Identification of determinants in E2 ubiquitin-conjugating enzymes required for hect E3 ubiquitin protein ligase interaction
Identification of determinants in E2 ubiquitin-conjugating enzymes required for hect E3 ubiquitin protein ligase interaction
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DOI：
10.1074/jbc.274.11.7576
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发表时间：
1999-03-12
影响因子：
4.8
通讯作者：
Scheffner, M
中科院分区：
文献类型：
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作者：
Nuber, U;Scheffner, M
hect结构域蛋白家族的成员的特征在于它们的C-末端区域与EG-AP（E3泛素-蛋白连接酶）的C-末端的序列相似性。E6-AP依赖性泛素化中的必要中间步骤是在不同的E2泛素缀合酶（包括人UbcH 5，E2的UBC 4/UBC 5亚家族的成员）存在下EG-AP和泛素之间形成硫酯复合物。类似地，几种hect结构域蛋白，包括酿酒酵母RSPB，形成泛素硫酯复合物，表明hect结构域蛋白通常具有E3活性。我们在这里显示，通过使用UbcH 5和其他E2 s之间产生的嵌合E2 s，UbcH 5的一个区域包含的催化位点半胱氨酸残基是至关重要的，其与E6-AP和RSP 5相互作用的能力。特别重要的是UbcH 5的62位的苯丙氨酸残基，其在UBC 4/UBC 5亚家族的成员中是保守的，但不存在于任何其他已知的E2中，而N-末端60个氨基酸对这些相互作用的特异性没有显著贡献。该苯丙氨酸残基在整个进化过程中的保守性强调了与hect结构域蛋白相互作用的能力对于UBC 4/UBC 5亚家族成员的细胞功能的重要性。
Members of the hect domain protein family are characterized by sequence similarity of their C-terminal regions to the C terminus of EG-AP, an E3 ubiquitin-protein ligase, An essential intermediate step in E6-AP-dependent ubiquitination is the formation of a thioester complex between EG-AP and ubiquitin in the presence of distinct E2 ubiquitin-conjugating enzymes including human UbcH5, a member of the UBC4/UBC5 subfamily of E2s. Similarly, several hect domain proteins, including Saccharomyces cerevisiae RSPB, form ubiquitin thioester complexes, indicating that hect domain proteins in general have E3 activity. We show here, by the use of chimeric E2s generated between UbcH5 and other E2s, that a region of UbcH5 encompassing the catalytic site cysteine residue is critical for its ability to interact with E6-AP and RSP5. Of particular importance is a phenylalanine residue at position 62 of UbcH5 that is con served among the members of the UBC4/UBC5 subfamily but is not present in any of the other known E2s, whereas the N-terminal 60 amino acids do not contribute significantly to the specificity of these interactions. The conservation of this phenylalanine residue throughout evolution underlines the importance of the ability to interact with hect domain proteins for the cellular function of UBC4/UBC5 subfamily members.