Role of RacC for the Regulation of WASP and Phosphatidylinositol 3-Kinase during Chemotaxis of Dictyostelium*
- ‡Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6600 and §Zentrum für Biochemie and Zentrum für Molekulare Medizin, Medizinische Fakultät, Universität zu Köln, Joseph-Stelzmann-Strasse 52, 50931 Köln, Germany
- 1 To whom correspondence should be addressed: 468 Robinson Research Bldg. (MRB I), 1215 21st Ave. South at Pierce, Nashville, TN 37232-6600. Tel.: 615-322-4956; Fax: 615-343-6532; E-mail: chang.chung{at}vanderbilt.edu.
Abstract
WASP family proteins are key players for connecting multiple signaling pathways to F-actin polymerization. To dissect the highly integrated signaling pathways controlling WASP activity, we identified a Rac protein that binds to the GTPase binding domain of WASP. Using two-hybrid and FRET-based functional assays, we identified RacC as a major regulator of WASP. RacC stimulates F-actin assembly in cell-free systems in a WASP-dependent manner. A FRET-based microscopy approach showed local activation of RacC at the leading edge of chemotaxing cells. Cells overexpressing RacC exhibit a significant increase in the level of F-actin polymerization upon cAMP stimulation, which can be blocked by a phosphatidylinositol (PI) 3-kinase inhibitor. Membrane translocation of PI 3-kinase and PI 3,4,5-trisphosphate reporter is absent in racC null cells. Cells overexpressing dominant negative RacC mutants and racC null cells move at a significantly slower speed and show a poor directionality during chemotaxis. Our results suggest that RacC plays an important role in PI 3-kinase activation and WASP activation for dynamic regulation of F-actin assembly during Dictyostelium chemotaxis.
Footnotes
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↵2 The abbreviations used are: GBD/CRIB, GTPase binding domain; CFP, cyan fluorescence protein; YFP, yellow fluorescence protein; TRITC, tetramethylrhodamine isothiocyanate; PH, pleckstrin homology; FRET, fluorescence resonance energy transfer; PI, phosphatidylinositol; PI3K, PI 3-kinase; GDPβS, guanyl-5′-yl thiophosphate; GST, glutathione S-transferase; GTPγS, guanosine 5′-3-O-(thio)triphosphate; GFP, green fluorescent protein; HSS, high speed supernatant; PIPES, 1,4-piperazinediethanesulfonic acid; PI(3,4,5)P3, PI 3,4,5-trisphosphate.
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↵3 C. Y. Chung and F. Rivero, unpublished data.
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↵* This work was supported in part by National Institutes of Health Grant GM068097 (to C. Y. C.), Deutsche Forschungsgemeinschaft Grant (RI 1034/2), and by the Köln Fortune program (to F. R.). 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.
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- Received June 22, 2006.
- Revision received September 7, 2006.
- The American Society for Biochemistry and Molecular Biology, Inc.
