The vasodilator-stimulated phosphoprotein is regulated by cyclic GMP-dependent protein kinase during neutrophil spreading

The expression and phosphorylation state of the vasodilator-stimulated phos phoprotein (VASP), a membrane-associated focal adhesion protein, was invest igated in human neutrophils. Adhesion and spreading of neutrophils induced the rapid phosphorylation of VASP. The phosphorylation of VASP was dependen t on cell spreading, as VASP was expressed as a dephosphorylated protein in round adherent cells and was phosphorylated at the onset of changes in cel l shape from round to spread cells. Immunofluorescence microscopy demonstra ted that VASP was localized at the cell cortex in round cells and redistrib uted to focal adhesions at the ventral surface of the cell body during cell spreading. Dual labeling of spread cells indicated that VASP was colocaliz ed with F-actin in filopodia and in focal adhesions, suggesting that the ph osphorylation of VASP during cell spreading may be involved in focal adhesi on complex organization and actin dynamics. VASP is a prominent substrate f or both cGMP-dependent protein kinase (cGK) and cAMP-dependent protein kina se. Evidence suggested that cGK regulated neutrophil spreading, as both VAS P phosphorylation and neutrophil spreading were inhibited by Rp-8-pCPT-cGMP S (cGK inhibitor), but not KT5720 (cAMP-dependent protein kinase inhibitor) . In contrast, neutrophil spreading was accelerated when cGMP levels were e levated with 8-Br-cGMP, a direct activator of cGK. Furthermore, the same co nditions that lead to VASP phosphorylation during neutrophil adherence and spreading induced significant elevations of cGMP in neutrophils. These resu lts indicate that cGMP/cGK signal transduction is required for neutrophil s preading, and that VASP is a target for cGK regulation.