Regulation of Oxidative Stress-Induced Calcium Release by PI3k & Btk in B Cells
PI3k 对氧化应激诱导的钙释放的调节
基本信息
- 批准号:6432638
- 负责人:
- 金额:--
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- 依托单位国家:美国
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- 财政年份:
- 资助国家:美国
- 起止时间:至
- 项目状态:未结题
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项目摘要
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.
过氧化氢刺激酪氨酸激酶依赖性钙从细胞内储存释放,这被认为是通过B细胞中酪氨酸磷酸化机制激活磷脂酶C γ 2(PLC γ 2)实现的。在这里,我们表明,过氧化氢诱导酪氨酸磷酸化的PLC γ 2和激活的磷脂酰肌醇3-激酶(PI 3 K)在B细胞和PI 3 K抑制剂,渥曼青霉素,部分抑制过氧化氢诱导的钙释放,而不影响酪氨酸磷酸化的PLC γ 2。 过表达的人布鲁顿酪氨酸激酶(Btk),这是激活过氧化氢,几乎完全克服了抑制钙释放渥曼青霉素。 通过提高Btk浓度来逆转Wortmannin的抑制似乎是过氧化氢介导的效应所特有的,因为Btk未能克服Wortmannin对B细胞受体触发的钙动员的抑制。免疫印迹分析表明,Btk形成稳定的复合物与几个酪氨酸磷酸化的蛋白质,包括PLC γ 2,只有在Btk过表达的细胞过氧化氢刺激后。 总之,我们的数据是一致的概念,即PIP 3和/或高浓度的Btk的目标激活PLC γ 2到其底物位点的最大催化efficiency.That Btk的过表达克服了渥曼青霉素对过氧化氢诱导的钙动员的抑制作用暗示了可能的作用,Btk在钙信号的调节。我们有Btk缺陷的DT 40细胞,并建立了在激酶(Arg 525至Gln)、Src同源2(SH 2,Arg 307至Ala)或普列克底物蛋白同源(PH,Arg 28至Cys)结构域中表达野生型Btk或Btk突变体的稳定细胞系。 利用这些突变体,我们正在调查的角色和结构功能的关系Btk过氧化氢诱导的钙动员。
项目成果
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{{ truncateString('Suofu E Qin', 18)}}的其他基金
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 - 财政年份:
- 资助金额:
-- - 项目类别:
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