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 geneti c suppressor of mutations in the Abelson tyrosine kinase and, more rec ently, as a member of the Ena/human vasodilator-stimulated phosphoprot ein (VASP) family of proteins. We have used genetic, biochemical, and cell biological approaches to demonstrate the functional relationship between Ena and human VASP. Ln 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 expresse d in cultured cells. To define sequences that are central to Ena funct ion, 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 substit ution in the EVH1 domain eliminated in vitro binding of Ena to the cyt oskeletal protein zyxin, a previously reported binding partner of VASP . A nonsense mutation that resulted in a C-terminally truncated Ena pr otein lacking the EVH2 domain failed to form multimeric complexes and exhibited reduced binding to zyxin and the Abelson Src homology 3 doma in. Our analysis demonstrates that Ena and VASP are functionally homol ogous and defines the conserved EVH1 and EVH2 domains as central to th e physiological activity of Ena.