Structural and kinetic analysis of the COP9-Signalosome activation and the cullin-RING ubiquitin ligase deneddylation cycle - PubMed (original) (raw)
(A) CSN5H138A is inactive and is a dominant-negative inhibitor of deneddylation. CSN, CSN5H138A, and substrate were used at 2 nM, 100 nM, and 75 nM, respectively. For reactions containing CSN and CSN5H138A, mutant enzyme was preincubated with substrate for 30 sec prior to initiating time-course by adding CSN. (B-E): The indicated proteins were mixed and allowed to equilibrate prior to determining the change in dansyl fluorescence. (B) CSN5H138A and dansylated, Nedd8-conjugated SCFSkp2. (C) CSN5H138A and dansylated, Nedd8-conjugated SCFFbxw7. Note that addition of Fbxw7–Skp1 greatly increased the variability in the measurement for unknown reasons. (D) CSN5H138A (first prep) and Cul1d/Rbx1, (E) CSN5H138A (second prep) and Cul1d/Rbx1, (F-I): The indicated CSN complexes were preincubated with Cul1d/Rbx1 for 10 min, followed by addition of unlabeled Cul1/Rbx1 chase and measurement of the decay in dansyl fluorescence over time. Final protein concentrations are listed for each experiment. (F) CSN (2000 nM), Cul1d/Rbx1 (200 nM), and Cul1/Rbx1 (3000 nM), (G) CSN5E104A (600 nM), Cul1d/Rbx1 (200 nM), and Cul1/Rbx1 (3000 nM), (H) CSN5E76A,5H138A (400 nM), Cul1d/Rbx1 (200 nM), and Cul1/Rbx1 (3000 nM), (I) CSN5E76A,5H138A (200 nM), Cul1d-N8/Rbx1 (100 nM), and Cul1/Rbx1 (1500 nM), (J–K): The indicated proteins were mixed and allowed to equilibrate prior to determining the change in dansyl fluorescence. (J) CSN5E76A, 5H138A and Cul1d/Rbx1, (K) CSN5H138A or CSN2∆N,5H138A and Cul1d-N8/Rbx1. All measurements in panels B–E and J–K were carried out in triplicate and error bars represent standard deviation. DOI: