Analysis of the T-cell activation signaling pathway mediated by tyrosine kinases, protein kinase C, and Ras protein, which is modulated by intracellular cyclic AMP

摘要

T-cell receptor (TCR) triggering by an anti-CD3 antibody or phytohemagglutinin (PHA) as well as the treatment with phorbol myristate acetate (PMA), a direct activator of protein kinase C (PKC), induces activation of Ras in T-lymphocytes (Downward, J. et al, (1990) Nature 364, 719-723), In this paper, we studied the role of Ras in the process of TCR-mediated T-cell activation using a human lymphomie Jurkut cell line. The stimulatory effect of TCR cross-linking on Ras activation was inhibited by herbimycin A, a specific inhibitor of protein lyrosine kinases (PTKs), whereas PMA-induced Ras activation was not affected. On the other hand, calphostin C, a specific inhibitor of PKC, blocked not only PMA-induced, but also TCR-mediated formation of Ras o GTP. Furthermore, down-regulation of PMA-sensitivc PKC severely impaired the activation of Ras in response to TCR-stimulation. Tyrosine-phosphorylation and translocation to the paniculate fraction of pliospholipase C-y 1 (PLC-y 1) were observed upon T-cell activation. Subcellular localization of PKC was also changed when the cells were stimulated with an anti-CD3 antibody or PMA. While TCR-stimulated translocation of PKC was observed only transiently, PMA-induced translocation of PKC was more sustained. These results suggest that the activation of PLC-y I by PTK and subsequent activation of PKC are important for TCR-mediated Ras activation in Jurkat cells. An activated form of Ras enhanced the activation of interlcukin 2 (IL-2) promoter by TCR stimulation or PMA treatment, although the activated Ras by itself was insufficient for IL-2 promoter activation. On the other hand, a dominant-inhibitory Ras diminished almost completely the activation of IL-2 promoter induced by PMA plus calcium ionophore, indicating that Ras is essential for transduction of T-cell activation signals. Cholera toxin (CTX), which directly activates Gsa, is shown to inhibit the activation of IL-2 promoter, TCR-mediated Ras activation, tyrosine phosphorylation and translocation of cellular proteins including ZAP-70, PLC-y I, and PKC. An activated Gs a mutant as well as dibutylyl cAMP (dBcAMP) also showed similar inhibitory effects.