Identification, purification, and characterization of a zyxin-related protein that binds the focal adhesion and microfilament protein VASP (vasodilator-stimulated phosphoprotein).

Abstract

VASP (vasodilator-stimulated phosphoprotein), an established substrate of cAMP-and cGMP-dependent protein kinases in vitro and in living cells, is associated with focal adhesions, microfilaments, and membrane regions of high dynamic activity. Here, the identification of an 83-kDa protein (p83) that specifically binds VASP in blot overlays of different cell homogenates is reported. With VASP overlays as a detection tool, p83 was purified from porcine platelets and used to generate monospecific polyclonal antibodies. VASP binding to purified p83 in solid-phase binding assays and the closely matching subcellular localization in double-label immunofluorescence analyses demonstrated that both proteins also directly interact as native proteins in vitro and possibly in living cells. The subcellular distribution, the biochemical properties, as well as microsequencing data revealed that porcine platelet p83 is related to chicken gizzard zyxin and most likely represents the mammalian equivalent of the chicken protein. The VASP-p83 interaction may contribute to the targeting ofVASP to focal adhesions, microfilaments, and dynamic membrane regions. Together with our recent identification of VASP as a natural ligand of the profilin poly-(L-proline) binding site, our present results suggest that, by linking profilin to zyxin/p83, VASP may participate in spatially confined profilin-regulated F-actin formation.Focal adhesions are transmembrane junctions between terminal portions of microfilaments and the underlying extracellular matrix with the heterodimeric integrins as the prevailing transmembrane adhesive receptors (1-3). Understanding of the molecular basis of integrin-dependent cell adhesion and associated signaling events (4, 5) requires elucidation of the focal adhesion architecture. Based mostly on in vitro assays, multiple low-to-moderate affinity interactions have been shown that allow the construction of several interdigitating routes that appear to link actin filaments to the transmembrane integrins and provide docking points for different regulatory proteins (for a review, see refs. 1-3). For instance, vinculin interacts with a-actinin and talin, which both bind to the cytoplasmic domains of integrin ,B subunits, and all three of them have actin-binding activity. Vinculin and a-actinin in addition have been recognized as paxillin-and zyxin-binding proteins, respectively. Although considerable insight into the molecular composition and structural organization of focal adhesions has been gained (1-3), current concepts concerning the functional relationships between individual constituents are still quite fragmentary.Originating from the analyses of cyclic nucleotide-dependent platelet inhibition, we characterized and purified the vasodilator-stimulated phosphoprotein (VASP) as an in vitro and in vivo substrate of cAMP-and cGMP-dependent protein kinases (6-8). Cyclic nucleotide-dependent VASP phosphorylation lies at a point of convergence of two signaling pathways that inhibit platelet activation and associate...

Induction of host signal transduction pathways by Helicobacter pylori

“…VASP is a known substrate for both cAMP-and cGMP-dependent protein kinases (27), placing VASP phosphorylation at the junction of two signal transduction pathways. VASP and profilin, which bind in vivo (28,29), appear to act conjointly to relay signal transduction to the actin cytoskeleton. In consideration of the involvement of VASP with PKG and its role in cellular signal transduction pathways, we investigated the effect of H. pylori attachment to gastric cells on VASP using IF microscopy.…”

cAMP-dependent Protein Kinase Phosphorylation of EVL, a Mena/VASP Relative, Regulates Its Interaction with Actin and SH3 Domains

“…The EVH1 domain is highly conserved and binds to a target sequence that has the consensus (E/ D)FPPPPXDE (11,12). Functional EVH1-binding motifs are present in the Listeria monocytogenes surface protein ActA (12); in the focal adhesion proteins vinculin and zyxin (13,14); in Fyb/SLAP (Fyn-binding protein/SLP76-associated protein), a component of the T-cell receptor pathway (15); and in the axon guidance proteins ROBO (16) and Semaphorin-6A-1. 2 In fibroblasts, the EVH1 domain mediates focal adhesion (1,12,17) and leading edge targeting (8), and a functional EVH1 domain is required for Ena function in Drosophila (18).…”

Analysis and Regulation of Vasodilator-stimulated Phosphoprotein Serine 239 Phosphorylation in Vitro and in Intact Cells Using a Phosphospecific Monoclonal Antibody

“…In platelets and many other cells including vascular smooth muscle cells, endothelial cells, and fibroblasts, VASP has been found to be associated with stress fibers, focal adhesions, cell-cell contacts, and highly dynamic membrane regions (16,17). VASP colocalizes with profilins and binds directly to their poly(L-proline) binding site (18), binds to and colocalizes with zyxin and vinculin (16,19), and also directly binds to Listeria monocytogenes surface protein ActA, which is essential for the actin polymerization-based intracellular motility of this pathogen (20). Functional evidence indicates that VASP is a crucial factor involved in the enhancement of spatially confined actin filament formation (16,20,21).…”