Splicing factor 3b subunit 4 binds BMPR-IA and inhibits osteochondral cell differentiation
Splicing factor 3b subunit 4 binds BMPR-IA and inhibits osteochondral cell differentiation
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DOI：
10.1074/jbc.m703292200
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发表时间：
2007-07-13
影响因子：
4.8
通讯作者：
Watanabe, Hideto
中科院分区：
文献类型：
--
作者：
Watanabe, Hiroki;Shionyu, Masafumi;Watanabe, Hideto
骨形态发生蛋白（BMP）-2/4在早期胚胎发生和骨骼发育中起关键作用。BMP-2/4信号通过两种类型的丝氨酸/苏氨酸激酶受体传导到细胞中，称为BMPR-I（IA和IB）和BMPR-II。在这里，我们确定剪接因子3b亚基4（SF 3b 4）作为一个分子，与BMPR-IA相互作用，使用酵母双杂交筛选与人胎脑cDNA文库。免疫共沉淀/免疫印迹分析证实了它们在哺乳动物细胞中的相互作用。通过分离细胞组分，SF 3b 4存在于具有BMPR-IA的细胞膜级分中以及细胞核中。SF 3b 4的过表达分别抑制BMP-2介导的C2 C12和ATDC 5细胞的成骨和软骨细胞分化，并且在ATDC 5细胞分化过程中SF 3b 4的内源性表达水平降低。通过报告基因测定，SF 3b 4抑制Id报告基因活性（特异于Smad 1/5/8途径），但不抑制TGF β介导的报告基因活性。生物素标记的细胞表面蛋白，然后通过免疫印迹显示，SF 3b 4降低细胞表面BMPRI-A水平。通过对BMPR-IA的细胞内结构域的分子建模的进一步分析，结合对其几种突变体的结合研究，表明SF 3b 4结合的位点与C-末端叶和活化片段不直接相关。综上所述，这些结果表明，SF 3b 4，已知定位在细胞核中，并参与RNA剪接，结合BMPR-IA和特异性抑制BMP介导的骨软骨细胞分化。
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 beta-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.