Regulation of VASP serine 157 phosphorylation in human neutrophils after stimulation by a chemoattractant.
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 hypothesis is that PKA regulates neutrophil migration through VASP Ser 157 phosphorylation. The objective of this study was to characterize VASP Ser 157 phosphorylation in chemoattractant-stimulated neutrophils. fMLF, IL-8, leukotriene B(4), 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 nonchemoattractant, proinflammatory cytokine TNF-alpha did not affect Ser 157 phosphorylation. The kinetics of fMLF-induced VASP Ser 157 phosphorylation levels closely matched the kinetics of the fold-change in F-actin levels in fMLF-stimulated neutrophils. fMLF-induced Ser 157 phosphorylation was abolished 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-dependent protein kinase II inhibitor KN-62. Inhibition of adhesion with EDTA or the anti-beta2-integrin antibody IB4 did not alter fMLF-induced VASP phosphorylation or dephosphorylation. These data show that chemoattractant stimulation of human neutrophils induces a rapid and transient PKA-dependent VASP Ser 157 phosphorylation. Adhesion does not appear to be an important regulator of the state of VASP Ser 157 phosphorylation in chemoattractant-stimulated neutrophils.
Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606, USA.
Rachael E
RE
Eckert
Samuel L
SL
Jones
D000199:Actins / Q000378:metabolism; D015153:Blotting, Western; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000037:antagonists & inhibitors / Q000502:physiology; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D002633:Chemotaxis; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D016209:Interleukin-8 / Q000494:pharmacology; D007975:Leukotriene B4 / Q000494:pharmacology; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000031:analogs & derivatives / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D010972:Platelet Activating Factor / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology; D012694:Serine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D000098662:Vasodilator-Stimulated Phosphoprotein
D000199:Actins; D015815:Cell Adhesion Molecules; D004791:Enzyme Inhibitors; D016209:Interleukin-8; D008840:Microfilament Proteins; D010750:Phosphoproteins; D010972:Platelet Activating Factor; D014409:Tumor Necrosis Factor-alpha; D000098662:Vasodilator-Stimulated Phosphoprotein; D007975:Leukotriene B4; D012694:Serine; D009240:N-Formylmethionine Leucyl-Phenylalanine; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D011493:Protein Kinase C; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D006152:Cyclic GMP