The interaction of the cell-contact proteins VASP and vinculin is regulated by phosphatidylinositol-4,5-bisphosphate

Abstract

Background: Focal adhesion sites are cell–matrix contacts that are regulated by phosphatidylinositol-4,5-bisphosphate (PIP₂)-dependent pathways. Vinculin is a major structural component of these sites and is thought to be engaged in multiple ligand interactions at the cytoplasmic face of these contacts. Cytoplasmic vinculin is considered to be inactive due to its closed conformation involving intramolecular head–tail interactions. Recently, the vasodilator-stimulated phosphoprotein (VASP), a substrate of cyclic AMP-dependent or cyclic GMP-dependent kinases and a component of focal adhesion sites, was shown to bind to vinculin.

Results: VASP–vinculin complexes could be immunoprecipitated from cell lysates and, using immunofluorescence, both proteins were found to colocalize in nascent focal adhesions. Consistent with the view that vinculin must be activated at these sites, we found that PIP₂, levels of which are elevated during the early stages of adhesion, bound to two discrete regions in the vinculin tail, disrupting the intramolecular head–tail interaction and inducing vinculin oligomerization. Vinculin–VASP complex formation was greatly enhanced by PIP₂ and both the EVH1 and EVH2 domains of VASP participated in vinculin binding.

Conclusions: Focal contact assembly involves interaction between VASP and vinculin, which is enhanced by PIP₂-induced vinculin activation and oligomerization. Given that vinculin and VASP both bind to F-actin, vinculin–VASP complexes might bundle the distal ends of actin filaments in focal contacts. We propose that PIP₂-dependent signalling modulates microfilament organization at cellular adhesion sites by regulating vinculin–VASP complexes.