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Deconjugation of Nedd8 from Cul1 Is Directly Regulated by Skp1-F-box and Substrate, and the COP9 Signalosome Inhibits Deneddylated SCF by a Noncatalytic Mechanism

Emberley, Ethan D. and Mosadeghi, Ruzbeh and Deshaies, Raymond J. (2012) Deconjugation of Nedd8 from Cul1 Is Directly Regulated by Skp1-F-box and Substrate, and the COP9 Signalosome Inhibits Deneddylated SCF by a Noncatalytic Mechanism. Journal of Biological Chemistry, 287 (35). pp. 29679-29689. ISSN 0021-9258. PMCID PMC3436198. https://resolver.caltech.edu/CaltechAUTHORS:20121009-110244928

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Abstract

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_mfor 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.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1074/jbc.M112.352484DOIArticle
http://www.jbc.org/content/287/35/29679PublisherArticle
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3436198/PubMed CentralArticle
ORCID:
AuthorORCID
Deshaies, Raymond J.0000-0002-3671-9354
Additional Information:© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Received February 15, 2012. Revision received June 24, 2012. This work was supported, in whole or in part, by National Institutes of Health Grant GM065997. These authors contributed equally to this work. Supported in part by a Canadian Institutes of Health Research postdoctoral fellowship. We thank N. Zheng for recombinant CSN, B. Schulman for Dcn1, W. den Besten for recombinant Ubxd7, B. Larimore and B. Clurman for phosphorylated cyclin E-Cdk2, A. Saha, and N. Pierce (Deshaies laboratory) for various purified proteins, N. S. Z.-Q. Pan for the FLAG-Csn2 cell line, and R. Enchev for baculovirus-expressed CSN. We are indebted to M. Peter and B. Schulman for communicating results prior to publication. We thank Jost Vielmetter and members of the Caltech Protein Expression Center for assistance with protein expression and members of the Deshaies lab for comments on the manuscript.
Funders:
Funding AgencyGrant Number
NIHGM065997
Canadian Institutes of Health Research (CIHR) postdoctoral fellowshipUNSPECIFIED
Issue or Number:35
PubMed Central ID:PMC3436198
Record Number:CaltechAUTHORS:20121009-110244928
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20121009-110244928
Official Citation:Deconjugation of Nedd8 from Cul1 Is Directly Regulated by Skp1-F-box and Substrate, and the COP9 Signalosome Inhibits Deneddylated SCF by a Noncatalytic Mechanism Ethan D. Emberley, Ruzbeh Mosadeghi, and Raymond J. Deshaies J. Biol. Chem. 2012 287: 29679-29689. First Published on July 5, 2012, doi:10.1074/jbc.M112.352484
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:34783
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:09 Oct 2012 18:26
Last Modified:03 Oct 2019 04:22

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