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. It binds numerous proteins, including the Ub E3 ligases and the deubiquitinating enzyme Ubp12p, 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.

The COP9 signalosome (CSN) occurs in all eukaryotic cells. It is a regulatory particle of the ubiquitin (Ub)/26S proteasome system. The eight subunits of the CSN possess sequence homologies with the polypeptides of the 26S proteasome lid complex and just like the lid, the CSN consists of six subunits with PCI (proteasome, COP9 signalosome, initiation factor 3) domains and two components with MPN (Mpr‐Pad1‐N‐terminal) domains. Here we show that the CSN directly interacts with the 26S proteasome and competes with the lid, which has consequences for the peptidase activity of the 26S proteasome in vitro. Flag‐CSN2 was permanently expressed in mouse B8 fibroblasts and Flag pull‐down experiments revealed the formation of an intact Flag‐CSN complex, which is associated with the 26S proteasome. In addition, the Flag pull‐downs also precipitated cullins indicating the existence of super‐complexes consisting of the CSN, the 26S proteasome and cullin‐based Ub ligases. Permanent expression of a chimerical subunit (Flag‐CSN2‐Rpn6) consisting of the N‐terminal 343 amino acids of CSN2 and of the PCI domain of S9/Rpn6, the paralog of CSN2 in the lid complex, did not lead to the assembly of an intact complex showing that the PCI domain of CSN2 is important for complex formation. The consequence of permanent Flag‐CSN2 overexpression was de‐novo assembly of the CSN complex connected with an accelerated degradation of p53 and stabilization of c‐Jun in B8 cells. The possible role of super‐complexes composed of the CSN, the 26S proteasome and of Ub ligases in the regulation of protein stability is discussed.

Background: The COP9/signalosome (CSN), a multiprotein complex consisting of eight subunits, is implicated in a wide variety of regulatory processes including cell cycle control, signal transduction, transcriptional activation, and plant photomorphogenesis. Some of these functions have been linked to CSN-associated enzymes, including kinases and an activity that removes the ubiquitin-like protein NEDD8/Rub1p from the cullin subunit of E3 ligases. CSN is highly conserved across species from fission yeast to humans, but sequence comparison has failed to identify the complex in budding yeast, except for a putative CSN5 subunit called Rri1p. Results: We show that disruption of four budding yeast genes, PCI8 and three previously uncharacterized ORFs, which encode proteins interacting with Rrr1p/Csn5p, each results in the accumulation of the cullin Cdc53p exclusively in the Rub1p-modified state. This phenotype, which resembles that of fission yeast csn mutants, is due to a biochemical defect in deneddylation that is complemented by wild-type cell lysate and by purified human CSN in vitro. Although three of the four genes encode proteins with PCI domains conserved in metazoan CSN proteins, their disruption does not confer the DNA damage sensitivity described in some fission yeast csn mutants. Conclusions: Our studies present unexpected evidence for the conservation of a functional homologue of the metazoan CSN, which mediates control of cullin neddylation in budding yeast.

The cyclooxygenase-2 (COX-2) enzyme is induced upon inflammation and in neoplastic tissues. It produces prostaglandins that stimulate tumor angiogenesis and tumor growth. Therefore, destruction and/or specific inhibition of COX-2 should be an important aspect of future tumor therapy. Recently, clinical application of specific COX-2 inhibitors called coxibs became doubtfully because they produce serious renal and cardiovascular complications under long term application. The exact underlying mechanisms are poorly understood and the different effects of diverse coxibs are not explained. It has been demonstrated before that COX-2 is degraded by the ubiquitin (Ub) proteasome system (UPS). However, how ubiquitination is accomplished and regulated was unclear. An important regulator of the UPS is the COP9 signalosome (CSN), which controls the stability of many proteins. Here we show that the proteasome-dependent degradation of COX-2 in HeLa cell lysate and in HeLa cells was stimulated by curcumin, an inhibitor of CSN-associated kinases. These data suggest a function of the CSN in the degradation of COX-2. In addition, proteolysis of COX-2 was significantly accelerated by parecoxib, but not by celecoxib or rofecoxib. By density gradient centrifugation and immunoprecipitation we demonstrate that COX-2 physically interacts with the CSN. Moreover, COX-2 is associated with large complexes consisting of the CSN, cullin-RING Ub ligases and the 26S proteasome. Pulldown experiments with Flag-COX-2 revealed cullin 1 and cullin 4 as components of the large super-complexes. Cullin 1 and 4 are scaffolding proteins of Ub ligases that presumably ubiquitinate COX-2. Treatment of HeLa cells with parecoxib results in an accelerated degradation of endogenous COX-2 accompanied by an increase of COX-2-Ub conjugates. In HeLa cells parecoxib is converted to the selective COX-2 inhibitor valdecoxib. Addition of valdecoxib also stimulates COX-2 degradation in HeLa cells. We therefore conclude that valdecoxib specifically interacts with COX-2 and induces a conformation accessible for ubiquitination and degradation.