Shimoji Tetsu, Murakami Kyoko, Sugiyama Yuichi, Matsuda Mami, Inubushi Sachiko, Nasu Junichi, Shirakura Masayuki, Suzuki Tetsuro, Wakita Takaji, Kishino Tatsuya, Hotta Hak, Miyamura Tatsuo, Shoji Ikuo
Department of Virology II, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan.
J Cell Biochem. 2009 Apr 15;106(6):1123-35. doi: 10.1002/jcb.22096.
E6-associated protein (E6AP) is a cellular ubiquitin protein ligase that mediates ubiquitylation and degradation of p53 in conjunction with the high-risk human papillomavirus E6 proteins. However, the physiological functions of E6AP are poorly understood. To identify a novel biological function of E6AP, we screened for binding partners of E6AP using GST pull-down and mass spectrometry. Here we identified annexin A1, a member of the annexin superfamily, as an E6AP-binding protein. Ectopic expression of E6AP enhanced the degradation of annexin A1 in vivo. RNAi-mediated downregulation of endogenous E6AP increased the levels of endogenous annexin A1 protein. E6AP interacted with annexin A1 and induced its ubiquitylation in a Ca(2+)-dependent manner. GST pull-down assay revealed that the annexin repeat domain III of annexin A1 is important for the E6AP binding. Taken together, our data suggest that annexin A1 is a novel substrate for E6AP-mediated ubiquitylation. Our findings raise the possibility that E6AP may play a role in controlling the diverse functions of annexin A1 through the ubiquitin-proteasome pathway.
E6相关蛋白（E6AP）是一种细胞泛素蛋白连接酶，它与高危型人乳头瘤病毒E6蛋白共同介导p53的泛素化和降解。然而，人们对E6AP的生理功能了解甚少。为了确定E6AP的一种新的生物学功能，我们利用谷胱甘肽S-转移酶（GST）沉降法和质谱分析法筛选E6AP的结合伴侣。在此，我们鉴定出膜联蛋白A1（annexin A1），它是膜联蛋白超家族的成员之一，为一种E6AP结合蛋白。E6AP的异位表达在体内增强了膜联蛋白A1的降解。RNA干扰介导的内源性E6AP下调增加了内源性膜联蛋白A1蛋白的水平。E6AP与膜联蛋白A1相互作用，并以Ca(2+)依赖的方式诱导其泛素化。GST沉降分析表明，膜联蛋白A1的膜联蛋白重复结构域III对E6AP结合很重要。综上所述，我们的数据表明膜联蛋白A1是E6AP介导的泛素化的新底物。我们的发现增加了一种可能性，即E6AP可能通过泛素-蛋白酶体途径在控制膜联蛋白A1的多种功能中发挥作用。
