A TYROSINE PHOSPHORYLATION REQUIREMENT FOR CYTOTOXIC T-LYMPHOCYTE DEGRANULATION

Phorbol myristate acetate (PMA) plus ionomycin induces the tyrosine ph osphorylation of several cytotoxic T lymphocyte (CTL) substrates, incl uding one with an apparent molecular weight of 100,000 (pp100) in clon ed murine CTL. cis-Unsaturated fatty acids and low concentrations of p henylarsine oxide specifically inhibit the tyrosine phosphorylation of pp100. Genistein also inhibits tyrosine phosphorylation of pp100, but not with the same specificity as cis-fatty acids or low concentration s of phenylarsine oxide. Degranulation triggered by PMA plus ionomycin is inhibited by cis-fatty acids, low concentrations of phenylarsine o xide, and genistein, under the same conditions that these agents inhib it tyrosine phosphorylation of pp100. Depleting CTL of protein kinase C (PKC) activity by prolonged exposure to PMA eliminates the increase in tyrosine phosphorylation when challenged by PMA plus ionomycin, but not when these PKC-depleted CTL are activated by cognate target cells , immobilized anti-T cell receptor (TCR) antibodies, or concanavalin A . Tyrosine phosphorylation of pp100 triggered by TCR engagement in PKC -depleted cells is inhibited by cis-fatty acids and phenylarsine oxide , indicating that the inhibitory mechanism of the tyrosine phosphoryla tion of pp100 is independent of PKC. Furthermore, because all three ty rosine phosphorylation inhibitors are unlikely to inhibit PKC, these r esults suggest that, in addition to PKC activation and a rise in intra cellular Ca2+, CTL degranulation requires the tyrosine phosphorylation of a CTL substrate(s), in addition to phospholipase C, and the presen t results are consistent with pp100 as that substrate. Taken together with previous studies, these results suggest that tyrosine phosphoryla tion of pp100 may play a central role in CTL function.