AMP-activated protein kinase impairs endothelial actin cytoskeleton assembly by phosphorylating vasodilator-stimulated phosphoprotein.
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.
Blume
Constanze
C
Institute for Clinical Biochemistry and Pathobiochemistry, Julius-Maximilians-University Würzburg, Josef-Schneider Strasse 2, D-97080 Würzburg, Germany.
Benz
Peter M
PM
Institute for Clinical Biochemistry and Pathobiochemistry, Julius-Maximilians-University Würzburg, Josef-Schneider Strasse 2, D-97080 Würzburg, Germany.
Walter
Ulrich
U
Institute for Clinical Biochemistry and Pathobiochemistry, Julius-Maximilians-University Würzburg, Josef-Schneider Strasse 2, D-97080 Würzburg, Germany.
Ha
Joohun
J
Department of Biochemistry and Molecular Biology, Kyung University College of Medicine, 1 Hoegi-dong, Tongdaemun-gu, Seoul, Korea 130-701, and.
Kemp
Bruce E
BE
St. Vincent's Institute of Medical Research, Fitzroy, Victoria 3065 and Commonwealth Scientific and Industrial Research Organization Molecular and Health Technologies, Parkville, Victoria 3052, Australia.
Renné
Thomas
T
Institute for Clinical Biochemistry and Pathobiochemistry, Julius-Maximilians-University Würzburg, Josef-Schneider Strasse 2, D-97080 Würzburg, Germany. Electronic address: thomas@renne.net.
D055372:AMP-Activated Protein Kinases; D000818:Animals; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism*; D003599:Cytoskeleton / Q000235:genetics / Q000378:metabolism*; D003921:Diabetes Mellitus, Experimental / Q000201:enzymology / Q000235:genetics; D003924:Diabetes Mellitus, Type 2 / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D042783:Endothelial Cells / Q000201:enzymology* / Q000473:pathology; D006801:Humans; D008297:Male; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D011924:Rats, Zucker; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D022502:Stress Fibers / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D000098662:Vasodilator-Stimulated Phosphoprotein
D015815:Cell Adhesion Molecules; D008840:Microfilament Proteins; D009097:Multienzyme Complexes; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D000098662:Vasodilator-Stimulated Phosphoprotein; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases