Gazdoiu Stefan, Yamoah Kosj, Wu Kenneth, Pan Zhen-Qiang
Department of Oncological Sciences, The Mount Sinai School of Medicine, New York, NY 10029-6574, USA.
Mol Cell Biol. 2007 Oct;27(20):7041-52. doi: 10.1128/MCB.00812-07. Epub 2007 Aug 13.
The Cdc34 E2 ubiquitin (Ub) conjugating enzyme catalyzes polyubiquitination of a substrate recruited by the Skp1-Cullin 1-F-box protein-ROC1 E3 Ub ligase. Using mutagenesis studies, we now show that human Cdc34 employs distinct sites to coordinate the transfer of Ub to a substrate and the assembly of polyubiquitin chains. Mutational disruption of the conserved charged stretch (residues 143 to 153) or the acidic loop residues D102 and D103 led to accumulation of monoubiquitinated IkappaBalpha while failing to yield polyubiquitin chains, due to a catalytic defect in Ub-Ub ligation. These results suggest an ability of human Cdc34 to position the attacking Ub for assembly of polyubiquitin chains. Analysis of Cdc34N85Q and Cdc34S138A revealed severe defects of these mutants in both poly- and monoubiquitination of IkappaBalpha, supporting a role for N85 in stabilizing the oxyanion and in coordinating, along with S138, the attacking lysine for catalysis. Finally, Cdc34S95D and Cdc34(E108A/E112A) abolished both poly- and monoubiquitination of IkappaBalpha. Unexpectedly, the catalytic defects of these mutants in di-Ub synthesis can be rescued by fusion of a glutathione S-transferase moiety at E2's N terminus. These findings support the hypothesis that human Cdc34 S95 and E108/E112 are required to position the donor Ub optimally for catalysis, in a manner that might depend on E2 dimerization.
Cdc34 E2泛素（Ub）结合酶催化由Skp1-Cullin 1-F-box蛋白-ROC1 E3 Ub连接酶招募的底物的多聚泛素化。通过诱变研究，我们现在表明，人类Cdc34利用不同的位点来协调Ub向底物的转移以及多聚泛素链的组装。保守的带电荷片段（第143至153位残基）或酸性环残基D102和D103的突变破坏导致单泛素化的IkappaBalpha积累，同时由于Ub-Ub连接中的催化缺陷而未能产生多聚泛素链。这些结果表明人类Cdc34具有定位进攻性Ub以组装多聚泛素链的能力。对Cdc34N85Q和Cdc34S138A的分析揭示了这些突变体在IkappaBalpha的多聚泛素化和单泛素化方面都存在严重缺陷，支持N85在稳定氧阴离子以及与S138一起协调催化的进攻性赖氨酸方面的作用。最后，Cdc34S95D和Cdc34（E108A/E112A）消除了IkappaBalpha的多聚泛素化和单泛素化。出乎意料的是，这些突变体在双Ub合成中的催化缺陷可以通过在E2的N末端融合谷胱甘肽S-转移酶部分来挽救。这些发现支持这样的假设，即人类Cdc34的S95和E108/E112需要以可能依赖于E2二聚化的方式将供体Ub最佳定位以进行催化。
