Regulation of vasP serine 157 phosphorylation in human neutrophils after stimulation by a chemoattractant

Abstract: Vasodilator-stimulated phosphoprotein (VASP) is a cAMP-dependent protein kinase A (PKA) substrate, which links cellular signaling to cytoskeletal organization and cellular movement. VASP is phosphorylated by PKA on serine 157 (Ser 157), which is required for VASP function in platelet adhesion and fibroblast motility. Our hy-pothesis is that PKA regulates neutrophil migration through VASP Ser 157 phosphorylation. The ob-jective of this study was to characterize VASP Ser 157 phosphorylation in chemoattractant-stimu-lated neutrophils. fMLF, IL-8, leukotriene B4, or platelet-activating factor stimulation resulted in an initial increase in VASP Ser 157 phosphorylation, which was maximal by 30 s and was followed by a return to baseline Ser 157 phosphorylation by 10 min. In contrast, stimulation with the nonchemoat-tractant, proinflammatory cytokine TNF- did not affect Ser 157 phosphorylation. The kinetics of fMLF-induced VASP Ser 157 phosphorylation lev-els closely matched the kinetics of the fold-change in F-actin levels in fMLF-stimulated neutrophils. fMLF-induced Ser 157 phosphorylation was abol-ished by pretreatment with the PKA inhibitor H89 and the adenylyl cyclase inhibitor SQ22536. In contrast, fMLF-induced Ser 157 phosphorylation was unaffected by the PKC inhibitors calphostin and staurosporine, the PKG inhibitors Rp-8-pCPT-cGMP and KT5823, and the calmodulin-depen-dent protein kinase II inhibitor KN-62. Inhibition of adhesion with EDTA or the anti-2-integrin an-tibody IB4 did not alter fMLF-induced VASP phos-phorylation or dephosphorylation. These data show that chemoattractant stimulation of human neutrophils induces a rapid and transient PKA-dependent VASP Ser 157 phosphorylation. Adhe-sion does not appear to be an important regulator of the state of VASP Ser 157 phosphorylation in chemoattractant-stimulated neutrophils. J. Leu-koc. Biol. 82: 000–000; 2007