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J. Biol. Chem., Vol. 282, Issue 11, 7799-7808, March 16, 2007

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Distant N- and C-terminal Domains Are Required for Intrinsic Kinase Activity of SMG-1, a Critical Component of Nonsense-mediated mRNA Decay^*

Tomoko Morita, Akio Yamashita¹, Isao Kashima², Kazuhiro Ogata^¶, Shoichi Ishiura, and Shigeo Ohno³

From the Department of Life Sciences, Graduate School of Arts and Science, The University of Tokyo 153-8902, Japan, and Department of Molecular Biology and ^¶Biochemistry, Yokohama City University School of Medicine, Yokohama 236-0004, Japan

Phosphatidylinositol 3-kinase-related kinases (PIKKs) consisting of SMG-1, ATM, ATR, DNA-PKcs, and mTOR are a family of proteins involved in the surveillance of gene expression in eukaryotic cells. They are involved in mechanisms responsible for genome stability, mRNA quality, and translation. They share a large N-terminal domain and a C-terminal FATC domain in addition to the unique serine/threonine protein kinase (PIKK) domain that is different from classical protein kinases. However, structure-function relationships of PIKKs remain unclear. Here we have focused on one of the PIKK members, SMG-1, which is involved in RNA surveillance, termed nonsense-mediated mRNA decay (NMD), to analyze the roles of conserved and SMG-1-specific sequences on the intrinsic kinase activity. Analyses of sets of point and deletion mutants of SMG-1 in a purified system and intact cells revealed that the long N-terminal region and the conserved leucine in the FATC domain were essential for SMG-1 kinase activity. However, the conserved tryptophan in the TOR SMG-1 (TS) homology domain and the FATC domain was not. In addition, the long insertion region between PIKK and FATC domains was not essential for SMG-1 kinase activity. These results indicated an unexpected feature of SMG-1, i.e. that distantly located N- and C-terminal sequences were essential for the intrinsic kinase activity.

Received for publication, October 30, 2006 , and in revised form, January 8, 2007.

^* This work was supported, in part, by grants (to S. O.) from the Japan Society for the Promotion of Science, Ministry of Education, Culture, Sports, Science, and Technology of Japan, Japan Science and Technology Corporation, and the Mitsubishi 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–4 and references.

¹ Supported by the Yokohama Foundation for Advancement of Medical Science.

² Research Fellow of the Japan Society for the Promotion of Sciences.

³ To whom correspondence should be addressed: Dept. of Molecular Biology, Yokohama City University School of Medicine, 3-9 Fuku-ura, Kanazawa-ku, Yokohama 236-0004, Japan. Tel.: 81-45-787-2596; Fax: 81-45-785-4140; E-mail: ohnos{at}med.yokohama-cu.ac.jp.

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