Analysis of the role of COP9 Signalosome (CSN) subunits in K562; the first link between CSN and autophagy
© Pearce et al; licensee BioMed Central Ltd. 2009
Received: 08 July 2008
Accepted: 28 April 2009
Published: 28 April 2009
The COP9/signalosome (CSN) is a highly conserved eight subunit complex that, by deneddylating cullins in cullin-based E3 ubiquitin ligases, regulates protein degradation. Although studied in model human cell lines such as HeLa, very little is known about the role of the CSN in haemopoietic cells.
Greater than 95% knockdown of the non-catalytic subunit CSN2 and the deneddylating subunit CSN5 of the CSN was achieved in the human myeloid progenitor cell line K562. CSN2 knockdown led to a reduction of both CSN5 protein and mRNA whilst CSN5 knockdown had little effect on CSN2. Both knockdowns inhibited CSN deneddylase function as demonstrated by accumulation of neddylated Cul1. Furthermore, both knockdowns resulted in the sequential loss of Skp2, Cdc4 and β-TrCP F-box proteins. These proteins were rescued by the proteasome inhibitor MG132, indicating the autocatalytic degradation of F-box proteins upon loss of CSN2 or CSN5. Interestingly, altered F-box protein gene expression was also observed in CSN2 and CSN5 knockdowns, suggesting a potential role of the CSN in regulating F-box protein transcription.
Loss of either CSN subunit dramatically reduced cell growth but resulted in distinct patterns of cell death. CSN5 knockdown caused mitotic defects, G2/M arrest and apoptotic cell death. CSN2 knockdown resulted in non-apoptotic cell death associated with accumulation of both the autophagy marker LC3-II and autophagic vacuoles. Treatment of vector control K562 cells with the autophagy inhibitors 3-methyladenine and bafilomycin A1 recapitulated the growth kinetics, vacuolar morphology and LC3-II accumulation of CSN2 knockdown cells indicating that the cellular phenotype of CSN2 cells arises from autophagy inhibition. Finally, loss of CSN2 was associated with the formation of a CSN5 containing subcomplex.
We conclude that CSN2 is required for CSN integrity and the stability of individual CSN subunits, and postulate that CSN2 loss results in a phenotype distinct from that of cells lacking CSN5 possibly as a consequence of altered CSN5 activity within a resultant CSN subcomplex. Our data present the first evidence for the sequential loss of F-box proteins upon CSN manipulation and are the first to identify a potential link between CSN function and autophagy.
The regulated expression and degradation of proteins are critical to all aspects of cell development and proliferation. The two main routes for eukaryotic intracellular protein clearance are the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway. A key component involved in regulating degradation of proteins by the UPS is the COP9 signalosome (CSN). The CSN is an eight-subunit (CSN1-8) protein complex, highly conserved amongst eukaryotes [1–5] originally identified in Arabidopsis as a negative regulator of photomorphogenesis . Through its function in the regulation of the UPS, the CSN has been implicated in the regulation of biological processes as diverse as DNA replication and repair, cell-cycle progression and cell development [7–9].
Degradation of cellular proteins by the 26S proteasome [10–13] is preceded by ubiquitination of target proteins , a process mediated by three enzyme complexes; a ubiquitin activating enzyme (E1), a ubiquitin conjugating enzyme (E2) and a ubiquitin ligase (E3) . The E3 ligase interacts with the protein substrate and thus confers the specificity of the UPS . The largest known class of E3 ubiquitin ligases comprises the Cullin-RING ligases (CRLs) of which the best studied is the SCF (Skp1, Cul1, F-box protein) complex . The cullin subunit (Cul1) of the SCF forms a scaffold to recruit and bring into close proximity the E2 and its substrate, thereby facilitating ubiquitin transfer from the E2 to target proteins (SCF structure reviewed in ). The RING protein (Hrt1/Roc1/Rbx1) is the fourth subunit of the SCF and is responsible for E2 recruitment, whilst the variable F-box protein subunit, recruited to the SCF complex via the adaptor protein Skp1, binds substrates selectively [17–19]. In yeast, over 19 F-box proteins are known, over 400 in A. thaliana, and ~70 in humans . Since each cullin (Cul1-5) forms complexes with a variable substrate recognition subunit (SRS) (F-box proteins for Cul1 as above, VHL box proteins for Cul2, BTB proteins for Cul3, WD40 proteins for Cul4 and SOCS box proteins for Cul5, reviewed in ) specificity in CRL target protein recruitment is achieved by the large number of variable SRS containing CRLs. It is thought that, altogether, the human genome may have the capacity to code for as many as 350 different CRLs.
Given the potential number and diversity of target proteins requiring CRL mediated ubiquitination for degradation, dynamic regulation of the CRL complex repertoire in a cell at any given time is essential. All cullins studied (Cul1-5) have been shown to be modified by neddylation , which facilitates their ubiquitin ligase activity  possibly via increased E2 affinity [23, 24]. The deneddylation of cullins is mediated by the CSN complex . Although initial studies indicated a negative role for deneddylation, further studies have implicated deneddylation in the positive regulation of CRL activity [3, 4, 26]. It has since been proposed by several groups that optimal CRL activity requires the cyclic neddylation and deneddylation of the cullin subunit [4, 7, 27]. Although the exact mechanisms are not fully understood, it is thought that F-box proteins themselves are targeted for degradation in part by autoubiquitination within the SCF complex . The deneddylation of cullins by the CSN is believed to regulate the autoubiquitination of SRSs [28, 29], thereby modulating CRL composition and activity. Furthermore, the CSN has been shown to be associated with a deubiquitinase activity which may further stabilize autoubiquitinated SRSs [27, 29]. The CSN complex is therefore an integral regulator of CRL activity and subsequent protein degradation.
In this present study we have investigated the effects of knocking down CSN2 and CSN5 in the model K562 cell line, a model of human erythrocyte and megakaryocyte progenitors [30, 31]. Whilst knockdown of either CSN2 or CSN5 resulted in common changes including the sequential loss of F-box proteins Skp2, Cdc4 and β-TrCP other important differences occurred. For example CSN5 knockdown resulted in apoptotic cell death associated with aberrant mitosis, whereas CSN2 knockdown cells underwent non-apoptotic cell death that was associated with both inhibition of autophagy and the formation of a novel CSN5 containing CSN subcomplex.
CSN2 and CSN5 knockdown and resulting aberrant SCF activity
The instability of particular CSN subunits in the absence of another subunit has been reported previously. For example, CSN8 knockdown has been shown to result in the loss of CSN3, CSN5 and CSN7 protein . We therefore determined CSN5 and CSN2 protein levels in CSN2 and CSN5 knockdown cells, respectively, by western blotting. A significant reduction in CSN5 protein was observed in cells lacking CSN2, whilst depletion of CSN5 had no effect on CSN2 protein or mRNA levels relative to vector control cells (Fig. 1C and data not shown). Interestingly, CSN2 knockdown not only resulted in loss of CSN5 protein, but also in the significant reduction of CSN5 mRNA as determined by QRT-PCR (Fig. 1D, P = 0.011).
Consistent with previous studies of CSN deregulation [28, 33], both CSN2 and CSN5 knockdowns were associated with accumulation of neddylated Cul1 (Fig. 1E) indicating functional dysregulation of the CSN following knockdown of either subunit. Studies demonstrating Cul1 hyperneddylation in CSN deficient cells have also demonstrated degradation of the F-box protein Skp2 [28, 33]. In agreement with these observations, complete loss of Skp2 protein was observed in both the CSN2 and CSN5 knockdown cells (Fig. 1E). Skp2 binds to and mediates the ubiquitination of multiple proteins, including the cyclin dependent kinase inhibitor p27kip1 . Consistent with loss of Skp2, knockdown of CSN2 and CSN5 resulted in the accumulation of p27 (Fig. 1E).
Loss of CSN2 and CSN5 results in the sequential loss of F-box proteins
In order to determine the mechanism of F-box protein loss in both CSN2 and CSN5 knockdown cells, we investigated the rescue of F-box proteins by the proteasome inhibitor MG132 (Fig. 2C) and measured the levels of F-box mRNA over time (Fig. 2D &2E). Treatment of both CSN2 and CSN5 knockdown cells with MG132 resulted in Skp2, Cdc4 and β-TrCP protein rescue (Fig. 2C), indicating involvement of the proteasome in the loss of these F-box proteins. However the strength of the observed rescue varied. Importantly, CSN subunit depletion also affected levels of F-box mRNA. CSN2 knockdown resulted in a significant reduction of Skp2 mRNA by day 2 post transfection. Thus it is likely that transcriptional changes also contribute to loss of Skp2 protein in these cells. In contrast Cdc4 and β-TrCP mRNAs were significantly increased following CSN2 knockdown despite clear loss of protein (Fig. 2D). CSN5 depletion resulted in a modest reduction of all three F-box protein mRNAs but not enough to account for the observed loss of protein (Fig. 2E). Collectively these data indicate that, with the possible exception of Skp2 in CSN2 knockdown cells, proteasomal degradation rather than transcriptional repression was the main driver of F-box protein loss in CSN2 and CSN5 knockdown K562 cells.
Both CSN2 and CSN5 knockdowns result in reduced cell growth and cell death
Knockdown of CSN5 but not CSN2 is associated with cell cycle arrest and defects in mitotic spindle formation
Knockdown of CSN5 but not CSN2 results in apoptotic cell death
CSN2 but not CSN5 knockdown is associated with autophagy
Loss of CSN2 results in the formation of an alternative CSN5 containing complex
Immunoblotting of extracts from shVC cells with either CSN2 or CSN5 antibody identified 2 major complexes, one of approximately 500 KDa, correlating in size with the CSN holocomplex, and the second of 750 KDa (Fig. 7A &7B). A multitude of proteins have been shown to associate with CSN subunits  and larger complexes have previously been observed. Monomeric CSN2 and CSN5 were also observed in the shVC cell extracts (Fig. 7C and data not shown). Following knockdown of CSN2, CSN2 protein was no longer detectable (Fig. 7A). CSN5 protein, as also shown in Fig. 1C, was greatly reduced in cells lacking CSN2, with significant loss of the CSN complex relative to vector controls (Fig. 7B). Interestingly, a longer exposure of the autoradiograph identified a CSN5 containing subcomplex of ~242 KDa in cells lacking CSN2 (Fig. 7C).
The achievement of almost complete CSN2 and CSN5 knockdown in this study has provided a powerful tool to study the function of these CSN subunits more closely. At the molecular level, CSN2 and CSN5 knockdowns resulted in aberrant SCF activity, with the accumulation of neddylated Cul-1, loss of the F-box protein Skp2 and an increase in the Skp2 target protein, p27. This complements another report in which CSN4 and CSN5 knockdown also resulted in increased neddylation of Cul-1 with a concomitant loss of Skp2 and increase in p27 protein in human epithelial cell lines rather than haemopoietic cells . Thus it appears that the CSN complex has highly conserved activities across cells from different cell lineages and that disruption of the complex by loss of any subunit causes derangement of these activities.
It was also observed that CSN2 knockdown not only results in loss of CSN5 protein but also results in a significant reduction of CSN5 mRNA. Moreover, both CSN2 and CSN5 knockdown resulted in temporal alterations of F-box protein mRNA. Together with other recent reports [32, 42], this data suggests that CSN subunits or the CSN complex as a whole may have a direct role in transcriptional regulation of CSN subunits and F-box proteins. However, it is also possible that the altered mRNA levels observed are due to secondary effects of aberrant CRL mediated protein degradation such as accumulation of proteins involved in transcriptional regulation.
Here we show for the first time sequential loss of F-box proteins following knockdown of CSN subunits. Moreover, protein levels were at least partially restored in both knockdowns upon treatment with the proteasome inhibitor MG132. These observations are in accordance with the finding that F-box proteins are autocatalytically degraded in the presence of hyperneddylated Cul-1 . However, the three f-box proteins studied were each lost at a different rate, with the loss of Skp2 protein being the most rapid. The sequential loss of F-box proteins is of great interest as it may explain published results which document the loss of particular F-box proteins at a specific time point post CSN manipulation, but no reduction in other F-box proteins [28, 32].
The CSN5 knockdown cultures contained aberrantly large cells and were associated with G2/M arrest and apoptosis. This data complements previous studies demonstrating that CSN5 loss inhibits proliferation and induces apoptosis [43–45]. Closer analysis of CSN5 knockdown cells identified disorganized condensed chromatids and abnormal mitotic spindles in the large cells. A recent report described stabilization of the microtubule end-binding protein 1 (EB1) by the CSN complex in human cells . EB1, which is a master regulator of microtubule dynamics, was shown to bind the CSN via CSN5, and was also shown to be reduced in cells lacking CSN1 or CSN3 . EB1 has recently been shown to directly interact with and regulate the activity of Aurora B, one essential component of the chromosomal passenger complex that is required for correct chromosomal alignment and spindle assembly checkpoint [47, 48]. Furthermore, the dynamic behaviour of Aurora B on mitotic chromosomes has been shown to be regulated by a Cul3 E3 ligase . Given that we observed hyperneddylated Cul3 in the CSN5 knockdown cells (data not shown), our data suggests that CSN5/the CSN complex is integral to the regulation of multiple components of the mitotic machinery.
K562 cells in which CSN2 had been knocked down did not display apoptosis markers as in CSN5 knockdown cells, but were instead associated with features of autophagy. Interestingly, autophagy inhibitors recapitulated the cell growth kinetics, vacuolar morphology and LC3-II accumulation of cells lacking CSN2, whilst treatment of CSN2 knockdown cells with one of these inhibitors (3-MA) had a comparatively mild effect on cell growth. These findings suggest that CSN2 knockdown K562 cells undergo autophagy inhibition resulting in non-apoptotic cell death. This is the first data to show an association between the CSN complex and autophagy.
The distinct phenotypes observed between CSN2 and CSN5 knockdowns may arise as a result of aberrant CSN5 activity within the observed CSN subcomplex in cells lacking CSN2. However, it is important to note that both the CSN subunits studied here have CSN independent functions [49–54], and that CSN5 has been shown to function within a CSN subcomplex in K562 . Therefore, we cannot rule out the possible contribution of the independent functions of CSN2 and CSN5 to the phenotypic differences observed between the knockdowns. It is also noteworthy that the subcomplex observed in this study may be a result of CSN complex breakdown in the absence of CSN2 , rather than a functional complex contributing to the observed phenotypic differences between knockdowns. Moreover, as we see no effect of CSN5 loss on the level of CSN2 protein, one possibility not investigated here is the formation of a CSN2 containing subcomplex in the absence of CSN5. This is an intriguing possibility, particularly given the recent findings of Su et al who demonstrated an increase in the proportion of CSN2 residing in mini-complexes upon CSN8 knockdown . It will be of great interest to determine the precise mechanism accounting for the divergent phenotypes encountered here, and is something which is currently under investigation.
In conclusion, we have shown that loss of either CSN2 or CSN5 in human K562 cells results in significant loss of viability but by very different mechanisms, potentially attributable to the formation of a CSN5 containing subcomplex in the absence of CSN2. Furthermore, we have provided data to suggest a possible function of the CSN complex in the transcriptional regulation of both its own components and CRL subunits. Finally, we have demonstrated here for the first time the sequential loss of F-box proteins in the absence of the CSN complex and have provided the first evidence of a link between the CSN complex and autophagy.
Cell culture and treatments
K562 cells were cultured in RPMI 1640 supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin and 10% v/v foetal bovine serum (Invitrogen, Gibco) and maintained at 37°C with 5% CO2. For proteasome inhibition, cells were treated with 10 μM MG132 for the final 18 hours of culturing. For autophagy inhibition, cells were treated in culture with either 10 mM 3-methyladenine from day 3-day 7 post transfection or 1 μM bafilomycin A1 for 48 hours day 5-day 7 post transfection.
The shRNA vector used was a modified pcDNA3.1 vector (pcDNA3.1-H1) developed by Heiko Lickert  (kind gift from Heiner Schrewe) in which the CMV promoter has been replaced by the human RNAse P RNA H1 promoter . CSN2 and CSN5 silencing sequences were selected using a siRNA design tool available on http://www1.qiagen.com/products/genesilencing/customsiRNA/siRNA designer.aspx and cloned into the Asp718 and XbaI restriction enzyme sites of pcDNA3.1-H1. The target sequences are as follows:
CSN2 knockdown 5'AAGCGGCATTAAGCAGTTTCC3'
CSN5 knockdown – 5'AAGGGCTACAAACCTCCTGAT3'
shRNA scramble control – 5'AAGCGGGATTCAGTAGTTACG3'
Transfections and cell sorting
Transfection efficiencies in K562 cells vary between 20–50%. Therefore, to allow enrichment of transfected cells, 5 × 106 K562 cells were electroporated in Nucleofector kit V solution (Amaxa) using a Nucleofector I (Amaxa) and file T16, with 5 μg pMACS Kk.II and 10 μg of the relevant knockdown pcDNA3.1-H1 plasmid according to manufacturer guidelines. The pMACS Kk.II produces a truncated murine MHC class I cell surface protein, H-2Kk, which lacks the cytoplasmic domain and is transiently expressed on the cell surface of transfected cells between 6 and 48 hours post-transfection. Transfected cells were sorted 24 hours post transfection using anti-H-2Kk antibody conjugated to magnetic beads, MACS MS columns and a MACS magnet (Miltenyi Biotec) according to manufacturer instructions. Post sorting, cells were set at 3 × 105/ml daily and cells harvested for protein and mRNA analysis as indicated in results.
Thymidine incorporation assay
2 × 104 cells were pulsed with 2 μCi/ml 3H-thymidine (Amersham) for the final 18 hours of culture leading up to each time point. Samples were transferred to a filter mat (Wallac) using a Skatron cell harvester (Skatron Instruments) and read using a beta-plate scintillation counter (Skatron Instruments).
Immunofluorescence and Jenner-Giemsa staining
Cytospins were made with 5 × 104 cells in 80 μl, using a Shandon cytospin 3 (Shandon). For immunofluorescence staining, cytospins were fixed in 4% paraformaldehyde and stained using anti-β-tubulin antibody (Sigma, 1/500 dilution) followed by FITC labelled secondary antibody (Jackson Laboratories, 1/500 dilution). DNA was counterstained using Hoescht 33342 (Sigma, 1/1000 dilution). All reagents were diluted in PBS and slides mounted using Mowiol (6 g glycerol, 2.4 g Moviol-4-88 (Sigma), 12 ml 0.2 M Tris HCl pH8.5, anti-fade crystal (Sigma), 6 ml distilled water). Slides were viewed using an Axioskop2 microscope (Zeiss) and images captured with a Q-imaging 12-bit QICAM (Media Cybernetics) and Openlab software (Improvision).
For Jenner-Giemsa staining, cytospins were air-dried, methanol fixed and stained; First with Jenner staining solution (VWR, UK) diluted 1/3 in 1 mM sodium phosphate buffer pH5.6 (5 mins) and second with Giemsa stain (VWR, UK) diluted 1/20 in 1 mM sodium phosphate buffer pH5.6 (10 mins). Slides were dried and then mounted onto coverslips using DePex (VWR, UK). Slides were viewed with an Olympus BX40 microscope (Olympus) and images captured using an Olympus Chameleon digital SLR (Olympus).
Staining of autophagosomes
For visualisation of autophagic vacuoles, 5 × 104 cells were incubated with 0.05 mM monodansylcadaverine (MDC, Sigma) in 0.5 ml PBS for 10 minutes at 37°C. Cells were washed four times with PBS, cytospins made as above and cells viewed immediately using a Leica DMIRE2 system.
Fluorescence flow cytometry
For Annexin V labelling, 1 × 105 cells were stained using Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences) according to manufacturer instructions, and staining analysed within 1 hour by flow cytometry. For cell cycle analysis 1 × 105 cells were resuspended in cell cycle buffer (10 μg/ml propidium iodide, 0.1 mM sodium chloride, 1% Triton X100) and samples analysed within 24 hours by flow cytometry. All staining was analysed using a FACS Calibur (Becton Dickinson) and the data evaluated using Cell Quest Pro software (Becton Dickinson).
Western blot analysis
Whole cell lysates were prepared using RIPA buffer (1% v/v NP40, 0.5% w/v sodium deoxycholate, 0.1% w/v 10% SDS, in distilled water) and protein quantified using the Dc protein assay according to manufacturer instructions (Bio-Rad). Forty micrograms of protein were boiled for 10 minutes in 1× SDS gel loading buffer (15.6 mM Tris HCl pH6.8, 6.25% v/v glycerol, 0.5% SDS, 1.25% v/v 2-mercaptoethanol, Bromophenol Blue, in distilled water). Proteins were separated by SDS-PAGE and transferred to PVDF membrane (Millipore). For western blot analysis, the following antibodies were used at 1:1000 dilution: CSN2 (Bethyl), CSN5 (Bethyl), Cul-1 (Zymed), Skp2 (Zymed), p27 (Santa Cruz), caspase-9 (Cell Signalling), LC-3 (Novus Biologicals) and β-actin (Sigma). Proteins recognized by these antibodies were detected using ant-mouse (Sigma, 1/1000 dilution) or anti-rabbit (Pierce, 1/1000 dilution) HRP conjugated secondary antibody followed by enhanced chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate, Pierce) and autoradiography (Kodak X-Omat LS film, Sigma). Quantitative analysis of western blots was carried out using ImageJ software http://rsb.info.nih.gov/ij/download.html and protein levels normalized by comparison to β-actin signals on the same membrane.
2-Dimensional gel analysis
Native protein extracts were obtained from 2.5 × 105 cells by resuspending cells in 50 μl mild lysis buffer (25% 4× NativePAGE sample buffer (Invitrogen), 1% digitonin, 10% 10× protease inhibitor, in distilled water). Extracts were separated out in the first dimension using a NativePAGE Novex Bis-Tris Gel System (Invitrogen) according to manufacturer instructions. The gel was then cut into individual lanes, proteins denatured by incubation in 1× SDS gel loading buffer and resolved in the second dimension by electrophoresis through 12.5% SDS-polyacrylamide gels. Proteins were transferred to PVDF membrane (Millipore) and immunoblotting performed as above.
Quantitative real-time PCR analysis (QRT-PCR)
RNA was extracted using the Qiagen RNeasy kit according to manufacturer instructions and cDNA generated using 1 μg RNA, random hexamers (Promega) and Superscript II reverse transcriptase (Invitrogen). Quantitative real-time PCR was carried out using either TAQMAN or SYBR-Green based assays. For TAQMAN assays, QRT-PCR was carried out in duplicate 20 μl reactions containing 1× qPCR Mastermix Plus (Eurogentec), 20–40 ng cDNA, 18 pmoles each primer and 2.5 pmoles FAM/TAMRA dual labeled probes. For SYBR-Green assays, QRT-PCR was carried out in duplicate 25 μl reactions containing 1× Sensimix (Quantace), 20–40 ng cDNA, 9 pmoles each primer, 1× SYBR-Green solution (Quantace), 4 mM MgCl2 and 0.5 units UNG (Quantace). QRT-PCR was carried out on an ABI Prism 7000 sequence detector (Applied Biosystems). The following primers (Sigma Genosys) and FAM/TAMRA labeled probes (Eurogentec) were used:
CSN2, 5'-CCTCATCCACTGATTATGGGAGT-3' (forward),
CSN5, 5'-ATATCCGCAGGGAAAG-3' (forward),
5'- TGGCGCCTTTAGGACATACCCAAAGG-3' (probe);
Skp2, 5'-CGCTGCCCACGATCATTT-3' (forward),
Cdc4, 5'-ACGACGCCGAATTACATCTGT-3' (forward),
β-Trcp, 5'-GAGGCATTGCCTGTTTGCA-3' (forward)
18S, 5'-GCCGCTAGAGGTGAAATTCTTG-3' (forward),
Preoptimised primers and probes to 18S ribosomal RNA were used as internal standards in TAQMAN QRT-PCR (Applied Biosystems). Cycle threshold (Ct) values were obtained graphically for test genes and 18S internal standards. ΔCt values were calculated by subtracting 18S Ct from test gene Ct, and average ΔCt values obtained from duplicates. Relative mRNA levels were determined by subtraction of mock transfection ΔCt values from shVC/shCSN2/shCSN5 ΔCt values to give a ΔΔCt value and conversion through 2-ΔΔCt.
We would like to acknowledge Heiner Schrewe (MPI, Berlin), Debbie Cunningham (Univ of Birmingham, UK) and Simon Johnston (Univ of Birmingham, UK) for technical assistance. CP is funded by a grant from the BBSRC (UK) and REH and FLK are funded by a Leukaemia Research (UK) grant.
- Wei N, Deng XW: Characterization and purification of the mammalian COP9 complex, a conserved nuclear regulator initially identified as a repressor of photomorphogenesis in higher plants. Photochemistry and photobiology. 1998, 68 (2): 237-241. 10.1111/j.1751-1097.1998.tb02495.x.View ArticlePubMedGoogle Scholar
- Mundt KE, Porte J, Murray JM, Brikos C, Christensen PU, Caspari T, Hagan IM, Millar JB, Simanis V, Hofmann K: The COP9/signalosome complex is conserved in fission yeast and has a role in S phase. Curr Biol. 1999, 9 (23): 1427-1430. 10.1016/S0960-9822(00)80091-3.View ArticlePubMedGoogle Scholar
- Doronkin S, Djagaeva I, Beckendorf SK: The COP9 signalosome promotes degradation of Cyclin E during early Drosophila oogenesis. Developmental cell. 2003, 4 (5): 699-710. 10.1016/S1534-5807(03)00121-7.View ArticlePubMedGoogle Scholar
- Pintard L, Kurz T, Glaser S, Willis JH, Peter M, Bowerman B: Neddylation and deneddylation of CUL-3 is required to target MEI-1/Katanin for degradation at the meiosis-to-mitosis transition in C. elegans. Curr Biol. 2003, 13 (11): 911-921. 10.1016/S0960-9822(03)00336-1.View ArticlePubMedGoogle Scholar
- Rosel D, Kimmel AR: The COP9 signalosome regulates cell proliferation of Dictyostelium discoideum. European journal of cell biology. 2006, 85 (9–10): 1023-1034. 10.1016/j.ejcb.2006.04.006.View ArticlePubMedGoogle Scholar
- Chamovitz DA, Wei N, Osterlund MT, von Arnim AG, Staub JM, Matsui M, Deng XW: The COP9 complex, a novel multisubunit nuclear regulator involved in light control of a plant developmental switch. Cell. 1996, 86 (1): 115-121. 10.1016/S0092-8674(00)80082-3.View ArticlePubMedGoogle Scholar
- Cope GA, Deshaies RJ: COP9 signalosome: a multifunctional regulator of SCF and other cullin-based ubiquitin ligases. Cell. 2003, 114 (6): 663-671. 10.1016/S0092-8674(03)00722-0.View ArticlePubMedGoogle Scholar
- Nielsen O: COP9 signalosome: a provider of DNA building blocks. Curr Biol. 2003, 13 (14): R565-567. 10.1016/S0960-9822(03)00475-5.View ArticlePubMedGoogle 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. 10.1016/S0092-8674(03)00316-7.View ArticlePubMedGoogle Scholar
- Hough R, Pratt G, Rechsteiner M: Ubiquitin-lysozyme conjugates. Identification and characterization of an ATP-dependent protease from rabbit reticulocyte lysates. The Journal of biological chemistry. 1986, 261 (5): 2400-2408.PubMedGoogle Scholar
- Peters JM: Proteasomes: protein degradation machines of the cell. Trends in biochemical sciences. 1994, 19 (9): 377-382. 10.1016/0968-0004(94)90115-5.View ArticlePubMedGoogle Scholar
- Rubin DM, Finley D: Proteolysis. The proteasome: a protein-degrading organelle?. Curr Biol. 1995, 5 (8): 854-858. 10.1016/S0960-9822(95)00172-2.View ArticlePubMedGoogle Scholar
- Voges D, Zwickl P, Baumeister W: The 26S proteasome: a molecular machine designed for controlled proteolysis. Annual review of biochemistry. 1999, 68: 1015-1068. 10.1146/annurev.biochem.68.1.1015.View ArticlePubMedGoogle Scholar
- Chau V, Tobias JW, Bachmair A, Marriott D, Ecker DJ, Gonda DK, Varshavsky A: A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein. Science. 1989, 243 (4898): 1576-1583. 10.1126/science.2538923.View ArticlePubMedGoogle Scholar
- Hershko A, Heller H, Elias S, Ciechanover A: Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown. The Journal of biological chemistry. 1983, 258 (13): 8206-8214.PubMedGoogle Scholar
- Petroski MD, Deshaies RJ: Function and regulation of cullin-RING ubiquitin ligases. Nature reviews. 2005, 6 (1): 9-20.View ArticlePubMedGoogle Scholar
- Bai C, Sen P, Hofmann K, Ma L, Goebl M, Harper JW, Elledge SJ: SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box. Cell. 1996, 86 (2): 263-274. 10.1016/S0092-8674(00)80098-7.View ArticlePubMedGoogle Scholar
- Skowyra D, Craig KL, Tyers M, Elledge SJ, Harper JW: F-box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex. Cell. 1997, 91 (2): 209-219. 10.1016/S0092-8674(00)80403-1.View ArticlePubMedGoogle Scholar
- Ho MS, Tsai PI, Chien CT: F-box proteins: the key to protein degradation. Journal of biomedical science. 2006, 13 (2): 181-191. 10.1007/s11373-005-9058-2.View ArticlePubMedGoogle Scholar
- Bosu DR, Kipreos ET: Cullin-RING ubiquitin ligases: global regulation and activation cycles. Cell division. 2008, 3: 7-10.1186/1747-1028-3-7.PubMed CentralView ArticlePubMedGoogle Scholar
- Hori T, Osaka F, Chiba T, Miyamoto C, Okabayashi K, Shimbara N, Kato S, Tanaka K: Covalent modification of all members of human cullin family proteins by NEDD8. Oncogene. 1999, 18 (48): 6829-6834. 10.1038/sj.onc.1203093.View ArticlePubMedGoogle Scholar
- Ohh M, Kim WY, Moslehi JJ, Chen Y, Chau V, Read MA, Kaelin WG: An intact NEDD8 pathway is required for Cullin-dependent ubiquitylation in mammalian cells. EMBO reports. 2002, 3 (2): 177-182. 10.1093/embo-reports/kvf028.PubMed CentralView ArticlePubMedGoogle Scholar
- Kawakami T, Chiba T, Suzuki T, Iwai K, Yamanaka K, Minato N, Suzuki H, Shimbara N, Hidaka Y, Osaka F: NEDD8 recruits E2-ubiquitin to SCF E3 ligase. The EMBO journal. 2001, 20 (15): 4003-4012. 10.1093/emboj/20.15.4003.PubMed CentralView ArticlePubMedGoogle Scholar
- Sakata E, Yamaguchi Y, Miyauchi Y, Iwai K, Chiba T, Saeki Y, Matsuda N, Tanaka K, Kato K: Direct interactions between NEDD8 and ubiquitin E2 conjugating enzymes upregulate cullin-based E3 ligase activity. Nature structural & molecular biology. 2007, 14 (2): 167-168. 10.1038/nsmb1191.View ArticleGoogle Scholar
- 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 (5520): 1382-1385. 10.1126/science.1059780.View ArticlePubMedGoogle Scholar
- Cope GA, Suh GS, Aravind L, Schwarz SE, Zipursky SL, Koonin EV, Deshaies RJ: Role of predicted metalloprotease motif of Jab1/Csn5 in cleavage of Nedd8 from Cul1. Science. 2002, 298 (5593): 608-611. 10.1126/science.1075901.View ArticlePubMedGoogle Scholar
- Wu JT, Chan YR, Chien CT: Protection of cullin-RING E3 ligases by CSN-UBP12. Trends in cell biology. 2006, 16 (7): 362-369. 10.1016/j.tcb.2006.05.001.View 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 biochemistry. 2006, 7: 1-10.1186/1471-2091-7-1.PubMed CentralView ArticlePubMedGoogle Scholar
- Wee S, Geyer RK, Toda T, Wolf DA: CSN facilitates Cullin-RING ubiquitin ligase function by counteracting autocatalytic adapter instability. Nature cell biology. 2005, 7 (4): 387-391. 10.1038/ncb1241.View ArticlePubMedGoogle Scholar
- Peng H, Du ZW, Zhang JW: Identification and characterization of a novel zinc finger protein (HZF1) gene and its function in erythroid and megakaryocytic differentiation of K562 cells. Leukemia. 2006, 20 (6): 1109-1116. 10.1038/sj.leu.2404212.View ArticlePubMedGoogle Scholar
- Jacquel A, Herrant M, Defamie V, Belhacene N, Colosetti P, Marchetti S, Legros L, Deckert M, Mari B, Cassuto JP: A survey of the signaling pathways involved in megakaryocytic differentiation of the human K562 leukemia cell line by molecular and c-DNA array analysis. Oncogene. 2006, 25 (5): 781-794. 10.1038/sj.onc.1209119.View ArticlePubMedGoogle Scholar
- Su H, Huang W, Wang X: The COP9 signalosome negatively regulates proteasome proteolytic function and is essential to transcription. The international journal of biochemistry & cell biology. 2009, 41 (3): 615-624. 10.1016/j.biocel.2008.07.008.View ArticleGoogle Scholar
- Denti S, Fernandez-Sanchez ME, Rogge L, Bianchi E: The COP9 signalosome regulates Skp2 levels and proliferation of human cells. The Journal of biological chemistry. 2006, 281 (43): 32188-32196. 10.1074/jbc.M604746200.View ArticlePubMedGoogle Scholar
- Tsvetkov LM, Yeh KH, Lee SJ, Sun H, Zhang H: p27(Kip1) ubiquitination and degradation is regulated by the SCF(Skp2) complex through phosphorylated Thr187 in p27. Curr Biol. 1999, 9 (12): 661-664. 10.1016/S0960-9822(99)80290-5.View ArticlePubMedGoogle Scholar
- Biederbick A, Kern HF, Elsasser HP: Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles. European journal of cell biology. 1995, 66 (1): 3-14.PubMedGoogle Scholar
- Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T: LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. The EMBO journal. 2000, 19 (21): 5720-5728. 10.1093/emboj/19.21.5720.PubMed CentralView ArticlePubMedGoogle Scholar
- Mizushima N, Yoshimori T: How to interpret LC3 immunoblotting. Autophagy. 2007, 3 (6): 542-545.View ArticlePubMedGoogle Scholar
- Schwechheimer C: The COP9 signalosome (CSN): an evolutionary conserved proteolysis regulator in eukaryotic development. Biochimica et biophysica acta. 2004, 1695 (1–3): 45-54.View ArticlePubMedGoogle Scholar
- Oren-Giladi P, Krieger O, Edgar BA, Chamovitz DA, Segal D: Cop9 signalosome subunit 8 (CSN8) is essential for Drosophila development. Genes Cells. 2008, 13 (3): 221-231. 10.1111/j.1365-2443.2008.01164.x.View ArticlePubMedGoogle Scholar
- Peth A, Berndt C, Henke W, Dubiel W: Downregulation of COP9 signalosome subunits differentially affects the CSN complex and target protein stability. BMC biochemistry. 2007, 8: 27-10.1186/1471-2091-8-27.PubMed CentralView ArticlePubMedGoogle Scholar
- Bech-Otschir D, Seeger M, Dubiel W: The COP9 signalosome: at the interface between signal transduction and ubiquitin-dependent proteolysis. Journal of cell science. 2002, 115 (Pt 3): 467-473.PubMedGoogle Scholar
- Ullah Z, Buckley MS, Arnosti DN, Henry RW: Retinoblastoma protein regulation by the COP9 signalosome. Molecular biology of the cell. 2007, 18 (4): 1179-1186. 10.1091/mbc.E06-09-0790.PubMed CentralView ArticlePubMedGoogle Scholar
- Fukumoto A, Tomoda K, Yoneda-Kato N, Nakajima Y, Kato JY: Depletion of Jab1 inhibits proliferation of pancreatic cancer cell lines. FEBS letters. 2006, 580 (25): 5836-5844. 10.1016/j.febslet.2006.09.042.View ArticlePubMedGoogle Scholar
- Panattoni M, Sanvito F, Basso V, Doglioni C, Casorati G, Montini E, Bender JR, Mondino A, Pardi R: Targeted inactivation of the COP9 signalosome impairs multiple stages of T cell development. The Journal of experimental medicine. 2008, 205 (2): 465-477. 10.1084/jem.20070725.PubMed CentralView ArticlePubMedGoogle Scholar
- Dohmann EM, Levesque MP, De Veylder L, Reichardt I, Jurgens G, Schmid M, Schwechheimer C: The Arabidopsis COP9 signalosome is essential for G2 phase progression and genomic stability. Development (Cambridge, England). 2008, 135 (11): 2013-2022.View ArticleGoogle 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. Journal of molecular biology. 2007, 368 (2): 550-563. 10.1016/j.jmb.2007.02.052.View ArticlePubMedGoogle Scholar
- Sun L, Gao J, Dong X, Liu M, Li D, Shi X, Dong JT, Lu X, Liu C, Zhou J: EB1 promotes Aurora-B kinase activity through blocking its inactivation by protein phosphatase 2A. Proceedings of the National Academy of Sciences of the United States of America. 2008, 105 (20): 7153-7158. 10.1073/pnas.0710018105.PubMed CentralView ArticlePubMedGoogle Scholar
- Sumara I, Quadroni M, Frei C, Olma MH, Sumara G, Ricci R, Peter M: A Cul3-based E3 ligase removes Aurora B from mitotic chromosomes, regulating mitotic progression and completion of cytokinesis in human cells. Developmental cell. 2007, 12 (6): 887-900. 10.1016/j.devcel.2007.03.019.View ArticlePubMedGoogle Scholar
- Chamovitz DA, Segal D: JAB1/CSN5 and the COP9 signalosome. A complex situation. EMBO reports. 2001, 2 (2): 96-101. 10.1093/embo-reports/kve028.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. 10.1038/383453a0.View ArticlePubMedGoogle Scholar
- Lee JW, Choi HS, Gyuris J, Brent R, Moore DD: Two classes of proteins dependent on either the presence or absence of thyroid hormone for interaction with the thyroid hormone receptor. Molecular endocrinology (Baltimore, Md. 1995, 9 (2): 243-254. 10.1210/me.9.2.243.Google Scholar
- Papaioannou M, Melle C, Baniahmad A: The coregulator Alien. Nuclear receptor signaling. 2007, 5: e008-PubMed CentralPubMedGoogle Scholar
- Tanguy G, Drevillon L, Arous N, Hasnain A, Hinzpeter A, Fritsch J, Goossens M, Fanen P: CSN5 binds to misfolded CFTR and promotes its degradation. Biochimica et biophysica acta. 2008, 1783 (6): 1189-1199.View ArticlePubMedGoogle Scholar
- Wei N, Serino G, Deng XW: The COP9 signalosome: more than a protease. Trends in biochemical sciences. 2008, 33 (12): 592-600. 10.1016/j.tibs.2008.09.004.View 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. 10.1182/blood-2004-04-1242.View ArticlePubMedGoogle Scholar
- Kunath T, Gish G, Lickert H, Jones N, Pawson T, Rossant J: Transgenic RNA interference in ES cell-derived embryos recapitulates a genetic null phenotype. Nature biotechnology. 2003, 21 (5): 559-561. 10.1038/nbt813.View ArticlePubMedGoogle 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.