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J. Biol. Chem., Vol. 282, Issue 8, 5871-5879, February 23, 2007

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Structure/Function Analysis of the Interaction of Phosphatidylinositol 4,5-Bisphosphate with Actin-capping Protein

IMPLICATIONS FOR HOW CAPPING PROTEIN BINDS THE ACTIN FILAMENT^*

Kyoungtae Kim¹, Michelle E. McCully¹², Nandini Bhattacharya³, Boyd Butler, David Sept, and John A. Cooper⁴

From the Department of Cell Biology and Physiology and the Department of Biomedical Engineering and Center for Computational Biology, Washington University, St. Louis, Missouri 63110

The heterodimeric actin-capping protein (CP) can be inhibited by polyphosphoinositides, which may be important for actin polymerization at membranes in cells. Here, we have identified a conserved set of basic residues on the surface of CP that are important for the interaction with phosphatidylinositol 4,5-bisphosphate (PIP₂). Computational docking studies predicted the identity of residues involved in this interaction, and functional and physical assays with site-directed mutants of CP confirmed the prediction. The PIP₂ binding site overlaps with the more important of the two known actin-binding sites of CP. Correspondingly, we observed that loss of PIP₂ binding correlated with loss of actin binding among the mutants. Using TIRF (total internal reflection fluorescence) microscopy, we observed that PIP₂ rapidly converted capped actin filaments to a growing state, consistent with uncapping. Together, these results extend our understanding of how CP binds to the barbed end of the actin filament, and they support the idea that CP can "wobble" when bound to the barbed end solely by the C-terminal "tentacle" of its -subunit.

Received for publication, October 19, 2006 , and in revised form, December 1, 2006.

^* This work was supported in part by National Institutes of Health Grants GM38542 (to J. A. C.) and GM067246 (to D. S.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables I and II, Fig. 1, and Movie 1.

¹ These authors made equal contributions to this work.

² Supported in part by a Washington University/Howard Hughes Medical Inst. summer undergraduate research fellowship funded by an Undergraduate Biological Sciences Education Program Grant from the Howard Hughes Medical Institute to Washington University.

³ Supported by National Institutes of Health Institutional Training Grant T32 HL007873.

⁴ To whom correspondence should be addressed: Campus Box 8228, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-3964; Fax: 314-362-7463; E-mail: jcooper{at}wustl.edu.

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