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J. Biol. Chem., Vol. 282, Issue 28, 20728-20738, July 13, 2007

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Splicing Factor 3b Subunit 4 Binds BMPR-IA and Inhibits Osteochondral Cell Differentiation^*

Hiroki Watanabe, Masafumi Shionyu^¶, Tomoatsu Kimura, Koji Kimata, and Hideto Watanabe¹

From the Institute for Molecular Science of Medicine, Aichi Medical University, Aichi 480-1195, Japan, the Department of Orthopaedic Surgery, Faculty of Medicine, University of Toyama, Toyama 930-0194, Japan, and the ^¶Faculty of Bio-Science, Nagahama Institute of Bio-Science and Technology, Shiga 526-0829, Japan

Bone morphogenetic protein (BMP)-2/4 play critical roles in early embryogenesis and skeletal development. BMP-2/4 signals conduct into cells via two types of serine/threonine kinase receptors, known as BMPR-I (IA and IB) and BMPR-II. Here we identified splicing factor 3b subunit 4 (SF3b4) as a molecule that interacts with BMPR-IA, using a yeast two-hybrid screening with a human fetal brain cDNA library. Co-immunoprecipitation/immunoblot analysis confirmed their interaction in mammalian cells. By separation of the cell components, SF3b4 was present in the cell membrane fraction with BMPR-IA as well as in the nucleus. Overexpression of SF3b4 inhibited BMP-2-mediated osteogenic and chondrocytic differentiation of C2C12 and ATDC5 cells, respectively, and the endogenous expression level of SF3b4 decreased during differentiation in ATDC5 cells. By reporter gene assay, SF3b4 suppressed Id reporter gene activity, specific to the Smad1/5/8 pathway, but not TGF-mediated reporter gene activity. Biotin labeling of the cell surface proteins followed by their immunoblot revealed that SF3b4 decreased the cell surface BMPRI-A levels. Further analysis by molecular modeling of the intracellular domain of BMPR-IA, coupled with binding studies of its several mutants, indicated that the site(s) for SF3b4 binding is not directly associated with the C-terminal lobe and the activation segment. Taken together, these results suggest that SF3b4, known to be localized in the nucleus and involved in RNA splicing, binds BMPR-IA and specifically inhibits BMP-mediated osteochondral cell differentiation.

Received for publication, April 19, 2007

^* This work is supported in part by a grant-in-aid for scientific research (C) (KAKENHI) (to H. W.). 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 To whom correspondence should be addressed: Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Aichi 480-1195, Japan. Tel.: 81-561-62-3311 (ext. 2086); Fax: 81-561-63-3532; E-mail: wannabee{at}aichi-med-u.ac.jp.

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