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Papers In Press, published online ahead of print November 1, 2003
Physiology Dept., University of Tennessee, Memphis, TN 38163
Corresponding Author: skhurana{at}utmem.edu
Summary Villin, an epithelial cell actin binding protein severs actin in vitro and in vivo. Previous studies report that phosphatidylinositol 4,5-bisphosphate (PIP2) regulates actin severing by villin, presumably by interaction with villin. However, direct association of villin with PIP2 has never been characterized. In this report, we present mutational analysis to identify the PIP2 binding sites in villin. Villin (human) binds PIP2 with a Kd of 39.5 mM, stoichiometry of 3.3 and a Hill coefficient of 1. We generated deletion mutants of villin lacking putative PIP2 binding sites and examined the impact of these mutations on PIP2 binding and actin dynamics. Our analysis revealed the presence of three PIP2 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 their association with PIP2. Using site-directed mutagenesis (arginine 138 to alanine) we demonstrated the presence of an identical F-actin and PIP2 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 PIP2 binding site in the actin crosslinking domain of villin. Consistent with this observation, association of villin with PIP2 inhibited the actin capping and severing functions of villin, and enhanced the actin bundling function of villin. Our studies reveal that structural changes induced by association with PIP2 could regulate the actin modifying functions of villin. This study provides biochemical proof of the functional significance of villin association with PIP2 as well as identifies the molecular mechanisms involved in the regulation of actin dynamics by villin and PIP2.
J. Biol. Chem, 10.1074/jbc.M308878200
Submitted on August 12, 2003
Revised on October 27, 2003
Accepted on November 1, 2003
Association of villin with phosphatidylinositol 4,5-bisphosphate regulates the actin cytoskeleton
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