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J. Biol. Chem., Vol. 279, Issue 4, 3096-3110, January 23, 2004

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Association of Villin with Phosphatidylinositol 4,5-Bisphosphate Regulates the Actin Cytoskeleton^*

Narendra Kumar, Peilin Zhao, Alok Tomar, Charles A. Galea, and Seema Khurana

From the Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee 38163 and the Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105

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 presented mutational analysis to identify the PIP₂-binding sites in villin. Villin (human) binds PIP₂ with a K_d of 39.5 µM, a 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 in their association with PIP₂. By 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 cross-linking 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 revealed that structural changes induced by association with PIP₂ could regulate the actin-modifying functions of villin. This study provided biochemical proof of the functional significance of villin association with PIP₂ and identified the molecular mechanisms involved in the regulation of actin dynamics by villin and PIP₂.

Received for publication, August 12, 2003 , and in revised form, October 27, 2003.

^* This work was supported by grants from the American Digestive Health Foundation (Industry Research Scholar Award) and by NIDDK Grant DK-54755 from the National Institutes of Health (to S. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: University of Tennessee Health Science Center, 894 Union Ave., Nash 402, Memphis, TN 38163. Tel.: 901-448-3410; Fax: 901-448-3505; E-mail: skhurana{at}utmem.edu.

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