Activation of protein phosphorylation by oxidants in vascular endothelial cells: Identification of tyrosine phosphorylation of caveolin
Activation of protein phosphorylation by oxidants in vascular endothelial cells: Identification of tyrosine phosphorylation of caveolin
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DOI：
10.1016/s0891-5849(96)00241-9
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发表时间：
1997-01-01
影响因子：
7.4
通讯作者：
Natarajan, V
中科院分区：
文献类型：
--
作者：
Vepa, S;Scribner, WM;Natarajan, V
氧化剂通过调节多种生化反应和信号转导途径在内皮细胞功能障碍中发挥重要作用。为了了解氧化剂在血管损伤中的作用，我们研究了过氧化氢（H2 O2），钒酸盐和过钒酸盐（V4+-OOH）对牛肺动脉内皮细胞（BPAEC）[P-32（i）]摄取和蛋白磷酸化的影响。标记的[P-32（i）]掺入BPAEC依赖于所用氧化剂的浓度和孵育时间。在测试的氧化剂中，过钒酸盐（10 μ M）诱导最大量的[P-32（i）]掺入细胞（超过对照的2 - 3倍），随后是H2 O2（1 mM）和钒酸盐（100 μ M），并且在对照和氧化剂处理的细胞之间注意到标记蛋白质谱的明显差异。通过SDS-PAGE分析的蛋白质显示出范围从21-205 kDa的标记模式的明显增加，如通过放射自显影所证明的。虽然大多数掺入的[P-32（i）]是在丝氨酸/苏氨酸残基，免疫沉淀和免疫印迹的细胞裂解物，使用抗磷酸酪氨酸抗体，显示氧化剂处理导致总蛋白酪氨酸磷酸化显着增加。最重要的是，免疫沉淀的细胞裂解物，从过钒酸盐处理显示出不同的酪氨酸磷酸化的22 kDa的蛋白质，这被确定为小窝蛋白，小窝的标记。过钒酸盐介导的磷酸化被有效地抑制星形孢菌素（5 μ M），而染料木素仅显示部分衰减。此外，H2 O2处理导致增强的24 kDa蛋白的磷酸化，这是减弱染料木素。此外，氧化剂处理的细胞表现出增加酪氨酸激酶活性和降低磷酸酶活性。这些数据显示了响应于不同氧化剂的蛋白质的标记谱的差异，表明不同蛋白激酶/磷酸酶的差异调节。版权所有（C）1996 Elsevier Science Inc.
Oxidants play a significant role in endothelial cell dysfunction through modulation of diverse biochemical reactions and signal transduction pathways. Towards understanding the role of oxidants in vascular injury, we studied the effect of hydrogen peroxide (H2O2), vanadate, and pervanadate (V4+-OOH) on [P-32(i)] uptake and protein phosphorylation in bovine pulmonary artery endothelial cells (BPAEC). The incorporation of labelled [P-32(i)] into BPAEC was dependent on the concentration of the oxidant employed and time of incubation. Of the oxidants tested, pervanadate (10 mu M) induced maximum incorporation of [P-32(i)] into cells (two- to threefold over control) followed by H2O2 (1 mM) and vanadate (100 mu M) and clear differences in labeled protein profiles were noticed between control and oxidant treated cells. The proteins, analyzed by SDS-PAGE, showed distinct increases in labeling patterns ranging from 21-205 kDa, as evidenced by autoradiography. While the majority of the incorporated [P-32(i)] was in serine/threonine residues, immunoprecipitation and immunoblotting of cell lysates, using an antiphosphotyrosine antibody, revealed that oxidant treatment resulted in significant increases in total protein tyrosine phosphorylation. Most significantly, immunoprecipitation of cell lysates, from pervanadate treatment showed distinct tyrosine phosphorylation of 22 kDa protein, which was identified as caveolin, a marker of caveolae. Pervanadate-mediated phosphorylation was effectively inhibited by staurosporine (5 mu M), while genistein showed only partial attenuation. Furthermore, H2O2 treatment resulted in enhanced phosphorylation of 24 kDa protein, which was attenuated by genistein. In addition, oxidant-treated cells exhibited increased tyrosine kinase activity and decreased phosphatase activity. These data show differences in labeling profiles of proteins in response to different oxidants, suggesting differential modulation of distinct protein kinases/phosphatases. Copyright (C) 1996 Elsevier Science Inc.