Koiwai Kotaro, Maezawa So, Hayano Takahide, Iitsuka Michiko, Koiwai Osamu
Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Noda, Chiba, Japan.
Genes Cells. 2008 Jun;13(6):593-607. doi: 10.1111/j.1365-2443.2008.01191.x. Epub 2008 Mar 4.
Bood POZ containing gene type 2 (BPOZ-2), which contains ankyrin repeats, NLS, BTB/POZ domains and LXXLL motifs, is an adaptor protein for the E3 ubiquitin ligase scaffold protein CUL3. We isolated a cDNA encoding eukaryotic elongation factor 1A1 (eEF1A1) as a BPOZ-2 binding protein by screening a human thymus cDNA library using a yeast two-hybrid system. eEF1A1 is essential for translation and is also involved in the 26S proteasome-dependent degradation of misfolded or unfolded proteins. The binding between BPOZ-2 and eEF1A1 was confirmed by pull-down and immunoprecipitation assays in vitro and in vivo, respectively. BPOZ-2 binds to eEF1A1 through the ankyrin repeats and both BTB/POZ domains in BPOZ-2 and Domains I and III in eEF1A1. BPOZ-2 and eEF1A1 over-expressed in HEK 293T cells co-localized as speckles within the cytoplasm. BPOZ-2 promoted eEF1A1 ubiquitylation and degradation, suggesting that eEF1A1 is a substrate of BPOZ-2. BPOZ-2 inhibited GTP binding to eEF1A1 and prevented translation in in vitro translation assay using rabbit reticulocytes.
含有2型基因的血液POZ（BPOZ - 2）含有锚蛋白重复序列、核定位信号、BTB/POZ结构域和LXXLL基序，是E3泛素连接酶支架蛋白CUL3的衔接蛋白。我们通过酵母双杂交系统筛选人胸腺cDNA文库，分离出一种编码真核延伸因子1A1（eEF1A1）的cDNA作为BPOZ - 2结合蛋白。eEF1A1对翻译至关重要，还参与错误折叠或未折叠蛋白的26S蛋白酶体依赖性降解。BPOZ - 2与eEF1A1之间的结合分别通过体外和体内的下拉和免疫沉淀试验得到证实。BPOZ - 2通过锚蛋白重复序列以及BPOZ - 2中的BTB/POZ结构域与eEF1A1中的结构域I和III结合。在HEK 293T细胞中过表达的BPOZ - 2和eEF1A1在细胞质内共定位成斑点。BPOZ - 2促进eEF1A1的泛素化和降解，表明eEF1A1是BPOZ - 2的底物。BPOZ - 2抑制GTP与eEF1A1的结合，并在使用兔网织红细胞的体外翻译试验中阻止翻译。
