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J. Biol. Chem., Vol. 283, Issue 21, 14801-14814, May 23, 2008

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Multiple Molecular Interactions Implicate the Connectin/Titin N2A Region as a Modulating Scaffold for p94/Calpain 3 Activity in Skeletal Muscle^*

Chikako Hayashi¹, Yasuko Ono¹², Naoko Doi^¶, Fujiko Kitamura, Mai Tagami, Reiko Mineki^||, Takao Arai, Hayao Taguchi, Mitsuaki Yanagida^**, Stephanie Hirner, Dietmar Labeit, Siegfried Labeit, and Hiroyuki Sorimachi^¶3

From the Department of Enzymatic Regulation for Cell Functions (Calpain Project), The Tokyo Metropolitan Institute of Medical Science (Rinshoken), Tokyo 113-8613, Japan, the Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Chiba 278-8570, Japan, ^¶CREST, Japan Science and Technology, Saitama 332-0012, Japan, ^||Biomedical Research Center, Juntendo University Graduate School of Medicine, Chiba 279-0021, Japan, the Institute für Anästhesiologie und Operative Intensivmedizin, Universitätsklinikum Mannheim, 68167 Mannheim, Germany, and the ^**Institute for Environmental and Gender Specific Medicine, Graduate School of Medicine, Juntendo University, Chiba 279-0021, Japan

p94/calpain 3 is a skeletal muscle-specific Ca²⁺-regulated cysteine protease (calpain), and genetic loss of p94 protease activity causes muscular dystrophy (calpainopathy). In addition, a small in-frame deletion in the N2A region of connectin/titin that impairs p94-connectin interaction causes a severe muscular dystrophy (mdm) in mice. Since p94 via its interaction with the N2A and M-line regions of connectin becomes part of the connectin filament system that serves as a molecular scaffold for the myofibril, it has been proposed that structural and functional integrity of the p94-connectin complex is essential for health and maintenance of myocytes. In this study, we have surveyed the interactions made by p94 and connectin N2A inside COS7 cells. This revealed that p94 binds to connectin at multiple sites, including newly identified loci in the N2A and PEVK regions of connectin. Functionally, p94-N2A interactions suppress p94 autolysis and protected connectin from proteolysis. The connectin N2A region also contains a binding site for the muscle ankyrin repeat proteins (MARPs), a protein family involved in the cellular stress responses. MARP2/Ankrd2 competed with p94 for binding to connectin and was also proteolyzed by p94. Intriguingly, a connectin N2A fragment with the mdm deletion possessed enhanced resistance to proteases, including p94, and its interaction with MARPs was weakened. Our data support a model in which MARP2-p94 signaling converges within the N2A connectin segment and the mdm deletion disrupts their coordination. These results also implicate the dynamic nature of connectin molecule as a regulatory scaffold of p94 functions.

Received for publication, October 4, 2007 , and in revised form, February 13, 2008.

^* This work was supported in part by MEXT.KAKENHI 17028055 and 18076007 (to H. S.), JSPS.KAKENHI 18770124 (to Y. O.), 18380085 (to H. S.), and Research Grant 17A-10 for Nervous and Mental Disorders from the Ministry of Health, Labor and Welfare, a Takeda Science Foundation grant (to H. S.), and by Deutsche Forschungsgemeinschaft Grants SFB753, La668/9-1+11-1 (to S. L.), and La1619/1-1 (to D. L.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ Both authors contributed equally to this work.

² To whom correspondence may be addressed: Dept. of Enzymatic Regulation for Cell Functions (Calpain Project), The Tokyo Metropolitan Institute of Medical Science (Rinshoken), 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan. Fax: 81-3-3823-2359; E-mail: yakoono{at}rinshoken.or.jp.

³ To whom correspondence may be addressed: Dept. of Enzymatic Regulation for Cell Functions (Calpain Project), The Tokyo Metropolitan Institute of Medical Science (Rinshoken), 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan. Fax: 81-3-3823-2359; E-mail: sorimach{at}rinshoken.or.jp.

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