Identification of HHR23A as a substrate for E6-associated protein-mediated ubiquitination
Identification of HHR23A as a substrate for E6-associated protein-mediated ubiquitination
复制标题
DOI：
10.1074/jbc.274.26.18785
复制
发表时间：
1999-06-25
影响因子：
4.8
通讯作者：
Howley, PM
中科院分区：
文献类型：
--
作者：
Kumar, S;Talis, AL;Howley, PM
人乳头瘤病毒E6相关蛋白(E6AP)在E6介导的P53泛素化过程中起泛素蛋白连接酶(E3)的作用。在没有E6的情况下，E6AP也是E3，但其正常的细胞底物尚未确定。在这里，我们报告了酵母DNA修复蛋白RAD23的人类同源物之一HHR23A被鉴定为E6AP的E6非依赖性靶标，HHR23A与E6AP结合并泛素化。以E6AP依赖的方式体外培养。在哺乳动物细胞中可以检测到泛素化形式的内源性HHR23A。野生型E6AP在体内的过表达增强了HHR23A的泛素化，而显性负性E6AP突变体抑制了HHR23A的泛素化。虽然HHR23A在非同步化细胞中是一种稳定的蛋白质，但其水平受到细胞周期依赖性的调节，特异性降解发生在S期。在体内，显性负性E6AP可阻断HHR23A的S时相降解，为E6AP参与HHR23A的调控提供了直接证据。与HHR23蛋白在DNA修复中的作用一致，紫外线诱导的DNA损伤抑制了HHR23A的降解。虽然HHR23蛋白在DNA修复和细胞周期进程中的确切作用尚不清楚，但我们的数据表明，E6AP介导的HHR23A泛素化可能在DNA修复和细胞周期进程中具有重要意义。
The human papilloma virus E6-associated protein (E6AP) functions as a ubiquitin protein ligase (E3) in the E6-mediated ubiquitination of p53. E6AP is also an E3 in the absence of E6, but its normal cellular substrates have not yet been identified. Here we report the identification of HHR23A, one of the human homologues of the yeast DNA repair protein Rad23, as an E6-independent target of E6AP, HHR23A binds E6AP and is ubiquitinated in. vitro in an E6AP dependent manner. Ubiquitinated forms of endogenous HHR23A are detectable in mammalian cells. Overexpression of wild-type E6AP in vivo enhances the ubiquitination of HHR23A, whereas a dominant negative E6AP mutant inhibits HHR23A ubiquitination. Although HHR23A is a stable protein in nonsynchronized cells, its levels are regulated in a cell cycle-dependent manner, with specific degradation occurring during S phase. The S phase degradation of HHR23A could be blocked in vivo by dominant negative E6AP, providing direct evidence for the involvement of E6AP in the regulation of HHR23A. Consistent with a role of the HHR23 proteins in DNA repair, UV-induced DNA damage inhibited HHR23A degradation. Although the precise role of HHR23 proteins in DNA repair and cell cycle progression remains to be elucidated, our data suggest that E6AP-mediated ubiquitination of HHR23A may have important implications in DNA repair and cell cycle progression.