The Zinc Finger of the CSN-Associated Deubiquitinating Enzyme USP15 Is Essential to Rescue the E3 Ligase Rbx1

• Published in: Current biology 2005-07, Vol.15 (13), p.1217-1221
• Main Authors: Hetfeld, Bettina K.J., Helfrich, Annett, Kapelari, Barbara, Scheel, Hartmut, Hofmann, Kay, Guterman, Adi, Glickman, Michael, Schade, Rüdiger, Kloetzel, Peter-Michael, Dubiel, Wolfgang
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Blotting, Western; Carrier Proteins - metabolism; COP9 Signalosome Complex; DNA, Complementary - genetics; Endopeptidases - genetics; Endopeptidases - metabolism; HeLa Cells; Humans; Microscopy, Electron; Molecular Sequence Data; Mucin-1 - genetics; Multiprotein Complexes - antagonists & inhibitors; Multiprotein Complexes - metabolism; Multiprotein Complexes - ultrastructure; Mutagenesis, Site-Directed; Mutation - genetics; Peptide Fragments - genetics; Peptide Hydrolases - metabolism; Peptide Hydrolases - ultrastructure; Phenanthrolines - pharmacology; Polyubiquitin - metabolism; Reverse Transcriptase Polymerase Chain Reaction; Ubiquitin - genetics; Ubiquitin-Protein Ligases - metabolism; Ubiquitin-Specific Proteases; Zinc Fingers - genetics
• ISSN: 0960-9822; 1879-0445
• DOI: 10.1016/j.cub.2005.05.059

The COP9 signalosome (CSN) is a conserved protein complex found in all eukaryotic cells and involved in the regulation of the ubiquitin (Ub)/26S proteasome system [ 1, 2]. It binds numerous proteins, including the Ub E3 ligases [ 3, 4] and the deubiquitinating enzyme Ubp12p [ 5], the S. pombe ortholog of human USP15. We found that USP15 copurified with the human CSN complex. Isolated CSN complex exhibited protease activity that deubiquitinated poly-Ub substrates and was completely inhibited by o-phenanthroline (OPT), a metal-chelating agent. Surprisingly, the recombinant USP15 was also not able to cleave isopeptide bonds of poly-Ub chains in presence of OPT. Detailed analysis of USP sequences led to the discovery of a novel zinc (Zn) finger in USP15 and related USPs. Mutation of a single conserved cysteine residue in the predicted Zn binding motif resulted in the loss of USP15 capability to degrade poly-Ub substrates, indicating that the Zn finger is essential for the cleavage of poly-Ub chains. Moreover, pulldown experiments demonstrated diminished binding of tetra-Ub to mutated USP15. Cotransfection of USP15 and the Ub ligase Rbx1 revealed that the wild-type deubiquitinating enzyme, but not the USP15 mutant with a defective Zn finger, stabilized Rbx1 toward the Ub system, most likely by reversing poly/autoubiquitination. In summary, a functional Zn finger of USP15 is needed to maintain a conformation essential for disassembling poly-Ub chains, a prerequisite for rescuing the E3 ligase Rbx1.