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J. Biol. Chem., Vol. 278, Issue 25, 23036-23045, June 20, 2003

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Membrane Ruffling Requires Coordination between Type I Phosphatidylinositol Phosphate Kinase and Rac Signaling^*

Renee L. Doughman, Ari J. Firestone, Michelle L. Wojtasiak, Matthew W. Bunce and Richard A. Anderson 

From the Molecular and Cellular Pharmacology Program, Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin 53706

Membrane ruffle formation requires remodeling of cortical actin filaments, a process dependent upon the small G-protein Rac. Growth factors stimulate actin remodeling and membrane ruffling by integration of signaling pathways that regulate actin-binding proteins. Phosphatidylinositol 4,5-bisphosphate (PIP₂) regulates the activity of many actin-binding proteins and is produced by the type I phosphatidylinositol phosphate kinases (PIPKIs). Here we show in MG-63 cells that only the PIPKI isoform is localized to platelet-derived growth factor (PDGF)-induced membrane ruffles. Further, expression of kinase dead PIPKI, which acts as a dominant negative mutant, blocked membrane ruffling, suggesting that PIPKI and PIP₂ participate in ruffling. To explore this, PIPKI was overexpressed in serum-starved cells and stimulated with PDGF. In serum-starved cells, PIPKI expression did not stimulate actin remodeling, but when these cells were stimulated with PDGF, actin rapidly reorganized into foci but not membrane ruffles. PIPKI-mediated formation of actin foci was independent of both Rac1 and phosphatidylinositol 3-kinase activities. Significantly, coexpression of dominant active Rac1 with PIPKI in PDGF-stimulated cells resulted in membrane ruffling. The PDGF- and Rac1-stimulated ruffling was inhibited by expression of kinase-dead PIPKI. Combined, these data support a model where the localized production of PIP₂ by PIPKI is necessary for actin remodeling, whereas formation of membrane ruffles required Rac signaling.

Received for publication, November 7, 2002 , and in revised form, April 3, 2003.

^* This work was supported by National Institutes of Health Grant GM-144-KW76 (to R. A. A.) and American Heart Association predoctoral Fellowships 133-DL14 (to R. L. D.) and 133-EF25 (to M. W. B.). 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 Pharmacology, 1300 University Ave., University of Wisconsin Medical School, Madison, WI 53706. Tel.: 608-262-3753; Fax: 608-262-1257; E-mail: raanders{at}facstaff.wisc.edu.

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