Bruton's tyrosine kinase is essential for hydrogen peroxide-induced calcium signaling

Using Btk-deficient DT40 cells and the transfectants expressing wild type Btk or Btk mutants in either kinase (Arg(525) to Gln), Src homology 2 (SH2, Arg(307) to Ala), or pleckstrin homology (PH Arg(28) to Cys) domains, we investigated the roles and structure-function relationships of Btk in hydrogen peroxide-induced calcium mobilization. Our genetic evidence showed that Btk deficiency resulted in a significant reduction in hydrogen peroxide-induced calcium response. This impaired calcium signaling is correlated with the complete elimination of IP3 production and the significantly reduced tyrosine phosphorylation of PLC gamma2 in Btk-deficient DT40 cells, All of these defects were fully restored by the expression of wildtype Btk in Btk-deficient DT40 cells. The data from the point mutation study revealed that a defect at any one of the three functional domains would prevent a full recovery of Btk-mediated hydrogen peroxide-induced intracellular calcium mobilization. However, mutation at either the SH2 or PH domain did not affect the hydrogen peroxide-induced activation of Btk. Mutation at the SH2 domain abrogates both IP3 generation and calcium release, while the mutant with the nonfunctional PH domain can partially activate PLC gamma2 and catalyze IP3 production bur fails to produce significant calcium mobilization. Thus, these observations suggest that Btk-dependent tyrosine phosphorylation of PLC gamma2 is required but not sufficient for hydrogen peroxide-induced calcium mobilization. Furthermore, hydrogen peroxide stimulates a Syk-, but not Btk-, dependent tyrosine phosphorylation of B cell linker protein BLNK, The overall results, together with those reported earlier [Qin et al. (2000) Proc, Natl. Acad. Sci, U.S.A, 97, 7118], are consistent with the notion that functional SH2 and PH domains are required for Btk to form a complex with PLC gamma2 through BLNK in order to position the Btk, PLC gamma2, and phosphatidylinositol 4,5-bisphosphate in close proximity for efficient activation of PLC gamma2 and to maximize its catalytic efficiency for IP3 production.