence for activation of phospholipase C via activation of pp60c-src.

The transforming protein of polyomavirus, middle T (mT), forms a complex with two cellular enzymes: the protein tyrosine kinase pp60c-src and a phosphatidylinositol (PtdIns) 3-kinase. A mutant virus, Py1178T, encodes an mT protein which associates with and activates pp60c-src to the same extent as the wild type but fails to associate with PtdIns 3-kinase. To investigate relationships between activation of pp60c-src, association of PtdIns 3-kinase, and cellular levels of the second messenger inositol 1,4,5-trisphosphate (InsP3), we examined the effects of wild-type and mutant mT proteins on inositol metabolism in rat and mouse fibroblasts. Expression of either wild-type or 1178T mT caused a 300 to 500% increase in the InsP3 level. Cells transformed by Rous sarcoma virus also showed similar increases in InsP3 levels. Mutant mT proteins which failed to activate pp60c-src (NG59 and 1387T) had no effect on InsP3 levels. Pulse-chase experiments with [3H]inositol showed that the turnover of phosphoinositides was increased in cells transformed by either wild-type polyomavirus or Py1178T as compared with the normal parent cell line. The turnover of inositol phosphates was unchanged upon transformation. These data indicate that cells expressing either wild-type or mutant 1178T mT or pp60v-src exhibit elevated levels of InsP3 because of activation of phospholipase C. This activation appears to depend, directly or indirectly, upon activation of pp60src protein kinase activity. Activation of pp60c-src and elevation of InsP3 content are not sufficient for full transformation. Full transformation also requires the association of mT-pp60c-src complexes with PtdIns 3-kinase.     

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.     

Middle tumor antigen of polyomavirus transformation-defective mutant NG59 is associated with pp60c-src.

We have found that lysis of mouse embryo cells infected with the polyomavirus host range transformation-defective (hr-t) mutant NG59 under gentle conditions that avoid ionic detergents results in detectable NG59-encoded middle tumor antigen (MTAg) associated with pp60c-src. This MTAg-pp60c-src complex could be immunoprecipitated from NG59-infected cell lysates by either sera from animals bearing polyomavirus-induced tumors or by monoclonal antibodies directed against MTAg. Immune complex kinase assays revealed that, whereas the pp60c-src associated with NG59 MTAg possessed tyrosyl kinase activity, the NG59 MTAg in this complex was not phosphorylated in these in vitro reactions. These results demonstrate that the point insertion mutation found in this transformation-deficient strain of polyomavirus encodes MTAg molecules capable of associating with pp60c-src and defines a limited region within MTAg which appears to be critical for stable MTAg-pp60c-src interactions.     

Enhancement of cellular src gene product associated tyrosyl kinase activity following polyoma virus infection and transformation

We examine the interaction between polyoma-virus-encoded middle tumor antigen and the cellular src gene product, pp60c-src, using a series of monoclonal antibodies that recognize mammalian pp60c-src. Our results show that infection of mouse cells with transformation-competent strains of polyoma virus results in the stimulation of pp60c-src kinase activity severalfold over that observed in uninfected mouse cells and mouse cells infected with transformation-deficient polyoma virus. A similar degree of enhancement of pp60c-src kinase activity was found in polyoma-virus-transformed rodent cells. No differences were detected in the level of pp60c-src synthesis in polyoma-virus-infected and uninfected mouse cells or polyoma-virus-transformed and normal rodent cells. These studies demonstrate that polyoma-virus-encoded middle tumor antigen is associated with pp60c-src in lysates of polyoma-virus-infected and polyoma-virus-transformed cells and suggest a novel mechanism for the functional activation of a cellular proto-oncogene product, namely, that the interaction between middle tumor antigen and pp60c-src leads to a stimulation of pp60c-src tyrosyl kinase activity.     

Tyrosine-specific phosphorylation of gpIIIa in platelet membranes

In vitro phosphorylation of platelet subcellular fractions revealed that most of the alkali-resistant phosphoproteins and the majority of pp60c-src were in the surface membrane fraction. An alkali-resistant phosphoprotein of about 100 kDa was also immune precipitated by an anti-phosphotyrosine antibody and comigrated with gpIIIa. The phosphorylation of gpIIIa, but not gpIIb, was confirmed by the comparison of reduced and non-reduced gels, and this protein was phosphorylated exclusively on tyrosine. In contrast, both gpIIb and gpIIIa were phosphorylated when the purified complex was added to immunopurified, immobilised pp60c-src. A synthetic peptide with partial homology to a putative tyrosine phosphorylation site in the cytoplasmic domain of gpIIIa was phosphorylated by antibody-purified pp60c-src. Our results indicate that tyrosine-specific phosphorylation of gpIIIa by pp60c-src may play a role in the regulation of platelet function.     

In vivo effect of sodium orthovanadate on pp60c-src kinase.

We have compared the tyrosine kinase activity of pp60c-src isolated from intact chicken embryo fibroblasts treated with micromolar sodium orthovanadate for 4 h and from untreated cells. We found an approximate 50% reduction in both autophosphorylation of pp60c-src and phosphorylation of casein when examined in the immune complex kinase assay. The reduction of in vitro enzymatic activity correlated with a vanadate-induced increase in in vivo phosphorylation of pp60c-src at the major site of tyrosine phosphorylation in the carboxyl-terminal half of the molecule and at serine in the amino-terminal half of the molecule. Our observations in vivo and those of Courtneidge in vitro (EMBO J. 4:1471-1477, 1985) suggest that vanadate may enhance a cellular regulatory mechanism that inhibits the activity of pp60c-src in normal cells. A likely candidate for this mechanism is phosphorylation at a tyrosine residue distinct from tyrosine 416, probably tyrosine 527 in the carboxyl-terminal sequence of amino acids unique to pp60c-src. The regulatory role, if any, of serine phosphorylation in pp60c-src remains unclear. The 36-kilodalton phosphoprotein, a substrate of pp60v-src, showed a significant phosphorylation at tyrosine after treatment of normal chicken embryo fibroblasts with vanadate. Assuming that pp60c-src is inhibited intracellularly by vanadate, either another tyrosine kinase is stimulated by vanadate (e.g., a growth factor receptor) or the 36-kilodalton phosphoprotein in normal cells is no longer rapidly dephosphorylated by a tyrosine phosphatase in the presence of vanadate.     

Common elements in growth factor stimulation and oncogenic transformation: 85 kd phosphoprotein and phosphatidylinositol kinase activity

The phosphorylation of proteins on tyrosine in vivo and in vitro was examined in 3T3 cells stimulated by platelet-derived growth factor (PDGF) and transformed by polyoma middle T antigen (MTAg) by using an antibody directed against phosphotyrosine (P-tyr). Two common events were observed upon PDGF stimulation or MTAg transformation of cells: the appearance in the immunoprecipitates of an 85 kd phosphoprotein, and increased phosphatidylinositol (PI) kinase activity. In PDGF-stimulated cells, the 85 kd phosphoprotein and PI kinase activity appeared rapidly, within 1 min of growth factor addition. The PI kinase activity and 85 kd phosphorylation were also increased in anti-P-tyr immunoprecipitates from cells transformed by v-fms and v-sis, but not by SV40 T antigen. The presence of the tyrosine-phosphorylated 85 kd protein correlated with PI kinase activity during several purification steps. These results suggest that the 85 kd phosphoprotein, a putative PI kinase, is a substrate for both the PDGF receptor and MTAg/pp60c-src tyrosine kinase activities.     

The complex of polyoma virus middle-T antigen and pp60c-src.

We have recently proposed that the transforming protein of polyoma virus, middle-T antigen, forms a complex with pp60c-src. Here we provide additional evidence for the existence of the complex using both monoclonal antibodies specific for middle-T and antibodies raised against synthetic peptides corresponding to sequences from both middle-T and pp60c-src. The complex was retained during partial purification of middle-T and was stable to incubation under various conditions. A survey of a number of mutants of middle-T antigen showed that there was a complete correlation between the ability of middle-T to accept phosphate in the in vitro kinase reaction and the presence of a middle-T: pp60c-src complex. This result is in accord with our hypothesis that middle-T itself is not a protein kinase but rather that pp60c-src phosphorylates middle-T. All mutant forms of middle-T antigen capable of transformation had associated pp60c-src. The middle-T of two non-transforming mutants (hr-t mutants) did not have associated pp60c-src, whereas other non-transforming middle-T species did associate with pp60c-src. We propose that the complex plays an essential role in transformation by polyoma virus, but that the existence of the complex per se may not be sufficient.