Characterization of the interaction between zyxin and members of the ena/vasodilator-stimulated phosphoprotein family of proteins

Zyxin contains a proline-rich N-terminal domain that is similar to the C-te rminal domain in the ActA protein of the bacteria, Listeria monocytogenes. We screened the entire amino acid sequence of human zyxin for Mena-interact ing peptides and found that, as with ActA, proline-rich sequences were the sole zyxin sequences capable of binding to Ena/vasodilator-stimulated phosp hoprotein (VASP) family members in vitro. From this information, we tested zyxin mutants in which the proline-rich sequences were altered. The reducti on in Mena/VASP binding was confirmed by peptide tests, immunoprecipitation , and ectopic expression of zyxin variants at the surface of mitochondria. By transfection assays we showed that zyxin interaction with Mena/VASP in v ivo enhances the production of actin-rich structures at the apical surface of cells. Microinjection into cells of peptides corresponding to the first proline-rich sequence of zyxin caused the loss of Mena/VASP from focal cont acts. Furthermore, these peptides reduced the degree of spreading of cells replated after trypsinization, We conclude that zyxin and proteins that har bor similar proline-rich repeats contribute to the positioning of Mena/VASP proteins. The positioning of Ena/VASP family members appears to be importa nt when the actin cytoskeleton is reorganized, such as during spreading.