Downregulation of COP9 signalosome subunits differentially affects the CSN complex and target protein stability
© Peth et al; licensee BioMed Central Ltd. 2007
Received: 03 September 2007
Accepted: 19 December 2007
Published: 19 December 2007
The COP9 signalosome (CSN) is a conserved protein complex in eukaryotic cells consisting of eight subunits (CSN1 to CSN8). Recent data demonstrate that the CSN is a regulator of the ubiquitin (Ub) proteasome system (UPS). It controls substrate ubiquitination by cullin-RING Ub ligases (CRLs), a process that determines substrate specificity of the UPS. The intrinsic deneddylating activity localized to CSN5 as well as the associated kinases and deubiquitinating activity are involved in the regulatory function of CSN. The exact mechanisms are unclear. In this study we knocked down CSN1 (siCSN1), CSN3 (siCSN3) and CSN5 (siCSN5) by specific siRNA oligos permanently expressed in HeLa cells. The analysis and comparison of siRNA cells revealed differential impact of individual subunits on CSN structure and function.
Permanent knockdowns of CSN1 and CSN3 led to a reduction of the subunits to approximately 40%, which is accompanied by a proportional decrease of the CSN holocomplex. In contrast, downregulation of CSN5 in HeLa cells reduced the CSN5 protein below 20% without significant effects on the remaining complex. The CRL component Rbx1 was characterized by accelerated proteolysis in siCSN1 and siCSN3 and also in siCSN5 cells, however, with lesser extent. Immunoprecipitated CSN complex from siCSN5 cells was less effective in phosphorylating c-Jun and p27. Accelerated degradation of c-Jun in siCSN5 cells was rescued by overexpression of CSN5 as well as of the deneddylation mutant CSN5D151N. Overexpression of CSN5 cannot rescue c-Jun destabilization in siCSN1.
There exists a coordinated downregulation of CSN subunits in the CSN1 and CSN3 knockdowns. The underlying regulatory mechanisms are obscure. CSN5 seems to possess a specific status in HeLa cells. Its reduction is not connected with coordinated downregulation of other subunits. CSN knockdowns confirm that the stabilization of the CRL component Rbx1 is a major CSN function. In addition, downregulation of CSN subunits influences the stability of important cellular regulators such as c-Jun and p27.
The COP9 signalosome (CSN) is a conserved protein complex, which controls eukaryotic protein degradation via the ubiquitin (Ub) proteasome system (UPS) [1, 2]. In mammals the core complex consists of 8 subunits (CSN1 to CSN8) , the exact function of which is not exactly known. CSN5 exhibits a MPN+/JAMM domain [4, 5] responsible for metalloprotease activity. As a complex-bound protein CSN5 removes NEDD8, an ubiquitin-like protein, from cullins. This cleavage of an isopeptide bond called deneddylation controls the ubiquitination by cullin-RING Ub ligases (CRLs), a large family of multisubunit E3s [6, 7]. Cullin proteins (Cul1 to Cul7) are components of CRLs functioning as scaffolds of the Ub ligase complexes. Cullin neddylation and deneddylation regulates the CRL complex assembly in a substrate dependent manner as it has been shown for Cul1-SCFSkp2 and p27Kip (p27) . In addition, CRLs contain RING-domain proteins, frequently Rbx1 or Rbx2, which are responsible for ubiquitination as well as for neddylation . Substrate specificity of the CRLs is determined by components called substrate receptors including F-box proteins in Cul1-based CRLs and BTB-domain proteins in Cul3-complexes .
The CSN is associated with a cysteine protease called Ub specific protease 15 (USP15), which belongs to the family of deubiquitinating enzymes (DUBs) [10, 11]. USP15 is able to cleave linear as well as branched Ub chains. The cleavage of lysine 48-linked poly-Ub chains requires a functional Zn finger . The cysteine protease regulates the activity of CRLs by protecting Rbx1  as well as F-box and BTB-domain proteins [11, 12] from autoubiquitination and degradation.
In addition, the CSN is associated with kinases such as CK2, PKD , Akt  and inositol 1,3,4-trisphosphate 5/6 kinase . The kinases modify substrates of the UPS and determine their stability . The CK2 binds to subunits CSN3 and CSN7 whereas the PKD interacts with CSN3 . CSN-associated CK2 phosphorylates the tumor suppressor p53  and the inhibitor of cyclin-dependent kinases p27 , which targets the proteins to degradation by the 26S proteasome. In contrast, phosphorylation of c-Jun stabilizes the transcription factor towards the UPS [13, 17]. Inhibitors of CSN-associated kinases such as curcumin or emodin elevate the amount of p53, which causes apoptosis in tumor cells . On the other hand, by blocking phosphorylation these compounds target c-Jun to rapid degradation by the UPS [13, 18]. Subunit CSN5 directly interacts with c-Jun , p27  as well as with p53 , which is presumably necessary for CSN-mediated phosphorylation and degradation of the proteins, although the exact interrelations are not yet clear. The impact of permanent downregulation of CSN5 in HeLa cells on the stability of c-Jun and p27 was investigated in the present study.
To analyze functional characteristics of genes in yeast, knockouts have been performed. Unfortunately knockouts of CSN genes in yeast possess only weak or no phenotypes . In contrast, CSN knockouts in Drosophila , in plants [2, 23] and in mammals [24, 25] are lethal. Recently functional genomic studies in mammalian cells include specific siRNA oligos that cause degradation of the target gene mRNAs. For example, the conditional knockdown of CSN5 expression in HEK293 cells using a doxycycline-inducible shRNA system led to the degradation of F-box proteins accompanied with reduced CRL activity . It has been shown that downregulation of Jab1/CSN5 in leukemia cell lines prevented p27 degradation . Recent knockdown studies in Drosophila revealed a role of the CSN in the cell cycle .
By using specific siRNA oligos against CSN1, CSN3 and CSN5 permanently expressed in HeLa cells, we studied the impact of CSN subunit knockdowns on the CSN complex stability and target protein degradation. We demonstrate that knockdowns of CSN1 or CSN3 led to a downregulation of the entire CSN complex, whereas in case of CSN5 downregulation the remaining complex was stable. The decrease of the binding subunit CSN5 in siCSN5 cells as well as diminished CSN complex in siCSN1 and siCSN3 cells led to reduced stability of Rbx1 and c-Jun, whereas p27 was stabilized in siCSN1 cells.
The CSN5 knockdown is unique compared to CSN1 or CSN3 knockdowns in HeLa cells
CSN knockdowns destabilize the RING-domain protein Rbx1
There are differential roles of CSN subunits in affecting target protein stability
The deneddylating MPN+-domain protein CSN5 interacts with a large number of ligands. It can be called a substrate receptor of CSN interacting proteins including important regulators such as c-Jun and p27 (for rev. see ). In contrast, CSN1 and CSN3 interact with CSN-associated kinases [13, 15]. One might postulate that knockdowns of CSN1, CSN3 and CSN5 should cause reduced CSN-mediated phosphorylation of c-Jun and of p27, however, due to different reasons.
A decreased phosphorylation in siCSN1 cells has been shown for the microtubule-end-binding protein 1 (EB1) . Just like c-Jun the protein binds to CSN5 and is stabilized by CSN-mediated phosphorylation. By in vitro pulldowns with purified CSN and recombinant GST-EB1 and by adding increasing amounts of recombinant CSN5 it was demonstrated that EB1 can be competed away from its CSN5 binding site (Fig. 5b). A similar experiment was performed in cells. CSN5wt or CSN5D151N mutant was overexpressed in HeLa cells and the binding and phosphorylation of endogenous EB1 to the CSN was indirectly estimated by EB1 stability. As seen in Fig. 5c, overexpression of CSN5wt led to an accelerated degradation of EB1, most likely because it was competed away from the CSN and was less phosphorylated. The same effect was obtained by overexpressing CSN5D151N indicating that the metalloprotease activity of CSN5 is not necessary for this competition.
In the present study we compared the effects of CSN subunit downregulation on the structure and functions of the holocomplex. The data presented here as well as in our recent work on EB1  demonstrate that downregulation of CSN1 and CSN3 by specific siRNA oligos permanently expressed in HeLa cells led to a proportional downregulation of the CSN holocomplex. In other words, knockdowns of CSN1 and CSN3 cause a proportional reduction of all determined CSN subunits paralleled by a decrease of the holocomplex. None of the CSN subunits were detected as free proteins under these conditions. Similar data were obtained by Kato and co-workers in murine embryonic fibroblasts were a reduction in the level of CSN3 protein with siRNA decreased the total amount of the holocomplex . The complete deletion of the CSN3 subunit in mice and also in other higher eukaryotes is lethal, which presumably can be due to the complete disappearance of the CSN complex . These data indicate that in mammalian cells exists a coordinated up and down regulation of CSN subunits accompanied by an assembly or disassembly process of the holocomplex.
On the contrary, downregulation of subunit CSN5 had little or no effect on other CSN subunits and did not lead to the proportional disappearance of the residual complex. Similar effects were observed by downregulation of CSN5 in Drosophila as well as in HeLa cells [31, 32]. In other words, in siCSN5 cells CSN complexes possess less or no CSN5. This might indicate that CSN5 has a different impact on the coordinated assembly of the CSN as compared to CSN1 and CSN3. On the other hand, a complete depletion of CSN5 in Arabidopsis resulted in CSN instability and the decay of various CSN components . Interestingly, overexpressed CSN5wt is most likely integrated into the CSN complex in siCSN5 cells, because it is able to restore deneddylation activity of the particle (see Fig. 1d).
Although CSN1, CSN3 as well as CSN5 knockdowns led to destabilization of Rbx1, the effects are different. Whereas in siCSN1 and siCSN3 cells Rbx1 degradation is significantly accelerated, in siCSN5 cells the RING-domain protein is less destabilized (see Fig. 2). The protection of Rbx1 from autoubiquitination and degradation is mostly due to the associated deubiquitinating enzyme USP15, which interacts predominantly via CSN7 with the CSN . This might explain why the stability of Rbx1 in siCSN5 cells is less affected as compared to siCSN1 or siCSN3 cells. Our siRNA studies confirm the hypothesis that the CSN stabilizes CRL components by protecting them against autoubiquitination [10, 12]. With less CSN and its associated USP15 in cells there is less protection of the CRLs.
We speculate that changed stabilities of c-Jun, p27 and EB1 in siCSN1, siCSN3 as well as siCSN5 cells can be due to reduced CSN-mediated phosphorylation of these proteins. In siCSN1 and siCSN3 cells the amount of the CSN platform itself together with its associated kinases was decreased. In CSN5 knockdown cells the reduction of the substrate receptor CSN5 might be sufficient to cause a diminished phosphorylation of the tested substrates (see Fig. 4). Immunoprecipitated CSN from siCSN5 cells was less efficient in phosphorylating c-Jun as well as p27 as compared to the control. This effect can be explained by the reduction of CSN5 and, at least in part, by reduced overall kinase activity (see Fig. 4a) perhaps caused by a changed CSN structure. In contrast to siCSN1 cells the accelerated degradation of c-Jun in siCSN5 cells was blocked by overexpression of CSN5wt as well as of CSN5D151N mutant (see Fig. 3). Based on these data we came to two conclusions. (i) The overexpressed CSN5wt was integrated into CSN5-deficient CSN complexes and acts as substrate receptor allowing normal substrate phosphorylation. (ii) The rescue of c-Jun stability by overexpressing CSN5D151N demonstrates that the mutant is as potent as the CSN5wt indicating that the metalloprotease activity of CSN5 was not responsible for the effect.
Presumably reduced phosphorylation of p27 in CSN knockdown cells resulted in a stabilization of the cell cycle inhibitor during serum starvation (see Fig. 5a). Thus, the CSN might control the ubiquitination of p27 by two different activities. First, it regulates the assembly of the appropriate Cul1-CRL complex for p27 ubiquitination by deneddylation . Second, it seems to be a platform for p27 phosphorylation that most likely accelerates p27 ubiquitination.
Our data also show that one should be cautioned with the overexpression of CSN5. In HeLa cells free CSN5 is undetectable under our conditions. Overexpression of CSN5 produces a free CSN5 protein pool, which stimulates the degradation of EB1. As shown by competition experiments in Fig. 5b and 5c, this is most likely due to the trapping of EB1 by free CSN5 and, as a result, to reduced CSN-mediated phosphorylation and destabilization of EB1. We speculate that overexpression of CSN5 might exert artificial effects on other substrates that interact with CSN5 as well.
Future knockdown experiments with other CSN subunits are necessary to fully understand the relationship between structure and function of the CSN complex. In addition, the integration of diverse mutated subunits into the CSN complex where possible will elucidate specific functions of subunit domains.
We have shown that downregulation of CSN1 and CSN3 reduced the CSN holocomplex. In contrast, downregulation of CSN5 did not change the remaining CSN complex, although CSN5 protein was reduced. All studied knockdown cells were characterized by a destabilization of the CRL component Rbx1 and of the transcription factor c-Jun. In CSN5 knockdowns c-Jun destabilization was rescued by CSN5 overexpression, demonstrating the substrate receptor role of CSN5.
Preparation of the CSN, cell culture and siRNA knockdown of CSN subunits
The human CSN complex was purified from red blood cells as outlined in detail . HeLa cells were cultured as described previously . siRNA-mediated knockdown of CSN1 and CSN3 was performed as outlined recently  using the pSUPER system. The knockdown of CSN5 was performed with the target sequences published by Groisman and co-workers . Synchronization of HeLa cells was carried out as described previously .
Transfection of HeLa cells, site directed mutagenesis and CHX chase experiments
HeLa cells were transfected with Lipofectamine2000 (Invitrogen) according to the manufacturers recommendations. The metalloprotease mutant CSN5D151N was created by site directed mutagenesis as outlined before . Expression of Flag-CSN5 and of Flag-CSN5D151N in HeLa cells was performed as described before . Rescue experiments of c-Jun degradation were performed after transfection with 2.5 μg of CSN5wt DNA or 5 μg of CSN5D151N DNA. CHX chases were carried out at a final concentration of 20 μg/ml CHX as in .
Immunoprecipitation, kinase assays, Western blotting, competition experiments and glycerol gradients
Immunprecipitations and glycerol gradient centrifugations were performed as described . The in vitro kinase assays were carried out as outlined previously . SDS-PAGE, Western blotting and ECL detection (GE Healthcare) were performed by standard procedures. Non-denaturing electrophoresis was carried out using the Phast-System (GE Healthcare). Competition experiments with EB1, CSN5 and the CSN complex were performed as described  using recombinant GST-EB1, recombinant His-CSN5 and purified CSN. In this study the following antibodies were used: Anti-CSN5 (provided by B. Christy), anti-Rbx1 (Zymed), anti-CSN8 (Biomol), anti-α 6 and anti-RPN2 (provided by C. Hendil), anti-c-Jun (Upstate), anti-p27 and anti-EB1 (Santa-Cruz), anti-β-tubulin (Covance), anti-CSN3 antibodies  and anti-Flag (Sigma).
List of abbreviations
ubiquitin proteasome system
cullin-RING ubiquitin ligases
This work was supported by a grant DU 229/6-3 and 9-1 from the Deutsche Forschungsgemeinschaft to W.D.
- Bech-Otschir D, Kapelari B, Dubiel W: The COP9 Signalosome: Its Possible Role in the Ubiquitin System. Protein Degradation. Edited by: Mayer R, Ciechanover A, Rechsteiner M. 2005, Weinheim , WILEY-VCH Verlag GmbH & Co. KGaA, 1: Ubiquitin and the Chemistry of Life: 348-369.View ArticleGoogle Scholar
- Wei N, Deng XW: The COP9 signalosome. Annu Rev Cell Dev Biol. 2003, 19: 261-286.View ArticlePubMedGoogle Scholar
- Deng XW, Dubiel W, Wei N, Hofmann K, Mundt K, Colicelli J, Kato J, Naumann M, Segal D, Seeger M, Carr A, Glickman M, Chamovitz DA: Unified nomenclature for the COP9 signalosome and its subunits: an essential regulator of development. Trends Genet. 2000, 16 (5): 202-203.View ArticlePubMedGoogle Scholar
- Maytal-Kivity V, Reis N, Hofmann K, Glickman MH: MPN+, a putative catalytic motif found in a subset of MPN domain proteins from eukaryotes and prokaryotes, is critical for Rpn11 function. BMC Biochem. 2002, 3 (1): 28-PubMed CentralView ArticlePubMedGoogle Scholar
- Verma R, Aravind L, Oania R, McDonald WH, Yates JR, Koonin EV, Deshaies RJ: Role of Rpn11 metalloprotease in deubiquitination and degradation by the 26S proteasome. Science. 2002, 298 (5593): 611-615.View ArticlePubMedGoogle Scholar
- Willems AR, Schwab M, Tyers M: A hitchhiker's guide to the cullin ubiquitin ligases: SCF and its kin. Biochim Biophys Acta. 2004, 1695 (1-3): 133-170.View ArticlePubMedGoogle Scholar
- Petroski MD, Deshaies RJ: Function and regulation of cullin-RING ubiquitin ligases. Nat Rev Mol Cell Biol. 2005, 6 (1): 9-20.View ArticlePubMedGoogle Scholar
- Bornstein G, Ganoth D, Hershko A: Regulation of neddylation and deneddylation of cullin1 in SCFSkp2 ubiquitin ligase by F-box protein and substrate. Proc Natl Acad Sci U S A. 2006, 103 (31): 11515-11520.PubMed CentralView ArticlePubMedGoogle Scholar
- Morimoto M, Nishida T, Nagayama Y, Yasuda H: Nedd8-modification of Cul1 is promoted by Roc1 as a Nedd8-E3 ligase and regulates its stability. Biochem Biophys Res Commun. 2003, 301 (2): 392-398.View ArticlePubMedGoogle Scholar
- Hetfeld BK, Helfrich A, Kapelari B, Scheel H, Hofmann K, Guterman A, Glickman M, Schade R, Kloetzel PM, Dubiel W: The zinc finger of the CSN-associated deubiquitinating enzyme USP15 is essential to rescue the E3 ligase Rbx1. Curr Biol. 2005, 15 (13): 1217-1221.View ArticlePubMedGoogle Scholar
- Zhou C, Wee S, Rhee E, Naumann M, Dubiel W, Wolf DA: Fission yeast COP9/signalosome suppresses cullin activity through recruitment of the deubiquitylating enzyme Ubp12p. Mol Cell. 2003, 11 (4): 927-938.View ArticlePubMedGoogle Scholar
- Wee S, Geyer RK, Toda T, Wolf DA: CSN facilitates Cullin-RING ubiquitin ligase function by counteracting autocatalytic adapter instability. Nat Cell Biol. 2005, 7 (4): 387-391.View ArticlePubMedGoogle Scholar
- Uhle S, Medalia O, Waldron R, Dumdey R, Henklein P, Bech-Otschir D, Huang X, Berse M, Sperling J, Schade R, Dubiel W: Protein kinase CK2 and protein kinase D are associated with the COP9 signalosome. EMBO J. 2003, 22 (6): 1302-1312.PubMed CentralView ArticlePubMedGoogle Scholar
- Huang X, Wagner E, Dumdey R, Peth A, Berse M, Dubiel W, Berndt C: Phosphorylation by COP9 signalosome-associated CK2 promotes degradation of p27 during the G1 cell cycle phase. Isr J Chem. 2006, 46 (2): 231-238.View ArticleGoogle Scholar
- Sun Y, Wilson MP, Majerus PW: Inositol 1,3,4-trisphosphate 5/6-kinase associates with the COP9 signalosome by binding to CSN1. J Biol Chem. 2002, 277 (48): 45759-45764.View ArticlePubMedGoogle Scholar
- Bech-Otschir D, Kraft R, Huang X, Henklein P, Kapelari B, Pollmann C, Dubiel W: COP9 signalosome-specific phosphorylation targets p53 to degradation by the ubiquitin system. EMBO J. 2001, 20 (7): 1630-1639.PubMed CentralView ArticlePubMedGoogle Scholar
- Naumann M, Bech-Otschir D, Huang X, Ferrell K, Dubiel W: COP9 signalosome-directed c-Jun activation/stabilization is independent of JNK. J Biol Chem. 1999, 274 (50): 35297-35300.View ArticlePubMedGoogle Scholar
- Fullbeck M, Huang X, Dumdey R, Frommel C, Dubiel W, Preissner R: Novel curcumin- and emodin-related compounds identified by in silico 2D/3D conformer screening induce apoptosis in tumor cells. BMC Cancer. 2005, 5: 97-PubMed CentralView ArticlePubMedGoogle Scholar
- Claret FX, Hibi M, Dhut S, Toda T, Karin M: A new group of conserved coactivators that increase the specificity of AP-1 transcription factors. Nature. 1996, 383 (6599): 453-457.View ArticlePubMedGoogle Scholar
- Tomoda K, Kubota Y, Kato J: Degradation of the cyclin-dependent-kinase inhibitor p27Kip1 is instigated by Jab1. Nature. 1999, 398 (6723): 160-165.View ArticlePubMedGoogle Scholar
- Mundt KE, Liu C, Carr AM: Deletion mutants in COP9/signalosome subunits in fission yeast Schizosaccharomyces pombe display distinct phenotypes. Mol Biol Cell. 2002, 13 (2): 493-502.PubMed CentralView ArticlePubMedGoogle Scholar
- Freilich S, Oron E, Kapp Y, Nevo-Caspi Y, Orgad S, Segal D, Chamovitz DA: The COP9 signalosome is essential for development of Drosophila melanogaster. Curr Biol. 1999, 9 (20): 1187-1190.View ArticlePubMedGoogle Scholar
- Dohmann EM, Kuhnle C, Schwechheimer C: Loss of the CONSTITUTIVE PHOTOMORPHOGENIC9 signalosome subunit 5 is sufficient to cause the cop/det/fus mutant phenotype in Arabidopsis. Plant Cell. 2005, 17 (7): 1967-1978.PubMed CentralView ArticlePubMedGoogle Scholar
- Yan J, Walz K, Nakamura H, Carattini-Rivera S, Zhao Q, Vogel H, Wei N, Justice MJ, Bradley A, Lupski JR: COP9 signalosome subunit 3 is essential for maintenance of cell proliferation in the mouse embryonic epiblast. Mol Cell Biol. 2003, 23 (19): 6798-6808.PubMed CentralView ArticlePubMedGoogle Scholar
- Lykke-Andersen K, Schaefer L, Menon S, Deng XW, Miller JB, Wei N: Disruption of the COP9 signalosome Csn2 subunit in mice causes deficient cell proliferation, accumulation of p53 and cyclin E, and early embryonic death. Mol Cell Biol. 2003, 23 (19): 6790-6797.PubMed CentralView ArticlePubMedGoogle Scholar
- Cope GA, Deshaies RJ: Targeted silencing of Jab1/Csn5 in human cells downregulates SCF activity through reduction of F-box protein levels. BMC Biochem. 2006, 7: 1-PubMed CentralView ArticlePubMedGoogle Scholar
- Tomoda K, Kato JY, Tatsumi E, Takahashi T, Matsuo Y, Yoneda-Kato N: The Jab1/COP9 signalosome subcomplex is a downstream mediator of Bcr-Abl kinase activity and facilitates cell-cycle progression. Blood. 2005, 105 (2): 775-783.View ArticlePubMedGoogle Scholar
- Bjorklund M, Taipale M, Varjosalo M, Saharinen J, Lahdenpera J, Taipale J: Identification of pathways regulating cell size and cell-cycle progression by RNAi. Nature. 2006, 439 (7079): 1009-1013.View ArticlePubMedGoogle Scholar
- Peth A, Boettcher JP, Dubiel W: Ubiquitin-dependent Proteolysis of the Microtubule End-binding Protein 1, EB1, Is Controlled by the COP9 Signalosome: Possible Consequences for Microtubule Filament Stability. J Mol Biol. 2007, 368 (2): 550-563.View ArticlePubMedGoogle Scholar
- Yoneda-Kato N, Tomoda K, Umehara M, Arata Y, Kato JY: Myeloid leukemia factor 1 regulates p53 by suppressing COP1 via COP9 signalosome subunit 3. EMBO J. 2005, 24 (9): 1739-1749.PubMed CentralView ArticlePubMedGoogle Scholar
- Oron E, Mannervik M, Rencus S, Harari-Steinberg O, Neuman-Silberberg S, Segal D, Chamovitz DA: COP9 signalosome subunits 4 and 5 regulate multiple pleiotropic pathways in Drosophila melanogaster. Development. 2002, 129 (19): 4399-4409.PubMedGoogle Scholar
- Groisman R, Polanowska J, Kuraoka I, Sawada J, Saijo M, Drapkin R, Kisselev AF, Tanaka K, Nakatani Y: The ubiquitin ligase activity in the DDB2 and CSA complexes is differentially regulated by the COP9 signalosome in response to DNA damage. Cell. 2003, 113 (3): 357-367.View ArticlePubMedGoogle Scholar
- Gusmaroli G, Figueroa P, Serino G, Deng XW: Role of the MPN subunits in COP9 signalosome assembly and activity, and their regulatory interaction with Arabidopsis Cullin3-based E3 ligases. Plant Cell. 2007, 19 (2): 564-581.PubMed CentralView ArticlePubMedGoogle Scholar
- Hetfeld BK, Bech-Otschir D, Dubiel W: Purification method of the COP9 signalosome from human erythrocytes. Methods Enzymol. 2005, 398: 481-491.View ArticlePubMedGoogle Scholar
- Seeger M, Kraft R, Ferrell K, Bech-Otschir D, Dumdey R, Schade R, Gordon C, Naumann M, Dubiel W: A novel protein complex involved in signal transduction possessing similarities to 26S proteasome subunits. FASEB J. 1998, 12 (6): 469-478.PubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.