CAND 1 and regulation of cullin RING ubiquitin ligases in C. elegans
Cullins function in multisubunit ubiquitin ligase (E3) complexes to promote the ubiquitinmediated proteolysis of substrates and regulate a wide range of cellular processes. CAND1 is a HEAT repeat protein that binds to cullins and regulates their ubiquitin ligase activity by inhibiting the formation of full E3 complexes. All cullins are modified by the covalent linkage of Nedd8, which is a ubiquitin like protein that is required for full cullin activity. CAND1 cannot bind a cullin that is neddylated but it forms tight complexes with the unneddylated form of cullins. It was originally believed that neddylation of CUL1 bound to CAND1 could dissociate CAND1 and trigger active SCF complex formation (Jidong Liu, Molecular Cell, December 2002) However, more recent biochemical analysis and the crystal structure of CAND1 bound to CUL1 revealed that neddylation can not decouple CAND1 from CUL1 (Goldenberg, Cell, November 2004) Currently, it is not known how CAND1 that is bound to a cullin can be dissociated, or the physiological significance of CAND1 binding to cullin proteins in vivo. In this study we determined the roles of CAND 1 in modulating the functions of the C. elegans cullins. C. elegans has only a single CAND1 ortholog: cand 1( Y102A5A.1) We have identified CAND 1 as a major component of CUL 2 and CUL 4 complexes. Analysis with the yeast two hybrid system indicates that both the Nand C terminal domains of CAND 1 interact with all six C. elegans cullin proteins. The mutant phenotype of cand 1 does not show any major cullin loss of function phenotypes. We have determined the neddylation level of CUL 2 and CUL 4 in cand 1 mutant and found that the level of neddylation increased substantially for both cullins, indicating that CAND 1 negatively regulates neddylation in vivo. We also carried out a genomic screen for cand 1 enhancers, and found 18 enhancers that are potential regulator(s) of CAND 1 and the CRLs (Cullin Ring ubiquitin Ligase) activation cycle. INDEX WORDS: Caenorhabditis elegans; CAND1; CUL 1; CUL 2; CUL 4; Skp1; F box protein; Rbx1; Nedd8; cand 1; Y102A5A.1; cell cycle; cullin; ubiquitin; ubiquitin ligase and CRL. CAND 1 AND REGULATION OF CULLIN RING UBIQUITIN LIGASES IN C. ELEGANS