CHARACTERIZATION OF P59(FYN)-MEDIATED SIGNAL-TRANSDUCTION ON T-CELL ACTIVATION

Protein tyrosine kinase p59(fyn) is associated with the TCR - CD3 comp lex and is suggested to play a role in T cell activation. To determine the molecular mechanism of p59(fyn)-mediated signal transduction in T cell activation, we established murine T cell hybridoma lines that ex pressed an elevated amount of wild type or mutant fyns. Clones that ex pressed high levels of normal p59(fyn) and active p59(fyn), encoded by wild-type and f-14 mutant fyn respectively, showed enhanced IL-2 prod uction upon stimulation by anti-CD3 antibodies or natural antigen. On the other hand, clones that expressed kinase negative p59(fyn) and p59 (fyn) with an SH2 (Src-homology 2) deletion encoded by t-1 mutant fyn showed little induction of IL-2 production upon stimulation. These dat a suggest that p59(fyn) is important in T cell signaling and that the SH2 sequence plays a critical role in the reaction. Induction of tyros ine phosphorylation of multiple proteins upon antigenic stimulation wa s augmented similarly in the cells that respectively expressed wild-ty pe and f-14 mutant fyns at elevated levels. The proteins that became h ighly tyrosine-phosphorylated included phospholipase C (PLC-gamma 1), p95(vava), ZAP-70, the MAP kinase, CD3 zeta and unidentified proteins of 120, 100 and 80 kDa. Tyrosine phosphorylation of the 120, 95 and 68 kDa proteins associated with PLC-gamma 1 was also observed in these c ells upon stimulation. In contrast, only the 100 kDa protein and the M AP kinase were increasingly tyrosine phosphorylated in the antigen-sti mulated cells expressing t-1 fyn. These data suggest that PLC-gamma 1, PLC-gamma 1 associated molecules, p95(vav), the 80 kDa protein, ZAP-7 0 and the CD3 zeta chain may be substrates of p59(fyn) or of other tyr osine kinases regulated by p59(fyn) and be important in T cell signali ng.