Synergistic activation of endothelial nitric-oxide synthase (eNOS) by HSP90 and Akt - Calcium-independent eNOS activation involves formation of an HSP90-Akt-CaM-BOUND eNOS complex
Synergistic activation of endothelial nitric-oxide synthase (eNOS) by HSP90 and Akt - Calcium-independent eNOS activation involves formation of an HSP90-Akt-CaM-BOUND eNOS complex
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DOI：
10.1074/jbc.m304471200
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发表时间：
2003-08-15
影响因子：
4.8
通讯作者：
Mendelsohn, ME
中科院分区：
文献类型：
--
作者：
Takahashi, S;Mendelsohn, ME
内皮型一氧化氮合酶（eNOS）产生内源性血管扩张剂一氧化氮（NO），受翻译后修饰和蛋白质相互作用的高度调节。我们最近使用纯化的蛋白质来表征热休克蛋白90（HSP 90）在低和高Ca 2+水平下增加eNOS活性的机制（Takahashi，S.和Mendelsohn，M. E.（2003）J.Biol.Chem.278，9339-9344）。在这里，我们扩展这些研究，探讨HSP 90，Akt和eNOS之间的相互作用。在纯化蛋白的研究中，HSP 90增加了Akt介导的eNOS磷酸化和激活的初始速率和最大程度，在低Ca 2+水平。在没有HSP 90的情况下，在eNOS复合物中未观察到Akt，但在HSP 90存在下，活性和非活性Akt均与eNOS相关。即使在没有eNOS的情况下也观察到Akt与HSP 90的直接结合。HSP 90也促进CaM与eNOS的结合，而与Akt的存在无关。格尔德霉素（GA）破坏HSP 90-eNOS结合，减少HSP 90刺激的CaM结合，并阻断Akt向eNOS复合物的募集和eNOS在Ser-1179的磷酸化。Akt磷酸化钙调素结合的eNOS，在HSP 90的独立方式。HSP 90和Akt协同增加eNOS活性，GA可逆转此作用。在牛主动脉内皮细胞（BAECs）中，比较了血管内皮生长因子（VEGF）和胰岛素对eNOS-HSP 90-Akt复合物形成和eNOS激活的影响。BAPTA-AM抑制VEGF诱导的eNOS-HSP 90-Akt复合物形成和eNOS磷酸化，但不抑制胰岛素诱导的eNOS-HSP 90-Akt复合物形成和eNOS磷酸化。胰岛素引起的eNOS活性的快速、短暂增加与eNOS-HSP 90-Akt复合物的形成在时间上相关。GA阻止胰岛素诱导的HSP 90、Akt和CaM与eNOS的结合，并抑制BAEC中eNOS的活化。血小板源性生长因子（PDGF）和胰岛素均可诱导BAEC中Akt的活化，但只有胰岛素可诱导HSP 90-Akt-eNOS结合和eNOS磷酸化。这些结果表明，HSP 90和Akt协同激活eNOS，并表明这种协同作用有助于响应于胰岛素的Ca 2+非依赖性eNOS激活。
Endothelial nitric-oxide synthase (eNOS), which generates the endogenous vasodilator, nitric oxide (NO), is highly regulated by post-translational modifications and protein interactions. We recently used purified proteins to characterize the mechanisms by which heat shock protein 90 (HSP90) increases eNOS activity at low and high Ca2+ levels (Takahashi, S. and Mendelsohn, M. E. (2003) J. Biol. Chem. 278, 9339-9344). Here we extend these studies to explore interactions between HSP90, Akt, and eNOS. In studies with purified proteins, HSP90 increased the initial rate and maximal extent of Akt-mediated eNOS phosphorylation and activation at low Ca2+ levels. Akt was not observed in the eNOS complex in the absence of HSP90, but both active and inactive Akt associated with eNOS in the presence of HSP90. Direct binding of Akt to HSP90 was observed even in the absence of eNOS. HSP90 also facilitated CaM binding to eNOS irrespective of Akt presence. Geldanamycin (GA) disrupted HSP90-eNOS binding, reduced HSP90-stimulated CaM binding, and blocked both recruitment of Akt to the eNOS complex and phosphorylation of eNOS at Ser-1179. Akt phosphorylated only CaM-bound eNOS, in an HSP90-independent manner. HSP90 and active Akt together increased eNOS activity synergistically, which was reversed by GA. In bovine aortic endothelial cells (BAECs), the effects of vascular endothelial growth factor (VEGF) and insulin on eNOS-HSP90-Akt complex formation and eNOS activation were compared. BAPTA-AM inhibited VEGF- but not insulin-induced eNOS-HSP90-Akt complex formation and eNOS phosphorylation. Insulin caused rapid, transient increase in eNOS activity correlated temporally with the formation of eNOS-HSP90-Akt complex. GA prevented insulin-induced association of HSP90, Akt and CaM with eNOS and inhibited eNOS activation in BAECs. Both platelet-derived growth factor (PDGF) and insulin induced activation of Akt in BAECs, but only insulin caused HSP90-Akt-eNOS association and eNOS phosphorylation. These results demonstrate that HSP90 and Akt synergistically activate eNOS and suggest that this synergy contributes to Ca2+-independent eNOS activation in response to insulin.