THE FOCAL-ADHESION VASODILATOR-STIMULATED PHOSPHOPROTEIN (VASP) BINDSTO THE PROLINE-RICH DOMAIN IN VINCULIN

In mammalian cells vasodilator-stimulated phosphoprotein (VASP) is loc alized to focal adhesions and areas of dynamic membrane activity where it is thought to have a role in actin-filament assembly. The proteins responsible for recruiting VASP to these sites within the cell are no t known. The bacterial protein ActA binds VASP via a proline-rich moti f that is very similar to a sequence in the proline-rich region of the focal-adhesion protein vinculin. We have examined the ability of VASP , synthesized using an in vitro transcription/translation system, to b ind to a series of vinculin peptides expressed as glutathione S-transf erase fusion proteins, and have shown that it binds specifically to th e proline-rich region in vinculin. Using immobilized peptides correspo nding to the two proline-rich motifs within this domain, the VASP-bind ing site was localized to proline-rich motif-1 (residues 839-850). Bin ding to this motif was not affected by the phosphorylation state of VA SP. The C-terminal region of VASP, which is known to be important in t argeting VASP to focal adhesions, was shown to be required for binding . These results identify vinculin as a VASP-binding protein likely to be important in recruiting VASP to focal adhesions and the cell membra ne.