Nedd8-modification of Cul1 is promoted by Roc1 as a Nedd8-E3 ligase and regulates its stability.
SCF is a ubiquitin ligase and is composed of Skp1, Cul1, F-box protein, and Roc1. The catalytic site of the SCF is the Cul1/Roc1 complex and RING-finger protein Roc1. It was shown earlier that when Cul1 was co-expressed with Roc1 in Sf-9 cells in a baculovirus protein expression system, Cul1 was highly neddylated in the cell, suggesting that Roc1 may function as a Nedd8-E3 ligase. However, there is no direct evidence that Roc1 is a Nedd8-E3 in an in vitro enzyme system. Here we have shown that Roc1 binds to Ubc12, E2 for Nedd8, but not to Ubc9, E2 for SUMO-1 and Roc1 RING-finger mutant, H77A, did not bind to Ubc12. In in vitro neddylation system using purified Cul1/Roc1 complex expressed in bacteria, Roc1 promotes neddylation of Cul1. These results demonstrate that Roc1 functions as a Nedd8-E3 ligase toward Cul1. Furthermore, Roc1 and Cul1 were ubiquitinylated in a manner dependent on the neddylation of Cul1 in vitro. In addition, Cul1 was degraded through the ubiquitin-proteasome pathway, and a non-neddylated mutant Cul1, K720R, was more stable than wild-type in intact cells. Thus, neddylation of Cul1 might regulate SCF function negatively via degradation of Cul1/Roc1 complex.
Morimoto
Mitsuru
M
Division of Molecular Biology, School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Nishida
Tamotsu
T
Nagayama
Yudai
Y
Yasuda
Hideyo
H
D000818:Animals; D020134:Catalytic Domain; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D044842:Cullin Proteins; D004795:Enzyme Stability; D006801:Humans; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D009097:Multienzyme Complexes; D000075687:NEDD8 Protein; D010453:Peptide Synthases / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D044843:SKP Cullin F-Box Protein Ligases; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism*
D018797:Cell Cycle Proteins; C100318:Cullin 1; D044842:Cullin Proteins; D009097:Multienzyme Complexes; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D014452:Ubiquitins; D044843:SKP Cullin F-Box Protein Ligases; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D010453:Peptide Synthases