Hydrogen Peroxide Induced Calcium Release in DT40 Cells

DT40 细胞中过氧化氢诱导钙释放

• 批准号: 6228003
• 负责人: Suofu E Qin
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6228003
• 关键词: calcium flux; enzyme activity; hydrogen peroxide; inositol phosphates; phosphatidylinositol 3 kinase; phospholipase C; tissue /cell culture

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

过氧化氢通过酪氨酸磷酸化激活磷脂酶C-γ-2(PLC-Gamma-2)，从而刺激细胞内钙离子的释放，依赖于酪氨酸激酶。遗传学研究表明，PLC-Gamma-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-Gamma-2的激活。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)部分(~40%)通过独立于其酪氨酸磷酸化水平的机制介导过氧化氢刺激PLC-伽马-2的活性。PI3K的特异性抑制剂Wortmannin部分抑制了过氧化氢诱导的钙释放和IP3的产生，但不能改变PLC-Gamma-2的酪氨酸磷酸化。由过氧化氢激活的Bruton酪氨酸激酶(BTK)的过表达几乎完全克服了Wortmannin对钙释放的抑制作用。BTK只有在过氧化氢刺激下过表达的细胞中才与几种酪氨酸磷酸化蛋白形成稳定的复合体。其中，PLC-Gamma-2与BTK物理上相关，BTK负责增强PLC-Gamma-2的酪氨酸磷酸化。总之，我们的发现为更好地理解过氧化氢激活PLC-Gamma-2的分子机制提供了明确的证据。--Bruton‘s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