Association of villin with phosphatidylinositol 4,5-bisphosphate regulates the actin cytoskeleton. Academic Article uri icon


  • Villin, an epithelial cell actin-binding protein, severs actin in vitro and in vivo. Previous studies report that phosphatidylinositol 4,5-bisphosphate (PIP(2)) regulates actin severing by villin, presumably by interaction with villin. However, direct association of villin with PIP(2) has never been characterized. In this report, we presented mutational analysis to identify the PIP(2)-binding sites in villin. Villin (human) binds PIP(2) with a K(d) of 39.5 microm, a stoichiometry of 3.3, and a Hill coefficient of 1. We generated deletion mutants of villin lacking putative PIP(2)-binding sites and examined the impact of these mutations on PIP(2) binding and actin dynamics. Our analysis revealed the presence of three PIP(2)-binding sites, two in the amino-terminal core and one in the carboxyl-terminal headpiece of human villin. Synthetic peptides analogous with these sites confirmed the binding domains. Circular dichroism and quenching of intrinsic tryptophan fluorescence revealed a significant conformational change in these peptides ensuing in their association with PIP(2). By using site-directed mutagenesis (arginine 138 to alanine), we demonstrated the presence of an identical F-actin and PIP(2)-binding site in the capping and severing domain of villin. In contrast, the mutants lysine 822 and 824 to alanine demonstrated the presence of an overlapping F-actin and PIP(2)-binding site in the actin cross-linking domain of villin. Consistent with this observation, association of villin with PIP(2) inhibited the actin capping and severing functions of villin and enhanced the actin bundling function of villin. Our studies revealed that structural changes induced by association with PIP(2) could regulate the actin-modifying functions of villin. This study provided biochemical proof of the functional significance of villin association with PIP(2) and identified the molecular mechanisms involved in the regulation of actin dynamics by villin and PIP(2).

published proceedings

  • J Biol Chem

author list (cited authors)

  • Kumar, N., Zhao, P., Tomar, A., Galea, C. A., & Khurana, S

citation count

  • 64

complete list of authors

  • Kumar, Narendra||Zhao, Peilin||Tomar, Alok||Galea, Charles A||Khurana, Seema

publication date

  • January 2004