Hsp90 ensures the transition from the early Ca2+-dependent to the late phosphorylation-dependent activation of the endothelial nitric-oxide synthase in vascular endothelial growth factor-exposed endothelial cells
Hsp90 ensures the transition from the early Ca2+-dependent to the late phosphorylation-dependent activation of the endothelial nitric-oxide synthase in vascular endothelial growth factor-exposed endothelial cells
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DOI：
10.1074/jbc.m101371200
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发表时间：
2001-08-31
影响因子：
4.8
通讯作者：
Feron, O
中科院分区：
文献类型：
--
作者：
Brouet, A;Sonveaux, P;Feron, O
血管内皮生长因子（VEGF）部分通过激活内皮一氧化氮合酶（eNOS）发挥其血管生成作用。最近显示，内皮细胞中 VEGF 刺激后，与热休克蛋白 90 (hsp90) 的关联和 Akt 的磷酸化可单独激活 eNOS。在这里，我们检查了 VEGF 暴露的内皮细胞中这些不同机制之间的相互作用。我们证明，hsp90 与 eNOS 的结合实际上是触发 eNOS 从 Ca2+ 依赖性激活转变为 VEGF 磷酸化介导的其活性增强的关键事件。因此，我们发现早期 VEGF 刺激首先导致 Caveolin-eNOS 复合物的 Ca2+/钙调蛋白破坏，并促进 eNOS 和 hsp90 之间的结合。然后，eNOS 结合的 hsp90 可以将 VEGF 激活（磷酸化）的 Akt 募集到复合物中，进而磷酸化 eNOS。在表达野生型或 S1177A 突变型 eNOS 的转染 COS 细胞中进行的进一步实验使我们确定丝氨酸 1177 是 hsp90 依赖性 Akt 介导的 eNOS 激活的关键残基。最后，我们记录了虽然 V-EGF 诱导的 eNOS 磷酸化导致 NO 的持续产生，与 [Ca2+](i) 的持续增加无关，但 eNOS 激活的这一后期阶段严格取决于最初 VEGF 诱导的 Ca2+ 依赖性酶刺激。这些数据建立了导致 VEGF 持续激活 eNOS 的关键事件时序，并提出了通过普遍存在的伴侣蛋白 hsp90 响应该细胞因子调节 NO 产生的新方法。
Vascular endothelial growth factor (VEGF) exerts its angiogenic effects partly through the activation of endothelial nitric-oxide synthase (eNOS). Association with heat shock protein 90 (hsp90) and phosphorylation by Akt were recently shown to separately activate eNOS upon VEGF stimulation in endothelial cells. Here, we examined the interplay between these different mechanisms in VEGF-exposed endothelial cells. We documented that hsp90 binding to eNOS is, in fact, the crucial event triggering the transition from the Ca2+-dependent activation of eNOS to the phosphorylation-mediated potentiation of its activity by VEGF. Accordingly, we showed that early VEGF stimulation first leads to the Ca2+/calmodulin disruption of the caveolin-eNOS complex and promotes the association between eNOS and hsp90. eNOS-bound hsp90 can then recruit VEGF-activated (phosphorylated) Akt to the complex, which in turn can phosphorylate eNOS. Further experiments in transfected COS cells expressing either wild-type or S1177A mutant eNOS led us to identify the serine 1177 as the critical residue for the hsp90-dependent Akt-mediated activation of eNOS. Finally, we documented that although the V-EGF-induced phosphorylation of eNOS leads to a sustained production of NO independently of a maintained increase in [Ca2+](i), this late stage of eNOS activation is strictly conditional on the initial VEGF-induced Ca2+-dependent stimulation of the enzyme. These data establish the critical temporal sequence of events leading to the sustained activation of eNOS by VEGF and suggest new ways of regulating the production of NO in response to this cytokine through the ubiquitous chaperone protein, hsp90.