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J. Biol. Chem., Vol. 279, Issue 9, 7988-7998, February 27, 2004

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Vacuolar H⁺-ATPase Binding to Microfilaments

REGULATION IN RESPONSE TO PHOSPHATIDYLINOSITOL 3-KINASE ACTIVITY AND DETAILED CHARACTERIZATION OF THE ACTIN-BINDING SITE IN SUBUNIT B^*

Shih-Hua Chen, Michael R. Bubb¶, Elena G. Yarmola¶, Jian Zuo, Jin Jiang||, Beth S. Lee**, Ming Lu¶, Stephen L. Gluck¶, I. Rita Hurst, and L. Shannon Holliday

From the Departments of Orthodontics and ^||Endodontics, University of Florida College of Dentistry, Gainesville, Florida 32610, The Research Service, Malcolm Randall Veterans Affairs Medical Center, Gainesville, Florida 32608, the Departments of ^¶Medicine and Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, Florida 32610, and the ^**Department of Physiology and Cell Biology, Ohio State University College of Medicine, Columbus, Ohio 43210

Vacuolar H⁺-ATPase (V-ATPase) binds microfilaments, and that interaction may be mediated by an actin binding domain in subunit B of the enzyme. To test for possible physiologic functions of the actin binding activity of V-ATPase, early responses of resorbing osteoclasts to inhibition of phosphatidylinositol 3-kinase activity by wortmannin and LY294002 were examined. Rapid co-localization between V-ATPase and F-actin was demonstrated by immunocytochemistry, and corresponding association between V-ATPase and F-actin in immunoprecipitations and pelleting assays was detected. This response was reversed as osteoclasts recovered resorptive activity after inhibitors were removed. By expressing and characterizing fusion proteins containing segments of the actin-binding amino-terminal regions of the B subunits of V-ATPase, we mapped the actin-binding site to a 44-amino acid domain. An 11-amino acid segment with a sequence similar to the actin-binding site of human profilin I was detected within this region. 13-Mers containing these profilin-like segments bound actin in fluorescent anisotropy studies and competed with profilin for binding to actin. Using site-directed mutagenesis, the 11-amino acid profilin-like actin-binding motifs (amino acids 49–59 of B1 and 55–65 of B2) were replaced with an 11-amino acid spacer with a sequence based on the homologous sequence from subunit B of Pyrococcus horikoshii, an organism that lacks an actin cytoskeleton. These substitutions eliminated the actin-binding activity of the B subunit fusion proteins. In summary, binding between V-ATPase and F-actin in osteoclasts occurs in response to blocking phosphatidylinositol 3-kinase activity. This response was fully reversible. The actin binding activities of the B subunits of V-ATPase required 11-amino acid actin-binding motifs that are similar in sequence to the actin-binding site of mammalian profilin I.

Received for publication, May 21, 2003 , and in revised form, October 7, 2003.

^* This work was supported by National Institutes of Health (NIH) Grant R01 AR47959 (to L. S. H.), a grant from the American Association of Orthodontists Foundation (to L. S. H.), and NIH Grant R01 DK 38848 (to S. L. G.). 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: Dept. of Orthodontics, J. Hillis Miller Health Center, D7-18, Campus Box 100444, University of Florida College of Dentistry, Gainesville, FL 32610. Tel.: 352-392-4135; Fax: 352-846-0459; E-mail: sholliday{at}dental.ufl.edu.

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