SULFHYDRYL OXIDATION DOWN-REGULATES T-CELL SIGNALING AND INHIBITS TYROSINE PHOSPHORYLATION OF PHOSPHOLIPASE C-GAMMA-1

Early events in both T-cell receptor (CD3)- and CD4-induced signal transduction pathways include tyrosine phosphorylation of protein substrates, the generation of phosphatidylinositol-phosphate breakdown products, and the mobilization of intracellular Ca2+. Oxidative stress in T cells mediated by sulfhydryl-reactive nonpolar maleimides was shown previously to down-regulate both receptor-mediated Ca2+ mobilization and interleukin 2 production. Here we show that N-ethylmaleimide suppresses both CD3- and CD4-induced Ca2+ responses in human T cells correlating with a reduction in the level of phospholipase C-gamma-1 (PLC-gamma-1) tyrosine phosphorylation. The inhibition of tyrosine phosphorylation of PLC-gamma-1 and additional protein substrates was observed at concentrations of N-ethylmaleimide above 20-mu-M, whereas lower concentrations of oxidant appeared to increase tyrosine kinase activity following cell stimulation. Sulfhydryl oxidation did not directly affect the catalytic activity of PLC-gamma-1, since immunopurified enzyme from N-ethylmaleimide-treated T cells was fully active. Although N-ethylmaleimide treatment of T cells did not cause a direct effect on total pp56lck kinase activity measured in vitro, the interaction between CD4 and pp56lck was oxidation-sensitive in vivo. However, CD3-induced signaling was inhibited at N-ethylmaleimide concentrations lower than that required for CD4/pp56lck dissociation, suggesting that CD3-associated tyrosine kinase activity involves acutely sensitive regulatory thiols. In addition to chemically induced sulfhydryl oxidation, naturally regulated cellular redox states appear to dictate the potential for T-cell responsiveness, since degranulating human peripheral blood neutrophils inhibited CD3-induced Ca2+ mobilization in T lymphocytes. These data indicate that signal transduction in T cells involves the activation of PLC-gamma-1 by tyrosine phosphorylation through an oxidation-sensitive intermediate between surface receptors and tyrosine kinases, perhaps including the interaction between CD4 and pp56lck.