Phosphorylation of the vasodilator-stimulated phosphoprotein regulates itsinteraction with actin

The vasodilator-stimulated phosphoprotein (VASP) is a major substrate for c yclic nucleotide-dependent kinases in platelets and other cardiovascular ce lls. It promotes actin nucleation and binds to actin filaments in vitro and associates with stress fibers in cells. The VASP-actin interaction is salt -sensitive, arguing for electrostatic interactions. Hence, phosphorylation may significantly alter the actin binding properties of VASP. This hypothes is was investigated by analyzing complex formation of recombinant murine VA SP with actin after phosphorylation with cAMP-dependent kinase in different assays. cAMP-dependent kinase phosphorylation had a negative effect on bot h actin nucleation and VASP interaction with actin filaments, with the acti n nucleating capacity being more affected than actin filament binding and b undling. Replacing VASP residues known to be phosphorylated in vivo by acid ic residues to mimic phosphorylation had similar although less dramatic eff ects on VASP-actin interactions. In contrast, phosphorylation had no signif icant effect on VASP oligomerization or its interaction with its known liga nds profilin, vinculin, and zyxin. When overexpressing VASP mutants in euka ryotic cells, they all showed targeting to focal contacts and stress fibers , Our results imply that VASP phosphorylation may act as an immediate negat ive regulator of actin dynamics.