Regulation of human endothelial cell focal adhesion sites and migration bycGMP-dependent protein kinase I

cGMP-dependent protein kinase type I (cGK I), a major constituent of the at rial natriuretic peptide (ANP)/nitric oxide/cGMP signal transduction pathwa y, phosphorylates the vasodilator-stimulated phosphoprotein (VASP), a membe r of the Ena/VASP family of proteins involved in regulation of the actin cy toskeleton. Here we demonstrate that stimulation of human umbilical vein en dothelial cells (HUVECs) by both ANP and 8-(4-chlorophenylthio)guanosine 3' :5'-monophosphate (8-pCPT-cGMP) activates transfected cGK I and causes deta chment of VASP and its known binding partner (zyxin) from focal adhesions i n >60% of cells after 30 min. The ANP effects, but not the 8-pCPT-cGMP effe cts, reversed after 3 h of treatment. In contrast, a catalytically inactive cGK I beta mutant (cGK I beta-K405A) was incapable of mediating these effe cts. VASP mutated (Ser/Thr to Ala) at all three of its established phosphor ylation sites (vesicular stomatitis virus-tagged VASP-AAA mutant) was not p hosphorylated by cGK I and was resistant to detaching from HUVEC focal adhe sions in response to 8-pCPT-cGMP. Furthermore, activation of cGK I, but not of mutant cGK I beta-K405A, caused a 1.5-2-fold inhibition of HUVEC migrat ion, a dynamic process highly dependent on focal adhesion formation and dis assembly. These results indicate that cGK I phosphorylation of VASP results in loss of VASP and zyxin from focal adhesions, a response that could cont ribute to cGK alteration of cytoskeleton-regulated processes such as cell m igration.