Characterization of p59^fyn-mediated signal transduction on T cell activation

Protein tyrosine kinase p59^fyn is associated with the TCR - CD3 complex and is suggested to play a role in T cell activation. To determine the molecular mechanism of p59^fyn-medlated signal transduction in T cell activation, we established murine T cell hybridoma lines that expressed an elevated amount of wild-type or mutant fyn. Clones that expressed high levels of normal p59^fyn and active p59^fyn, encoded by wild-type and f-14 mutant fyn respectively, showed enhanced IL-2 production 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 data suggest that p59^fyn is important in T cell signaling and that the SH2 sequence plays a critical role in the reaction. Induction of tyrosine phosphorylatlon of multiple proteins upon antigenic stimulation was augmented similarly in the cells that respectively expressed wild-type and f-14 mutant fyn at elevated levels. The proteins that became highly tyrosine-phosphorylated included phospholipase C (PLC-γ1), P95^vav, ZAP-70, the MAP kinase, CD3ζ and unidentified proteins of 120, 100 and 80 kDa. Tyrosine phosphorylation of the 120, 95 and 68 kDa proteins associated with PLC-γ1 was also observed in these cells upon stimulation. In contrast, only the 100 kDa protein and the MAP kinase were increasingly tyrosine phosphorylated in the antigen-stimulated cells expressing t-1 fyn. These data suggest that PLC-γ1, PLC-γ1 associated molecules, p95^vav, the 80 kDa protein, ZAP-70 and the CD3ζ chain may be substrates of p59^fyn or of other tyrosine kJnases regulated by p59^fyn and be important in T cell signaling.