POLYOMAVIRUS MIDDLE T-ANTIGEN AS A PROBE FOR T-CELL ANTIGEN RECEPTOR-COUPLED SIGNALING PATHWAYS

Stimulation of the T cell antigen receptor (TCR) triggers a complex se ries of signaling events that culminate in T cell. activation and prol iferation. The complex structure of the TCR has hindered efforts to li nk specific signaling events induced by TCR cross-linkage to downstrea m activation responses, such as interleukin-2 (IL-2) gene transcriptio n. Previous studies have shown that the polyomavirus-derived oncoprote in, middle T antigen (mT), transforms rodent fibroblasts by interactin g with and activating several cytoplasmic signaling proteins (Src kina ses, phospholipase C (PLC)-gamma 1, Shc, and phosphoinositide 3-kinase (PIS-K) implicated in cell growth control. In this study, we demonstr ate that expression of mT activates Jurkat T cells, as measured by inc reases in IL-2 promoter-and NFAT (nuclear factor of activated T cells) -dependent reporter gene transcription. The transcriptional response p rovoked by mT was blocked by the immunosuppressive drug FK506, a poten t inhibitor of TCR-mediated IL-2 gene expression, Mutations that disru pted the binding of mT to Src kinases or PLC-yl abrogated the ability of mT to deliver the signals needed for IL-2 promoter activation. In c ontrast, a mT mutant that failed to bind PI3-K induced a markedly elev ated transcriptional response in Jurkat cells, whereas mutation of the Shc binding site in mT had little effect on the transactivating poten tial of this viral oncoprotein, Additional studies demonstrated that t he association of mT with PLC-gamma 1 was necessary and sufficient to activate both Ca2+- and Ras dependent signaling cascades in Jurkat cel ls. These results indicate that PLC-yl activation plays pivotal and pl eiotropic roles in the stimulation of IL-2 gene expression, whereas ac tivation of PI3-K negatively modulates this response in Jurkat T cells .