AMP激活蛋白激酶与沉默调节蛋白1对皮层肌动蛋白的共同调节作用有助于内皮功能。
AMP-Activated Protein Kinase and Sirtuin 1 Coregulation of Cortactin Contributes to Endothelial Function.
作者信息
Shentu Tzu-Pin, He Ming, Sun Xiaoli, Zhang Jianlin, Zhang Fan, Gongol Brendan, Marin Traci L, Zhang Jiao, Wen Liang, Wang Yinsheng, Geary Gregory G, Zhu Yi, Johnson David A, Shyy John Y-J
机构信息
From the Division of Cardiology, Department of Medicine, University of California, San Diego, La Jolla (T.-P.S., M.H., J.Z., J.Z.; L.W., J.Y.-J.S.); Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing, China (X.S., Y.Z.); Department of Chemistry, University of California, Riverside (F.Z., Y.W.); Department of Cardiopulmonary Sciences, Schools of Allied Health, Loma Linda University, CA (B.G., T.L.M.); Department of Kinesiology and Health Sciences, California State University, San Bernardino (G.G.G.); and Division of Biomedical Sciences, University of California, Riverside (D.A.J.).
出版信息
Arterioscler Thromb Vasc Biol. 2016 Dec;36(12):2358-2368. doi: 10.1161/ATVBAHA.116.307871. Epub 2016 Oct 6.
OBJECTIVE
Cortactin translocates to the cell periphery in vascular endothelial cells (ECs) on cortical-actin assembly in response to pulsatile shear stress. Because cortactin has putative sites for AMP-activated protein kinase (AMPK) phosphorylation and sirtuin 1 (SIRT1) deacetylation, we examined the hypothesis that AMPK and SIRT1 coregulate cortactin dynamics in response to shear stress.
APPROACH AND RESULTS
Analysis of the ability of AMPK to phosphorylate recombinant cortactin and oligopeptides whose sequences matched AMPK consensus sequences in cortactin pointed to Thr-401 as the site of AMPK phosphorylation. Mass spectrometry confirmed Thr-401 as the site of AMPK phosphorylation. Immunoblot analysis with AMPK siRNA and SIRT1 siRNA in human umbilical vein ECs and EC-specific AMPKα2 knockout mice showed that AMPK phosphorylation of cortactin primes SIRT1 deacetylation in response to shear stress. Immunoblot analyses with cortactin siRNA in human umbilical vein ECs, phospho-deficient T401A and phospho-mimetic T401D mutant, or aceto-deficient (9K/R) and aceto-mimetic (9K/Q) showed that cortactin regulates endothelial nitric oxide synthase activity. Confocal imaging and sucrose-density gradient analyses revealed that the phosphorylated/deacetylated cortactin translocates to the EC periphery facilitating endothelial nitric oxide synthase translocation from lipid to nonlipid raft domains. Knockdown of cortactin in vitro or genetic reduction of cortactin expression in vivo in mice substantially decreased the endothelial nitric oxide synthase-derived NO bioavailability. In vivo, atherosclerotic lesions increase in ApoE/cortactin mice, when compared with ApoE/cortactin littermates.
CONCLUSIONS
AMPK phosphorylation of cortactin followed by SIRT1 deacetylation modulates the interaction of cortactin and cortical-actin in response to shear stress. Functionally, this AMPK/SIRT1 coregulated cortactin-F-actin dynamics is required for endothelial nitric oxide synthase subcellular translocation/activation and is atheroprotective.
目的
在搏动性剪切应力作用下，皮层肌动蛋白组装时，皮层肌动蛋白结合蛋白（Cortactin）会转位至血管内皮细胞（ECs）的细胞周边。由于Cortactin具有假定的AMP激活蛋白激酶（AMPK）磷酸化位点和沉默调节蛋白1（SIRT1）去乙酰化位点，我们检验了以下假设：AMPK和SIRT1共同调节Cortactin在剪切应力作用下的动力学。
方法与结果
分析AMPK对重组Cortactin和序列与Cortactin中AMPK共有序列匹配的寡肽进行磷酸化的能力，结果表明苏氨酸-401（Thr-401）是AMPK磷酸化位点。质谱分析证实Thr-401是AMPK磷酸化位点。在人脐静脉内皮细胞和内皮细胞特异性AMPKα2基因敲除小鼠中，用AMPK siRNA和SIRT1 siRNA进行免疫印迹分析表明，在剪切应力作用下，Cortactin的AMPK磷酸化引发SIRT1去乙酰化。在人脐静脉内皮细胞中用Cortactin siRNA、磷酸化缺陷型T401A和磷酸化模拟型T401D突变体或乙酰化缺陷型（9K/R）和乙酰化模拟型（9K/Q）进行免疫印迹分析表明，Cortactin调节内皮型一氧化氮合酶活性。共聚焦成像和蔗糖密度梯度分析显示，磷酸化/去乙酰化的Cortactin转位至内皮细胞周边，促进内皮型一氧化氮合酶从脂筏结构域向非脂筏结构域转位。在体外敲低Cortactin或在体内基因降低小鼠Cortactin表达，会显著降低内皮型一氧化氮合酶衍生的NO生物利用度。在体内，与ApoE/Cortactin同窝小鼠相比，ApoE/Cortactin小鼠的动脉粥样硬化病变增加。
结论
Cortactin先经AMPK磷酸化再经SIRT1去乙酰化，可调节Cortactin与皮层肌动蛋白在剪切应力作用下的相互作用。在功能上，这种由AMPK/SIRT1共同调节的Cortactin - F - 肌动蛋白动力学是内皮型一氧化氮合酶亚细胞转位/激活所必需的，且具有抗动脉粥样硬化作用。
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