Tyrosine phosphorylation of phospholipase C␥2 (PLC␥2) is a crucial activation switch that initiates and maintains intracellular calcium mobilization in response to B cell antigen receptor (BCR) engagement. Although members from three distinct families of nonreceptor tyrosine kinases can phosphorylate PLC␥ in vitro, the specific kinase(s) controlling BCR-dependent PLC␥ activation in vivo remains unknown. Bruton's tyrosine kinase (Btk)-deficient human B cells exhibit diminished inositol 1,4,5-trisphosphate production and calcium signaling despite a normal inducible level of total PLC␥2 tyrosine phosphorylation. This suggested that Btk might modify a critical subset of residues essential for PLC␥2 activity. To evaluate this hypothesis, we generated site-specific phosphotyrosine antibodies recognizing four putative regulatory residues within PLC␥2. Whereas all four sites were rapidly modified in response to BCR engagement in normal B cells, Btkdeficient B cells exhibited a marked reduction in phosphorylation of the Src homology 2 (SH2)-SH3 linker region sites, Tyr 753 and Tyr 759 . Phosphorylation of both sites was restored by expression of Tec, but not Syk, family kinases. In contrast, phosphorylation of the PLC␥2 carboxyl-terminal sites, Tyr 1197 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 PLC␥2 SH2-SH3 linker.Signals generated by the pre-B and mature B cell receptors are essential for B cell development, activation, and maintenance of mature B cell populations (1). Engagement of the BCR 1 initiates the formation of a lipid-raft associated signaling complex, or "signalosome," containing tyrosine and serine/threonine kinases, adapter molecules, and lipid hydrolases including phospholipase C␥ isoforms. Together, these events promote a series of downstream signals including a sustained increase in intracellular calcium concentrations. PLC␥ is essential for antigen receptor-mediated calcium mobilization (2-4). Activated PLC␥ hydrolyzes its substrate, phosphatidylinositol 4,5-bisphosphate, generating the second messengers inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol (5, 6). IP 3 acts to open intracellular calcium stores promoting an initial, transient rise in intracellular calcium. Depletion of intracellular calcium stores triggers the opening of plasma membrane store-operated calcium channels (7), resulting in an influx of extracellular calcium and a secondary, sustained calcium signal. The amplitude and duration of this sustained calcium signal is a key determinant of the specific transcription program initiated in response to antigen receptor engagement (8 -10).The most abundantly expressed PLC␥ isoform in B lineage cells is PLC␥2. Chicken B lymphoma cells lacking PLC␥2 are unable to generate IP 3 in response to BCR engagement, resulting in the abrogation of receptor-mediated calcium mobilization (11). Similarly, mice deficient in...

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