Takayama S, Krajewski S, Krajewska M, Kitada S, Zapata J M, Kochel K, Knee D, Scudiero D, Tudor G, Miller G J, Miyashita T, Yamada M, Reed J C
The Burnham Institute, La Jolla, California 92037, USA.
Cancer Res. 1998 Jul 15;58(14):3116-31.
BAG-1 is a multifunctional protein that blocks apoptosis and interacts with several types of proteins, including Bcl-2 family proteins, the kinase Raf-1, certain tyrosine kinase growth factor receptors, and steroid hormone receptors, possibly by virtue of its ability to regulate the Hsp70/Hsc70 family of molecular chaperones. Two major forms of the human and mouse BAG-1 proteins were detected by immunoblotting. The longer human and mouse BAG-1 proteins (BAG-1L) appear to arise through translation initiation at noncanonical CTG codons located upstream of and in-frame with the usual ATG codon used for production of the originally described BAG-1 protein. Immunoblotting experiments using normal tissues revealed that BAG-1L is far more restricted in its expression and is present at lower levels than the more prevalent BAG-1 protein. Human but not mouse tissues also produce small amounts of an additional isoform of BAG-1 of intermediate size (BAG-1M) that probably arises through translation initiation at yet another site involving an ATG codon. All three isoforms of human BAG-1 (BAG-1, BAG-1M, and BAG-1L) retained the ability to bind Hsc70. Subcellular fractionation and immunofluorescence confocal microscopy studies indicated that BAG-1L often resides in the nucleus, consistent with the presence of a nuclear localization sequence in the NH2-terminal unique domain of this protein. In immunohistochemical assays, BAG-1 immunoreactivity was detected in a wide variety of types of cells in normal adult tissues and was localized to either cytosol, nucleus, or both, depending on the particular type of cell. In some cases, cytosolic BAG-1 immunostaining was clearly associated with organelles resembling mitochondria, consistent with the reported interaction of BAG-1 with Bcl-2 and related proteins. Furthermore, experiments using a green fluorescence protein (GFP)-BAG-1 fusion protein demonstrated that overexpression of Bcl-2 in cultured cells can cause intracellular redistribution of GFP-BAG-1, producing a membranous pattern typical of Bcl-2 family proteins. The BAG-1 protein was found at high levels in several types of human tumor cell lines among the 67 tested, particularly leukemias, breast, prostate, and colon cancers. In contrast to normal tissues, which only rarely expressed BAG-1L, tumor cell lines commonly contained BAG-1L protein, including most prostate, breast, and leukemia cell lines, suggesting that a change in BAG-1 mRNA translation frequently accompanies malignant transformation.
BAG-1是一种多功能蛋白,它能阻止细胞凋亡,并与多种类型的蛋白相互作用,包括Bcl-2家族蛋白、激酶Raf-1、某些酪氨酸激酶生长因子受体以及类固醇激素受体,这可能得益于其调节分子伴侣Hsp70/Hsc70家族的能力。通过免疫印迹法检测到人和小鼠的BAG-1蛋白有两种主要形式。较长的人和小鼠BAG-1蛋白(BAG-1L)似乎是通过在非经典CTG密码子处起始翻译产生的,这些密码子位于最初描述的BAG-1蛋白产生所使用的常规ATG密码子的上游且读码框一致。使用正常组织进行的免疫印迹实验表明,BAG-1L的表达受到更严格的限制,且其水平低于更普遍的BAG-1蛋白。人类组织而非小鼠组织还会产生少量中等大小的BAG-1额外异构体(BAG-1M),它可能是通过在另一个涉及ATG密码子的位点起始翻译产生的。人类BAG-1的所有三种异构体(BAG-1、BAG-1M和BAG-1L)都保留了结合Hsc70的能力。亚细胞分级分离和免疫荧光共聚焦显微镜研究表明,BAG-1L常定位于细胞核,这与该蛋白NH2末端独特结构域中存在核定位序列一致。在免疫组织化学分析中,在正常成人组织的多种细胞类型中检测到BAG-1免疫反应性,其定位取决于特定细胞类型,可定位于细胞质、细胞核或两者。在某些情况下,细胞质中的BAG-1免疫染色明显与类似线粒体的细胞器相关,这与报道的BAG-1与Bcl-2及相关蛋白的相互作用一致。此外,使用绿色荧光蛋白(GFP)-BAG-1融合蛋白的实验表明,在培养细胞中过表达Bcl-2可导致GFP-BAG-1在细胞内重新分布,产生Bcl-2家族蛋白特有的膜状模式。在所测试的67种人类肿瘤细胞系中,在几种类型的细胞系中发现BAG-1蛋白水平较高,尤其是白血病、乳腺癌、前列腺癌和结肠癌。与仅很少表达BAG-1L的正常组织不同,肿瘤细胞系通常含有BAG-1L蛋白,包括大多数前列腺癌、乳腺癌和白血病细胞系,这表明BAG-1 mRNA翻译的改变常伴随恶性转化。