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

过氧化氢通过激活B细胞中的磷脂酶CGamma2(PLCGamma2)，通过酪氨酸磷酸化机制，刺激细胞内依赖酪氨酸激酶的钙释放。在本研究中，我们发现过氧化氢可诱导B细胞中PLCGamma2上的酪氨酸磷酸化和磷脂酰肌醇3-激酶(PI3K)的激活，而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。由过氧化氢激活的人Bruton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。由于BTK不能克服Wortmannin对B细胞受体触发的钙动员的抑制作用，因此通过提高BTK浓度来逆转Wortmannin的抑制似乎是过氧化氢介导的作用所特有的。免疫印迹分析表明，在过氧化氢刺激下，BTK只与包括PLCGamma2在内的几种酪氨酸磷酸化蛋白形成稳定的复合体。总之，我们的数据与PIP3和/或高浓度的BTK将激活的PLCGamma2靶向其底物位置以获得最大催化效率的观点是一致的。BTK的表达克服了Wortmannin对过氧化氢诱导的钙动员的抑制作用，暗示BTK可能在钙信号调节中发挥作用。我们获得了BTK缺失的DT40细胞，并建立了稳定表达野生型BTK或BTK突变体的细胞系，这些突变体的结构域包括KK(Arg525到Gln)、Src同源2(SH2，Arg307到Ala)或Pleckstrin同源(PH，Arg28到Cys)。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及其结构与功能的关系。

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.