Subcellular localization and ubiquitin-conjugating enzyme (E2) interactions of mammalian HECT family ubiquitin protein ligases
Subcellular localization and ubiquitin-conjugating enzyme (E2) interactions of mammalian HECT family ubiquitin protein ligases
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DOI：
10.1074/jbc.272.24.15085
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发表时间：
1997-06-13
影响因子：
4.8
通讯作者：
Weissman, AM
中科院分区：
文献类型：
--
作者：
Hatakeyama, S;Jensen, JP;Weissman, AM
在大多数情况下，泛素向靶蛋白的转移是通过泛素蛋白连接酶（E3）的作用来催化的。克隆了编码鼠E6相关蛋白（mE 6-AP）以及Nedd-4（在其C末端与E6-AP同源的蛋白）的全长cDNA。Nedd-4和小鼠E6-AP都是具有酶活性的E3，并且与E2的UbcH 5家族的成员一起起作用，小鼠E6-AP与其人类对应物一样，在人乳头瘤病毒E6蛋白存在下泛素化p53，而Nedd-4则不。与其在p53泛素化中的作用一致，mE 6-AP在细胞核和胞质溶胶中均被发现，而Nedd-4仅在胞质溶胶中被发现，结合研究表明，mE 6-AP和Nedd-4之间有40%相同的150个氨基酸的区域作为E2酶（UbcH 5 B）C末端部分的结合位点，Nedd-4被确定具有第二个非重叠的E2结合位点，其识别UbcH 5 B的前67个氨基酸，但不识别该E2的更C末端部分。这些发现首次证明了哺乳动物E2和E3之间的物理相互作用，并建立了这些相互作用独立于泛素和完整的E3催化结构域。此外，Nedd-4内两个E2结合位点的存在表明了涉及与E3相关的多种E2酶的泛素化模型。
In most instances, the transfer of ubiquitin to target proteins is catalyzed by the action of ubiquitin protein ligases (E3s), Full-length cDNAs encoding murine E6-associated protein (mE6-AP) as well as Nedd-4, a protein that is homologous to E6-AP in its C terminus, were cloned. Nedd-4 and mouse E6-AP are both enzymatically active E3s and function with members of the UbcH5 family of E2s, Mouse E6-AP, like its human counterpart, ubiquitinates p53 in the presence of human papilloma virus E6 protein, while Nedd-4 does not, Consistent with its role in p53 ubiquitination, mE6-AP was found both in the nucleus and cytosol, while Nedd-4 was found only in the cytosol, Binding studies implicate a 150-amino acid region that is 40% identical between mE6-AP and Nedd-4 as a binding site for the C terminal portion of an E2 enzyme (UbcH5B), Nedd-4 was determined to have a second nonoverlapping E2 binding site that recognizes the first 67 amino acids of UbcH5B but not the more C-terminal portion of this E2. These findings provide the first demonstration of physical interactions between mammalian E2s and E3s and establish that these interactions occur independently of ubiquitin and an intact E3 catalytic domain, Furthermore, the presence of two E2 binding sites within Nedd-4 suggests models for ubiquitination involving multiple E2 enzymes associated with E3s.