PHOSPHOLIPASE C-GAMMA-1 INTERACTS WITH CONSERVED PHOSPHOTYROSYL RESIDUES IN THE LINKER REGION OF SYK AND IS A SUBSTRATE FOR SYK

Antigen receptor ligation on lymphocytes activates protein tyrosine ki nases and phospholipase C-gamma (PLC-gamma) isoforms, Glutathione S-tr ansferase fusion proteins containing the C-terminal Src-homology 2 [SH 2(C)] domain of PLC-gamma 1 bound to tyrosyl phosphorylated Syk, Syk i solated from antigen receptor-activated B cells phosphorylated PLC-gam ma 1 on Tyr-771 and the key regulatory residue Tyr-783 in vitro, where as Lyn from the same B cells phosphorylated PLC-gamma 1 only on Tyr-77 1, The ability of Syk to phosphorylate PLC-gamma 1 required antigen re ceptor ligation, while Lyn was constitutively active, An mCD8-Syk cDNA construct could be expressed as a tyrosyl-phosphorylated chimeric pro tein tyrosine kinase in COS cells, was recognized by PLC-gamma 1 SH2(C ) in vitro, and induced tyrosyl phosphorylation of endogenous PLC-gamm a 1 in vivo. Substitution of Tyr-525 and Tyr-526 at the autophosphoryl ation site of Syk in mCD8-Syk substantially reduced the kinase activit y and the binding of this variant chimera to PLC-gamma SH2(C) in vitro ; it also failed to induce tyrosyl phosphorylation of PLC-gamma 1 in v ivo, In contrast, substitution of Tyr-348 and Tyr-352 in the linker re gion of Syk in mCD8-Syk did not affect the kinase activity of this var iant chimera but almost completely eliminated its binding to PLC-gamma 1 SH(C) and completely eliminated its ability to induce tyrosyl phosp horylation of PLC-gamma 1 in vivo. Thus, an optimal kinase activity of Syk and an interaction between the linker region of Syk with PLC-gamm a 1 are required for the tyrosyl phosphorylation of PLC-gamma 1.