Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.
J Biol Chem. 2012 Aug 24;287(35):29679-89. doi: 10.1074/jbc.M112.352484. Epub 2012 Jul 5.
COP9 signalosome (CSN) mediates deconjugation of the ubiquitin-like protein Nedd8 from the cullin subunits of SCF and other cullin-RING ubiquitin ligases (CRLs). This process is essential to maintain the proper activity of CRLs in cells. Here, we report a detailed kinetic characterization of CSN-mediated deconjugation of Nedd8 from SCF. CSN is an efficient enzyme, with a k(cat) of ~1 s(-1) and K(m) for neddylated Cul1-Rbx1 of ~200 nm, yielding a k(cat)/K(m) near the anticipated diffusion-controlled limit. Assembly with an F-box-Skp1 complex markedly inhibited deneddylation, although the magnitude varied considerably, with Fbw7-Skp1 inhibiting by ~5-fold but Skp2-Cks1-Skp1 by only ~15%. Deneddylation of both SCF(Fbw7) and SCF(Skp2-Cks1) was further inhibited ~2.5-fold by the addition of substrate. Combined, the inhibition by Fbw7-Skp1 plus its substrate cyclin E was greater than 10-fold. Unexpectedly, our results also uncover significant product inhibition by deconjugated Cul1, which results from the ability of Cul1 to bind tightly to CSN. Reciprocally, CSN inhibits the ubiquitin ligase activity of deneddylated Cul1. We propose a model in which assembled CRL complexes engaged with substrate are normally refractory to deneddylation. Upon consumption of substrate and subsequent deneddylation, CSN can remain stably bound to the CRL and hold it in low state of reduced activity.
COP9 信号体（CSN）介导将泛素样蛋白 Nedd8 从 SCF 和其他 Cullin-RING 泛素连接酶（CRL）的 Cullin 亚基上解偶联。这个过程对于维持细胞中 CRL 的适当活性是必不可少的。在这里，我们报告了 CSN 介导的 Nedd8 从 SCF 上解偶联的详细动力学特征。CSN 是一种高效的酶，其 k(cat)约为 1 s(-1)，对 neddylated Cul1-Rbx1 的 K(m)约为 200nm，产生接近预期扩散控制极限的 k(cat)/K(m)。与 F-box-Skp1 复合物组装显著抑制去泛素化，尽管抑制程度差异很大，Fbw7-Skp1 抑制约 5 倍，但 Skp2-Cks1-Skp1 仅抑制约 15%。添加底物进一步抑制了 SCF(Fbw7)和 SCF(Skp2-Cks1)的去泛素化，抑制程度约为 2.5 倍。结合 Fbw7-Skp1 和其底物细胞周期蛋白 E 的抑制作用大于 10 倍。出乎意料的是，我们的结果还揭示了去泛素化 Cul1 对反应的显著产物抑制，这是由于 Cul1 能够与 CSN 紧密结合。相反，CSN 抑制去泛素化 Cul1 的泛素连接酶活性。我们提出了一个模型，其中组装的 CRL 复合物与底物结合通常不易发生去泛素化。在底物消耗和随后的去泛素化之后，CSN 可以稳定地与 CRL 结合，并将其保持在低活性状态。
