Okamoto Kazuki, Isohashi Fumihide
Department of Biochemistry, St. Marianna University School of Medicine, Sugao, Miyamae-ku, Kawasaki, Kanagawa 216-8511, Japan.
J Biol Chem. 2005 Nov 4;280(44):36986-93. doi: 10.1074/jbc.M506056200. Epub 2005 Sep 2.
Macromolecular translocation inhibitor II (MTI-II), which was first identified as an in vitro inhibitor of binding between the highly purified glucocorticoid receptor (GR) and isolated nuclei, is an 11.5-kDa Zn(2+)-binding protein that is also known as ZnBP or parathymosin. MTI-II is a small nuclear acidic protein that is highly conserved in rats, cows, and humans and widely distributed in mammalian tissues, yet its physiological function is unknown. To elucidate its in vivo function in relation to GR, we transiently transfected mammalian cells with an expression plasmid encoding MTI-II. Unexpectedly, we found that the expression of MTI-II enhances the transcriptional activity of GR. The magnitude of the transcriptional enhancement induced by MTI-II is comparable with that induced by the steroid receptor coactivator SRC-1. In contrast, MTI-II had little effect on the transcriptional activity of estrogen receptor. Immunoprecipitation analysis showed that in the presence of glucocorticoid hormone, GR coprecipitates with MTI-II, and, vice versa, MTI-II coprecipitates with GR. The expression of various deletion mutants of MTI-II revealed that the central acidic domain is essential for the enhancement of GR-dependent transcription. Microscopic analysis of MTI-II fused to green fluorescent protein and GR fused to red fluorescent protein in living HeLa cells showed that MTI-II colocalizes with GR in discrete subnuclear domains in a hormone-dependent manner. Coexpression of MTI-II with the coactivator SRC-1 or p300 further enhances GR-dependent transcription. Immunoprecipitation analysis showed that in the presence of glucocorticoid hormone, p300 and CREB-binding protein are coprecipitated with MTI-II. Furthermore, the knockdown of endogenous MTI-II by RNAi reduces the transcriptional activity of GR in cells. Moreover, expression of MTI-II enhances the glucocorticoid-dependent transcription of the endogenous glucocorticoid-inducible enzyme in cells. Taken together, these results indicate that MTI-II enhances GR-dependent transcription via a direct interaction with GR in vivo. Thus, MTI-II is a new member of the GR-coactivator complex.
大分子易位抑制剂II（MTI-II）最初被鉴定为高度纯化的糖皮质激素受体（GR）与分离细胞核之间结合的体外抑制剂，是一种11.5 kDa的锌结合蛋白，也被称为锌结合蛋白（ZnBP）或胸腺旁素。MTI-II是一种小核酸性蛋白，在大鼠、牛和人类中高度保守，广泛分布于哺乳动物组织中，但其生理功能尚不清楚。为了阐明其与GR相关的体内功能，我们用编码MTI-II的表达质粒瞬时转染哺乳动物细胞。出乎意料的是，我们发现MTI-II的表达增强了GR的转录活性。MTI-II诱导的转录增强幅度与类固醇受体共激活因子SRC-1诱导的相当。相反，MTI-II对雌激素受体的转录活性影响很小。免疫沉淀分析表明，在糖皮质激素存在的情况下，GR与MTI-II共沉淀，反之亦然，MTI-II与GR共沉淀。MTI-II各种缺失突变体的表达表明，中央酸性结构域对于增强GR依赖性转录至关重要。对活的HeLa细胞中与绿色荧光蛋白融合的MTI-II和与红色荧光蛋白融合的GR进行显微镜分析表明，MTI-II以激素依赖的方式与GR在离散的核内亚结构域中共定位。MTI-II与共激活因子SRC-1或p300共表达进一步增强GR依赖性转录。免疫沉淀分析表明，在糖皮质激素存在的情况下，p300和CREB结合蛋白与MTI-II共沉淀。此外，RNA干扰对内源MTI-II的敲低降低了细胞中GR的转录活性。此外，MTI-II的表达增强了细胞中内源性糖皮质激素诱导酶的糖皮质激素依赖性转录。综上所述，这些结果表明MTI-II在体内通过与GR的直接相互作用增强GR依赖性转录。因此，MTI-II是GR-共激活因子复合物的新成员。
