Regulation of human endothelial cell focal adhesion sites and migration by cGMP-dependent protein kinase I.
cGMP-dependent protein kinase type I (cGK I), a major constituent of the atrial natriuretic peptide (ANP)/nitric oxide/cGMP signal transduction pathway, phosphorylates the vasodilator-stimulated phosphoprotein (VASP), a member of the Ena/VASP family of proteins involved in regulation of the actin cytoskeleton. Here we demonstrate that stimulation of human umbilical vein endothelial cells (HUVECs) by both ANP and 8-(4-chlorophenylthio)guanosine 3':5'-monophosphate (8-pCPT-cGMP) activates transfected cGK I and causes detachment of VASP and its known binding partner (zyxin) from focal adhesions in >60% of cells after 30 min. The ANP effects, but not the 8-pCPT-cGMP effects, reversed after 3 h of treatment. In contrast, a catalytically inactive cGK Ibeta mutant (cGK Ibeta-K405A) was incapable of mediating these effects. VASP mutated (Ser/Thr to Ala) at all three of its established phosphorylation sites (vesicular stomatitis virus-tagged VASP-AAA mutant) was not phosphorylated by cGK I and was resistant to detaching from HUVEC focal adhesions in response to 8-pCPT-cGMP. Furthermore, activation of cGK I, but not of mutant cGK Ibeta-K405A, caused a 1.5-2-fold inhibition of HUVEC migration, a dynamic process highly dependent on focal adhesion formation and disassembly. These results indicate that cGK I phosphorylation of VASP results in loss of VASP and zyxin from focal adhesions, a response that could contribute to cGK alteration of cytoskeleton-regulated processes such as cell migration.
Smolenski
A
A
Institut für Klinische Biochemie und Pathobiochemie, Medizinische Universitätsklinik, Würzburg, Germany.
Poller
W
W
Walter
U
U
Lohmann
S M
SM
D000256:Adenoviridae / Q000235:genetics; D009320:Atrial Natriuretic Factor / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D002384:Catalysis; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002460:Cell Line; D002465:Cell Movement; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006023:Glycoproteins; D006801:Humans; D008667:Metalloproteins / Q000378:metabolism; D008840:Microfilament Proteins; D016296:Mutagenesis; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011233:Precipitin Tests; D013873:Thionucleotides / Q000494:pharmacology; D013997:Time Factors; D014471:Umbilical Veins / Q000378:metabolism; D016596:Vinculin / Q000378:metabolism; D060589:Zyxin; D000098662:Vasodilator-Stimulated Phosphoprotein
D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; D005353:Fibronectins; D006023:Glycoproteins; D008667:Metalloproteins; D008840:Microfilament Proteins; D010750:Phosphoproteins; D010975:Platelet Aggregation Inhibitors; D013873:Thionucleotides; C092842:ZYX protein, human; D060589:Zyxin; D000098662:Vasodilator-Stimulated Phosphoprotein; D016596:Vinculin; C055656:8-((4-chlorophenyl)thio)cyclic-3',5'-GMP; D009320:Atrial Natriuretic Factor; D005340:Fibrinogen; D003094:Collagen; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP