Blume Constanze, Benz Peter M, Walter Ulrich, Ha Joohun, Kemp Bruce E, Renné Thomas
Institute for Clinical Biochemistry and Pathobiochemistry, Julius-Maximilians-University Würzburg, Josef-Schneider Strasse 2, D-97080 Würzburg, Germany.
Department of Biochemistry and Molecular Biology, Kyung University College of Medicine, 1 Hoegi-dong, Tongdaemun-gu, Seoul, Korea 130-701, and.
J Biol Chem. 2007 Feb 16;282(7):4601-4612. doi: 10.1074/jbc.M608866200. Epub 2006 Nov 2.
Vasodilator-stimulated phosphoprotein (VASP) is an actin regulatory protein that links signaling pathways to remodeling of the cytoskeleton. VASP functions are modulated by protein kinases, which phosphorylate the sites Ser-157, Ser-239, and Thr-278. The kinase responsible for Thr-278 phosphorylation, biological functions of the phosphorylation, and association with disease states have remained enigmatic. Using VASP phosphorylation status-specific antibodies, we identified AMP-activated protein kinase (AMPK), a serine-threonine kinase and fundamental sensor of energy homeostasis, in a screen for kinases that phosphorylate the Thr-278 site of VASP in endothelial cells. Pharmacological AMPK inhibitors and activators and AMPK mutants revealed that the kinase specifically targets residue Thr-278 but not Ser-157 or Ser-239. Quantitative fluorescence-activated cell sorter analysis and serum response factor transcriptional reporter assays, which quantify the cellular F-/G-actin equilibrium, indicated that AMPK-mediated VASP phosphorylation impaired actin stress fiber formation and altered cell morphology. In the Zucker Diabetic Fatty (ZDF) rat model for type II diabetes, AMPK activity and Thr-278 phosphorylation were substantially reduced in arterial vessel walls. These findings suggest that VASP is a new AMPK substrate, that VASP Thr-278 phosphorylation translates metabolic signals into actin cytoskeleton rearrangements, and that this signaling system becomes down-regulated in diabetic vessels.
血管舒张刺激磷蛋白（VASP）是一种肌动蛋白调节蛋白，它将信号通路与细胞骨架重塑联系起来。VASP的功能受蛋白激酶调节，这些激酶使丝氨酸157、丝氨酸239和苏氨酸278位点磷酸化。负责苏氨酸278磷酸化的激酶、磷酸化的生物学功能以及与疾病状态的关联一直不明确。我们使用VASP磷酸化状态特异性抗体，在内皮细胞中筛选磷酸化VASP苏氨酸278位点的激酶时，鉴定出了AMP激活的蛋白激酶（AMPK），一种丝氨酸 - 苏氨酸激酶和能量稳态的基本传感器。药理学上的AMPK抑制剂和激活剂以及AMPK突变体表明，该激酶特异性靶向苏氨酸278残基，而不是丝氨酸157或丝氨酸239。定量荧光激活细胞分选分析和血清反应因子转录报告分析（用于量化F - /G - 肌动蛋白平衡）表明，AMPK介导的VASP磷酸化损害了肌动蛋白应激纤维的形成并改变了细胞形态。在II型糖尿病的Zucker糖尿病脂肪（ZDF）大鼠模型中，动脉血管壁中的AMPK活性和苏氨酸278磷酸化显著降低。这些发现表明，VASP是一种新的AMPK底物，VASP苏氨酸278磷酸化将代谢信号转化为肌动蛋白细胞骨架重排，并且这种信号系统在糖尿病血管中下调。
