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 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.

VASP is a CXCR2-interacting protein that regulates CXCR2-mediated polarization and chemotaxis

“…1B). It has previously been demonstrated that VASP is phosphorylated on Ser157 upon stimulation with the chemoattractant fMLP (Eckert and Jones, 2007). Western blot analysis on lysates from vehicleand CXCL8-stimulated cells using a Ser157-P VASP-specific antibody confirmed that the shifted VASP band was Ser157-P VASP (Fig.…”

Prasugrel Metabolites Inhibit Neutrophil Functions

“…Alternatively, prasugrel metabolites could activate a different receptor on neutrophils, which elevates intracellular cyclic adenosine monophosphate (cAMP) levels and inhibits neutrophil functions. We believe that this is unlikely as prasugrel metabolites also failed to cause phosphorylation of vasodilator-stimulated phosphoprotein (downstream of cAMP signaling [Eckert and Jones, 2007]) in neutrophils (data not shown). Hence, further investigations are required to evaluate these possibilities.…”

β2-Adrenergic Receptor Agonists Modulate Human Airway Smooth Muscle Cell Migration via Vasodilator-Stimulated Phosphoprotein

“…The PKA-dependent of VASP was demonstrated in rat aortic vascular smooth muscle cells (20). The stimulation of human neutrophils by a chemoattractant induces the PKA-dependent phosphorylation of VASP at Ser157 (21). VASP also plays a critical role in neuronal function by modulating the initiation of neurites and the outgrowth and guidance of axons (22).…”