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

Abstract

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 Py8O8A, 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 pp60`cs 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"sc, 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.Transformation of cells by polyomavirus requires the interaction of the polyomavirus middle T antigen with the cellular proto-oncogene product pp6Oc-src (15,16). This interaction leads to the activation of the intrinsic tyrosine kinase activity of pp60c-src and ultimately to transformation (10,14). Although pp60c-src activation is obligatory for transformation, mutant middle T's exist which interact with and activate pp6Oc-src yet fail to transform infected cells. Full transformation in culture and tumor induction in mice may require the presence of an additional activity in middle T-pp60csrc immune complexes which phosphorylates phosphatidylinositol (PI) (27, 51; D. Talmage, R. Freund, A. Young, C. Dawe, and T. Benjamin, manuscript in preparation).Polyomavirus hr-t mutants encoding defective middle T and small T antigens grow poorly on NIH 3T3 cells, yielding bursts which are only 2 to 5% the size of those of wild-type virus (22). The low burst size results from inefficient or unstable assembly of viral components into infectious virions and is associated with underphosphorylation of the major viral capsid protein, VP1 (19,20). Thus, the role of the hr-t gene in viral growth lies in inducing cellular "permissivity factors" which act, at least in part, to bring about stable phosphorylation of VP1. Activation of this phosphorylation pathway in NIH 3T3 cells does not require detectable interaction of middle T with pp60c-src and therefore represents a middle T function which is separate from its known function during transformation (R. Garcea, D. Talmage, R. Freund, A. Harmatz, and T. Benjamin, manuscript in preparation).The ribosomal protein S6 is highly phosphorylated in growing cell...

Studies of Partially Transforming Polyomavirus Mutants Establish a Role for Phosphatidylinositol 3-Kinase in Activation of pp70 S6 Kinase

“…This study extends an earlier report on polyomavirus-induced elevation of ribosomal protein S6 phosphorylation (68) by demonstrating activation of pp70 S6K in a pathway mediated by mT and PI3K. Genetic and biochemical evidence supports the conclusion that PI3K binding to mT is required for activation of both the cytoplasmic (70-kDa) and nuclear (85-kDa) forms of S6 kinase.…”

“…Previous work has shown that two mT-dependent mechanisms contribute to increased S6 phosphorylation. One involves activation of an S6 kinase(s) and is dependent on mT activation of pp60 c-src ; the other is independent of mT-pp60 c-src interaction and may reflect negative regulation of an S6 phosphatase (68). How mT exerts these effects, and specifically whether pp70 S6K is in-volved, is unknown.…”

Protein kinase B/Akt is activated by polyomavirus middle-T antigen via a phosphatidylinositol 3-kinase-dependent mechanism

“…Another kinase associated with growth factor stimulation, p70 S6-kinase , is activated by constitutively active mutants of PKB/Akt, but is not a direct substrate (Burgering and Co er, 1995). p70 S6-kinase activation has also been observed as a consequence of polyomavirus infection (Talmage et al, 1988;Dahl et al, 1996). The involvement of PKB/Akt in growth regulation is further supported by data obtained with an oncogenic form of PKB/Akt expressed as a Gag-PKB/Akt fusion protein by the acute retrovirus AKT8 (Bellacosa et al, 1991).…”

Polyomavirus middle-sized tumor antigen modulates c-Jun phosphorylation and transcriptional activity.

“…The (39,40) and by expression of ras antisense RNA (41) or a dominant inhibitory mutant of ras (42), suggesting that both Src and Ras are required for MT-mediated transformation. Raf-1 and Rsk kinase activities are elevated in MT-transformed cells, but their involvement in MT-mediated alterations in gene expression and transformation has not been defined (16,17).…”

“…These mutants recently were shown to be defective in binding the SH2-containing adaptor molecule, SHC, which is phosphorylated on tyrosine in MT-transformed cells and binds to GRB2, providing a plausible link between MT and Ras activation (15). Polyoma-transformed cells show increases in the activities of protein serine/threonine kinases, including Raf-1 (16) and ribosomal protein S6 kinase (17), and an increase in the activities of transcriptional regulators, PEA1/ AP-1 and PEA3/c-ets (18,19). Therefore, it seems likely that association of MT with proteins in the cell membrane induces signaling pathways that activate serine/threonine kinases and transduce signals to transcriptional regulators in the nucleus.…”