scholarly journals Transformation by Polyomavirus Middle T Antigen Involves a Unique Bimodal Interaction with the Hippo Effector YAP

2016 ◽  
Vol 90 (16) ◽  
pp. 7032-7045 ◽  
Author(s):  
Cecile Rouleau ◽  
Arun T. Pores Fernando ◽  
Justin H. Hwang ◽  
Nathalie Faure ◽  
Tao Jiang ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
T Antigen ◽  
Tumor Formation ◽  
Ras Signaling ◽  
Hippo Signaling ◽  
Control Mechanisms ◽  
Positive Contribution ◽  
Activation Functions ◽  
Wild Type ◽  
Middle T Antigen

ABSTRACTMurine polyomavirus has repeatedly provided insights into tumorigenesis, revealing key control mechanisms such as tyrosine phosphorylation and phosphoinositide 3-kinase (PI3K) signaling. We recently demonstrated that polyomavirus small T antigen (ST) binds YAP, a major effector of Hippo signaling, to regulate differentiation. Here we characterize YAP as a target of middle T antigen (MT) important for transformation. Through a surface including residues R103 and D182, wild-type MT binds to the YAP WW domains. Mutation of either R103 or D182 of MT abrogates YAP binding without affecting binding to other signaling molecules or the strength of PI3K or Ras signaling. Either genetic abrogation of YAP binding to MT or silencing of YAP via short hairpin RNA (shRNA) reduced MT transformation, suggesting that YAP makes a positive contribution to the transformed phenotype. MT targets YAP both by activating signaling pathways that affect it and by binding to it. MT signaling, whether from wild-type MT or the YAP-binding MT mutant, promoted YAP phosphorylation at S127 and S381/397 (YAP2/YAP1). Consistent with the known functions of these phosphorylated serines, MT signaling leads to the loss of YAP from the nucleus and degradation. Binding of YAP to MT brings it together with protein phosphatase 2A (PP2A), leading to the dephosphorylation of YAP in the MT complex. It also leads to the enrichment of YAP in membranes. Taken together, these results indicate that YAP promotes MT transformation via mechanisms that may depart from YAP's canonical oncogenic transcriptional activation functions.IMPORTANCEThe highly conserved Hippo/YAP pathway is important for tissue development and homeostasis. Increasingly, changes in this pathway are being associated with cancer. Middle T antigen (MT) is the primary polyomavirus oncogene responsible for tumor formation. In this study, we show that MT signaling promotes YAP phosphorylation, loss from the nucleus, and increased turnover. Notably, MT genetics demonstrate that YAP binding to MT is important for transformation. Because MT also binds PP2A, YAP bound to MT is dephosphorylated, stabilized, and localized to membranes. Taken together, these results indicate that YAP promotes MT transformation via mechanisms that depart from YAP's canonical oncogenic transcriptional activation functions.

scholarly journals Role of the Three Polyoma Virus Early Proteins in Tumorigenesis

1983 ◽  
Vol 3 (8) ◽  
pp. 1451-1459 ◽  
Author(s):  
Claude Asselin ◽  
Celine Gelinas ◽  
Marcel Bastin
Keyword(s):  
Cultured Cells ◽  
Virus Genome ◽  
T Antigen ◽  
Polyoma Virus ◽  
Transformed Cells ◽  
Newborn Rats ◽  
Wild Type ◽  
Complementary Function ◽  
Small T Antigen ◽  
Middle T Antigen

A modified polyoma virus genome which can encode the middle T protein but not the large or small T proteins transforms rat cells in culture with an efficiency about 20% that of the wild-type genome. Although middle T-transformed cells grow as tumors when transplanted into nude mice or syngeneic rats, the middle T gene alone is totally inactive when used in a more stringent and rigorous assay for tumorigenicity such as the injection of DNA into newborn rats. Thus, functions other than those expressed by middle T antigen are required for the elaboration of all the properties associated with tumorigenesis. To assess whether a complementary function could be exerted by the large or the small T antigen, we constructed plasmids containing two modified early regions which independently encoded middle T and one of the two other proteins. Both recombinants were tumorigenic in newborn rats. Cell lines derived by transfer of these plasmids under no special selective conditions did not acquire the property of growth in low-serum medium but exhibited the same tumorigenic properties as wild-type polyoma DNA-transformed cells. Furthermore, a recombinant which encoded the middle and small T antigens, but not the large T antigen, was tumorigenic in newborn rats. Although the small T antigen provides a complementary function for tumorigenicity, it cannot complement the middle T antigen for an efficient induction of transformation of cultured cells. This suggests that the complementary function exerted by the small T antigen is different from that of the N-terminal fragment of the large T protein.

scholarly journals PDTM-29. STRUCTURE FUNCTION AND CELL TYPE DETERMINANTS OF C11orf95-RELA DRIVEN TUMORS IN MICE

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi193-vi193
Author(s):  
Jesse Dunnack ◽  
Ericka Randazzo ◽  
Jahmique Caines ◽  
Jame He ◽  
Joseph LoTurco
Keyword(s):  
Nuclear Localization ◽  
Transcriptional Activation ◽  
Point Mutations ◽  
Cell Types ◽  
Tumor Formation ◽  
Wild Type ◽  
Nuclear Localization Signals ◽  
Transcriptional Activation Domain ◽  
Neuronal Progenitor ◽  
Glial Progenitor

Abstract We used a new mouse model to better understand the cellular and molecular determinants of tumors driven by the C11orf95-RELA fusion. Our approach makes use of in utero electroporation and a binary transposase system to introduce human C11orf95-RELA sequence, wild type and mutant, into neural progenitors, and drive expression of the fusion in different glial and neuronal progenitor cell types. Our results indicate that truncations or point mutations in C11orf95 sequence which interfere with nuclear localization result in a complete loss of tumor-inducing activity. The mutations include truncations of the first 60 amino acids, internal truncations that delete possible mono and bipartite nuclear localization signals, and point mutations of two cysteines and histidines that make up a possible zinc finger domain in C11orf95. Interestingly, all of the mutations that block tumorigenesis also block signal independent nuclear localization of the wild type fusion, without blocking induction of NFKB response genes. We further found that over-expression of the NFKB1 subunit P50 which lacks a transcriptional activation domain significantly inhibits tumor formation by the fusion. In addition, we find that driving expression of the wild type fusion in glial progenitor types using promoters for either astrocytes or oligodendrocytes results in the formation of tumors with transcriptomes displaying significant similarities to human supratentorial ependymoma (ST-EPN), but with distinct patterns depending upon the glial progenitor promoter utilized. In contrast, promoters driving expression selectively in neuron restricted progenitors do not result in the formation of ST-EPN. Together our results reveal three new features of C11orf95-RELA driven tumorigenesis: i) multiple sequences within the C11orf95 domain are required for oncogenic driver activity of the fusion, ii) the P50 subunit of NFKB1 can inhibit fusion induced tumorigenesis, and iii) neuron-restricted precursors are less competent than glia-restricted precursors to form tumors induced by C11orf95-RELA.

scholarly journals A Transformation-Defective Polyomavirus Middle T Antigen with a Novel Defect in PI3 Kinase Signaling

2016 ◽  
Vol 91 (2) ◽  
Author(s):  
Deborah Denis ◽  
Cecile Rouleau ◽  
Brian S. Schaffhausen
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
T Antigen ◽  
Pi3 Kinase ◽  
Binding Motif ◽  
Wild Type ◽  
Pi3k Activity ◽  
Murine Polyomavirus ◽  
Middle T Antigen

ABSTRACT Middle T antigen (MT), the principal oncoprotein of murine polyomavirus, transforms by association with cellular proteins. Protein phosphatase 2A (PP2A), YAP, Src family tyrosine kinases, Shc, phosphatidylinositol 3-kinase (PI3K), and phospholipase C-γ1 (PLCγ1) have all been implicated in MT transformation. Mutant dl1015, with deletion of residues 338 to 347 in the C-terminal region, has been an enigma, because the basis for its transformation defect has not been apparent. This work probes the dl1015 region of MT. Because the region is proline rich, the hypothesis that it targets Src homology domain 3 (SH3) domains was tested, but mutation of the putative SH3 binding motif did not affect transformation. During this work, two point mutants, W348R and E349K, were identified as transformation defective. Extensive analysis of the E349K mutant is described here. Similar to wild-type MT, the E349K mutant associates with PP2A, YAP, tyrosine kinases, Shc, PI3 kinase, and PLCγ1. The E349K mutant was examined to determine the mechanism for its transformation defect. Assays of cell localization and membrane targeting showed no obvious difference in localization. Src association was normal as assayed by in vitro kinase and MT phosphopeptide mapping. Shc activation was confirmed by its tyrosine phosphorylation. Association of type 1 PI3K with MT was demonstrated by coimmunoprecipitation, showing both PI3K subunits and in vitro activity. Nonetheless, expression of the mutants failed to lead to the activation of two known downstream targets of PI3K, Akt and Rac-1. Strikingly, despite normal association of the E349K mutant with PI3K, cells expressing the mutant failed to elevate phosphatidylinositol (3,4,5)-trisphosphate (PIP3) in mutant-expressing cells. These results indicate a novel unsuspected aspect to PI3K control. IMPORTANCE The gene coding for middle T antigen (MT) is the murine polyomavirus oncogene most responsible for tumor formation. Its study has a history of uncovering novel aspects of mammalian cell regulation. The importance of PI3K activity and tyrosine phosphorylation are two examples of insights coming from MT. This study describes new mutants unable to transform like the wild type that point to novel regulation of PI3K signaling. Previous mutants were defective in PI3K because they failed to bind the enzyme and bring the activity to the membrane. These mutants recruit PI3K activity like the wild type, but fail to elevate the cellular level of PIP3, the product used to signal downstream of PI3K. As a result, they fail to activate either Akt or Rac1, explaining the transformation defect.

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.

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.

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.

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