Hydrogen Peroxide Induced Calcium Release in DT40 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，这一过程以前被认为是通过酪氨酸磷酸化激活磷脂酶c - γ -2 （PLC- γ -2）来实现的。遗传学研究表明，plc - γ -2的酪氨酸磷酸化水平与其酶活性没有很好的相关性，这表明其他因素也参与了过氧化氢诱导的plc - γ -2活化。在这里，我们提供了药理学和遗传学证据，表明磷脂酰肌醇3-激酶（PI3K）通过独立于酪氨酸磷酸化水平的机制部分（约40%）介导过氧化氢刺激的plc - γ -2活性。Wortmannin是PI3K的特异性抑制剂，部分抑制过氧化氢诱导的钙释放和IP3的产生，但不能改变plc - γ -2的酪氨酸磷酸化。过氧化氢激活的布鲁顿酪氨酸激酶（Brutons tyrosine kinase, Btk）过表达几乎完全克服了Wortmannin对钙释放的抑制作用。过氧化氢刺激下，Btk仅在Btk过表达细胞中与几种酪氨酸磷酸化蛋白形成稳定的复合物。其中，PLC-gamma-2与Btk存在物理关联，Btk是PLC-gamma-2酪氨酸磷酸化增强的原因。总之，我们的发现为更好地理解过氧化氢激活plc - γ -2的分子机制提供了明确的证据。-布鲁顿酪氨酸激酶，钙释放，肌醇三磷酸，磷脂酰肌醇3-激酶，磷脂酶c - γ -2

Hydrogen peroxide stimulates a tyrosine kinase-dependent calcium release from intracellular stores, which has been previously assumed to be achieved through the activation of phospholipase C-gamma-2 (PLC- gamma-2) via tyrosine phosphorylation. Genetic studies revealed that the tyrosine phosphorylation level of PLC-gamma-2 is not well correlated with its enzymatic activity, suggesting that other factors are also involved in mediating hydrogen peroxide-induced activation of PLC-gamma-2. Here, we provide pharmacological and genetic evidence showing that phosphatidylinositol 3-kinase (PI3K) partially (~40 percent) mediated hydrogen peroxide-stimulated PLC-gamma-2 activity through a mechanism independent of its tyrosine phosphorylation level. Wortmannin, a specific inhibitor of PI3K, partially inhibited hydrogen peroxide-induced calcium release and IP3 production, but failed to alter tyrosine phosphorylation of PLC-gamma-2. Overexpression of Brutons tyrosine kinase (Btk), which was activated by hydrogen peroxide, almost completely overcame the inhibitory effect of Wortmannin on calcium release. Btk formed a stable complex with several tyrosine-phosphorylated proteins only in Btk overexpressing cells upon hydrogen peroxide stimulation. Among them, PLC-gamma-2 was physically associated with Btk which was responsible for the enhanced tyrosine phosphorylation of PLC-gamma-2. Together, our findings provide clear evidence for a better understanding of the molecular mechanisms by which hydrogen peroxide activates PLC-gamma-2. - Bruton's tyrosine kinase, calcium release, inositol trisphosphate, phosphatidylinositol 3-kinase, phospholipase C-gamma-2