Tyrosine residues in phospholipase Cγ2 essential for the enzyme function in B-cell signaling

Phospholipase C gamma (PLC gamma) isoforms are regulated through activation of tyrosine kinase-linked receptors. The importance of growth factor-stimulated phosphorylation of specific tyrosine residues has been documented for PLC gamma1; however, despite the critical importance of PLC gamma2 in B-cell signal transduction, neither the tyrosine kinase(s) that directly phosphorylate PLC gamma2 nor the sites in PLC gamma2 that become phosphorylated after stimulation are known. By measuring the ability of human PLC gamma2 to restore calcium responses to the B-cell receptor stimulation or oxidative stress in a B-cell line (DT40) deficient in PLC gamma2, we have demonstrated that two tyrosine residues, Tyr(753) and Tyr(759), were important for the PLC gamma2 signaling function. Furthermore, the double mutation Y753F/Y759F in PLC gamma2 resulted in a loss of tyrosine phosphorylation in stimulated DT40 cells. Of the two kinases that previously have been proposed to phosphorylate PLC gamma2, Btk, and Syk, purified Btk had much greater ability to phosphorylate recombinant PLC gamma2 in vitro, whereas Syk efficiently phosphorylated adapter protein BLNK. Using purified proteins to analyze the formation of complexes, we suggest that function of Syk is to phosphorylate BLNK, providing binding sites for PLC gamma2. Further analysis of PLC gamma2 tyrosine residues phosphorylated by Btk and several kinases from the Src family has suggested multiple sites of phosphorylation and, in the context of a peptide incorporating residues Tyr(753) and Tyr(759), shown preferential phosphorylation of Tyr(753).