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，这先前被认为是通过酪氨酸磷酸化激活磷脂酶C-γ-2（PLC-γ-2）实现的。遗传研究表明，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介导过氧化氢诱导的PLC-γ-2活化。在这里，我们提供的药理学和遗传学证据表明，磷脂酰肌醇3-激酶（PI 3 K）部分（约40%）介导过氧化氢刺激PLC-γ-2活性通过一种机制，独立于其酪氨酸磷酸化水平。磷脂酰肌醇3激酶的特异性抑制剂渥曼青霉素部分抑制过氧化氢诱导的钙释放和IP 3的产生，但未能改变PLC-γ-2的酪氨酸磷酸化。过表达的Brutons酪氨酸激酶（Btk），这是激活过氧化氢，几乎完全克服了渥曼青霉素对钙释放的抑制作用。Btk形成了一个稳定的复合物与几个酪氨酸磷酸化的蛋白质，只有在Btk过表达细胞过氧化氢刺激后。其中，PLC-γ-2与Btk物理相关，Btk负责PLC-γ-2的增强的酪氨酸磷酸化。总之，我们的研究结果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、三磷酸肌醇、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