Amino-terminal regions of polyomavirus middle T antigen are required for interactions with protein phosphatase 2A.

Polyomavirus middle T antigen (MT) is the major transforming protein of the virus. It functions through interactions with a number of cellular proteins involved in cell proliferation. MT forms complexes with protein phosphatase 2A (PP2A), pp60c-src, phosphatidylinositol 3-kinase, and Shc. We introduced both deletion and point mutations into three regions of MT and examined their ability to associate with PP2A and pp60c-src. The first 25 amino acid residues of MT are required for association with PP2A and pp60c-src. Amino acids 105 to 111, comprising the sequence Cys-Arg-Met-Pro-Leu-Thr-Cys, is also required for complex formation between MT and PP2A. However, the sequence Asp-Lys-Gly-Gly (amino acids 44 to 47), also found in the B subunit of PP2A, is dispensable for complex formation between MT and PP2A. We find a strict correlation between the ability of MT to associate with PP2A and the ability of MT to associate with pp60c-src. One mutant, L5E, associates with a phosphatase other than PP2A, pp60c-src, and phosphatidylinositol 3-kinase in a manner similar to that of wild-type MT yet is reduced in its transforming ability on NIH 3T3 cells.     

Catalytically Inactive Protein Phosphatase 2A Can Bind to Polyomavirus Middle Tumor Antigen and Support Complex Formation with pp60c-src

Interaction between the heterodimeric form of protein phosphatase 2A (PP2A) and polyomavirus middle T antigen (MT) is required for the subsequent assembly of a transformation-competent MT complex. To investigate the role of PP2A catalytic activity in MT complex formation, we undertook a mutational analysis of the PP2A 36-kDa catalytic C subunit. Several residues likely to be involved in the dephosphorylation mechanism were identified and mutated. The resultant catalytically inactive C subunit mutants were then analyzed for their ability to associate with a cellular (B subunit) or a viral (MT) B-type subunit. Strikingly, while all of the inactive mutants were severely impaired in their interaction with B subunit, most of these mutants formed complexes with polyomavirus MT. These findings indicate a potential role for these catalytically important residues in complex formation with cellular B subunit, but not in complex formation with MT. Transformation-competent MT is known to associate with, and modulate the activity of, several cellular proteins, including pp60(c-src) family kinases. To determine whether association of MT with an active PP2A A-C heterodimer is necessary for subsequent association with pp60(c-src), catalytically inactive C subunits were examined for their ability to form complexes containing pp60(c-src) in MT-expressing cells. Two catalytically inactive C subunit mutants that efficiently formed complexes with MT also formed complexes that included an active pp60(c-src) kinase, demonstrating that PP2A activity is not essential in cis in MT complexes for subsequent pp60(c-src) association.     

pp60c-src binding to polyomavirus middle T-antigen (MT) requires residues 185 to 210 of the MT sequence.

Interaction with the src family of tyrosine kinases is crucial to the transforming action of polyomavirus middle T-antigen (MT). Association with MT activates the tyrosine kinase activity of pp60(c-src) and, through subsequent MT phosphorylation, creates binding sites for signalling molecules whose stimulation culminates in cell transformation. Despite this importance, and many studies, little is known of the mechanisms by which pp60(c-src) binds to MT. We report here isolation of the first MT mutants that disrupt pp60(c-src) binding without affecting the interaction between MT and protein phosphatase 2A (PP2A). Through deletion analysis we established that interaction with pp60(c-src) requires the sequences between amino acids 185 and 210 of MT, but these residues have no effect on PP2A binding. Cells expressing these mutants showed few altered properties, indicating that the PP2A-MT interaction alone has little influence on cell phenotype. Subcellular location of these mutant MT molecules was indistinguishable by immunofluorescence analysis from that of wild-type MT but was altered markedly on loss of PP2A binding. This suggests a possible role for PP2A in specifying subcellular distribution.     

Identification of regions in polyomavirus middle T and small t antigens important for association with protein phosphatase 2A.

Two subunits of protein phosphatase 2A (PP2A) have been shown previously to bind to the small t and middle T antigens (ST and MT, respectively) of polyomavirus. To determine sequences important for binding of PP2A to ST and MT, we first constructed a series of ST mutants in regions known to be important for biological activity of ST and MT. Several mutations in two small regions just amino terminal to the Cys-X-Cys-X-X-Cys motifs of ST and MT abolished PP2A binding to ST in vitro. Parallel mutations were constructed in MT to investigate the role of PP2A binding in the function of polyomavirus MT. Wild-type and mutant MT proteins were stably expressed in NIH 3T3 cells and analyzed (i) for their ability to induce transformation and (ii) for associated cellular proteins and corresponding enzymatic activities previously described as associating with wild-type MT. A number of the mutant MTs were found to be defective in binding of PP2A as assayed by coimmunoprecipitation. In contrast, a deletion of the highly conserved stretch of amino acids 42 to 47 (His-Pro-Asp-Lys-Gly-Gly) in the ST-MT-large T antigen common region did not affect PP2A binding to MT. MT mutants defective for PP2A binding were also defective in transformation, providing further evidence that association with PP2A is important for the ability of MT to transform cells. All mutants which were impaired for PP2A binding were similarly or more dramatically impaired for associated protein and lipid kinase activities, supporting the possibility that PP2A binding is necessary for the formation and/or stability of an MT-pp60c-src complex.     

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.     

Mutation of amino acids in pp60c-src that are phosphorylated by protein kinases C and A.

The product of the c-src proto-oncogene, pp60c-src, is phosphorylated at Ser-17 by cyclic AMP-dependent protein kinase A and at Ser-12 by calcium-phospholipid-dependent protein kinase C (when stimulated by 12-O-tetradecanoyl phorbol acetate). We tested the effects of Ser----Ala and Ser----Glu mutations at these sites in pp60c-src and in pp60c-src(F527) (a mutant whose transforming activities are enhanced by Tyr-527----Phe mutation) by transfecting single-, double-, and triple-mutant src expression plasmids into NIH 3T3 cells. Tryptic phosphopeptide analyses of the mutant proteins confirmed prior biochemical identifications of the phosphorylation sites and showed that neither separate nor coordinate mutations at Ser-12 and Ser-17 affected Tyr-416, Tyr-527, or Ser-48 phosphorylation or prevented mitosis-specific phosphorylations of either pp60c-src or pp60c-src(F527). Ser-12 mutation did not affect phosphorylation of the Ser-17-containing peptide, but mutation of Ser-17 significantly increased phosphorylation at Ser-12. Specific kinase activities (both with and without in vivo 12-O-tetradecanoyl phorbol acetate treatment) and the abilities of pp60c-src and pp60c-src(F527) to induce foci, transformed morphologies, and anchorage-independent growth were unaffected by any of the serine mutations. Thus, pp60c-src transforming activity in NIH 3T3 cells is relatively insensitive to phosphorylation at these sites, but there is a suggestion that Ser-17 phosphorylation may have a subtle regulatory effect.     

Myristylation of pp60c-src is not required for complex formation with polyomavirus middle-T antigen.

Middle-T antigen (middle-T), the transforming gene product of polyomavirus, associates with several cellular tyrosine kinases, such as pp60c-src. Complex formation leads to kinase activation and is essential for cell transformation. Middle-T-associated as well as uncomplexed pp60c-src is predominantly found in the plasma membrane. We transfected mouse 3T3 fibroblasts with a mutated c-src gene (2Ac-src), allowing the expression of a protein containing alanine instead of glycine in position 2 of the primary translation product. Contrary to the wild-type c-src gene product, pp60c-src(2A) was not myristylated and accumulated in the cytoplasm instead of being transferred to cellular membranes. The mutant protein was able to associate with middle-T and was activated similarly to the wild-type c-src gene product. Both wild-type and 2A mutant protein were membrane associated upon complex formation with middle-T. This finding suggests that the putative carboxy-terminal membrane anchor sequence of middle-T is sufficient to hold middle-T-associated pp60c-src(2A) in the plasma membrane.     

The sites of phosphorylation by protein kinase C and an intact SH2 domain are required for the enhanced response to beta-adrenergic agonists in cells overexpressing c-src.

Previously we demonstrated that C3H10T1/2 murine fibroblasts overexpressing avian c-src exhibit elevated levels of cyclic AMP (cAMP) in response to beta-adrenergic agonists compared with that in control cells and that this enhanced response requires c-src kinase activity (W. A. Bushman, L. K. Wilson, D. K. Luttrell, J. S. Moyers, and S. J. Parsons, Proc. Natl. Acad. Sci. USA 87:7462-7466, 1990). However, it is not yet known which components of the beta-adrenergic receptor pathway, if any, interact with pp60c-src. It has recently been shown that immune complexes of pp60c-src phosphorylate recombinant G alpha proteins in vitro to stoichiometric levels, resulting in alterations of GTP binding and GTPase activity (W. P. Hausdorff, J. A. Pitcher, D. K. Luttrell, M. E. Linder, H. Kurose, S. J. Parsons, M. G. Caron, and R. J. Lefkowitz, Proc. Natl. Acad. Sci. USA 89:5720-5724, 1992), raising the possibility that the Gs alpha protein may be an in vivo target for the interaction with pp60c-src. To further characterize the involvement of pp60c-src in the beta-adrenergic signalling pathway, we have overexpressed, in 10T1/2 cells, pp60c-src containing mutations in several domains which are believed to be important for signalling processes. In this study we show that the sites of phosphorylation by protein kinase C (PKC) (Ser-12 and Ser-48) as well as the SH2 region of pp60c-src are required for the enhanced response of c-src overexpressors to beta-agonist stimulation. Mutation at the site of myristylation (Gly-2) results in a decrease in the enhanced response, while mutation at the site of phosphorylation by cAMP-dependent protein kinase (Ser-17) has no effect. Two-dimensional phosphotryptic analyses indicate that phosphorylation on Ser-12 and Ser-48 in unstimulated cells is associated with the ability of overexpressed pp60c-src to potentiate beta-adrenergic signalling. Cells overexpressing wild-type c-src also exhibit enhanced cAMP accumulation upon treatment with cholera toxin, an effect that is abated in cells overexpressing pp60c-src defective in the kinase or SH2 domains or altered at the sites of phosphorylation by PKC. These studies provide the first evidence for the physiological significance of the pp60c-src sites of PKC phosphorylation. In addition, they show that the SH2, Ser-12/48, and myristylation regions may be important for efficient interaction of pp60c-src with components of the beta-adrenergic pathway. Our data also support the possibility that the Gs alpha protein may be an in vivo target for alteration by pp60c-src.     

Transformation-defective mutants of polyomavirus middle T antigen associate with phosphatidylinositol 3-kinase (PI 3-kinase) but are unable to maintain wild-type levels of PI 3-kinase products in intact cells.

Middle T antigen (MT) of polyomavirus causes transformation by associating with a number of cellular proteins. The association with and activation of two such proteins, phosphatidylinositol 3-kinase (PI 3-kinase) and pp60c-src, appears to be necessary for transformation by MT. The tyrosine kinase activity of MT-associated pp60c-src is significantly increased when assayed in vitro, and levels of phosphotyrosine-containing proteins are elevated in vivo. Similarly, levels of the PI 3-kinase products phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] and phosphatiylinositol-3,4,5-trisphosphate [PI(3,4,5)P3] are constitutively elevated in MT-transformed cells. However, the formation of a complete MT/cellular protein complex and the activation of tyrosine kinase are not sufficient to cause transformation, since the transformation-defective mutants 248m and dl1015 associate with all wild-type MT-associated proteins, including PI 3-kinase and pp60c-src, and neither mutant appears to be defective in MT-associated tyrosine kinase activity. Studies presented here compared (i) the amount of PI 3-kinase activity associated with the MT complex and (ii) levels of [3H]inositol incorporation into PI 3-kinase products in cells expressing mutant or wild-type MT. The results show that dl1015 is defective in both assays, whereas 248m is defective only for incorporation of [3H]inositol into PI(3,4,5)P2 and PI(3,4)P3. These findings identify a biochemical defect in the 248m mutant and corroborate previous results correlating transformation and elevated levels of PI 3-kinase products in vivo. In addition, they indicate that PI 3-kinase product levels are affected by factors other than simply the amount of PI 3-kinase activity associated with the MT complex.     

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 the hamster polyomavirus middle T antigen.

Hamster polyomavirus (HaPV) is associated with lymphoid and hair follicle tumors in Syrian hamsters. The early region of HaPV has the potential to encode three polypeptides (which are related to the mouse polyomavirus early proteins) and can transform fibroblasts in vitro. We identified the HaPV middle T antigen (HamT) as a 45-kDa protein. Like its murine counterpart, HamT was associated with serine/threonine phosphatase, phosphatidylinositol-3 kinase, and protein tyrosine kinase activities. However, whereas mouse middle T antigen associates predominantly with pp60c-src and pp62c-yes, HamT was associated with a different tyrosine kinase, p59fyn. The ability of HaPV to cause lymphoid tumors may therefore reside in its ability to associate with p59fyn, a potentially important tyrosine kinase in lymphocytes.     

Signaling from Polyomavirus Middle T and Small T Defines Different Roles for Protein Phosphatase 2A

Polyomavirus causes a broad spectrum of tumors as the result of the action of its early proteins. This work compares signaling from middle T antigen (MT), the major transforming protein, to that from small T antigen (ST). The abilities of MT mutants to promote cell cycle progression in serum-starved NIH 3T3 cells were compared. Transformation-defective mutants lacking association with SHC or with phosphatidylinositol 3-kinase (PI3-K) retained the ability to induce DNA synthesis as measured by bromodeoxyuridine incorporation. Only when both interactions were lost in the Y250F/Y315F double mutant was MT inactive. ST promoted cell cycle progression in a manner dependent on its binding of protein phosphatase 2A (PP2A). Since the Y250F/Y315F MT mutant was wild type for PP2A binding yet unable to promote cell cycle progression, while ST was capable of promoting cell cycle progression, these experiments revealed a functional difference in MT and ST signaling via PP2A. Assays testing the abilities of MT and ST to induce the c-fos promoter and to activate c-jun kinase led to the same conclusion. ST, but not Y250F/Y315F MT, was able to activate the c-fos promoter through its interaction with PP2A. In contrast, MT, but not ST, was able to activate c-jun kinase by virtue of its interaction with PP2A.     

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.     

pp60c-src tyrosine kinase, myristylation, and modulatory domains are required for enhanced mitogenic responsiveness to epidermal growth factor seen in cells overexpressing c-src.

In previous studies examining the potential role of pp60c-src in cellular proliferation, we demonstrated that C3H10T1/2 murine embryo fibroblasts overexpressing transfected chicken genomic c-src displayed an epidermal growth factor (EGF)-induced mitogenic response which was 200 to 500% of the response exhibited by parental control cells (Luttrell et al., Mol. Cell. Biol. 8:497-501, 1988). In order to examine specific structural and functional requirements for pp60c-src in this event, 10T1/2 cells were transfected with chicken c-src genes encoding pp60c-src deficient in tyrosine kinase activity (pm430), myristylation, (pm2A), or a domain hypothesized to modulate the interaction with substrates or regulatory components (dl155). Neomycin-resistant clonal cell lines overexpressing each of the mutated c-src genes were assayed for EGF mitogenic responsiveness by measuring [3H]thymidine incorporation into acid-precipitable material or into labeled nuclei. The results were compared with those obtained with lines overexpressing the cDNA form of wild-type (wt) c-src or control cells transfected with the neomycin resistance gene only. As previously described for cells overexpressing wt genomic c-src (Luttrell et al., 1988), clones overexpressing wt cDNA c-src also exhibited enhanced EGF mitogenic responses ranging from approximately 300 to 400% of the control cell response. In contrast, clones overexpressing unmyristylated, modulation-defective, or kinase-deficient c-src not only failed to support an augmented response to EGF but also exhibited EGF responses lower than that of the control cells. Furthermore, there were no significant differences in the mitogenic responses to 10% fetal calf serum among any of the cells tested. These results indicate that pp60(c-scr) can potentiate mitogenic signaling generated by EGF but not all growth factors. This potentiation requires the utilization of pp60(c-scr) myristylation, and modulatory and tyrosine kinase domains and can me mediated by cDNA-encoded as well as by genome-encoded wt pp60(c-scr).     

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.     

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.     

Analysis of pp60c-src tyrosine kinase activity and phosphotyrosyl phosphatase activity in human colon carcinoma and normal human colon mucosal cells

We have compared the level of phosphotyrosyl phosphatase activity in lysates from normal human colon mucosal cells and human colon carcinoma cells and analyzed the effect of incubating these cells with sodium orthovanadate, an inhibitor of phosphotyrosyl phosphatase activity, on the relative abundance of acid-stable phosphotyrosine and on in vitro protein kinase activity of pp60c-src. Additionally, we compared the effect of lysing these cells in buffer containing only nonionic detergents with RIPA buffer, which contains both sodium dodecyl sulfate and deoxycholate, on the in vitro kinase activity of pp60c-src. Our results show that the level of detectable phosphotyrosyl phosphatase activity in lysates derived from normal colon cells and colon carcinoma cells is very similar. Additionally, the abundance of acid-stable phosphotyrosine in these cells cultured in the absence or presence of vanadate is not significantly different. However, incubation of these cells with vanadate significantly stimulates the activity of pp60c-src derived from the normal colon cells in immune-complex kinase assays, while having no detectable effect on the activity of pp60c-src from the colon tumor cells. The in vitro protein kinase activity of pp60c-src derived from RIPA buffer lysates of colon carcinoma cells was found to be elevated five- to sevenfold when compared with pp60c-src from these same cells lysed in buffer containing only Nonidet-P 40 as a detergent. The type of lysis buffer did not effect the activity of pp60c-src from normal colon mucosal cells. These results provide additional evidence that the activity of pp60c-src may be regulated differently in colon carcinoma and normal colon mucosal cells.     

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.     

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.