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.     

Membrane antigens associated with infection,transformation, and tumorigenesis by polyoma virus

Conclusion Infection, transformation, or tumorigenesis by Py virus leads inter alia to modifications in the membrane antigens of the affected cells. The modifications include antigenic gains or losses or quantitative changes in both directions. Although there is a pronounced common denominator in the antigenic alterations in the three distinct Py-induced biological processes, it is nontheless possible that each of them is characterized by specific antigenic modifications. This possibility has yet to be analyzed. Specific antigenic modifications, if they occur, are probably the result of different selective processes and adaptions to these pressures.In this brief review, we have attempted to survey the literature pertinent to this aspect. While doing so, we discovered that most researchers have not considered the possibility that differences could exist between antigens of cells infected by Py, cells transformed by this virus, and Py-induced tumor cells. We feel that a comprehensive antigenic comparison between these cells utilizing well-defined reagents is an essential prerequisite to understanding of the successful immunological surveillance against Py-induced malignancy.Incumbent: The David Furman Chair of Cancer Immunobiology     

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.     

Characterization of protein kinase activity associated with the transforming gene product of Fujinami sarcoma virus

Fujinami sarcoma virus (FSV), a newly characterized avian sarcoma virus, produces a protein of 140,000 daltons (p140) in infected cells. p140 is the product of a fused gene consisting of a part of the gag gene of avian retrovirus and FSV-unique sequences which are not related to the src sequences of Rous sarcoma virus. In vivo, p140 was found to be phosphorylated at both serine and tyrosine residues. Immunoprecipitates of p140 with antiserum against gag gene-coded proteins had a cyclic nucleotide-independent protein kinase activity which phosphorylated p140 itself, rabbit IgG of the immune complex and alpha-casein, an externally added soluble protein substrate. The phosphorylation was specific to tyrosine of the substrate proteins. p140 was phosphorylated in vitro at the same two tyrosine residues that were phosphorylated in vivo. The phosphate transferred to tyrosine residues of p140 forms a stable bond: it does not turn over during the kinase reaction, and the 32P-phosphate of p140 labeled in vitro or in vivo is not transferred to alpha-casein. FSV-p140 differs from p60src, the transforming protein of Rous sarcoma virus, in its marked preference of Mn2+ to Mg2+ ions, and in its inability to use GTP instead of ATP as the donor of gamma-phosphate.     

Amino acid alterations within a highly conserved region of the Rous sarcoma virus src gene product pp60src inactivate tyrosine protein kinase activity.

Bisulfite mutagenesis techniques have been used to introduce single-point mutations within a region of the Rous sarcoma virus src gene defined by a BglI restriction endonuclease cleavage site. The mutants of Rous sarcoma virus that are produced by these techniques encode src proteins which contain single amino acid changes within a highly conserved amino acid sequence encompassing residues 430 to 433. DNA from the mutants CHpm26 ( Ala430 to Val), CHpm9 ( Pro431 to Ser), CHpm6 ( Glu432 to Lys), and CHpm65 ( Ala433 to Thr) each failed to transform chicken cells upon transfection, whereas DNA from CHpm59 (a third base alteration in the codon for Glu432 ) readily transformed chicken cells. Analysis of immune complexes containing the altered src proteins indicates that these proteins have decreased tyrosine protein kinase activity in vitro. In vivo labeling of cells infected with the mutant virus revealed diminished levels of the tyrosine-phosphorylated 34,000-molecular-weight protein. These data indicate that mutations within the sequence Ala430 - Pro431 - Glu432 - Ala433 lead to alterations in pp60src-specific tyrosine protein kinase activity and a concomitant loss of transforming potential of the mutant virus.     

Evidence that the Rous sarcoma virus transforming gene product is associated with glycerol kinase activity

This communication provides biochemical, immunological, and genetic evidence that pp60src, the Rous sarcoma virus transforming gene product, is associated with glycerol kinase activity. Our investigations demonstrated that the compound phosphorylated by pp60src or by glycerol kinase (EC 2.7.1.30) from Candida mycoderma share the same electrophoretic and chromatographic mobilities. The glycerol kinase and protein kinase activities of pp60src were inhibited similarly by preincubation with immune IgG. Both activities were reduced 6-9-fold in pp60src preparations derived by immunoaffinity chromatography from cells which were infected with NY68, a temperature-sensitive transformation mutant of Rous sarcoma virus. The thermolability at 41 degrees C of the glycerol kinase activity of pp60src from the mutant virus-infected cells was greater (t/2 = 1.3 min) than the same activity in pp60src preparations from wild type virus-infected cells (t/2 = 4.8 min).     

Small deletion in src of Rous sarcoma virus modifying transformation phenotypes: identification of 207-nucleotide deletion and its smaller product with protein kinase activity.

Partial deletion in the src gene and the gene product were characterized in a deletion mutant, dl5, isolated from the Prague strain of Rous sarcoma virus. The mutant induced fusiform-like transformed cells, unlike the parental Prague strain, which induced round transformed cells. Determination of the total nucleotide sequences of src in dl5 and the Prague strain of Rous sarcoma virus demonstrated that in the former two deletions of 196 and 11 nucleotides had occurred at positions 403 and 696, respectively, from the 5' end of src. A protein with a molecular weight of 52,000 (p52src) was detected in cells infected with dl5, as predicted from the deletion size in src. From the nucleotide sequence, it was predicted that p52src had two deletions of 65 and 4 amino acids at positions 135 and 232, respectively, from the N-terminal methionine of p60src and also had 33 amino acid changes between these two deletion sites due to alteration of the reading frame. p52src, which contained deletions and alterations of amino acids near the N-terminus, showed protein kinase activity similar to that of p60src and functioned in the infected cells. These results strongly suggest that changes in the N-terminal region of p60src modified its transforming ability, causing induction of the fusiform-like transformation phenotype.     

Half-life of the Rous sarcoma virus transforming protein pp60src and its associated kinase activity.

The half-life of metabolically labeled pp60src of the Prague A strain of Rous sarcoma virus and of several transformation-defective, temperature-sensitive mutants was investigated by pulse-labeling infected cells with [35S]methionine, chasing for different times, and immunoprecipitating pp60src with tumor-bearing rabbit serum. These experiments showed that pp60src has a short half-life of approximately 60 min under normal physiological conditions and that the mutant pp60src proteins have similar half-lives to the wild type, irrespective of whether the cells are kept at the nonpermissive (42 degrees C) or permissive (35 degrees C) temperature. The half-life of the pp60src -associated kinase activity was determined by monitoring its decay by the immunoglobulin G heavy chain assay after the cells had been treated with several inhibitors of protein synthesis. In these experiments the kinase half-life was much longer than expected from the half-life of pp60src. The apparent contradiction between the half-lives of the kinase activity and the [35S]methionine-labeled pp60src protein could be resolved by the observation that treatment of cells with inhibitors of protein synthesis stabilized pp60src, resulting in a greatly extended half-life. Inhibitors of protein synthesis also extended the half-life of the gag precursor polypeptide, Pr76, suggesting that a host factor(s) may be required for the efficient intracellular processing of this polypeptide to the gag proteins.     

Site-directed point mutation in the src gene oF rous sarcoma virus results in an inactive src gene product

Site-directed mutagenesis techniques were used to construct defined point mutations within the src gene of the Prague A strain of Rous sarcoma virus. Bisulfite mutagenesis at a Bg/I restriction site in the src gene yielded three mutations which contained the same single base change, a guanine-to-adenine transition. The resulting genomes encoded an src protein containing a substitution of threonine for alanine at amino acid position 433. Transfection of chicken cells with mutagenized DNA did not result in cellular transformation even though the cells produced a pp60src. Immune complexes containing mutant pp60src did not phosphorylate immunoglobulin G heavy chain or casein.     

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.     

Encapsidation and expression of the herpes thymidine kinase gene in polyoma virus.

A recombinant DNA of 5,150 base pairs was prepared containing the intact early region of polyoma virus, including the viral origin of replication and the structural sequences of the herpes simplex virus type 1 thymidine kinase gene. Although no thymidine kinase activity was detected when herpes structural sequences alone were transfected into cells, activity was produced when the structural gene followed the polyoma early region. The recombinant DNA was encapsidated into polyoma virions when cotransfected into mouse 3T6 cells with helper DNA from an early polyoma virus mutant. Herpes thymidine kinase activity was detected by a rapid in situ autoradiographic assay in which [125]iododeoxycytidine was utilized as a substrate for the viral but not the cellular enzyme.     

Product of in vitro translation of the Rous sarcoma virus src gene has protein kinase activity.

In vitro translation of Rous sarcoma virus virion RNA resulted in the synthesis of a protein kinase which, when immunoprecipitated with antitumor serum, phosphorylated the immunoglobulin heavy chain. Even though in vitro translation of virion RNA resulted in the synthesis of a number of polypeptides which were recognized by antitumor serum, control experiments demonstrated that an immunoprecipitable protein kinase activity was found only when an immunoprecipitable p60src, the polypeptide product of the src gene, was synthesized. A protein kinase with similar properties was therefore intimately associated with p60src which was synthesized in vitro in the reticulocyte lysate, just as it is with p60src which is obtained from transformed chick and mammalian cells. It is therefore highly unlikely that this association is artifactual. ts NY68 is a mutant of Rous sarcoma virus which is able to transform cells at 36 but not at 41 degrees C. In vitro translation of ts NY68 virion RNA at 30 degrees C resulted in efficient synthesis of immunoprecipitable p60src, but very inefficient synthesis of an immunoprecipitable protein kinase. The p60src obtained by in vitro translation of wild-type virion RNA was more than 20-fold more active as a protein kinase than was that obtained from ts NY68 RNA. The correlation in the case of ts NY68 of a deficiency in protein kinase activity with an inability to transform cells at high temperature suggests that the protein kinase activity associated with p60src is indeed critical to cellular transformation.     

Enhancement of the interferon-induced double-stranded RNA-dependent protein kinase activity by Sindbis virus infection and heat-shock stress

In extracts of FL cells that were infected with Sindbis virus or treated with heat-shock stress, dsRNA-dependent phosphorylation of 77K protein was markedly increased. The 77K phosphoprotein was indistinguishable from the autophosphorylated and activated form of interferon (IFN)-induced dsRNA-dependent protein kinase (PK-I) by two-dimensional gel electrophoresis, and was immunologically related to P68 (Galabru, J. and Hovanessian, A., J. Biol. Chem. 262, 15538 (1987], the HeLa cell counterpart of PK-I. Immunoblotting experiments using monoclonal antibody against PK-I revealed that control cell extracts contained a substantial amount of PK-I protein, although they showed no measurable PK-I activity even when dsRNA was added. The amount of PK-I protein did not increase during a transient dsRNA-dependent enhancement of PK-I activity caused by Sindbis virus infection and heat-shock stress. This implies that the conversion of PK-I protein from a dsRNA-unresponsive form to a responsive form may be important in the regulation of PK-I activity. A similar mode of PK-I regulatory mechanism was operative in the early stages of IFN treatment, although after a prolonged treatment a net synthesis of the PK-I protein did take place.     

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.     

Mutants of Fujinami sarcoma virus which are temperature sensitive for cellular transformation and protein kinase activity.

Two temperature-sensitive mutants of Fujinami sarcoma virus were isolated and characterized. Cells infected with the mutants were temperature sensitive in focus formation, colony formation, increased sugar uptake, and synthesis of plasminogen activator. The changes between transformed and nontransformed states of cultures were completely reversible by shifting the temperature. A Fujinami sarcoma virus-specific protein of 130,000 daltons, p130, was synthesized in mutant-infected cells regardless of the temperature, but the immunoprecipitates of p130 from extracts of infected cells were active in protein kinase only when cells had been incubated at the permissive temperature. These results appear to indicate that p130 is the transforming protein of Fujinami sarcoma virus, and that its protein kinase activity plays a crucial role in cell transformation by this virus.