Characterization of p59fyn-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 signaltransduction in T cell activation, we established murine T cellhybridoma lines that expressed an elevated amount of wild-typeor mutant fyn. Clones that expressed high levels of normal p59^fynand active p59^fyn, encoded by wild-type and f-14 mutant fynrespectively, showed enhanced IL-2 production upon stimulationby anti-CD3 antibodies or natural antigen. On the other hand,clones that expressed kinase negative p59^fyn and p59^fyn withan SH2 (Src-homology 2) deletion encoded by t-1 mutant fyn showedlittle induction of IL-2 production upon stimulation. Thesedata suggest that p59^fyn is important in T cell signaling andthat the SH2 sequence plays a critical role in the reaction.Induction of tyrosine phosphorylatlon of multiple proteins uponantigenic stimulation was augmented similarly in the cells thatrespectively expressed wild-type and f-14 mutant fyn at elevatedlevels. The proteins that became highly tyrosine-phosphorylatedincluded phospholipase C (PLC-1), P95^vav, ZAP-70, the MAP kinase,CD3 and unidentified proteins of 120, 100 and 80 kDa. Tyrosinephosphorylation of the 120, 95 and 68 kDa proteins associatedwith PLC-1 was also observed in these cells upon stimulation.In contrast, only the 100 kDa protein and the MAP kinase wereincreasingly tyrosine phosphorylated in the antigen-stimulatedcells expressing t-1 fyn. These data suggest that PLC-1, PLC-1associated molecules, p95^vav, the 80 kDa protein, ZAP-70 andthe CD3 chain may be substrates of p59^fyn or of other tyrosinekJnases regulated by p59^fyn and be important in T cell signaling.