Proteins of the Ena/VASP family are implicated in processes that require dynamic actin remodeling such as axon guidance and platelet activation. In this work, we explored some of the pathways that likely regulate actin dynamics in part via EVL (Ena/VASP-like protein). Two isoforms, EVL and EVL-I, were highly expressed in hematopoietic cells of thymus and spleen. In CD3-activated T-cells, EVL was found in F-actin-rich patches and at the distal tips of the microspikes that formed on the activated side of the T-cells. Like the other family members, EVL localized to focal adhesions and the leading edge of lamellipodia when expressed in fibroblasts. EVL was a substrate for the cAMP-dependent protein kinase, and this phosphorylation regulated several of the interactions between EVL and its ligands. Unlike VASP, EVL nucleated actin polymerization under physiological conditions, whereas phosphorylation of both EVL and VASP decreased their nucleating activity. EVL bound directly to the Abl, Lyn, and nSrc SH3 domains; the FE65 WW domain; and profilin, likely via its proline-rich core. Binding of Abl and nSrc SH3 domains, but not profilin or other SH3 domains, was abolished by cAMP-dependent protein kinase phosphorylation of EVL. We show strong cooperative binding of two profilin dimers on the polyproline sequence of EVL. Additionally, profilin competed with the SH3 domains for binding to partially overlapping binding sites. These data suggest that the function of EVL could be modulated in a complex manner by its interactions with multiple ligands and through phosphorylation by cyclic nucleotide dependent kinases.To respond properly to environmental cues, cells possess multiple complex signal transduction networks. Many pathways lead to dynamic changes of the actin cytoskeleton that form the basis for cell movement in a wide variety of biological phenomena. In recent years, many proteins participating in one or more signal transduction pathways have been identified. One group of multifunctional proteins, involved in actin-based motility, is the Ena/VASP family of proteins that include Drosophila Ena (Enabled), Mena (mammalian Ena), VASP (vasodilator-stimulated phosphoprotein), and EVL (Ena/VASP-like protein) (1). Ena was identified through genetic interactions with the Drosophila Abl homologue (2, 3), whereas VASP was identified as a prominent target for cAMP (PKA) 1 -and cGMPdependent protein kinases in platelets (4). Mena and EVL were identified by similarity to Ena (1). Ena, Mena, and VASP are important in processes that require highly dynamic actin reorganization, including axon guidance (5), platelet aggregation (6, 7), and fibroblast motility (8). The proteins are concentrated in regions of the cell associated with movement and adhesion, including the leading edge of lamellipodia, focal adhesions, and adherens junctions (1, 9, 10).The Ena/VASP proteins share a common domain structure that consists of an amino-terminal Ena/VASP homology (EVH) 1 domain, a carboxyl-terminal EVH2 domain, and a central proline-rich domain...

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Vertebrate nonmuscle cells express two actin isoforms: cytoplasmic ␤-and ␥-actin. Because of the presence and localized translation of ␤-actin at the leading edge, this isoform is generally accepted to specifically generate protrusive forces for cell migration. Recent evidence also implicates ␤-actin in gene regulation. Cell migration without ␤-actin has remained unstudied until recently and it is unclear whether other actin isoforms can compensate for this cytoplasmic function and/or for its nuclear role. Primary mouse embryonic fibroblasts lacking ␤-actin display compensatory expression of other actin isoforms. Consistent with this preservation of polymerization capacity, ␤-actin knockout cells have unchanged lamellipodial protrusion rates despite a severe migration defect. To solve this paradox we applied quantitative proteomics revealing a broad genetic reprogramming of ␤-actin knockout cells. This also explains why reintroducing ␤-actin in knockout cells does not restore the affected cell migration. Pathway analysis suggested increased Rho-ROCK signaling, consistent with observed phenotypic changes. We therefore developed and tested a model explaining the phenotypes in ␤-actin knockout cells based on increased Rho-ROCK signaling and increased TGF␤ production resulting in increased adhesion and contractility in the knockout cells. Inhibiting ROCK or myosin restores migration of ␤-actin knockout cells indicating that other actins compensate for ␤-actin in this process. Consequently, isoactins act redundantly in providing propulsive forces for cell migration, but ␤-actin has a unique nuclear function, regulating expression on transcriptional and post-translational levels, thereby preventing myogenic differentiation.