Vasodilator-stimulated phosphoprotein (VASP) is a member of the Ena/VASP family of proteins that are implicated in regulation of the actin cytoskeleton. All family members share a tripartite structural organization, comprising an N-terminal Ena/VASP homology (EVH) 1 domain, a more divergent proline-rich central part, and a common C-terminal EVH2 region of about 160 -190 amino acids. Using chemical cross-linking, sucrose gradient sedimentation, and gel filtration analyses of different truncated VASP constructs, we demonstrate that the VASP EVH2 region is both necessary and sufficient for tetramerization. Moreover, co-sedimentation and fluorescent phalloidin staining showed that the EVH2 region binds and bundles F-actin in vitro and localizes to stress fibers in transfected cells. Analysis of the functional contribution of highly conserved blocks within this region indicated that residues 259 -276 of human VASP are essential for the interaction with F-actin, whereas residues 343-380 are required for tetramerization, probably via coiled-coil formation. Interactions with F-actin are enhanced by VASP tetramerization. The results demonstrate that the C-terminal EVH2 segment is not only conserved in sequence but also forms a distinct functional entity. The data suggest that the EVH2 segment represents a novel oligomerization and F-actin binding domain.The mammalian vasodilator-stimulated phosphoprotein (VASP) 1 (1) and Drosophila Enabled (Ena) (2) are the founding members of the Ena/VASP family of proteins. While Ena is a substrate of the Abelson tyrosine kinase (Abl) and is also genetically linked to the Abl signaling pathway (2), VASP is a substrate of both cGMP-and cAMP-dependent protein kinases (Ref. 3; for a review see Ref. 4). Three common cGMP-dependent protein kinase/cAMP-dependent protein kinase phosphorylation sites have been biochemically identified in human VASP (Ser-157, Ser-239, and Thr-278) (5, 6), two of which are also conserved in Mena (mammalian Enabled) and one in Evl (Ena-VASP-like), two other family members (7). VASP phosphorylation in response to cyclic nucleotide regulating vasodilators (i.e. cAMP-elevating prostaglandins and cGMP-elevating NO donors) closely correlates with platelet inhibition and in particular with the inhibition of fibrinogen binding to the human platelet integrin ␣ IIb ␤ 3 (3,8). In agreement with these earlier studies, recent analysis of platelets from VASP-deficient mice support the concept that VASP is an important component in modulating agonist-induced integrin ␣ IIb ␤ 3 and P-selectin activation (9, 10). Similar to Ena (11) and its mammalian homologue Mena (7), VASP is an actin filament-associated protein that is predominantly localized at stress fibers, cell-matrix and cell-cell adherens junctions, and highly dynamic membrane areas (12, 13). There is a large body of evidence that both VASP and Mena are involved in the facilitation of spatially confined actin filament formation (for a review see Refs. 14 and 15).All Ena/VASP family members share a tripartite structural ...

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