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.     

Stimulation of pp60c-src tyrosyl kinase activity in polyoma virus-infected mouse cells is closely associated with polyoma middle tumor antigen synthesis

We have examined the effect of polyoma virus infection of primary mouse embryo cells on the tyrosyl kinase activity associated with the cellular src gene product, pp60c-src. The results of our studies demonstrate that infection of mouse cells with wild-type polyoma virus or viral mutants capable of transforming rodent cells in culture and inducing tumors in animals results in the stimulation of pp60c-src tyrosyl kinase activity. The level of pp60c-src kinase stimulation in infected cells was found to be proportional to both the oncogenic potential of the virus strain used for infection and the characteristic phenotype of rodent cells transformed by the various strains of polyoma virus. Stimulation of pp60c-src kinase activity was not observed in mouse cells infected with transformation-defective strains of polyoma virus. In examining the kinetics of pp60c-src kinase stimulation in mouse cells at various times following wild-type polyoma virus infection, we found that the level of pp60c-src kinase activity correlated directly with the synthesis of polyoma virus-encoded tumor antigens. By comparing wild-type polyoma virus with other viral mutants in these experiments, we conclude that the stimulation of pp60c-src kinase activity in mouse cells following polyoma virus infection is associated with the synthesis of middle tumor antigen.     

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.     

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.     

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.     

An 81 kd protein complexed with middle T antigen and pp60c-src: a possible phosphatidylinositol kinase

It has previously been shown that a proportion of middle T antigen molecules exist in a stable complex with pp60c-src. Here we show that there appears to be a third component to the complex, a protein of molecular mass 81 kd (p81). p81 was phosphorylated exclusively on tyrosine residues in kinase assays performed using immunoprecipitates from polyoma virus-transformed cells and antibodies to both middle T and pp60c-src, and was also detected when immunoprecipitates were made from lysates of 32P-labeled cells. p81 was bound to middle T and pp60c-src in cell lines containing transforming mutants of middle T, but not (in phosphorylated form) to all nontransforming mutants. A parallel investigation of phosphatidylinositol kinase activity in immune complexes containing these middle T mutants revealed a complete coincidence between the presence of p81 and phosphatidylinositol kinase activity. We therefore suggest that p81 is a phosphatidylinositol kinase.     

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.     

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.     

Purified polyoma virus medium T antigen has tyrosine-specific protein kinase activity but no significant phosphatidylinositol kinase activity.

Medium T antigen, the transforming protein of polyoma virus, is associated with pp60c-src and strongly activates its tyrosine-specific protein kinase activity. We investigated whether the medium T-pp60c-src complex is also associated with an activity that phosphorylates the membrane phospholipid phosphatidylinositol, as shown for pp60v-src and p68v-ros, the transforming proteins of Rous sarcoma virus and avian sarcoma virus UR2, respectively. Medium T was purified by affinity chromatography from extracts of polyoma virus-infected mouse fibroblasts. It was bound to antibodies against a peptide corresponding to the carboxy terminus of medium T and released from the immune complex with an excess of the same peptide. In a second step, the partially purified medium T was bound to antibodies against another peptide corresponding to an internal region of medium T and released with excess peptide. Further purification was carried out with a monoclonal antibody against pp60c-src. Samples from each purification step were examined for protein kinase and phosphatidylinositol kinase activity. The highly purified preparations of the medium T-pp60c-src complex showed very low levels of phosphatidylinositol kinase activity, and no difference between medium T from transforming viruses and nontransforming hr-t mutants was detected. In contrast, protein kinase activity was associated with medium T purified from transforming viruses but not from hr-t mutants.     

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.     

Cooperation of p53 and polyoma virus middle T antigen in the transformation of primary rat embryo fibroblasts.

Cell transformation in vivo seems to be a multistep process. In in vitro studies certain combinations of two oncogenes, a cytoplasmic gene product together with a nuclear gene product, are sufficient to transform primary rodent cells. Polyoma virus large T antigen can immortalize and, in cooperation with polyoma virus middle T antigen, transform primary cells. On the other hand mutant mouse p53 can also immortalize and, in cooperation with an activated Ha-ras oncogene, transform primary cells. In the present study we analyzed whether mutant p53 can replace polyoma virus large T antigen in a cell transformation assay with polyoma virus middle T antigen. Transfection of mutant p53 alone resulted in a cell line which had retained the actin cable network, grew poorly in medium with low concentration of serum, and failed to grow in semisolid agar. Cotransfection of mutant p53 together with polyoma virus middle T led to cells which grew in medium containing low serum concentration, grew well in semisolid agar, and displayed an altered morphology with the tendency to overgrow the normal monolayer. By these criteria these cells were considered fully transformed. The rate of p53 synthesis was similar in both cell lines. However, only p53 from the transformed cell line turned out to be stable. Cells transformed by mutant p53 and polyoma virus middle T expressed nearly the same amount of the c-src-encoded pp60c-src protein as cells transformed by the same p53 and cotransfected activated Ha-ras oncogene. However, only the polyoma virus middle T/p53-transformed cells exhibited an elevated level of pp60c-src-specific tyrosine kinase activity. Thus, despite different mechanisms leading to cell transformation, mutant p53 can replace polyoma virus large T antigen and polyoma virus middle T can replace the activated Ha-ras oncogene in cell transformation.     

Simultaneous overexpression of avian pp60c-src and polyomavirus middle T antigen in mammalian cells.

Recombinant adenoviruses bearing the avian c-src gene and polyomavirus middle-T-antigen gene were isolated and used to simultaneously overexpress both proteins in human 293 cells. Cells overexpressing both proteins had greater middle-T-antigen-associated tyrosine kinase activity than cells overexpressing only middle T antigen. By contrast, the intrinsic pp60c-src tyrosine kinase activity was not greater in cells overexpressing both proteins than in cells overexpressing only pp60c-src. This system of simultaneous overexpression provides a means of obtaining large quantities of pp60c-src, middle T antigen, and the complex between them.     

Mechanisms of transformation by polyoma virus middle T antigen

This review addresses a fundamental question of polyoma virus biology: What is the molecular mechanism by which the polyoma virus middle T antigen (MTAg) transforms cells in culture? Since MTAg has no known intrinsic biochemical activity, it is believed to act by modulating the properties of the host cell's proteins (see review by Courtneidge [26]). Experiments to date have largely focused on the interaction of MTAg with the cellular tyrosine kinase, pp60c-src. However, recent data from a number of laboratories have demonstrated the importance of other MTAg-associating cellular proteins in MTAg-mediated transformation, including pp62c-yes and a phosphatidylinositol kinase. In this review, we will summarize what is presently known about the proteins interacting with MTAg. The extent to which the currently known details of the biochemistry of MTAg and its associated proteins can explain the transforming properties of the various mutant alleles of MTAg will be assessed.     

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.     

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.     

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.     

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.     

The amino terminus of polyomavirus middle T antigen is required for transformation.

In polyomavirus-transformed cells, pp60c-src is activated by association with polyomavirus middle T antigen. These complexes have a higher tyrosine kinase activity compared with that of unassociated pp60c-src. Genetic analyses have revealed that the carboxy-terminal 15 amino acids of pp60c-src and the amino-terminal half of middle T antigen are required for this association and consequent activation of the tyrosine kinase. To define in greater detail the borders of the domain in middle T antigen required for activation of pp60c-src, we constructed a set of unidirectional amino-terminal deletion mutants of middle T antigen. Analysis of these mutants revealed that the first six amino acids of middle T antigen are required for it to activate the kinase activity of pp60c-src and to transform Rat-1 fibroblasts. Analysis of a series of insertion and substitution mutants confirmed these observations and further revealed that mutations affecting the first four amino acids of middle T antigen reduced or abolished its capacity to activate the kinase activity of pp60c-src and to transform Rat-1 cells in culture. Our results suggest that the first four amino acids of middle T antigen constitute part of a domain required for activation of the pp60c-src tyrosyl kinase activity and for consequent cellular transformation.     

Phosphatidylinositol 3-phosphate is present in normal and transformed fibroblasts and is resistant to hydrolysis by bovine brain phospholipase C II

The transforming protein of polyoma virus, middle T antigen, associates with two cellular enzymes, pp60c-src, a protein tyrosine kinase, and a phosphatidylinositol kinase that forms phosphatidylinositol 3-phosphate. The formation of a ternary complex of these proteins is essential for complete transformation and maximal tumor induction by the virus. A mutant virus encoding an altered middle T protein that activates pp60c-src but fails to bind phosphatidylinositol kinase is partially defective in transformation. We have confirmed, using an enzymological method, that the product of the in vitro reaction catalyzed by middle T-pp60c-src-phosphatidylinositol kinase complexes is phosphatidylinositol 3-phosphate (PtdIns(3)P), as previously reported (Whitman, M., Downes, C. P., Keeler, M., Keller, T., and Cantley, L. (1988) Nature 332, 644-646). PtdIns(3)P is present in normal as well as virus-infected and transformed cells at levels ranging from 0.6 to 2.6% of the major phosphatidylinositol phosphate isomer, phosphatidylinositol 4-phosphate (PtdIns(4)P). Steady-state levels of PtdIns(3)P do not appear to be affected by the expression of middle T in cells. PtdIns(3)P is not hydrolyzed by bovine brain phospholipase C II, which readily cleaves PtdIns(4)P and other phosphatidylinositols. This result underscores the likelihood that the metabolism of PtdIns(3)P is distinct from that of PtdIns(4)P and raises further questions regarding a possible role of PtdIns(3)P in normal and neoplastic cell growth.