title: 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.
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(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.
authors: Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.
authors: Ethan D
authors: ED
authors: Emberley
authors: Ruzbeh
authors: R
authors: Mosadeghi
authors: Raymond J
authors: RJ
authors: Deshaies
mesh_terms: D000075686:COP9 Signalosome Complex; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D044842:Cullin Proteins / Q000235:genetics / Q000378:metabolism*; D019927:Cyclin E / Q000235:genetics / Q000378:metabolism; D044783:F-Box Proteins / Q000235:genetics / Q000378:metabolism*; D000073979:F-Box-WD Repeat-Containing Protein 7; D006801:Humans; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D000075687:NEDD8 Protein; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D044786:S-Phase Kinase-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism*
chemical_list: C475820:CCNE1 protein, human; D018797:Cell Cycle Proteins; C100318:Cullin 1; D044842:Cullin Proteins; D019927:Cyclin E; D044783:F-Box Proteins; D000073979:F-Box-WD Repeat-Containing Protein 7; C513273:FBXW7 protein, human; D046912:Multiprotein Complexes; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; D015513:Oncogene Proteins; D044786:S-Phase Kinase-Associated Proteins; C097351:SKP1 protein, human; D014452:Ubiquitins; D044767:Ubiquitin-Protein Ligases; D010447:Peptide Hydrolases; D000075686:COP9 Signalosome Complex