Role of Predicted Metalloprotease Motif of Jab1/Csn5 in Cleavage of Nedd8 from Cul1 Gregory A. Cope, 1 Greg S. B. Suh, 3 * L. Aravind, 4 Sylvia E. Schwarz, 1 † S. Lawrence Zipursky, 2,3 Eugene V. Koonin, 4 Raymond J. Deshaies 1,2 ‡ COP9 signalosome (CSN) cleaves the ubiquitin-like protein Nedd8 from the Cul1 subunit of SCF ubiquitin ligases. The Jab1/MPN domain metalloenzyme (JAMM) motif in the Jab1/Csn5 subunit was found to underlie CSN’s Nedd8 isopeptidase activity. JAMM is found in proteins from archaea, bacteria, and eukaryotes,includingtheRpn11subunitofthe26S proteasome.Metalchelators andpointmutationswithinJAMMabolishedCSN-dependentcleavageofNedd8 from Cul1, yet had little effect on CSN complex assembly. Optimal SCF activity in yeast and both viability and proper photoreceptor cell (R cell) development in Drosophila melanogaster required an intact Csn5 JAMM domain. We propose that JAMM isopeptidases play important roles in a variety of physiological pathways. SCF ubiquitin ligases (1, 2) regulate cellular function through the ubiquitination of numer- ous substrates, including p27 and IB (3). SCF complexes consist of four essential sub- units: Cul1, the RING-H2 protein Hrt1/Roc1/ Rbx1, Skp1, and a substrate-binding F-box protein (3). In addition, the ubiquitin-like protein Nedd8 is conjugated to Cul1. This modification (neddylation) is essential in fis- sion yeast (4 ) and stimulates recruitment of E2 to SCF ubiquitin ligase (5). The COP9 signalosome (CSN), which is a highly conserved complex related to both the proteasome lid and the eukaryotic initiation factor 3, promotes deneddylation of Cul1 (6, 7 ). CSN may also regulate a protein kinase that phosphorylates IB and p53 (8). CSN has been implicated in a diverse range of physiological processes, including plant and animal development (9–11), transcription (12), and signaling (13, 14 ), but the relation- ship between the biochemical and physiolog- ical functions of CSN remains unclear. Individual deletion of all known de-ubiq- uitinating enzymes in Saccharomyces cerevi- siae (15) and mutation of a candidate active- site cysteine in the Csn5/Jab1 subunit of CSN had no effect on deneddyation of Cul1 (15, 16 ). Thus, to elucidate the mechanism of deneddylation, we sought to identify a motif within CSN that might underlie isopeptidase activity. Iterated PSI-BLAST analysis (17 ) and sequence alignment revealed a set of eukaryotic and prokaryotic Pad1/Jab1/MPN/ Mov34 domain (hereafter referred to as Jab1/ MPN domain) proteins previously identified as homologs of Csn5 and Csn6 (18–20). Al- most all prokaryotic Jab1/MPN domains pos- sess a His-X-His-X 10 -Asp motif (where X indicates any residue) accompanied by an upstream conserved glutamate (Fig. 1). Al- 1 Department of Biology, California Institute of Tech- nology (CalTech), Pasadena, CA 91125, USA. 2 Howard Hughes Medical Institute, 3 Department of Biological Chemistry, The School of Medicine, University of California at Los Angeles (UCLA), Los Angeles, CA 90095, USA. 4 National Center for Biotechnology In- formation, National Library of Medicine, National In- stitutes of Health, Bethesda, MD 20894, USA. *Present address: Department of Biology, California Institute of Technology, Pasadena, CA 91125, USA. †Present address: G2M Cancer Drugs AG, Frankfurt/ Main, Germany. ‡To whom correspondence should be addressed. E- mail: deshaies@caltech.edu Fig. 1. Alignment of predicted JAMM do- mains ( 34). Selected se- quences were aligned using T-Coffee ( 35) and modified manually to ensure correct superpo- sition of the conserved motifs. A more thor- ough alignment is avail- able (fig. S1). Eukaryotic proteins are grouped to reflect orthologous re- lationships. Position of the aligned region in the sequence is shown by numbers; poorly con- served spacers are not shown and are desig- nated by numbers. The consensus includes ami- no acid residues con- served in 80% of the aligned sequences; l in- dicates aliphatic resi- dues (A, I, L, V; yellow shading), h indicates hydrophobic residues (F, Y, W, A, I, L, V, M; yellow shading), and s indicates small residues (G, A, C, S, D, N, V, P; blue letters). Predicted metal-chelating and catalytic residues are shown in yellow against a dark blue background. Secondary structure prediction was made using the PHD program with the multiple alignment submitted as the query ( 36); E indicates extended conformation ( -strand), and H indicates -helix. Each protein is denoted by the GenBank identifier followed by the gene name and abbreviated species name. Species abbreviations: Hsa, Homo sapiens; Dme, Drosophila melanogaster; Cel, Caenorhabditis elegans; Ath, Arabidopsis thaliana; Sce, Saccharomyces cerevisiae; Afu, Archaeoglobus fulgidus; Pho, Pyrococcus horikoshii; Hsp, Halobacterium sp.; Aae, Aquifex aeolicus; Nsp, Nostoc sp.; Dra, Deinococcus radiodurans; Psa, Pseudomonas aeruginosa; Vch, Vibrio cholerae; Rso, Ralstonia solanacearum; Eco, Escherichia coli; Bsu, Bacillus subtilis; Mac, Methanosarcina acetivorans. R EPORTS 18 OCTOBER 2002 VOL 298 SCIENCE www.sciencemag.org 608