Identification of profilin and src homology 3 domains as binding partners for Drosophila enabled.

Drosophila Enabled (Ena) was first identified as a genetic suppressor of mutations in the Abelson tyrosine kinase and subsequently was shown to be a member of the Ena/vasodilator-stimulated phosphoprotein family of proteins. All members of this family have a conserved domain organization, bind the focal adhesion protein zyxin, and localize to focal adhesions and stress fibers. Members of this family are thought to be involved in the regulation of cytoskeleton dynamics. The Ena protein sequence has multiple poly-(L-proline) residues with similarity to both profilin and src homology 3 binding sites. Here, we show that Ena can bind directly to the Drosophila homolog of profilin, chickadee. Furthermore, Ena and profilin were colocalized in spreading cultured cells. We report that the proline-rich region of Ena is responsible for this interaction as well as for mediating binding to the src homology 3 domain of the Abelson tyrosine kinase. These data support the hypothesis that Ena provides a regulated link between signal transduction and cytoskeleton assembly in the developing Drosophila embryo.

Mutations in Drosophila enabled and rescue by human vasodilator-stimulated phosphoprotein (VASP) indicate important functional roles for Ena/VASP homology domain 1 (EVH1) and EVH2 domains.

Drosophila Enabled (Ena) was initially identified as a dominant genetic suppressor of mutations in the Abelson tyrosine kinase and, more recently, as a member of the Ena/human vasodilator-stimulated phosphoprotein (VASP) family of proteins. We have used genetic, biochemical, and cell biological approaches to demonstrate the functional relationship between Ena and human VASP. In addition, we have defined the roles of Ena domains identified as essential for its activity in vivo. We have demonstrated that VASP rescues the embryonic lethality associated with loss of Ena function in Drosophila and have shown that Ena, like VASP, is associated with actin filaments and focal adhesions when expressed in cultured cells. To define sequences that are central to Ena function, we have characterized the molecular lesions present in two lethal ena mutant alleles that affected the Ena/VASP homology domain 1 (EVH1) and EVH2. A missense mutation that resulted in an amino acid substitution in the EVH1 domain eliminated in vitro binding of Ena to the cytoskeletal protein zyxin, a previously reported binding partner of VASP. A nonsense mutation that resulted in a C-terminally truncated Ena protein lacking the EVH2 domain failed to form multimeric complexes and exhibited reduced binding to zyxin and the Abelson Src homology 3 domain. Our analysis demonstrates that Ena and VASP are functionally homologous and defines the conserved EVH1 and EVH2 domains as central to the physiological activity of Ena.