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Protein stability: The COP9 signalosome gets in on the act
Dispatch
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Protein stability: The COP9 signalosome gets in on the act Michael Seeger*, Colin Gordon* and Wolfgang Dubiel†
The COP9 signalosome is a multiprotein complex somewhat similar to the lid component of the 26S proteasome. Recent studies suggest that it regulates the stability of proteins by interfering with the ubiquitin–proteasome pathway via deneddylation and phosphorylation. Addresses: *MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh, EH4 2XU, UK. †Department of Surgery, Division of Molecular Biology, Medical Faculty Charité, Humboldt University, Monbijoustr. 2, D-10117 Berlin, Germany. E-mail: [email protected]; [email protected] Current Biology 2001, 11:R643–R646 0960-9822/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved.
Degradation of regulatory proteins by the ubiquitin system plays a crucial role in many signalling pathways. Ubiquitin modifies target proteins to tag them for degradation by the proteasome (for review see [1]). The conjugation of ubiquitin — itself a small protein — to a substrate protein is accomplished by a set of ubiquitinating enzymes. Ubiquitin is first activated by a ubiquitin-activating enzyme (E1), and then ligated to the substrate by a ubiquitin-conjugating enzyme (E2) assisted by a ubiquitin protein ligase (E3). Proteins that carry a chain of ubiquitin molecules are recognised by the 26S proteasome and rapidly degraded. There is a remarkable similarity between the lid component of the proteasome and the COP9 signalosome, a multiprotein complex first discovered in plants but now known to occur generally in eukaryotes. Each of the eight proteasome lid subunits shows significant sequence similarity to a COP9 signalosome component [2–4], and even the quaternary structures of the complexes resemble each other [5]. There is an increasing body of evidence that the two machineries, the ubiquitin system and the COP9 signalosome, cooperate in regulating the stability of important cellular proteins. Two recent studies [6,7] in particular have shed light on the mechanism of this cooperation. Interactions of COP9 signalosome subunits
The COP9 signalosome was first identified in the model plant Arabidopsis, the discovery coming from work on mutant plants with defective light-dependent development [8]. These mutants failed to switch off photomorphogenesis when grown in the dark, as a result of their inability to accumulate a protein dubbed COP1, for ‘constitutive photomorphogenesis 1’ in the nucleus. It was therefore reasoned that COP1 acts as repressor of photomorphogenic development, and that the epistatic mutations in the COP9
and COP11 genes must be upstream of COP1 in the signalling pathway. In gel-filtration experiments, COP9 and COP11 proteins co-eluted in high molecular weight fraction; further purification identified a 560 kDa multiprotein complex containing those proteins. The isolation of a highly purified homologous complex — the COP9 signalosome — from mammalian cells enabled detailed characterisation of its subunit composition [2,3]. Eight subunits were identified, now called CSN1 to CSN8 according to their electrophoretic mobility, with CSN1 moving the slowest [9]. Some of the COP9 signalosome subunits had come up in other contexts, in particular they include ‘Jun activation domain binding protein 1’ (Jab1) and ‘thyroid receptor interacting protein 15’ (TRIP15), recently renamed CSN5 and CSN2, respectively. Further studies in various species have provided valuable information about subunit–subunit interactions and about a range of proteins that interact with the COP9 signalosome (reviewed in [10]). These data indicated that binding to the COP9 signalosome affects the subcellular localisation and stability of certain regulatory proteins. Binding to CSN5 was shown to stabilise proteins such as the progesterone receptor, the steroid receptor [11] and the IκB protein Bcl-3 [12], but it also promotes nucleocytoplasmic translocation and degradation of the cyclin-dependent kinase (Cdk) inhibitor p27kip [13]. Overproduction of CSN5 was found to stabilise the precursor of the lutropin/choriogonadotropin receptor [14] and the transcription factor c-Jun [15]. Kinase activity of the COP9 signalosome
CSN5 was originally described as a coactivator, Jab1, that increases the specificity of AP-1 transcription factors by binding to c-Jun or JunD [15]. The identification of Jab1 as a COP9 signalosome subunit led to the re-evaluation of its described effects on the c-Jun pathway [2,3,16]. A serine/threonine kinase activity associated with the COP9 signalosome was detected, and its specificity for c-Jun and IκB characterised [2]. COP9 signalosome-dependent phosphorylation could be inhibited efficiently by curcurmin [17], known to have anti-tumorigenic and anti-angiogenic effects [18]. De novo formation of the COP9 signalosome in HeLa cells, induced by overproduction of CSN2, was found to stabilise c-Jun and increase AP-1 activity [16]. These observations suggested the existence of a COP9 signalosome-directed c-Jun signalling pathway [16]. The tumour suppressor protein p53 has now been identified as another target of the COP9 signalosome-associated kinase activity [19]. COP9 signalosome-specific phosphorylation of p53 between amino acids 145 and 160 promotes
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Figure 1 SCF complex
COP9 signalosome
26S proteasome
Ub
Stabilisation P Substrate A
E1
Substrate A
Ub
Nedd8
Nedd8
Cul1 Degradation E2 Substrate B
P
Substrate B Ub Ub
Substrate B
Ub Ub
P F-box
Ub Substrate B Ub Ub Ub Ub Current Biology
A speculative model for how the phosphorylation and deneddylation activities that have been shown to be associated with the COP9 signalosome might act in concert to regulate substrate ubiquitination by the SCF. See text for details.
degradation of the protein by the ubiquitin–proteasome pathway. Inhibition of this phosphorylation, either by curcurmin or a phosphorylated p53(145–164) peptide, stabilised p53 and led to accumulation of the endogenous protein in vivo. It appears that substrates of the COP9 signalosome kinase activity must bind to one of its subunits in order to be phosphorylated. CSN5, for example, recruits c-Jun [15] and p53 [19] to the COP9 signalosome, whereas phosphorylation of the interferon consensus sequencebinding protein (ICSBP) requires binding to CSN2 [20]. Deneddylation activity of the COP9 signalosome
The transcription factor HY5, which induces the expression of genes responsible for photomorphogenic development in plants, is stabilised upon phosphorylation. Recent studies have shown that COP1 promotes the proteasomedependent degradation of unphosphorylated HY5 [21]. Interestingly, COP1 contains a RING finger domain, characteristic of a family of ubiquitin ligases, which indicates that it might be a HY5-specific ubiquitin ligase. As the COP9 signalosome regulates COP1 activity, this was the first hint that the signalosome might control components of the ubiquitination machinery. Co-precipitation studies have now revealed that COP9 signalosome associates with SCF, a multimeric ubiquitin ligase responsible for ubiquitination of p27kip, IκB, a subset of cyclins and other regulatory proteins [6,7]. SCF contains Skp1, Hrt1, substrate-binding F-box proteins and a RINGfinger domain protein of the cullin family as major components. Cullin is modified by the covalent attachment of the
ubiquitin-like protein Nedd8 [22,23]. Cells of the fission yeast Schizosaccharomyces pombe deleted for csn1+ showed an accumulation of Nedd8-modified — neddylated — cullin species, accompanied by a significant increase of SCF activity [6]. Together with the results of reconstitution experiments using purified mammalian COP9 signalosome, the data suggest that the COP9 signalosome complex mediates cleavage of Nedd8–cullin conjugates. Studies in Arabidopsis showed that reduced levels of CSN5 cause insensitivity to the plant hormone auxin [7]. In the CSN5-deficient plants, SCF-mediated degradation of PSIAA6, a regulatory protein of the auxin response pathway, is compromised and an accumulation of neddylated cullin was observed. These findings indicate that tight regulation of neddylation and deneddylation events might be important for the degradation of certain SCF substrates. In fission yeast, however, deletion of csn1+ did not affect the stability of the SCF substrate Rum1 [20]. Moreover, although fission yeast cells deleted for csn1+ and csn2+ display a cell-cycle-related phenotype they are still viable, indicating that COP9 signalosome function is not essential in S. pombe [24]. Interestingly, deletion of ned8+ is lethal in fission yeast [25]. It therefore appears that, although neddylation is crucial for viability in fission yeast, COP9 signalosome function is not. In the budding yeast Saccharomyces cerevisiae, the picture is slightly different as, apart from CSN5, no other orthologues of COP signalosome subunits have been found, and deletion of the gene encoding the Nedd8 orthologue, RUB1, is not lethal [22–24]. It is therefore conceivable that Nedd8 modification
Dispatch
and COP9 signalosome function developed as an additional control mechanism for the degradation of certain SCF substrates. Why not both deneddylation and phosphorylation?
Inhibition of the COP9 signalosome deneddylation activity by the alkylating agent NEM suggested that removal of Nedd8 is accomplished by cysteine protease activity [6]. Sequence comparison studies indicate that CSN5 is the only subunit of the complex with homology to cysteine proteases. Moreover, deletion of the CSN5 orthologue in budding yeast led to accumulation of modified cullin [6]. CSN5 is therefore a strong candidate for being the deneddylase. Two-hybrid studies have shown that the cullin CUL1 binds to CSN2 and CSN6, but no interaction could be detected between CSN5 and CUL1 or any other components of the SCF [6,7]. That implies that CSN5 would have to cleave CUL1–Nedd8 without binding it directly. It would be interesting to find out whether recombinant CSN5 protein can cleave CUL1–Nedd8 conjugates, and whether mutation of the conserved cysteine residue in the putative protease active site of CSN5 leads to the accumulation of Nedd8-modified cullins. The question is whether the COP9 signalosome deneddylation activity can be assigned to one of its eight subunits — perhaps CSN5 as suggested above — or to a protein that co-purifies with the complex. There is a similar problem with the COP9 signalosome kinase activity. None of the COP9 signalosome subunits shows significant sequence similarity to any known kinase, or has been shown capable of phosphorylating model substrates on its own. One explanation for this would be that the kinase copurifies with the COP9 signalosome complex. Another possibility, however, is that the kinase activity might need the cooperation of several COP9 signalosome subunits. The same might be true for the deneddylation activity of the COP9 signalosome. It is conceivable that CSN2 binds to CUL1–Nedd8, which then becomes accessible to CSN5. In budding yeast, other proteins might mediate the interaction with CSN5. Therefore, both phosphorylation and deneddylation might be catalysed by the COP9 signalosome, which would allow the complex to interfere with the pathway at two points — the substrate and the E3 ubiquitin ligase. A possible model of COP9 signalosome action is shown in Figure 1. In this model, COP9 signalosome-specific phosphorylation of a substrate protein triggers either its release from the complex and stabilisation, or the association of the COP9 signalosome with neddylated SCF. The COP9 signalosome—SCF interaction results in transfer of the phosphorylated substrate to the glycine rich repeat region of the F-box protein, and its ubiquitination. When, after a number of conjugation steps, the ubiquitin chain has reached a certain length, the cullin is deneddylated by the
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COP9 signalosome, causing inactivation of SCF and transfer of the multiubiquitinylated substrate to the 26S proteasome. Taking all the data together, it appears that the COP9 signalosome acts as an interface that links various signalling pathways with the ubiquitination machinery. Future work will provide further insights into how the kinase and deneddylase activities of the COP9 signalosome complex regulate ubiquitin-dependent proteolysis. References 1. Hershko A, Ciechanover A: The ubiquitin system. Annu Rev Biochem 1998, 67:425-479. 2. 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, 12:469-478. 3. Wei N, Tsuge T, Serino G, Dohmae N, Takio K, Matsui M, Deng XW: The COP9 complex is conserved between plants and mammals and is related to the 26S proteasome regulatory complex. Curr Biol 1998, 8:919-922. 4. Glickman MH, Rubin DM, Coux O, Wefes I, Pfeifer G, Cjeka Z, Baumeister W, Fried VA, Finley D: A subcomplex of the proteasome regulatory particle required for ubiquitin-conjugate degradation and related to the COP9-signalosome and eIF3. Cell 1998, 94:615-623. 5. Kapelari B, Bech-Otschir D, Hegerl R, Schade R, Dumdey R, Dubiel W: Electron microscopy and subunit-subunit interaction studies reveal a first architecture of COP9 signalosome. J Mol Biol 2000, 300:1169-1178. 6. Lyapina S, Cope G, Shevchenko A, Serino G, Tsuge T, Zhou C, Wolf DA, Wei N, Shevchenko A, Deshaies RJ: Promotion of NEDD-CUL1 conjugate cleavage by COP9 signalosome. Science 2001, 292:1382-1385. 7. Schwechheimer C, Serino G, Callis J, Crosby WL, Lyapina S, Deshaies RJ, Gray WM, Estelle M, Deng XW: Interactions of the COP9 signalosome with the E3 ubiquitin ligase SCFTIRI in mediating auxin response. Science 2001, 292:1379-1382. 8. Wei N, Chamovitz DA, Deng XW: Arabidopsis COP9 is a component of a novel signaling complex mediating light control of development. Cell 1994, 78:117-124. 9. Deng X-W, Dubiel W, Wei N, Hofmann K, Mundt K, Colicelli J, Kato J-Y, Naumann M, Segal D, Seeger M, et al.: Unified nomenclature for the COP9 signalosome and its subunits: an essential regulator of development. Trends Genet 2000, 16:202-203. 10. Chamovitz DA, Segal D: JAB1/CSN5 and the COP9 signalosome. A complex situation. EMBO Rep 2001, 2:96-101. 11. Chauchereau A, Georgiakaki M, Perrin-Wolff M, Milgrom E, Loosfelt H: JAB1 interacts with both the progesterone receptor and SRC-1. J Biol Chem 2000, 275:8540-8548. 12. Dechend R, Hirano F, Lehmann K, Heissmeyer V, Ansieau S, Wulczyn FG, Scheidereit C, Leutz A: The Bcl-3 oncoprotein acts as a bridging factor between NF-kappaB/Rel and nuclear co-regulators. Oncogene 1999, 18:3316-3323. 13. Tomoda K, Kubota Y, Kato J: Degradation of the cyclin-dependentkinase inhibitor p27Kip1 is instigated by Jab1. Nature 1999, 398:160-165. 14. Li S, Liu X, Ascoli M: p38JAB1 binds to the intracellular precursor of the lutropin/choriogonadotropin receptor and promotes its degradation. J Biol Chem 2000, 275:13386-13393. 15. 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, 383:453-457. 16. 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, 274:35297-35300. 17. Henke W, Ferrell K, Bech-Otschir D, Seeger M, Schade R, Jungblut P, Naumann M, Dubiel W: Comparison of human COP9 signalosome and 26S proteasome lid. Mol Biol Rep 1999, 26:29-34.
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18. Huang MT, Ma W, Lu YP, Chang RL, Fisher C, Manchand PS, Newmark HL, Conney A: Effects of curcumin, demethoxycurcumin, bisdemethoxycurcumin and tetrahydrocurcumin of 12-Otetradecanoylphorbol-13-acetate-induced tumor promotion. Carcinogenesis 1995, 16:2493-2497. 19. 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, 20:1630-1639. 20. Cohen H, Azriel A, Cohen T, Meraro D, Hashmueli S, Bech-Otschir D, Kraft R, Dubiel W, Levi BZ: Interaction between interferon consensus sequence-binding protein and COP9/signalosome subunit CSN2 (Trip15). A possible link between interferon regulatory factor signaling and the COP9/signalosome. J Biol Chem 2000, 275:39081-39089. 21. Hardtke CS, Gohda K, Osterlund MT, Oyama T, Okada K, Deng XW: HY5 stability and activity in Arabidopsis is regulated by phosphorylation in its COP1 binding domain. EMBO J 2000, 19:4997-5006. 22. Lammer D, Mathias N, Laplaza JM, Jiang W, Liu Y, Callis J, Goebl M, Estelle M: Modification of yeast Cdc53p by the ubiquitin-related protein rub1p affects function of the SCFCdc4 complex. Genes Dev 1998, 12:914-926. 23. Liakopoulos D, Doenges G, Matuschewski K, Jentsch S: A novel protein modification pathway related to the ubiquitin system. EMBO J 1998, 17:2208-2214. 24. Mundt KE, Porte J, Murray JM, Brikos C, Christensen PU, Caspari T, Hagan IM, Millar JB, Simanis V, Hofmann K, Carr AM: The COP9/signalosome complex is conserved in fission yeast and has a role in S phase. Curr Biol 1999, 9:1427-1430. 25. Osaka F, Saeki M, Katayama S, Aida N, Toh-EA, Kominami K, Toda T, Suzuki T, Chiba T, Tanaka K, Kato S: Covalent modifier NEDD8 is essential for SCN ubiquitin ligase function in fission yeast. EMBO J 2000, 19:3475-3484.
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Protein stability: The COP9 signalosome gets in on the act Michael Seeger*, Colin Gordon* and Wolfgang Dubiel†
The COP9 signalosome is a multiprotein complex somewhat similar to the lid component of the 26S proteasome. Recent studies suggest that it regulates the stability of proteins by interfering with the ubiquitin–proteasome pathway via deneddylation and phosphorylation. Addresses: *MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh, EH4 2XU, UK. †Department of Surgery, Division of Molecular Biology, Medical Faculty Charité, Humboldt University, Monbijoustr. 2, D-10117 Berlin, Germany. E-mail: [email protected]; [email protected] Current Biology 2001, 11:R643–R646 0960-9822/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved.
Degradation of regulatory proteins by the ubiquitin system plays a crucial role in many signalling pathways. Ubiquitin modifies target proteins to tag them for degradation by the proteasome (for review see [1]). The conjugation of ubiquitin — itself a small protein — to a substrate protein is accomplished by a set of ubiquitinating enzymes. Ubiquitin is first activated by a ubiquitin-activating enzyme (E1), and then ligated to the substrate by a ubiquitin-conjugating enzyme (E2) assisted by a ubiquitin protein ligase (E3). Proteins that carry a chain of ubiquitin molecules are recognised by the 26S proteasome and rapidly degraded. There is a remarkable similarity between the lid component of the proteasome and the COP9 signalosome, a multiprotein complex first discovered in plants but now known to occur generally in eukaryotes. Each of the eight proteasome lid subunits shows significant sequence similarity to a COP9 signalosome component [2–4], and even the quaternary structures of the complexes resemble each other [5]. There is an increasing body of evidence that the two machineries, the ubiquitin system and the COP9 signalosome, cooperate in regulating the stability of important cellular proteins. Two recent studies [6,7] in particular have shed light on the mechanism of this cooperation. Interactions of COP9 signalosome subunits
The COP9 signalosome was first identified in the model plant Arabidopsis, the discovery coming from work on mutant plants with defective light-dependent development [8]. These mutants failed to switch off photomorphogenesis when grown in the dark, as a result of their inability to accumulate a protein dubbed COP1, for ‘constitutive photomorphogenesis 1’ in the nucleus. It was therefore reasoned that COP1 acts as repressor of photomorphogenic development, and that the epistatic mutations in the COP9
and COP11 genes must be upstream of COP1 in the signalling pathway. In gel-filtration experiments, COP9 and COP11 proteins co-eluted in high molecular weight fraction; further purification identified a 560 kDa multiprotein complex containing those proteins. The isolation of a highly purified homologous complex — the COP9 signalosome — from mammalian cells enabled detailed characterisation of its subunit composition [2,3]. Eight subunits were identified, now called CSN1 to CSN8 according to their electrophoretic mobility, with CSN1 moving the slowest [9]. Some of the COP9 signalosome subunits had come up in other contexts, in particular they include ‘Jun activation domain binding protein 1’ (Jab1) and ‘thyroid receptor interacting protein 15’ (TRIP15), recently renamed CSN5 and CSN2, respectively. Further studies in various species have provided valuable information about subunit–subunit interactions and about a range of proteins that interact with the COP9 signalosome (reviewed in [10]). These data indicated that binding to the COP9 signalosome affects the subcellular localisation and stability of certain regulatory proteins. Binding to CSN5 was shown to stabilise proteins such as the progesterone receptor, the steroid receptor [11] and the IκB protein Bcl-3 [12], but it also promotes nucleocytoplasmic translocation and degradation of the cyclin-dependent kinase (Cdk) inhibitor p27kip [13]. Overproduction of CSN5 was found to stabilise the precursor of the lutropin/choriogonadotropin receptor [14] and the transcription factor c-Jun [15]. Kinase activity of the COP9 signalosome
CSN5 was originally described as a coactivator, Jab1, that increases the specificity of AP-1 transcription factors by binding to c-Jun or JunD [15]. The identification of Jab1 as a COP9 signalosome subunit led to the re-evaluation of its described effects on the c-Jun pathway [2,3,16]. A serine/threonine kinase activity associated with the COP9 signalosome was detected, and its specificity for c-Jun and IκB characterised [2]. COP9 signalosome-dependent phosphorylation could be inhibited efficiently by curcurmin [17], known to have anti-tumorigenic and anti-angiogenic effects [18]. De novo formation of the COP9 signalosome in HeLa cells, induced by overproduction of CSN2, was found to stabilise c-Jun and increase AP-1 activity [16]. These observations suggested the existence of a COP9 signalosome-directed c-Jun signalling pathway [16]. The tumour suppressor protein p53 has now been identified as another target of the COP9 signalosome-associated kinase activity [19]. COP9 signalosome-specific phosphorylation of p53 between amino acids 145 and 160 promotes
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Figure 1 SCF complex
COP9 signalosome
26S proteasome
Ub
Stabilisation P Substrate A
E1
Substrate A
Ub
Nedd8
Nedd8
Cul1 Degradation E2 Substrate B
P
Substrate B Ub Ub
Substrate B
Ub Ub
P F-box
Ub Substrate B Ub Ub Ub Ub Current Biology
A speculative model for how the phosphorylation and deneddylation activities that have been shown to be associated with the COP9 signalosome might act in concert to regulate substrate ubiquitination by the SCF. See text for details.
degradation of the protein by the ubiquitin–proteasome pathway. Inhibition of this phosphorylation, either by curcurmin or a phosphorylated p53(145–164) peptide, stabilised p53 and led to accumulation of the endogenous protein in vivo. It appears that substrates of the COP9 signalosome kinase activity must bind to one of its subunits in order to be phosphorylated. CSN5, for example, recruits c-Jun [15] and p53 [19] to the COP9 signalosome, whereas phosphorylation of the interferon consensus sequencebinding protein (ICSBP) requires binding to CSN2 [20]. Deneddylation activity of the COP9 signalosome
The transcription factor HY5, which induces the expression of genes responsible for photomorphogenic development in plants, is stabilised upon phosphorylation. Recent studies have shown that COP1 promotes the proteasomedependent degradation of unphosphorylated HY5 [21]. Interestingly, COP1 contains a RING finger domain, characteristic of a family of ubiquitin ligases, which indicates that it might be a HY5-specific ubiquitin ligase. As the COP9 signalosome regulates COP1 activity, this was the first hint that the signalosome might control components of the ubiquitination machinery. Co-precipitation studies have now revealed that COP9 signalosome associates with SCF, a multimeric ubiquitin ligase responsible for ubiquitination of p27kip, IκB, a subset of cyclins and other regulatory proteins [6,7]. SCF contains Skp1, Hrt1, substrate-binding F-box proteins and a RINGfinger domain protein of the cullin family as major components. Cullin is modified by the covalent attachment of the
ubiquitin-like protein Nedd8 [22,23]. Cells of the fission yeast Schizosaccharomyces pombe deleted for csn1+ showed an accumulation of Nedd8-modified — neddylated — cullin species, accompanied by a significant increase of SCF activity [6]. Together with the results of reconstitution experiments using purified mammalian COP9 signalosome, the data suggest that the COP9 signalosome complex mediates cleavage of Nedd8–cullin conjugates. Studies in Arabidopsis showed that reduced levels of CSN5 cause insensitivity to the plant hormone auxin [7]. In the CSN5-deficient plants, SCF-mediated degradation of PSIAA6, a regulatory protein of the auxin response pathway, is compromised and an accumulation of neddylated cullin was observed. These findings indicate that tight regulation of neddylation and deneddylation events might be important for the degradation of certain SCF substrates. In fission yeast, however, deletion of csn1+ did not affect the stability of the SCF substrate Rum1 [20]. Moreover, although fission yeast cells deleted for csn1+ and csn2+ display a cell-cycle-related phenotype they are still viable, indicating that COP9 signalosome function is not essential in S. pombe [24]. Interestingly, deletion of ned8+ is lethal in fission yeast [25]. It therefore appears that, although neddylation is crucial for viability in fission yeast, COP9 signalosome function is not. In the budding yeast Saccharomyces cerevisiae, the picture is slightly different as, apart from CSN5, no other orthologues of COP signalosome subunits have been found, and deletion of the gene encoding the Nedd8 orthologue, RUB1, is not lethal [22–24]. It is therefore conceivable that Nedd8 modification
Dispatch
and COP9 signalosome function developed as an additional control mechanism for the degradation of certain SCF substrates. Why not both deneddylation and phosphorylation?
Inhibition of the COP9 signalosome deneddylation activity by the alkylating agent NEM suggested that removal of Nedd8 is accomplished by cysteine protease activity [6]. Sequence comparison studies indicate that CSN5 is the only subunit of the complex with homology to cysteine proteases. Moreover, deletion of the CSN5 orthologue in budding yeast led to accumulation of modified cullin [6]. CSN5 is therefore a strong candidate for being the deneddylase. Two-hybrid studies have shown that the cullin CUL1 binds to CSN2 and CSN6, but no interaction could be detected between CSN5 and CUL1 or any other components of the SCF [6,7]. That implies that CSN5 would have to cleave CUL1–Nedd8 without binding it directly. It would be interesting to find out whether recombinant CSN5 protein can cleave CUL1–Nedd8 conjugates, and whether mutation of the conserved cysteine residue in the putative protease active site of CSN5 leads to the accumulation of Nedd8-modified cullins. The question is whether the COP9 signalosome deneddylation activity can be assigned to one of its eight subunits — perhaps CSN5 as suggested above — or to a protein that co-purifies with the complex. There is a similar problem with the COP9 signalosome kinase activity. None of the COP9 signalosome subunits shows significant sequence similarity to any known kinase, or has been shown capable of phosphorylating model substrates on its own. One explanation for this would be that the kinase copurifies with the COP9 signalosome complex. Another possibility, however, is that the kinase activity might need the cooperation of several COP9 signalosome subunits. The same might be true for the deneddylation activity of the COP9 signalosome. It is conceivable that CSN2 binds to CUL1–Nedd8, which then becomes accessible to CSN5. In budding yeast, other proteins might mediate the interaction with CSN5. Therefore, both phosphorylation and deneddylation might be catalysed by the COP9 signalosome, which would allow the complex to interfere with the pathway at two points — the substrate and the E3 ubiquitin ligase. A possible model of COP9 signalosome action is shown in Figure 1. In this model, COP9 signalosome-specific phosphorylation of a substrate protein triggers either its release from the complex and stabilisation, or the association of the COP9 signalosome with neddylated SCF. The COP9 signalosome—SCF interaction results in transfer of the phosphorylated substrate to the glycine rich repeat region of the F-box protein, and its ubiquitination. When, after a number of conjugation steps, the ubiquitin chain has reached a certain length, the cullin is deneddylated by the
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COP9 signalosome, causing inactivation of SCF and transfer of the multiubiquitinylated substrate to the 26S proteasome. Taking all the data together, it appears that the COP9 signalosome acts as an interface that links various signalling pathways with the ubiquitination machinery. Future work will provide further insights into how the kinase and deneddylase activities of the COP9 signalosome complex regulate ubiquitin-dependent proteolysis. References 1. Hershko A, Ciechanover A: The ubiquitin system. Annu Rev Biochem 1998, 67:425-479. 2. 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, 12:469-478. 3. Wei N, Tsuge T, Serino G, Dohmae N, Takio K, Matsui M, Deng XW: The COP9 complex is conserved between plants and mammals and is related to the 26S proteasome regulatory complex. Curr Biol 1998, 8:919-922. 4. Glickman MH, Rubin DM, Coux O, Wefes I, Pfeifer G, Cjeka Z, Baumeister W, Fried VA, Finley D: A subcomplex of the proteasome regulatory particle required for ubiquitin-conjugate degradation and related to the COP9-signalosome and eIF3. Cell 1998, 94:615-623. 5. Kapelari B, Bech-Otschir D, Hegerl R, Schade R, Dumdey R, Dubiel W: Electron microscopy and subunit-subunit interaction studies reveal a first architecture of COP9 signalosome. J Mol Biol 2000, 300:1169-1178. 6. Lyapina S, Cope G, Shevchenko A, Serino G, Tsuge T, Zhou C, Wolf DA, Wei N, Shevchenko A, Deshaies RJ: Promotion of NEDD-CUL1 conjugate cleavage by COP9 signalosome. Science 2001, 292:1382-1385. 7. Schwechheimer C, Serino G, Callis J, Crosby WL, Lyapina S, Deshaies RJ, Gray WM, Estelle M, Deng XW: Interactions of the COP9 signalosome with the E3 ubiquitin ligase SCFTIRI in mediating auxin response. Science 2001, 292:1379-1382. 8. Wei N, Chamovitz DA, Deng XW: Arabidopsis COP9 is a component of a novel signaling complex mediating light control of development. Cell 1994, 78:117-124. 9. Deng X-W, Dubiel W, Wei N, Hofmann K, Mundt K, Colicelli J, Kato J-Y, Naumann M, Segal D, Seeger M, et al.: Unified nomenclature for the COP9 signalosome and its subunits: an essential regulator of development. Trends Genet 2000, 16:202-203. 10. Chamovitz DA, Segal D: JAB1/CSN5 and the COP9 signalosome. A complex situation. EMBO Rep 2001, 2:96-101. 11. Chauchereau A, Georgiakaki M, Perrin-Wolff M, Milgrom E, Loosfelt H: JAB1 interacts with both the progesterone receptor and SRC-1. J Biol Chem 2000, 275:8540-8548. 12. Dechend R, Hirano F, Lehmann K, Heissmeyer V, Ansieau S, Wulczyn FG, Scheidereit C, Leutz A: The Bcl-3 oncoprotein acts as a bridging factor between NF-kappaB/Rel and nuclear co-regulators. Oncogene 1999, 18:3316-3323. 13. Tomoda K, Kubota Y, Kato J: Degradation of the cyclin-dependentkinase inhibitor p27Kip1 is instigated by Jab1. Nature 1999, 398:160-165. 14. Li S, Liu X, Ascoli M: p38JAB1 binds to the intracellular precursor of the lutropin/choriogonadotropin receptor and promotes its degradation. J Biol Chem 2000, 275:13386-13393. 15. 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, 383:453-457. 16. 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, 274:35297-35300. 17. Henke W, Ferrell K, Bech-Otschir D, Seeger M, Schade R, Jungblut P, Naumann M, Dubiel W: Comparison of human COP9 signalosome and 26S proteasome lid. Mol Biol Rep 1999, 26:29-34.
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18. Huang MT, Ma W, Lu YP, Chang RL, Fisher C, Manchand PS, Newmark HL, Conney A: Effects of curcumin, demethoxycurcumin, bisdemethoxycurcumin and tetrahydrocurcumin of 12-Otetradecanoylphorbol-13-acetate-induced tumor promotion. Carcinogenesis 1995, 16:2493-2497. 19. 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, 20:1630-1639. 20. Cohen H, Azriel A, Cohen T, Meraro D, Hashmueli S, Bech-Otschir D, Kraft R, Dubiel W, Levi BZ: Interaction between interferon consensus sequence-binding protein and COP9/signalosome subunit CSN2 (Trip15). A possible link between interferon regulatory factor signaling and the COP9/signalosome. J Biol Chem 2000, 275:39081-39089. 21. Hardtke CS, Gohda K, Osterlund MT, Oyama T, Okada K, Deng XW: HY5 stability and activity in Arabidopsis is regulated by phosphorylation in its COP1 binding domain. EMBO J 2000, 19:4997-5006. 22. Lammer D, Mathias N, Laplaza JM, Jiang W, Liu Y, Callis J, Goebl M, Estelle M: Modification of yeast Cdc53p by the ubiquitin-related protein rub1p affects function of the SCFCdc4 complex. Genes Dev 1998, 12:914-926. 23. Liakopoulos D, Doenges G, Matuschewski K, Jentsch S: A novel protein modification pathway related to the ubiquitin system. EMBO J 1998, 17:2208-2214. 24. Mundt KE, Porte J, Murray JM, Brikos C, Christensen PU, Caspari T, Hagan IM, Millar JB, Simanis V, Hofmann K, Carr AM: The COP9/signalosome complex is conserved in fission yeast and has a role in S phase. Curr Biol 1999, 9:1427-1430. 25. Osaka F, Saeki M, Katayama S, Aida N, Toh-EA, Kominami K, Toda T, Suzuki T, Chiba T, Tanaka K, Kato S: Covalent modifier NEDD8 is essential for SCN ubiquitin ligase function in fission yeast. EMBO J 2000, 19:3475-3484.