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.     

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.     

Regulation of pp60c-src and its interaction with polyomavirus middle T antigen in insect cells.

High yields of soluble, biologically active pp60c-src and middle t antigen (MTAg) of polyomavirus were produced in insect cells, using a baculovirus expression system. In mammalian cells, pp60c-src undergoes a regulatory phosphorylation on Tyr-527 in vivo and is autophosphorylated on Tyr-416 in vitro. In insect cells, pp60c-src was phosphorylated primarily on Tyr-416, although Tyr-527 was detectable at a low level. A kinase-negative mutant of pp60c-src was not phosphorylated on either Tyr-527 or Tyr-416 in insect cells and thus is an excellent biochemical reagent to search for the regulatory kinase that usually phosphorylates Tyr-527 in mammalian cells. MTAg synthesized in insect cells was not phosphorylated on tyrosine residues in vivo or in vitro, suggesting that it did not associate with any endogenous tyrosine kinases. However, MTAg isolated from cells coinfected with viruses encoding both MTAg and pp60c-src was phosphorylated on tyrosine residues both in vivo and in vitro.     

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.     

Analysis of middle tumor antigen and pp60c-src interactions in polyomavirus-transformed rat cells.

The relative abundance of pp60c-src molecules associated with polyomavirus (Py) middle tumor antigen (MTAg) and the relative abundance of MTAg associated with pp60c-src in a variety of Py-transformed rat cells was determined by quantitative immunoblot analyses which detect pp60c-src or Py MTAg. The results demonstrate that approximately 5 to 10% of the total immunoprecipitable pp60c-src molecules in Py-transformed rat cells are stably associated with MTAg and have elevated protein kinase activities. In these same cells, it was found that approximately 10 to 15% of the detectable MTAg molecules are stably associated with pp60c-src. Other results presented in this report demonstrate that approximately 50 to 75% of the total MTAg-associated cellular tyrosine kinase activity potentially represents the enzymatic activity of pp60c-src, while the remaining 25 to 50% represents the activity of other cellular tyrosine kinases. Our results also show that most pp60c-src molecules associated with Py MTAg do not possess electrophoretic mobilities that are altered from those of pp60c-src molecules not associated with MTAg or pp60c-src molecules obtained from normal rodent cells.     

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.     

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.     

Phosphorylation of polyoma middle T antigen and cellular proteins in purified plasma membranes of polyoma virus-infected cells.

We have studied phosphorylation carried out by purified plasma membranes from polyoma virus-infected cells. When isolated membranes are incubated with [gamma-32P]ATP, polyoma virus middle T antigen (mT) becomes phosphorylated on tyrosine. Partial proteolysis mapping shows the same pattern as previously noted for mT labeled in immune complexes. Membranes labeled in vitro were also extracted and immunoprecipitated with anti-T or anti-src antibody. With either antibody, both mT and pp60c-src were brought down and shown to be labeled on tyrosine. The mT of an hr-t mutant (NG59) showed only a trace amount of labeling in membranes under the same conditions. Proteins from infected and uninfected cell membranes labeled in vitro were separated on two-dimensional gels. An acidic 40-kd phosphoprotein was labeled in uninfected cell membranes, but was not seen using membranes from wild-type virus-infected cells. Neither NG59, which encodes a defective but membrane-associated mT, nor a mutant encoding a truncated mT that fails to associate with membranes, alters the level of the 40-kd phosphoprotein in membranes labeled in vitro. These results suggest that mT, acting through pp60c-src and possibly other cellular kinases and phosphatases, can affect cell protein phosphorylation as part of the transformation process.     

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.     

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.     

Analysis of polyomavirus middle-T-antigen-transformed rat cell variants expressing different levels of pp60c-src.

We characterize two independent variant cellular clones which arose following in vitro passage of polyomavirus middle-T-antigen (MTAg)-transformed FR3T3 cells expressing RNA complementary to c-src mRNA. These clones were initially flat and underwent morphologic transformation at a high frequency to a phenotype indistinguishable from that of parental MTAg-transformed FR3T3 cells. Biochemical analysis of the flat clones prior to phenotypic conversion revealed that these cells synthesized little detectable pp60c-src and had correspondingly low levels of pp60c-src protein kinase activity and MTAg-associated protein kinase activity. The flat cell clones did not possess detectable focus-forming activity, were not capable of detectable anchorage-independent growth, and had saturation densities and doubling times below those normally observed for FR3T3 cells. Following conversion of the flat clones to a shape resembling that of typical MTAg-transformed cells, the abundance of pp60c-src, pp60c-src kinase activity, and MTAg-associated in vitro protein kinase activity were all restored to the levels found in the parental MTAg transformants. These cells had growth rates, focus-forming activities, anchorage-independent growth rates, and saturation densities similar to those of the parental MTAg-transformed rat cells. These data provide additional evidence that maintenance of a transformed phenotype by polyomavirus MTAg in established rat cell lines depends, at least in part, on a minimal threshold level of pp60c-src.     

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.     

Protein kinase C phosphorylates pp60src at a novel site

The transforming protein of Rous sarcoma virus (pp60v-src) and its normal cellular homolog (pp60c-src) are demonstrated to be phosphorylated at serine 12 in vivo under certain conditions. We propose that protein kinase C is responsible for this modification based on the following evidence. First, the tumor promoters, 12-O-tetradecanoylphorbol-13-acetate and teleocidin, and synthetic diacylglycerol, known activators of protein kinase C in vivo, cause nearly complete phosphorylation of pp60src at serine 12. Second, among five purified serine/threonine-specific protein kinases tested, only protein kinase C phosphorylates pp60c-src and pp60v-src in vitro at serine 12. Third, purified protein kinase C phosphorylates a synthetic peptide corresponding to the N-terminal 20 amino acids of pp60c-src at serine 12. The physiological significance of this novel phosphorylation is discussed.     

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.     

Neural tissues express high levels of the cellular src gene product pp60c-src.

Chicken embryo tissues were examined for the expression of pp60c-src, the normal cellular homolog of the transforming protein of Rous sarcoma virus. Three assays, including a solid-phase radioimmunoassay, a competitive radioimmunoprecipitation assay, and an immune complex protein kinase assay, were employed. Elevated levels of pp60c-src were detected in lysates from several neural tissues, including brain, retina, and spinal ganglia. Other tissues contained 8- to 10-fold-lower levels of pp60c-src, levels comparable to those found in chicken embryo fibroblasts. Expression of pp60c-src in brain tissues was also shown to vary with the developmental stage of the embryo.     

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.     

Dephosphorylation or antibody binding to the carboxy terminus stimulates pp60c-src.

Phosphorylation of pp60c-src at Tyr-527, six residues from the carboxy terminus, has been implicated in regulation of the protein-tyrosine kinase activity of pp60c-src. Here we show that dephosphorylation of pp60c-src by phosphatase treatment in vitro caused a 10- to 20-fold increase in pp60c-src protein-tyrosine kinase activity. Binding of specific antibody to the region of pp60c-src which contains phosphotyrosine-527 also increased kinase activity. Each treatment increased phosphorylation of added substrates and of Tyr-416 within pp60c-src by a similar mechanism that involved altered interactions with ATP and increased catalytic rate. We suggest that the phosphorylated carboxy terminus acts as an inhibitor of the protein kinase domain of pp60c-src, unless its conformation is altered by either dephosphorylation or antibody binding. The antibody additionally stimulated the phosphorylation of forms of pp60c-src that had reduced gel mobility, much like those phosphorylated in kinase reactions containing pp60c-src activated by polyomavirus medium tumor antigen. These in vitro experiments provide models for the activation of pp60c-src in cells transformed by polyomavirus. We also show that autophosphorylation of pp60c-src at Tyr-527 occurs only to a very limited extent in vitro, even when Tyr-527 is made available for phosphorylation by treatment with phosphatase. This suggests that other protein-tyrosine kinases may normally phosphorylate Tyr-527 and regulate pp60c-src in the cell.     

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.     

The carboxy terminus of pp60c-src is a regulatory domain and is involved in complex formation with the middle-T antigen of polyomavirus.

A large number of mutations were introduced into the carboxy-terminal domain of pp60c-src. The level of phosphorylation on Tyr-416 and Tyr-527, the transforming activity (as measured by focus formation on NIH 3T3 cells), kinase activity, and the ability of the mutant pp60c-src to associate with the middle-T antigen of polyomavirus were examined. The results indicate that Tyr-527 is a major carboxy-terminal element responsible for regulating pp60c-src in vivo. A good but not perfect correlation exists between lack of phosphorylation at Tyr-527 and increased phosphorylation at Tyr-416, between elevated phosphorylation on Tyr-416 and activated kinase activity, and between activated kinase activity and transforming activity. Phosphorylation of Tyr-527 was insensitive to the mutation of adjacent residues, indicating that the primary sequence only has a minor role in recognition by kinases or phosphatases which regulate it in vivo. Three mutants which have in common a modified Glu-524 residue were phosphorylated on Tyr-416 and Tyr-527 and were weakly transforming. This suggests that other mechanisms besides complete dephosphorylation of Tyr-527 can lead to increased phosphorylation of Tyr-416 and activation of the transforming activity of pp60c-src. Furthermore, the residues between Asp-518 and Pro-525 were required to form a stable complex with middle-T antigen. The proximity of these sequences to Tyr-527 suggests a model in which middle-T activates pp60c-src by binding directly to this region of the molecular and thereby preventing phosphorylation of Tyr-527. Alternatively, middle-T binding may mediate a conformational change in this region, which in turn induces an alteration in the level of phosphorylation at Tyr-527 and Tyr-416.