Gazdoiu Stefan, Yamoah Kosj, Wu Kenneth, Escalante Carlos R, Tappin Inger, Bermudez Vladimir, Aggarwal Aneel K, Hurwitz Jerard, Pan Zhen-Qiang
Department of Oncological Sciences, Mount Sinai School of Medicine, New York, NY 10029-6574, USA.
Proc Natl Acad Sci U S A. 2005 Oct 18;102(42):15053-8. doi: 10.1073/pnas.0507646102. Epub 2005 Oct 6.
Cdc34 is an E2-conjugating enzyme required for catalyzing the polyubiquitination reaction mediated by the Skp1.CUL1.F-box (SCF) protein E3 ubiquitin (Ub) ligase. Here, we show that the activity of human Cdc34 in the Ub-Ub ligation reaction was enhanced dramatically by SCF's core Ub ligase module, composed of a heterodimeric complex formed by the ROC1 RING finger protein and the CUL1 C terminus that contains a Nedd8 moiety covalently conjugated at K720. Unexpectedly, we found that N-terminal fusion of a GST moiety to human Cdc34 generated dimeric GST-Cdc34 that was constitutively active in supporting the assembly of K48-linked polyUb chains independently of SCF. Furthermore, fusion of a FK506-binding protein (FKBP) to the N terminus of human Cdc34 yielded FKBP-Cdc34 that was induced to form a dimer upon treatment with the chemical inducer AP20187. The AP20187-induced dimeric form of FKBP-Cdc34 was substantially more active than the monomer in catalyzing Ub-Ub ligation. Thus, juxtaposition of human Cdc34 activates its catalytic capability, suggesting that the SCF-mediated polyubiquitination reaction may require the conversion of Cdc34 from an inactive monomer to a highly active dimeric form.
Cdc34是一种E2缀合酶，催化由Skp1.CUL1.F-box（SCF）蛋白E3泛素（Ub）连接酶介导的多聚泛素化反应。在此，我们表明，由ROC1环指蛋白和CUL1 C末端形成的异二聚体复合物组成的SCF核心Ub连接酶模块，可显著增强人Cdc34在Ub-Ub连接反应中的活性，该CUL1 C末端在K720处含有一个共价缀合的Nedd8部分。出乎意料的是，我们发现将GST部分与人类Cdc34进行N端融合会产生二聚体GST-Cdc34，其在支持K48连接的多聚泛素链组装方面具有组成型活性，且不依赖于SCF。此外，将FK506结合蛋白（FKBP）与人类Cdc34的N端融合，得到FKBP-Cdc34，在用化学诱导剂AP20187处理后会诱导形成二聚体。AP20187诱导的FKBP-Cdc34二聚体形式在催化Ub-Ub连接方面比单体活性高得多。因此，人Cdc34的并列激活了其催化能力，这表明SCF介导的多聚泛素化反应可能需要将Cdc34从无活性的单体转化为高活性的二聚体形式。
