Four tyrosine residues in phospholipase C-gamma 2, identified as Btk-dependent phosphorylation sites, are required for B cell antigen receptor-coupled calcium signaling

Activation of phospholipase C-gamma2 (PLC gamma2) is the critical step in B cell antigen receptor (BCR)-coupled calcium signaling. Although genetic di ssection experiments on B cells have demonstrated that Bruton's tyrosine ki nase (Btk) and Syk are required for activating PLC gamma2, the exact activa tion mechanism of PLC gamma2 by these kinases has not been established. We identify the tyrosine residues 753, 759, 1197, and 1217 in rat PLC gamma2 a s Btk-dependent phosphorylation sites by using an in vitro kinase assay. To evaluate the role of these tyrosine residues in phosphorylation-dependent activation of PLC gamma2, PLC gamma2-deficient DT40 cells were reconstitute d with a series of mutant PLC gamma 2s in which the phenylalanine was subst ituted for tyrosine. Substitution of all four tyrosine residues almost comp letely eliminated the BCR-induced PLC gamma2 phosphorylation, indicating th at these residues include the major phosphorylation sites upon BCR engageme nt. Cells expressing PLC gamma2 with a single substitution exhibited some e xtent of reduction in calcium mobilization, whereas those expressing quadru ple mutant PLC gamma2 showed greatly reduced calcium response. These findin gs indicate that the phosphorylations of the tyrosine residues 753, 759, 11 97, and 1217, which have been identified as Btk-dependent phosphorylation s ites in vitro, coordinately contribute to BCR-induced activation of PLC gam ma2.