Takahashi Satoru, Mendelsohn Michael E
Department of Medicine and Division of Cardiology, Molecular Cardiology Research Institute New England Medical Center Hospitals and Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
J Biol Chem. 2003 Aug 15;278(33):30821-7. doi: 10.1074/jbc.M304471200. Epub 2003 Jun 10.
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.
内皮型一氧化氮合酶（eNOS）可生成内源性血管舒张剂一氧化氮（NO），其活性受到翻译后修饰和蛋白质相互作用的高度调控。我们最近使用纯化蛋白来表征热休克蛋白90（HSP90）在低钙和高钙水平下增加eNOS活性的机制（高桥，S.和门德尔松，M.E.（2003年）《生物化学杂志》278，9339 - 9344）。在此，我们扩展这些研究以探讨HSP90、Akt和eNOS之间的相互作用。在对纯化蛋白的研究中，HSP90增加了Akt介导的eNOS在低钙水平下磷酸化和激活的初始速率及最大程度。在没有HSP90的情况下，未在eNOS复合物中观察到Akt，但在有HSP90的情况下，活性和非活性Akt均与eNOS相关联。即使在没有eNOS的情况下，也观察到Akt与HSP90的直接结合。无论是否存在Akt，HSP90都促进钙调蛋白（CaM）与eNOS的结合。格尔德霉素（GA）破坏HSP90 - eNOS结合，减少HSP90刺激的CaM结合，并阻断Akt募集到eNOS复合物以及eNOS在丝氨酸1179处的磷酸化。Akt仅以不依赖HSP90的方式使与CaM结合的eNOS磷酸化。HSP90和活性Akt共同协同增加eNOS活性，这被GA逆转。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 - HSP90 - Akt复合物形成及eNOS激活的影响。1,2 -双（2 -氨基苯氧基）乙烷 - N,N,N',N'-四乙酸四乙酰甲酯（BAPTA - AM）抑制VEGF诱导的而非胰岛素诱导的eNOS - HSP90 - Akt复合物形成及eNOS磷酸化。胰岛素导致eNOS活性迅速、短暂增加，在时间上与eNOS - HSP90 - Akt复合物的形成相关。GA阻止胰岛素诱导的HSP90、Akt和CaM与eNOS的结合，并抑制BAECs中eNOS的激活。血小板衍生生长因子（PDGF）和胰岛素均诱导BAECs中Akt的激活，但只有胰岛素导致HSP90 - Akt - eNOS结合及eNOS磷酸化。这些结果表明HSP90和Akt协同激活eNOS，并提示这种协同作用有助于胰岛素诱导的不依赖钙的eNOS激活。
