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.     

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.     

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 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.     

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.     

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.     

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.     

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.     

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.     

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.     

Location of sites in human lipocortin I that are phosphorylated by protein tyrosine kinases and protein kinases A and C

Lipocortin I is a 39-kilodalton membrane-associated protein that in A431 cells is phosphorylated on tyrosine in response to epidermal growth factor (EGF). We have used recombinant human lipocortin I as a substrate for several protein kinases and identified phosphorylated residues by a combination of peptide mapping and sequence analysis. Lipocortin I was phosphorylated near the amino terminus at Tyr-21 by recombinant pp60c-src. The same tyrosine residue was phosphorylated by polyoma middle T/pp60c-src complex, by recombinant pp50v-abl, and with A431 cell membranes by the EGF receptor/kinase. The primary site of phosphorylation by protein kinase C was also near the amino terminus at Ser-27. The major site of phosphorylation by adenosine cyclic 3',5'-phosphate dependent protein kinase was on the carboxy-terminal half of the molecule at Thr-216. These sites are compared to the phosphorylation sites previously located in the structurally related protein lipocortin II.     

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.     

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.     

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.     

Retrovirus shuttle vector for study of kinase activities of pp60c-src synthesized in vitro and overproduced in vivo.

We have constructed a recombinant murine retrovirus which efficiently transduces avian pp60c-src into murine cells and which is easily rescued from infected cells in plasmid form. To characterize the virus, several randomly selected NIH 3T3 lines were isolated after infection with recombinant retroviral stocks. All lines overproduced avian pp60c-src and appeared morphologically normal. Immunoprecipitates made from these lines with antisera specific for pp60c-src were tested for their kinase activities in vitro. We find that both autokinase and enolase kinase activities increase proportionately with the level of pp60c-src in the immunoprecipitates. To further test the authenticity of the pp60c-src encoded by the retroviral vector, these analyses were repeated in the presence of polyomavirus middle T antigen. Avian pp60c-src was activated as a protein kinase, indicating that the virally encoded pp60c-src interacts normally with middle T antigen. Interestingly, by increasing the intracellular levels of pp60c-src 15-fold over normal endogenous levels, we were unable to obtain a proportionate increase in the amount of middle-T-antigen-pp60c-src complex. Finally, using the shuttle features designed into the vector, we have isolated the first fully processed cDNA encoding functional avian pp60c-src X pp60c-src synthesized in vitro with this cDNA had intrinsic protein kinase activity and no detectable phosphatidylinositol kinase activity.     

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.     

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.     

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.