Abstract
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.
Highlights
Proteins coordinate different modes of actin organization including actin filament assembly, cross-linking, and bundling
The present study identifies Vasodilator-stimulated phosphoprotein (VASP) as a novel AMPK substrate in vitro in living cells and in a rat model for diabetes
We demonstrate that AMPK-mediated phosphorylation of the residue Thr-278 reduces filamentous actin (F-actin) accumulation activity of VASP and impairs stress fiber formation
Summary
Ser-239 (pSer-239) in cyclic-nucleotide dependent-kinase signaling cascades was established in other cardiovascular cells including aortic smooth muscle cells [10] and endothelial cells [11], in vessels of animal models, and in humans (for review, see Ref. 12). To quantify the magnitude of phosphorylation at these sites, phosphorylation-specific antibodies anti-pSer-157 (5C6) and anti-pSer-239 (16C2) were developed [11] Analyses based on these antibodies revealed that reduced Ser-157/Ser-239 phosphorylation was associated with defective nitric oxide (NO)/ cGMP signaling and endothelial dysfunction [13]. AMPK-mediated VASP Thr278 phosphorylation reduced F-actin assembly, resulting in defective stress fiber formation and altered cell morphology. In arterial vessel walls from Zucker Diabetic Fatty (ZDF) rats, which serve as a model for type II diabetes mellitus, AMPK activity and VASP Thr-278 phosphorylation were severely reduced. Our data show that metabolic signals via AMPK-mediated VASP Thr-278 phosphorylation may inhibit actin polymerization. In this way our study establishes a novel signaling pathway that directly links cytoskeleton dynamics to energy metabolism that is impaired in diabetic vessels
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