Vinexin: A novel vinculin-binding protein with multiple SH3 domains enhances actin cytoskeletal organization

Using the yeast two-hybrid system and an in vitro binding assay, we have id entified a novel protein termed vinexin as a vinculin-binding protein. By N orthern blotting, we identified two types of vinexin mRNA that were 3 and 2 kb in length. Screening for full-length cDNA clones and sequencing indicat ed that the two mRNA encode 82- and 37-kD polypeptides termed vinexin alpha and beta, respectively. Both forms of vinexin share a common carboxyl-term inal sequence containing three SH3 domains. The larger vinexin alpha contai ns an additional amino-terminal sequence. The interaction between vinexin a nd vinculin was mediated by two SH3 domains of vinexin and the proline-rich region of vinculin. When expressed, vinexin alpha and beta localized to fo cal adhesions in NIH 3T3 fibroblasts, and to cell-cell junctions in epithel ial LLC-PK1 cells. Furthermore, expression of vinexin increased focal adhes ion size. Vinexin alpha also promoted upregulation of actin stress fiber fo rmation. In addition, cell lines stably expressing vinexin beta showed enha nced cell spreading on fibronectin. These data identify vinexin as a novel focal adhesion and cell-cell adhesion protein that binds via SH3 domains to the hinge region of vinculin, which can enhance actin cytoskeletal organiz ation and cell spreading.