Protein kinase C increases the activity of the polyoma virus middle T antigen-associated phosphatidylinositol kinase

Exposure of polyoma virus-transformed fibroblasts to the protein kinase C-stimulating phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) is known to increase the transforming potential of the virus's middle T antigen. Here it is shown that this TPA treatment also stimulates an 85 kDa phosphatidylinositol kinase associated with the middle T antigen. Since activation of this kinase is known to be necessary, although not by itself sufficient for the transformation of cells by polyoma virus, bursts of protein kinase C activity, triggered by TPA or various cellular receptors, might enhance the oncogenicity of polyoma virus by stimulating this middle T antigen-associated phosphatidylinositol kinase.     

Protein kinase activity associated with polyoma virus middle T antigen in vitro.

A protein kinase activity can be detected in immunoprecipitates of extracts from polyoma virus (Py)-infected cells using antiserum raised against Py-transformed cells (anti-T serum). The activity is not detected in uninfected cells or when using control serum. Using rat anti-T serum both Py middle T and the heavy chain of rat IgG are phosphorylated, whereas using hamster anti-T serum only Py middle T is phosphorylated. Experiments using a number of different mutants of Py indicate that the kinase activity detected is under viral control and is associated with Py middle T. Consistent with this the kinase, like middle T, can be detected in purified preparations of plasma membranes. The kinase can also be detected in a large number of Py-transformed cells, but not in untransformed cells or in cells transformed by other viruses. Some of the Pytransformed cells which contain kinase activity lack full sized Py large T but all contain middle T. Kinase activity is not detected in a cell line (18.37) which contains integrated viral DNA of a nontransforming hr-t deletion mutant and which contains Py large T but not middle T or small t. These results show that Py middle T or a protein which specifically binds to it has protein kinase activity in vitro. Although these results raise the possibility that protein kinases play an essential role in Py-induced transformation, however, thus far we have no data which show unequivocally that the results are physiologically significant.     

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.     

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.     

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.     

Inhibition of polyoma virus middle T antigen-associated tyrosyl kinase activity by N-ethylmaleimide

The middle T antigen (MT Ag) encoded by polyoma virus has an associated protein kinase activity which transfers a phosphoryl group from ATP or GTP to a tyrosine residue on MT Ag in immunoprecipitates formed between polyoma virus-infected or transformed cell extracts and serum from animals bearing polyoma-induced tumors. Incubation of such immunoprecipitates or polyoma-transformed cell extracts prior to immunoprecipitation with the sulfhydryl reagent, N-ethylmaleimide (NEM), resulted in a significant inhibition of MT Ag-associated kinase activity. Inactivation of this enzyme activity by NEM was found to be dependent upon the incubation pH, time of incubation, and NEM concentration. ATP, GTP, and ADP in the presence of Mg2+ were found to decrease the rate of NEM-mediated inactivation of MT Ag-associated kinase activity, while CTP and UTP did not detectably alter the rate of enzyme inhibition by NEM. These results suggest that the MT Ag-associated kinase possesses at least one NEM-sensitive sulfhydryl group essential for phosphotransferase activity which may be present at or near the enzyme catalytic site.     

Protein kinase activities associated with distinct antigenic forms of polyoma virus middle T-antigen

The tyrosine-specific protein kinase activity previously described in T-antigens of polyoma virus immunoprecipitated with anti-tumour sera has been investigated using monoclonal antibodies. This activity is associated with middle T-antigen but it can be separated by selective antibody precipitation from the majority of this protein. The difference between active and inactive forms can be accounted for by an antigenic difference at the N terminus of middle T-antigen molecules. Moreover, the two different mol. wt. forms of middle T-antigen that can act as phosphoacceptors have been separated by antibody precipitation and therefore shown to be immunologically distinct. The binding position of the antibody used for immunoprecipitation has been observed to have a quantitative influence on the in vitro protein kinase reaction, in one case appearing to stimulate the activity. The detection of the in vitro protein kinase activity in immunoprecipitates obtained with several different monoclonal antibodies directed against the middle T-antigen indicates that the activity is a property tightly associated with this polyoma virus-coded protein.     

Expression of biologically active middle T antigen of polyoma virus from recombinant baculoviruses.

Two different recombinant baculoviruses have been generated for expressing the middle T antigen (MT) of polyoma virus in insect (Sf9) cells. One (pAcI-PyMT) produces moderate levels of MT and the other (pVL-PyMT) high levels. Indirect immunofluorescence and cellular fractionation studies with pAcI-PyMT infected Sf9 cells give results similar to those observed with wild type polyoma virus infected mouse cells, and show MT to be mainly associated with cytoplasmic membranes in the insect cell. In the latter, a sub-population of MT is phosphorylated in in vitro protein kinase assays. The yields of MT from pVL-PyMT infected cells are high enough to suggest that this protein can now be produced by this method in sufficient amounts for definitive biochemical and crystallographic analyses.     

Deletion mapping of a short polyoma virus middle T antigen segment important for transformation.

Viable polyoma virus mutants were constructed that had small deletions in the early region of the genome. The deletions together removed most of the segment missing from the genome of the nontransforming mutant dl23 (N. Smolar and B. E. Griffin, J. Virol. 38:958-967, 1981). The transformation properties, as measured by colony formation in soft agar, of mutants with overlapping or contiguous deletions showed that part or all of the middle T antigen segment, consisting of the short amino acid sequence Glu4-Tyr-Met-Pro-Met, was essential for the activity of the protein in transformation. However, the segment could be deleted without significant effect on the in vitro protein kinase activity associated with the middle T antigen.     

Antibody to the nonapeptide Glu-Glu-Glu-Glu-Tyr-Met-Pro-Met-Glu is specific for polyoma middle T antigen and inhibits in vitro kinase activity

Middle T antigen of polyoma virus has an associated tyrosine kinase activity which phosphorylates tyrosine residue 315 on middle T in immunoprecipitates. A peptide representing the sequence of middle T from residue 311 to 319 has been synthesized. This peptide acts as a weak inhibitor of the kinase reaction. An antiserum has been raised against this peptide after conjugation to bovine serum albumin. The antibody is middle T-specific. Middle T antigen precipitated by this serum is largely inactive in the kinase reaction. Dissociation of the immune complex with peptide releases middle T in a kinase-active form.     

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.     

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.     

Cooperation of mitogenic growth factors with polyoma virus middle T antigen in transformation of secondary cultured rat cells

Platelet derived growth factor cooperated with middle T antigen in inducing growth in agarose medium of secondary cultured rat embryo cells transfected with a polyoma virus middle T antigen cDNA clone. In contrast, epidermal growth factor and a conditioned medium containing transforming growth factor did not stimulate the colony-forming efficiency of such cells in the agarose medium.     

Large-scale production of polyoma middle T antigen by using genetically engineered tumors.

A recombinant plasmid containing a metallothionein promoter-polyoma middle T cDNA fusion was constructed and used to transfect NIH 3T3 cells. Transformed cells expressing middle T were injected into nude mice. Within 3 weeks, each mouse produced tumors containing middle T equivalent to that in 250 to 1,000 100-mm dishes of polyomavirus-infected cells. This middle T, partially purified by immunoaffinity chromatography, retained activity as measured by its ability to be phosphorylated in vitro. The combined approach of fusing strong promoters to genes of interest and utilizing nude mice to grow large quantities of cells expressing the gene provides a quick, inexpensive alternative to other expression systems.     

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.     

Infection of CV1 cells expressing the polyoma virus middle T antigen or the SV40 agnogene product with simian virus 40 host-range mutants

Summary SV40 viruses bearing mutations at the carboxy-terminus of large T antigen exhibit a host-range phenotype: such viruses are able to grow in BSC monkey kidney cells at 37° C, but give at least 10 000-fold lower yields than wild type virus in BSC cells at 32° C or in CV1 monkey kidney cells at either temperature. The block to infection in the nonpermissive cell type occurs after the onset of viral DNA replication. Infectious progeny virions are produced at very low efficiency. Although capsid proteins are synthesized at decreased levels, this does not account for the magnitude of the defect. Presumably some step of virion assembly or maturation is affected in these mutants. We have previously reported that the viral agnogene product, a protein throught to be involved in viral assembly or release, fails to accumulate in CV1 cells infected with host-range mutants. In polyoma virus the middle T antigen plays a role in virion maturation by influencing the phosphorylation of capsid proteins. In this communication we show that host-range mutants fail to undergo productive infection of CV1 cells expressing middle T antigen. These mutants do form plaques on an agnoprotein-expressing cell line. However, the agnoprotein does not seem to act by correcting the mutational block but rather increases the efficiency of plaque formation. This work was supported by grants CA40586 and BRSG 2S07RR07084-23 to J. M. P. and grant CA33079 to L. T., from the National Institutes of Health, Bethesda, MD.     

ATP phosphohydrolase (ATPase) activity of a polyoma virus T antigen

Among the various polyoma virus T antigens which have so far been identified, only the large-T and a 63 000-Mr polypeptide were found to bind to double-stranded calf thymus DNA. The proteins were not retained on single-stranded DNA-cellulose columns, and a purification procedure was designed on the basis of this observation. Purified fractions (approx. 1000-fold) exhibited an enzymatic activity which converts ATP into ADP and Pi. This activity was quantitatively inhibited after preincubation in the presence of anti-(polyoma T antigen) immunoglobulins and was shown to be dependent on a virus-coded gene product (alpha gene) on the basis of the following observations: (a) ATPase activity from cells infected with tsa mutants of polyoma was reduced after a shift to the restrictive temperature; (b) the enzyme purified from tsa-infected cells maintained at the permissive temperature was more thermolabile in vitro than that prepared in parallel from cells infected with wild-type virus.