Regulation of oxidative stress-induced calcium release by phosphatidylinositol 3-kinase and Bruton's tyrosine kinase in B cells

Hydrogen peroxide stimulates a tyrosine kinase-dependent calcium release fr om intracellular stores, which is assumed to be achieved through the activa tion of phospholipase C gamma 2 (PLC gamma 2) via a tyrosine phosphorylatio n mechanism in B cells. Here we show that H2O2 induces both tyrosine phosph orylation on PLC gamma 2 and the activation of phosphatidylinositol 3-kinas e (PI3K) in B cells, and that the phosphatidylinositol 3-kinase inhibitor, Wortmannin, partially inhibited the H2O2-induced calcium release without af fecting tyrosine phosphorylation on PLC gamma 2. Overexpression of human Br uton's tyrosine kinase (Btk), which was activated by H2O2, almost completel y overcame the inhibition of calcium release by Wortmannin, The reversal of Wortmannin's inhibition by enhancing Btk concentration seemed unique to th e H2O2-mediated effect, because Btk failed to overcome the inhibition of Wo rtmannin on B cell receptor-triggered calcium mobilization. Immunoblot anal ysis revealed that Btk formed stable complexes with several tyrosine-phosph orylated proteins, including PLC gamma 2, only in Btk-overexpressed cells o n H2O2 stimulation. Together, our data are consistent with the notion that PIP3 and/or a high concentration of Btk target the activated PLC gamma 2 to its substrate site for maximal catalytic efficiency.