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.     

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.     

Requirement for the C-terminal region of middle T-antigen in cellular transformation by polyoma virus

Deletions of polyoma virus DNA around the region that codes for the C-terminus of the viral middle T-antigen were created using a transforming fragment (BamH I/EcoR I) of viral DNA cloned in the plasmid vector pAT153. These species were recloned and assayed for their ability to transform Rat-1 cells in culture. Our results showed that whereas the DNA sequence between the presumed translational termination codon for the viral middle T-antigen and the single viral EcoR I site could be removed with no apparent effect on transformation, the removal of the termination codon itself or any amino acid coding sequences of this protein caused a drastic decrease in the transforming ability of the DNA. Transfection of Rat-1 cells with plasmids that contained viral DNA with deletions which corresponded to the last fourteen or more amino acids of the middle T-antigen never gave rise to cellular 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.     

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 in immune complexes of simian virus 40 large T-antigen and transformation-associated cellular p53 protein.

Immune complex kinase assays in the simian virus 40 system were performed by incubation of immunoprecipitates containing tumor antigens with [gamma-32P]ATP, followed by analysis of any phosphoacceptor proteins. These assays yielded mainly the viral large T-antigen and, in particular, the associated cellular p53 as endogenous substrates. The nature of these substrates was confirmed by proteolysis techniques. Under specific conditions, casein could be used as an exogenous substrate as well. The kinase reactions showed preference for ATP and MgCl2 instead of GTP or MnCl2. Both phosphoserine and phosphothreonine, but in no case phosphotyrosine, were detected after an immune complex kinase reaction. Apparently, several in vivo phosphorylation sites were recognized in vitro in both large T-antigen and p53, but the presence of some artifactual sites could not be completely excluded. Although contaminating kinases were detectable in the immune complexes, at least the p53 molecules were phosphorylated in vitro in a more specific way. This followed from several characteristics of the immune complex kinase reactions and especially from the strong inhibition of p53 phosphorylation by two anti-large-T monoclonal antibodies. It was shown that large T-antigen showed associated kinase activity, although none of our results could unambiguously demonstrate an intrinsic kinase activity of this protein. Finally, anti-p53 monoclonal antibodies only slightly affected in vitro phosphorylation reactions, whereas a p53 molecule from a simian virus 40-free, chemically transformed human cell line was not phosphorylated in vitro under any condition tested. Thus, it is highly unlikely that the p53 molecule per se carries intrinsic or even associated kinase activities.     

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.     

T-antigen expression by polyoma mutants with modified RNA splicing

Polyoma virus mutants were constructed that could not express all the three T-antigens. The mutagenesis was directed to the two 5' splice sites utilized in the maturation of early RNA. The mutant bc1051 had a base change at the splice site of large T-antigen mRNA, and the mutants dl1061 and dl1062 had deletions at the corresponding splice point of small and middle T-antigen mRNA. The site was removed in mutant dl1061 and altered by fusion to upstream sequences in mutant dl1062. Analysis of viral RNA showed that dl1061 and dl1062 formed only large T-antigen mRNA, whereas bc1051 did not produce this RNA-species. However, the biological properties of dl1062 suggested that it also produced mRNA directing the synthesis of a small T-antigen-related polypeptide, at least in low amounts. Only mutant bc1051 could induce transformation of rat cells. In mouse 3T3 cells dl1062 multiplied to a limited extent, while bc1051 and dl1061 failed to produce virus. However, dl1061 DNA was synthesized at a low rate which could be increased to normal levels by co-transfection with mutant bc1051. This result suggests that polyoma small and middle T-antigen have a previously unrecognized function in the early phase of the infection process, or in viral DNA-synthesis.     

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.     

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.     

Protein kinase associated with ribosomes of streptomycetes

Protein kinases can be classified into two main superfamilies on the basis of their sequence similarity and substrate specificity. The protein His kinase superfamily which autophosphorylate a His residue, and superfamily Ser/Thr and Tyr protein kinases, which phosphorylate Ser, Thr or Tyr residues. During the last years genes encoding Ser/Thr protein kinases have been identified in several microorganisms. Phosphorylation of proteins on Ser/Thr residues can be involved in many functions of prokaryotic cells including cell differentiation, signal transduction and protein biosynthesis. Phosphorylation of prokaryotic protein-synthesizing systems showed that the phosphorylation of initiation and elongation factors is subject to alteration during cell differentiation or bacteriophage infection. Protein kinase associated with ribosomes of streptomycetes phosphorylate the elongation factor Tu and 11 ribosomal proteins even in bacteriophage-uninfected cells. After phosphorylation of ribosomal proteins, ribosomes lose about 30% of their activity at the translation of poly(U). Presented at theSymposium on Regulation of Translation of Genetic Information by Protein Phosphorylation, 21st Congress of the Czechoslovak Society for Microbiology, Hradec Králové (Czech Republic), September 6–10, 1998.     

Enzyme activities associated with an invertebrate iridovirus: protein kinase activity associated with iridescent virus type 6 (chilo iridescent virus)

Iridescent virus type 6 was found to contain an endogenous protein kinase activity which can phosphorylate some viral proteins and exogenous basic proteins. The enzyme required a divalent metal ion but was not stimulated by cyclic nucleotides. Procedures which are known to solubilize the viral envelope indicated that the protein kinase was an internal component of the virion. Conditions for protein kinase activity are described.     

Protein kinase activities associated with actin cytoskeleton in the oocytes and eggs of African clawed frog

Protein phosphorylation with specific protein kinases plays the key role in the regulation of meiotic maturation of oocytes. However, little is known about the contribution of kinases to the temporal and positional regulation of the cytoskeleton rearrangement in maturing oocytes, including the actin cytoskeleton. In order to study a relationship between the kinase activities and actin cytoskeleton rearrangement, we analyzed protein phosphorylation in the isolated actin cytoskeleton of Xenopus laevis oocytes. Analysis of the full grown oocytes and eggs injected with [gamma-32P] "P has revealed phosphorylation of many proteins associated with the actin cytoskeleton and shown the appearance of three additional major phosphoproteins, 20, 43, and 69 kDa, during oocyte maturation. A significant number of these phosphoproteins were also found after incubation of the isolated cytoskeleton with [gamma-32P] "P in vitro, thus confirming that the kinases modifying these substrates are also specifically associated with actin. The in vivo and in vitro kinase activities were also stimulated during maturation. Analysis of kinase self-phosphorylation in situ and protein phosphorylation in solutions and substrate containing gels revealed a set of actin-associated kinases, including cAMP- and Ca(2+)-dependent kinases, as well as MAP, p34cdc2, and tyrosine kinase activities. Their level was the highest in the eggs. The involvement of kinases in the actin cytoskeleton rearrangement during oocyte maturation is discussed.     

Subcellular localisation of the middle and large T-antigens of polyoma virus.

The distribution of two of the polyoma virus early proteins (the large and middle T-antigens) in lytically infected mouse cells and transformed rat cells has been investigated by indirect immunofluorescence and immuno-electron microscopy using well-characterised monoclonal antibodies. By these techniques, the viral large T-antigen was found almost exclusively in the nucleus, sometimes in association with nuclear pores, but never in the nucleolus. In lytically infected, but not transformed cells, fluorescence was detected in discrete areas ('hot spots') within the nucleus and, in a minor population of lytically infected cells, cytoplasmic immunoreactive material was observed. The viral middle T-antigen was found in association with most cytoplasmic membranes and in the majority of cells mainly in the endoplasmic reticulum. Only a fraction of the staining was observed in the plasma membrane and no staining in the nucleoplasm was observed. The data suggest that the site of action of the major transforming activity of polyoma virus need not be at the plasma membrane. Functions associated with the viral antigens are discussed in terms of their subcellular distributions within cells.     

Protein kinase activities in Neurospora crassa

Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) has been purified from human erythrocytes using a simple chromatographic procedure. Purified enzyme was obtained from individuals who were homozygous for the principal isozyme (ADA 1) as well as from individuals who were heterogyzous for the major variant (ADA 2-1). Although ADA 1 and ADA 2-1 are electrophoretically distinguishable, they have many common physical and catalytic properties. No significant differences between the two isozymic forms were found in measurements of molecular weight, catalytic activity in the presence of various substrates and inhibitors, pH optimum, turnover number, and stability in conditions of both high and low pH. ADA 2-1 was, however, substantially less stable than ADA 1 with respect to thermal denaturation. These studies support the idea that adenosine deaminase activity in erythrocytes is lower in those individuals who possess the variant form of the enzyme.     

Mutational analysis of simian virus 40 T-antigen primosome activities in viral DNA replication

The recruitment of DNA polymerase alpha-primase (pol-prim) is a crucial step in the establishment of a functional replication complex in eukaryotic cells, but the mechanism of pol-prim loading and the composition of the eukaryotic primosome are poorly understood. In the model system for simian virus 40 (SV40) DNA replication in vitro, synthesis of RNA primers at the origin of replication requires only the viral tumor (T) antigen, replication protein A (RPA), pol-prim, and topoisomerase I. On RPA-coated single-stranded DNA (ssDNA), T antigen alone mediates priming by pol-prim, constituting a relatively simple primosome. T-antigen activities proposed to participate in its primosome function include DNA helicase and protein-protein interactions with RPA and pol-prim. To test the role of these activities of T antigen in mediating priming by pol-prim, three replication-defective T antigens with mutations in the ATPase or helicase domain have been characterized. All three mutant proteins interacted physically and functionally with RPA and pol-prim and bound ssDNA, and two of them displayed some helicase activity. However, only one of these, 5030, mediated primer synthesis and elongation by pol-prim on RPA-coated ssDNA. The results suggest that a novel activity, present in 5030 T antigen and absent in the other two mutants, is required for T-antigen primosome function.     

Multiple forms of polyoma virus tumor antigens from infected and transformed cells.

At least three distinct forms of polyoma virus tumor antigens were isolated from productively infected and transformed hamster cells by immunoprecipitation with anti-T serum. These proteins had approximate molecular weights of 105,000 (large T antigen), 63,000 (middle T antigen), and 20,000 (small T antigen) as estimated by acrylamide gel electrophoresis. An examination of the appearance of these antigens in polyoma-infected mouse cells showed that all three polypeptides were synthesized maximally at approximately the same time after infection. Analysis of the methionine-containing tryptic peptides of these proteins indicated that the large, middle, and small forms of polyoma T antigens contained five similar or identical peptides. In addition, the 63,000- and 20,000-dalton antigens contained two other methionine peptides absent from the large T-antigen species. Other methionine peptides were found only in the large or middle T-antigen forms. These results and results obtained previously suggested that the three T-antigen species have the same NH2-terminal end regions but different COOH termini. A model is presented describing the synthesis of these polypeptides from different regions of the polyoma virus genome.