Phosphorylation sites of protein kinase C δ in H2O2-treated cells and its activation by tyrosine kinase in vitro
Phosphorylation sites of protein kinase C δ in H2O2-treated cells and its activation by tyrosine kinase in vitro
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DOI：
10.1073/pnas.111158798
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发表时间：
2001-06-05
影响因子：
11.1
通讯作者：
Nishizuka, Y
中科院分区：
文献类型：
--
作者：
Konishi, H;Yamauchi, E;Nishizuka, Y
蛋白激酶C δ（PKC δ）通常被由肌醇磷脂的受体介导的水解产生的二酰基甘油激活。在用H2 O2刺激细胞时，该酶被酪氨酸磷酸化，伴随着酶活性的增加。这种激活似乎并不伴随其易位到膜。在本研究中，在H2 O2处理的细胞中的PKC δ的酪氨酸磷酸化位点被确定为Tyr-311，Tyr-332，和Tyr-512通过质谱分析与使用的荧光扫描方法和通过免疫印迹分析与使用磷酸化位点特异性抗体。其中Tyr-311是主要的修饰位点。在体外研究中，通过Lck（一种非受体型酪氨酸激酶）在该位点磷酸化。在甘油二酯存在下，基础酶活性增强，最大酶速提高。Tyr-311突变为苯丙氨酸可阻止该酶最大酶活的增加，但替换另外两个酪氨酸残基并不阻止这种作用。结果表明，在调节和催化结构域之间的Tyr-311的磷酸化是响应H2 O2产生活性PKC δ的关键步骤。
Protein kinase C delta (PKC delta) is normally activated by diacylglycerol produced from receptor-mediated hydrolysis of inositol phospholipids. On stimulation of cells with H2O2, the enzyme is tyrosine phosphorylated, with a concomitant increase in enzymatic activity. This activation does not appear to accompany its translocation to membranes. In the present study, the tyrosine phosphorylation sites of PKC delta in the H2O2-treated cells were identified as Tyr-311, Tyr-332, and Tyr-512 by mass spectrometric analysis with the use of the precursor-scan method and by immunoblot analysis with the use of phosphorylation site-specific antibodies. Tyr-311 was the predominant modification site among them. In an in vitro study, phosphorylation at this site by Lck, a non-receptor-type tyrosine kinase. enhanced the basal enzymatic activity and elevated its maximal velocity in the presence of diacylglycerol, The mutation of Tyr-311 to phenylalanine prevented the increase in this maximal activity, but replacement of the other two tyrosine residues did not block such an effect. The results indicate that phosphorylation at Tyr-311 between the regulatory and catalytic domains is a critical step for generation of the active PKC delta in response to H2O2.