Tec kinases mediate sustained calcium influx via site-specific tyrosine phosphorylation of the phospholipase Cγ Src homology 2-Src homology 3 linker

Abstract

Tyrosine phosphorylation of phospholipase Cγ2 (PLCγ2) is a crucial activation switch that initiates and maintains intracellular calcium imobilization in response to B cell antigen receptor (BCR) engagement. Although members from three distinct families of non-receptor tyrosine kinases can phosphorylate PLCγ in vitro, the specific kinase(s) controlling BCR-dependent PLCγ activation in vivo remains unknown. Bruton's tyrosine kinase (Btk)-deficient human B cells exhibit diminished inositol 1,4,5-trisphosphiate production and calcium signaling despite a normal inducible level of total PLCγ2 tyrosine phosphorylation. This suggested that Btk might modify a critical subset of residues essential for PLCγ2 activity. To evaluate this hypothesis, we generated site-specific phosphotyrosine antibodies recognizing four putative regulatory residues within PLCγ2. Whereas all four sites were rapidly modified in response to BCR engagement in normal B cells, Btk-deficient B cells exhibited a marked reduction in phosphoryiation of the Src homology 2 (SH2)-SH3 linker region sites, Tyr 753 and Tyr758. Phosphorylation of both sites was restored by expression of Tec, but not Syk, family kinases. In contrast, phosphorylation of the PLCγS carboxyl-terminal sites, Tyr1197 and Tyr 1217, was unaffected by the absence of functional Btk. Together, these data support a model whereby Btk/Tec kinases control sustained calcium signaling via site-specific phosphorylation of key residues within the PLCγ2 SH2-SH3 linker.