Regulation of Oxidative Stress-Induced Calcium Release by PI3k & Btk in B Cells

过氧化氢刺激酪氨酸激酶依赖性钙从细胞内储存中释放，这被认为是通过B细胞中酪氨酸磷酸化机制激活磷脂酶Cgamma2 （PLCgamma2）来实现的。本研究表明，过氧化氢可诱导PLCgamma2上的酪氨酸磷酸化和B细胞中磷脂酰肌醇3-激酶（PI3K）的激活，PI3K抑制剂Wortmannin可部分抑制过氧化氢诱导的钙释放，但不影响PLCgamma2上的酪氨酸磷酸化。过氧化氢激活的人布鲁顿酪氨酸激酶（Btk）的过表达几乎完全克服了Wortmannin对钙释放的抑制作用。通过提高Btk浓度逆转Wortmannin的抑制作用似乎是过氧化氢介导的唯一作用，因为Btk未能克服Wortmannin对B细胞受体触发的钙动员的抑制。免疫印迹分析显示，只有在过氧化氢刺激下Btk过表达的细胞中，Btk与几种酪氨酸磷酸化蛋白（包括PLCgamma2）形成稳定的复合物。总之，我们的数据与PIP3和/或高浓度Btk将活化的PLCgamma2靶向其底物位点以获得最大催化效率的观点一致。Btk过表达克服了Wortmannin对过氧化氢诱导的钙动员的抑制作用，提示Btk可能在钙信号的调控中起作用。我们有Btk缺陷的DT40细胞，并建立了在激酶（Arg525 - Gln）、Src同源性2 （SH2, Arg307 - Ala）或pleckstrin同源性（PH, Arg28 - Cys）结构域表达野生型Btk或Btk突变体的稳定细胞系。利用这些突变体，我们正在研究Btk在过氧化氢诱导的钙动员中的作用和结构-功能关系。

Hydrogen peroxide stimulates a tyrosine kinase-dependent calcium release from intracellular stores, which is assumed to be achieved through the activation of phospholipase Cgamma2 (PLCgamma2) via a tyrosine phosphorylation mechanism in B cells. Here we show that hydrogen peroxide induces both tyrosine phosphorylation on PLCgamma2 and the activation of phosphatidylinositol 3-kinase (PI3K) in B cells and that the PI3K inhibitor, Wortmannin, partially inhibited the hydrogen peroxide-induced calcium release without affecting tyrosine phosphorylation on PLCgamma2. Overexpression of human Bruton's tyrosine kinase (Btk), which was activated by hydrogen peroxide, almost completely overcame the inhibition of calcium release by Wortmannin. The reversal of Wortmannin's inhibition by enhancing Btk concentration seemed unique to the hydrogen peroxide-mediated effect because Btk failed to overcome the inhibition of Wortmannin on B cell receptor-triggered calcium mobilization. Immunoblot analysis revealed that Btk formed stable complexes with several tyrosine-phosphorylated proteins, including PLCgamma2, only in Btk overexpressed cells upon hydrogen peroxide stimulation. Together, our data are consistent with the notion that PIP3 and/or a high concentration of Btk targets the activated PLCgamma2 to its substrate site for maximal catalytic efficiency.That Btk overexpression overcomes the inhibitory effect of Wortmannin on hydrogen peroxide-induced calcium mobilization implicates a possible role of Btk in the regulation of calcium signaling. We have Btk-deficient DT40 cells and established the stable cell lines expressing wild-type Btk or Btk mutants either in kinase (Arg525 to Gln), Src homology 2 (SH2, Arg307 to Ala), or pleckstrin homology (PH, Arg28 to Cys) domains. Using these mutants, we are investigating the roles and structure-function relationship of Btk in hydrogen peroxide-induced calcium mobilization.