Kato Yukio, Kato Takako, Ono Tetsuo, Susa Takao, Kitahara Kousuke, Matsumoto Kaori
Division of Life Science, Graduate School of Agriculture, Meiji University, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan.
J Cell Biochem. 2009 Apr 1;106(5):912-9. doi: 10.1002/jcb.22066.
We have previously cloned cofactor CLIM2 (Ldb1/NL1) as a binding protein for LIM homeodomain transcription factor and now seek a protein interacting with CLIM2. Ultimately, SSBP2 was cloned as CLIM2 binding protein from the adult porcine pituitary cDNA library by the Yeast Two-Hybrid System. The amino acid sequence of porcine SSBP2 shows a high identity (99%) with those of other mammalian species, man, and mouse. Using fluorescence protein-fused SSBP2 and its deletion mutants, we observed that SSBP2 overexpressed in CHO cells predominantly localizes in mitochondria. Expression of mutant SSBP2s demonstrated that the first 241 amino acid residues are responsive for the mitochondrial localization. When CLIM2 vector was co-transfected, SSBP2 changed its location to nuclei. The similar translocation was also observed when CLIM2 vector was transfected 17 h after the transfection of SSBP2 vector. The first 120 residues of SSBP2 are responsible for the nuclear localization by guidance with CLIM2. RT-PCR demonstrated that SSBP2 was expressed in the porcine pituitary from fetal 40 days to postnatal 230 days in both genders and in the variety of pituitary and non-pituitary tumor cell lines, indicating that SSBP2 is present ubiquitously and plays a universal function during fetal and postnatal pituitary development.
我们之前已克隆出辅因子CLIM2（Ldb1/NL1）作为LIM同源域转录因子的结合蛋白，现在要寻找与CLIM2相互作用的蛋白。最终，通过酵母双杂交系统从成年猪垂体cDNA文库中克隆出SSBP2作为CLIM2结合蛋白。猪SSBP2的氨基酸序列与其他哺乳动物物种（人类和小鼠）的序列具有高度同源性（99%）。使用荧光蛋白融合的SSBP2及其缺失突变体，我们观察到在CHO细胞中过表达的SSBP2主要定位于线粒体。突变型SSBP2的表达表明，前241个氨基酸残基负责线粒体定位。当共转染CLIM2载体时，SSBP2会转移至细胞核。在转染SSBP2载体17小时后转染CLIM2载体时，也观察到了类似的转位现象。SSBP2的前120个残基在CLIM2的引导下负责细胞核定位。RT-PCR表明，SSBP2在40天胎儿期至出生后230天的猪垂体中均有表达，且在两性以及多种垂体和非垂体肿瘤细胞系中均有表达，这表明SSBP2广泛存在，并在胎儿期和出生后垂体发育过程中发挥普遍作用。
