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

Using Btk-deficient DT40 cells and the transfectants expressing wild type B tk 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 in vestigated the roles and structure-function relationships of Btk in hydroge n peroxide-induced calcium mobilization. Our genetic evidence showed that B tk deficiency resulted in a significant reduction in hydrogen peroxide-indu ced calcium response. This impaired calcium signaling is correlated with th e complete elimination of IP3 production and the significantly reduced tyro sine phosphorylation of PLC gamma2 in Btk-deficient DT40 cells, All of thes e defects were fully restored by the expression of wildtype Btk in Btk-defi cient DT40 cells. The data from the point mutation study revealed that a de fect at any one of the three functional domains would prevent a full recove ry of Btk-mediated hydrogen peroxide-induced intracellular calcium mobiliza tion. However, mutation at either the SH2 or PH domain did not affect the h ydrogen peroxide-induced activation of Btk. Mutation at the SH2 domain abro gates both IP3 generation and calcium release, while the mutant with the no nfunctional PH domain can partially activate PLC gamma2 and catalyze IP3 pr oduction bur fails to produce significant calcium mobilization. Thus, these observations suggest that Btk-dependent tyrosine phosphorylation of PLC ga mma2 is required but not sufficient for hydrogen peroxide-induced calcium m obilization. Furthermore, hydrogen peroxide stimulates a Syk-, but not Btk- , dependent tyrosine phosphorylation of B cell linker protein BLNK, The ove rall 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 fun ctional 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 phosphati dylinositol 4,5-bisphosphate in close proximity for efficient activation of PLC gamma2 and to maximize its catalytic efficiency for IP3 production.