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J. Biol. Chem., Vol. 283, Issue 13, 8746-8755, March 28, 2008

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Biochemical Characterization of the Rho GTPase-regulated Actin Assembly by Diaphanous-related Formins, mDia1 and Daam1, in Platelets^*

Tomohito Higashi¹, Tomoyuki Ikeda, Ryutaro Shirakawa¹, Hirokazu Kondo, Mitsunori Kawato, Masahito Horiguchi, Tomohiko Okuda, Katsuya Okawa^¶, Shuya Fukai^||**, Osamu Nureki^**, Toru Kita, and Hisanori Horiuchi²

From the Department of Cardiovascular Medicine, COE Formation for Genomic Analysis of Disease Model Animals with Multiple Genetic Alterations, and ^¶Frontier Technology Center, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan, ^||Life Science Division, Synchrotron Radiation Research Organization, University of Tokyo, Tokyo 113-0032, Japan, ^**Department of Biological Information, Graduate School of Bioscience and Biotechnology and Center for Biological Resources and Informatics, Tokyo Institute of Technology, Yokohama 226-8501, Japan, and RIKEN Genomics Sciences Center, Yokohama 230-0045, Japan

The diaphanous-related formins are actin nucleating and elongating factors. They are kept in an inactive state by an intramolecular interaction between the diaphanous inhibitory domain (DID) and the diaphanous-autoregulatory domain (DAD). It is considered that the dissociation of this autoinhibitory interaction upon binding of GTP-bound Rho to the GTPase binding domain next to DID induces exposure of the FH1-FH2 domains, which assemble actin filaments. Here, we isolated two diaphanous-related formins, mDia1 and Daam1, in platelet extracts by GTP-RhoA affinity column chromatography. We characterized them by a novel assay, where beads coated with the FH1-FH2-DAD domains of either mDia1 or Daam1 were incubated with platelet cytosol, and the assembled actin filaments were observed after staining with rhodamine-phalloidin. Both formins generated fluorescent filamentous structures on the beads. Quantification of the fluorescence intensity of the beads revealed that the initial velocity in the presence of mDia1 was more than 10 times faster than in the presence of Daam1. The actin assembly activities of both FH1-FH2-DADs were inhibited by adding cognate DID domains. GTP-RhoA, -RhoB, and -RhoC, but not GTP-Rac1 or -Cdc42, bound to both mDia1 and Daam1 and efficiently neutralized the inhibition by the DID domains. The association between RhoA and Daam1 was induced by thrombin stimulation in platelets, and RhoA-bound endogenous formins induced actin assembly, which was inhibited by the DID domains of Daam1 and mDia1. Thus, mDia1 and Daam1 are platelet actin assembly factors having distinct efficiencies, and they are directly regulated by Rho GTPases.

Received for publication, September 19, 2007 , and in revised form, December 31, 2007.

^* This work was supported in part by a research grant from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. This study was also supported in part by grants from the Takeda Science Foundation and Mitsubishi Pharma Research Foundation. 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.

¹ Recipients of the Japan Society for the Promotion of Science Research Fellowship for Young Scientists.

² To whom correspondence should be addressed: Dept. of Cardiovascular Medicine, Kyoto University, 54 Shogoinkawahara-cho, Sakyo, Kyoto 606-8507, Japan. Tel.: 81-75-751-3195; Fax: 81-75-751-3203; E-mail: horiuchi{at}kuhp.kyoto-u.ac.jp.

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