scholarly journals Polyomavirus middle T antigen induces ribosomal protein S6 phosphorylation through pp60c-src-dependent and -independent pathways.

1988 ◽  
Vol 8 (6) ◽  
pp. 2309-2315 ◽  
Author(s):  
D A Talmage ◽  
J Blenis ◽  
T L Benjamin
Keyword(s):  
Plasma Membrane ◽  
Ribosomal Protein ◽  
Kinase Activity ◽  
T Antigen ◽  
Wild Type ◽  
S6 Kinase ◽  
Ribosomal Protein S6 ◽  
Infected Cells ◽  
Polyomavirus Middle T Antigen ◽  
Middle T Antigen

Phosphorylation of ribosomal protein S6 is elevated in polyomavirus-infected cells. This elevation results only in part from activation of S6 kinase activity. These effects appear to reflect independent activities of wild-type middle T antigen. Hr-t mutant NG59, encoding a defective middle T protein, and mutant Py808A, encoding no middle T protein, were unable to induce S6 kinase activity or elevate S6 phosphorylation. Two other site-directed mutants encoding altered middle T proteins did elevate S6 phosphorylation while only weakly stimulating S6 kinase activity. These results suggest at least two independent pathways leading to elevation of S6 phosphorylation. One pathway leads to induction of S6 kinase activity following activation of pp60c-src by transformation-competent middle T antigen. Another pathway operates independently of S6 kinase induction and can be regulated by transformation-defective middle T mutants such as Py1387T. This mutant, encoding a truncated middle T protein that failed to associate with the plasma membrane and to activate pp60c-src, caused increased levels of S6 phosphorylation without detectably increasing S6 kinase activity. The ability of mutants such as Py1387T to induce S6 phosphorylation correlated with their ability to increase phosphorylation of VP1, an event linked to maturation of infectious virions.

Polyomavirus middle T antigen induces ribosomal protein S6 phosphorylation through pp60c-src-dependent and -independent pathways

1988 ◽  
Vol 8 (6) ◽  
pp. 2309-2315
Author(s):  
D A Talmage ◽  
J Blenis ◽  
T L Benjamin
Keyword(s):  
Plasma Membrane ◽  
Ribosomal Protein ◽  
Kinase Activity ◽  
T Antigen ◽  
Wild Type ◽  
S6 Kinase ◽  
Ribosomal Protein S6 ◽  
Infected Cells ◽  
Polyomavirus Middle T Antigen ◽  
Middle T Antigen

Phosphorylation of ribosomal protein S6 is elevated in polyomavirus-infected cells. This elevation results only in part from activation of S6 kinase activity. These effects appear to reflect independent activities of wild-type middle T antigen. Hr-t mutant NG59, encoding a defective middle T protein, and mutant Py808A, encoding no middle T protein, were unable to induce S6 kinase activity or elevate S6 phosphorylation. Two other site-directed mutants encoding altered middle T proteins did elevate S6 phosphorylation while only weakly stimulating S6 kinase activity. These results suggest at least two independent pathways leading to elevation of S6 phosphorylation. One pathway leads to induction of S6 kinase activity following activation of pp60c-src by transformation-competent middle T antigen. Another pathway operates independently of S6 kinase induction and can be regulated by transformation-defective middle T mutants such as Py1387T. This mutant, encoding a truncated middle T protein that failed to associate with the plasma membrane and to activate pp60c-src, caused increased levels of S6 phosphorylation without detectably increasing S6 kinase activity. The ability of mutants such as Py1387T to induce S6 phosphorylation correlated with their ability to increase phosphorylation of VP1, an event linked to maturation of infectious virions.

Polyomavirus middle T antigen downregulates junctional cell-to-cell communication

1987 ◽  
Vol 7 (2) ◽  
pp. 946-950
Author(s):  
R Azarnia ◽  
W R Loewenstein
Keyword(s):  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Recombinant Dna ◽  
Cell Communication ◽  
T Antigen ◽  
Conditional Mutant ◽  
F Cells ◽  
Junctional Permeability ◽  
Polyomavirus Middle T Antigen ◽  
Middle T Antigen

We examined the effect of polyomavirus middle T antigen on cell-to-cell communication in rat F cells transfected with an inducible middle T recombinant DNA or infected with a conditional mutant virus. Junctional permeability fell (reversibly) when middle T transcription was induced or when middle T was switched to the transformation+ form. The effect correlates with the rise of protein tyrosine kinase activity.

scholarly journals Retrovirus shuttle vector for study of kinase activities of pp60c-src synthesized in vitro and overproduced in vivo.

1986 ◽  
Vol 6 (6) ◽  
pp. 2033-2040 ◽  
Author(s):  
H Piwnica-Worms ◽  
D R Kaplan ◽  
M Whitman ◽  
T M Roberts
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Shuttle Vector ◽  
T Antigen ◽  
Protein Kinase Activity ◽  
Polyomavirus Middle T Antigen ◽  
Nih 3T3 ◽  
Middle T Antigen

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.

scholarly journals Serine 257 Phosphorylation Regulates Association of Polyomavirus Middle T Antigen with 14-3-3 Proteins

1998 ◽  
Vol 72 (1) ◽  
pp. 558-563 ◽  
Author(s):  
Xavier Culleré ◽  
Paul Rose ◽  
Usha Thathamangalam ◽  
Alakananda Chatterjee ◽  
Karen P. Mullane ◽  
...  
Keyword(s):  
Phosphorylation Site ◽  
T Antigen ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Major Site ◽  
Reporter Assays ◽  
Polyomavirus Middle T Antigen ◽  
Cycle Regulation ◽  
Middle T Antigen

ABSTRACT Polyomavirus middle T antigen (MT) is phosphorylated on serine residues. Partial proteolytic mapping and Edman degradation identified serine 257 as a major site of phosphorylation. This was confirmed by site-directed mutagenesis. Isoelectric focusing of immunoprecipitated MT from transfected 293T cells showed that phosphorylation on wild-type MT occurred at near molar stoichiometry at S257. MT was previously shown to be associated with 14-3-3 proteins, which have been connected to cell cycle regulation and signaling. The association of 14-3-3 proteins with MT depended on the serine 257 phosphorylation site. This has been demonstrated by comparing wild-type and S257A mutant MTs expressed with transfected 293T cells or with Sf9 cells infected with recombinant baculoviruses. The 257 site is not critical for transformation of fibroblasts in vitro, since S257A and S257C mutant MTs retained the ability to form foci or colonies in agar. The tumor profile of a virus expressing S257C MT showed a striking deficiency in the induction of salivary gland tumors. The basis for this defect is uncertain. However, differences in activity for the wild type and mutant MT lacking the 14-3-3 binding site have been observed in transient reporter assays.

scholarly journals Evidence of a Role for Phosphatidylinositol 3-Kinase Activation in the Blocking of Apoptosis by Polyomavirus Middle T Antigen

1998 ◽  
Vol 72 (4) ◽  
pp. 3221-3226 ◽  
Author(s):  
Jean Dahl ◽  
Annmarie Jurczak ◽  
Linda A. Cheng ◽  
David C. Baker ◽  
Thomas L. Benjamin
Keyword(s):  
T Antigen ◽  
Wild Type Virus ◽  
Temperature Sensitive ◽  
Type Virus ◽  
Wild Type ◽  
Phosphatidylinositol 3 Kinase ◽  
Tumor Viruses ◽  
Polyomavirus Middle T Antigen ◽  
Middle T Antigen ◽  
Phosphatidylinositol 3

ABSTRACT A polyomavirus mutant (315YF) blocked in binding phosphatidylinositol 3-kinase (PI 3-kinase) has previously been shown to be partially deficient in transformation and to induce fewer tumors and with a significant delay compared to wild-type virus. The role of polyomavirus middle T antigen-activated PI 3-kinase in apoptosis was investigated as a possible cause of this behavior. When grown in medium containing 1d-3-deoxy-3-fluoro-myo-inositol to block formation of 3′-phosphorylated phosphatidylinositols, F111 rat fibroblasts transformed by wild-type polyomavirus (PyF), but not normal F111 cells, showed a marked loss of viability with evidence of apoptosis. Similarly, treatment with wortmannin, an inhibitor of PI 3-kinase, stimulated apoptosis in PyF cells but not in normal cells. Activation of Akt, a serine/threonine kinase whose activity has been correlated with regulation of apoptosis, was roughly twofold higher in F111 cells transformed by either wild-type virus or mutant 250YS blocked in binding Shc compared to cells transformed by mutant 315YF. In the same cells, levels of apoptosis were inversely correlated with Akt activity. Apoptosis induced by serum withdrawal in Rat-1 cells expressing a temperature-sensitive p53 was shown to be at least partially p53 independent. Expression of either wild-type or 250YS middle T antigen inhibited apoptosis in serum-starved Rat-1 cells at both permissive and restrictive temperatures for p53. Mutant 315YF middle T antigen was partially defective for inhibition of apoptosis in these cells. The results indicate that unlike other DNA tumor viruses which block apoptosis by inactivation of p53, polyomavirus achieves protection from apoptotic death through a middle T antigen–PI 3-kinase–Akt pathway that is at least partially p53 independent.

Interleukin 2- and polyomavirus middle T antigen-induced modification of phosphatidylinositol 3-kinase activity in activated T lymphocytes

1991 ◽  
Vol 11 (9) ◽  
pp. 4431-4440
Author(s):  
J A Augustine ◽  
S L Sutor ◽  
R T Abraham
Keyword(s):  
T Cells ◽  
T Lymphocytes ◽  
Kinase Activity ◽  
Interleukin 2 ◽  
T Antigen ◽  
Threefold Increase ◽  
Activated T Lymphocytes ◽  
Polyomavirus Middle T Antigen ◽  
Middle T Antigen ◽  
Stimulation Of

Stimulation of activated T lymphocytes with interleukin 2 (IL-2) results in rapid increases in intracellular protein tyrosine phosphorylation. Both the identity of the protein tyrosine kinase (PTK) activated by IL-2 receptor ligation and the identities of the critical target proteins for this PTK remain largely undefined. In this article, we demonstrate that stimulation of activated murine or human T cells with IL-2 for 10 to 30 min induces two- to threefold increases in the level of phosphatidylinositol (PtdIns) 3-kinase activity present in antiphosphotyrosine (p-Tyr) antibody immunoprecipitates from these cells. Furthermore, substantial levels of PtdIns 3-kinase activity were coprecipitated from IL-2-deprived T cells by antibodies to the src-related PTK p59fyn. Cellular stimulation with IL-2 induced a two- to threefold increase in the level of p59fyn-associated PtdIns 3-kinase activity. To examine the effect of a constitutive increase in PtdIns 3-kinase activity on the growth factor responsiveness of activated T cells, murine CTLL-2 cells were transfected with a polyomavirus middle T antigen (MTAg) expression vector. Anti-p-Tyr and anti-p59fyn immunoprecipitates from MTAg-transfected CTLL-2 cells contained three- to sixfold higher levels of PtdIns 3-kinase activity than wild-type cells. Immune complex kinase assays revealed that MTAg expression concomitantly induced a constitutive threefold increase in the PTK activity of p59fyn in these cells. However, stable MTAg expression did not abrogate the dependence of CTLL-2 cells on exogenous IL-2 for continued growth and proliferation.

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