Cullin-RING ligases (CRIB) regulate diverse cellular functions such as cell cycle progression and cytokine signaling by ubiquitinating key regulatory proteins. The activity of CRIB is controlled by Nedd8 modification of the cullin subunits. Recent reports have Suggested that CAND1, which specifically binds to unmodified CUL1 but not to neddylated one, is required for the in vivo function of SCFs. the CULL-containing CRLs. We show here that CAND1 and COP9 signalosome (CSN), the major deneddylase of cullins. bind to unneddylated CUL1 in a mutually exclusive way. The Suppression of CAND1 expression by small inhibitory RNA enhanced the interaction between CULL and CSN, suggesting that CAND1 inhibited the binding of CSN to CULL. We found that the binding of CSN to CUL1 required the four helix bundle in CUL1 C-terminal domain, which was wrapped around by CAND1 in the CAND1-CULL-Rbxl complex. CAND1 greatly facilitated CSN-mediated deneddylation of CUL1 in vitro, which was dependent on its binding to CULL Our data suggest that enhancement of CSN-mediated deneddylation by CAND1 may contribute to its function as a positive regulator of SCFs in vivo. (c) 2005 Elsevier Inc. All rights reserved.