Pleiotropic contributions of phospholipase C-gamma 1 (PLC-gamma 1) to T-cell antigen receptor-mediated signaling: Reconstitution studies of a PLC-gamma 1-deficient Jurkat T-cell line

Phospholipase C-gamma1 (PLC-gamma1) plays a crucial role in the coupling of T-cell antigen receptor (TCR) ligation to interleukin-2 (IL-2) gene expres sion in activated T lymphocytes. In this study, we have isolated and charac terized two novel, PLC-gamma1-deficient sublines derived from the Jurkat T- leukemic cell line. The P98 subline displays a > 90% reduction in PLC-gamma 1 expression, while the J.gamma1 subline contains no detectable PLC-gamma1 protein. The lack of PLC-gamma1 expression in J.gamma1 cells caused profoun d defects in TCR-dependent Ca2+ mobilization and NFAT activation. In contra st, both of these responses occurred at normal levels in PLC-gamma1-deficie nt P98 cells. Unexpectedly, the P98 cells displayed significant and selecti ve defects in the activation of both the composite CD28 response element (R E/AP) and the full-length IL-2 promoter following costimulation with anti-T CR antibodies and phorbol ester, These transcriptional defects were reverse d by transfection of P98 cells with a wild-type PLC-gamma1 expression vecto r but not by expression of mutated PLC-gamma1 constructs that lacked a func tional, carboxyl-terminal SH2 [SH2(C)] domain or the major Tyr(783) phospho rylation site. On the other hand, the amino-terminal SH2 [SH2(N)] domain wa s not essential for reconstitution of RE/AP- or IL-2 promoter-dependent tra nscription but was required for the association of PLC-gamma1 with LAT, as well as the tyrosine phosphorylation of PLC-gamma1 itself, in activated P98 cells. These studies demonstrate that the PLC-gamma1 SH2(N) and SH2(C) dom ains play functionally distinct roles during TCR-mediated signaling and ide ntify a non-Ca2+-related signaling function linked to the SH2(C) domain, wh ich couples TCR plus phorbol ester-CD28 costimulation to the activation of the IL-2 promoter in T lymphocytes.