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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In higher eukaryotic cells, the p53 protein is degraded by the ubiquitin-26S proteasome system mediated by Mdm2 or the human papilloma virus E6 protein. Here we show that COP9 signalosome (CSN)-specific phosphorylation targets human p53 to ubiquitin-26S proteasome-dependent degradation. As visualized by electron microscopy, p53 binds with high affinity to the native CSN complex p53 interacts via its N-terminus with CSN subunit 5/Jab1 as shown by far-western and pull-down assays. The CSN-specific phosphorylation sites were mapped to the core domain of p53 including Thr155. A phosphorylated peptide, Ap53(l45-l64), specifically inhibits CSN- mediated phosphorylation and p53 degradation. Curcumin, a CSN kinase inhibitor, blocks E6-dependent p53 degradation in reticulocyte lysates. Mutation of Thr155 to valine is sufficient to stabilize p53 against E6-dependent degradation in reticutocyte lysates and to reduce binding to Mdm2. The p53T155V mutant accumulates in both HeLa and HL 60 cells and exhi- bits a mutant (PAb 240+) conformation. It induces the cyclin-dependent inhibitor p21. In HeLa and MCF-7 cells, inhibition of CSN kinase by curcumin or Δp53(145-164) results in accumulation of endogenous p53. [ABSTRACT FROM AUTHOR]

Deneddylases remove the ubiquitin-like protein Nedd8 from modified proteins. An increased deneddylase activity has been associated with various human cancers. In contrast, we show here that a mutant strain of the model fungus Aspergillus nidulans deficient in two deneddylases is viable but can only grow as a filament and is highly impaired for multicellular development. The DEN1/DenA and the COP9 signalosome (CSN) deneddylases physically interact in A. nidulans as well as in human cells, and CSN targets DEN1/DenA for protein degradation. Fungal development responds to light and requires both deneddylases for an appropriate light reaction. In contrast to CSN, which is necessary for sexual development, DEN1/DenA is required for asexual development. The CSN-DEN1/DenA interaction that affects DEN1/DenA protein levels presumably balances cellular deneddylase activity. A deneddylase disequilibrium impairs multicellular development and suggests that control of deneddylase activity is important for multicellular development. [ABSTRACT FROM AUTHOR]

The COP9 signalosome (CSN) purified from human erythrocytes possesses kinase activity that phosphorylates proteins such as c-Jun and p53 with consequence for their ubiquitin (Ub)-dependent degradation. Here we show that protein kinase CK2 (CK2) and protein kinase D (P1(D) co-purify with CSN. Immunoprecipitation and far-western blots reveal that CK2 and PKD are in fact associated with CSN. As indicated by electron microscopy with gold-labeled ATP, at least 10% of CSN particles are associated with kinases. Kinase activity, most likely due to CK2 and PKD, co-immuno- precipitates with CSN from HeLa cells. CK2 binds to ACSN3(111-403) and CSN7, whereas PKD interacts with full-length CSN3. CK2 phosphorylates CSN2 and CSN7, and PKD modifies CSN7. Both CK2 and PKD phosphorylate c-Jun as well as p53. CK2 phosphorylates Thr155 which targets p53 to degradation by the Ub system. Curcumin, emodin, DRB and resveratrol block CSN-associated kinases and induce degradation of c-Jun in HeLa cells. Curcumin treatment results in elevated amounts of c-Jun-Ub conjugates. We conclude that CK2 and PKD are recruited by CSN in order to regulate Ub conjugate formation. [ABSTRACT FROM AUTHOR]