Regulation of Cross-linking of Actin Filament by IQGAP1, a Target for Cdc42*
- Masaki Fukata‡§,
- Shinya Kuroda‡¶,
- Katsuhiko Fujii‡,
- Tomoko Nakamura‡,
- Ikuo Shoji‖,
- Yoshiharu Matsuura‖,
- Katsuya Okawa**,
- Akihiro Iwamatsu**,
- Akira Kikuchi§ and
- Kozo Kaibuchi‡‡
- From the ‡Division of Signal Transduction, Nara Institute of Science and Technology, Ikoma 630-01, the§Department of Biochemistry, Hiroshima University School of Medicine, Hiroshima 734, the ¶Inheritance and Variation Group, Precursory Research for Embryonic Science and Technology, Kyoto 619-02, the ‖Department of Virology II, National Institute of Infectious Diseases, Tokyo 162, and the **Central Laboratories for Key Technology, Kirin Brewery Company, Ltd., Yokohama 236, Japan
Abstract
We have previously shown that IQGAP1, a recently identified target for Cdc42 and Rac1 small GTPases, showed a distribution similar to that of cortical actin cytoskeleton at the membrane ruffling area induced by insulin and Rac1val12 (Kuroda, S., Fukata, M., Kobayashi, K., Nakafuku, M., Nomura, N., Iwamatsu, A., and Kaibuchi, K. (1996) J. Biol. Chem. 271, 23363–23367). Here we identified an IQGAP1-interacting molecule with molecular mass of 43 kDa (p43) from bovine brain cytosol, using glutathione S-transferase (GST)-IQGAP1 affinity column chromatography. The amino acid sequencing of the protein revealed that p43 was identical to β- and γ-actin. IQGAP1 was cosedimentated with filamentous actin (F-actin). The amino-terminal domain (amino acids 1–216) of IQGAP1 was responsible for the interaction with F-actin. Falling ball viscometry assay revealed that IQGAP1 cross-linked the F-actin. This IQGAP1 activity was further enhanced by guanosine 5′-(3-O-thio)triphosphate (GTPγS)·GST-Cdc42 but not by GDP·GST-Cdc42. The gel filtration analysis of IQGAP1 revealed that IQGAP1 appeared as oligomers and that GTPγS·GST-Cdc42 but not GDP·GST-Cdc42 enhanced the oligomerization of IQGAP1. These results strongly suggest that IQGAP1, acting downstream of Cdc42, can cross-link the actin filament through its oligomerization.
Footnotes
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↵* This study was supported by grants-in-aid for Scientific Research and for Cancer Research from the Ministry of Education, Science, and Culture of Japan (1996) and by grants from Kirin Brewery Co. Ltd. and from the Mitsubishi Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The 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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↵‡ To whom correspondence should be addressed. Tel.: 81-7437-2-5440; Fax: 81-7437-2-5449; E-mail:kaibuchi{at}bs.aist-nara.ac.jp.
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↵1 The abbreviations used are: WASP, Wiskott-Aldrich syndrome protein; CHD, calponin homology domain; F-actin, filamentous actin; GTPγS, guanosine 5′-(3-O-thio)triphosphate; GST, glutathione S-transferase; aa, amino acid; PAGE, polyacrylamide gel electrophoresis.
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↵2 Identification of p41 will be described elsewhere (study in progress).
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- Received July 7, 1997.
- Revision received September 4, 1997.
