cAMP-dependent protein kinase phosphorylation of EVL, a mena/VASP relative, regulates its interaction with actin and SH3 domains

Proteins of the Ena/VASP family are implicated in processes that require dy namic actin remodeling such as axon guidance and platelet activation. In th is work, we explored some of the pathways that likely regulate actin dynami cs in part via EVL (Ena/VASP-like protein). Two isoforms, EVL and EVL-I, we re highly expressed in hematopoietic cells of thymus and spleen. In CDS-act ivated T-cells, EVL was found in F-actin-rich patches and at the distal tip s 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 fo r the cAMP-dependent protein kinase, and this phosphorylation regulated sev eral of the interactions between EVL and its ligands. Unlike VASP, EVL nucl eated actin polymerization under physiological conditions, whereas phosphor ylation 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 pr ofilin, likely via its proline-rich core. Binding of Abl and nSrc SH3 domai ns, but not profilin or other SH3 domains, was abolished by cAMP-dependent protein kinase phosphorylation of EVL. We show strong cooperative binding o f two profilin dimers on the polyproline sequence of EVL, Additionally, pro filin competed with the SH3 domains for binding to partially overlapping bi nding 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.