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Originally published In Press as doi:10.1074/jbc.M604746200 on August 30, 2006

J. Biol. Chem., Vol. 281, Issue 43, 32188-32196, October 27, 2006
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The COP9 Signalosome Regulates Skp2 Levels and Proliferation of Human Cells*Formula

Simona Denti12, Maria Elena Fernandez-Sanchez1, Lars Rogge, and Elisabetta Bianchi3

From the Immunoregulation Laboratory, Department of Immunology, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris, France

Received for publication, May 17, 2006 , and in revised form, July 14, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The COP9 signalosome (CSN) is a conserved, multisubunit complex first identified as a developmental regulator in plants. Gene inactivation of single CSN subunits results in early embryonic lethality in mice, indicating that the CSN is essential for mammalian development. The pleiotropic function of the CSN may be related to its ability to remove the ubiquitin-like peptide Nedd8 from cullin-RING ubiquitin ligases, such as the SCF complex, and therefore regulate their activity. However, the mechanism of CSN regulatory action on cullins has been debated, since, paradoxically, the CSN has an inhibitory role in vitro, while genetic evidence supports a positive regulatory role in vivo. We have targeted expression of CSN subunits 4 and 5 in human cells by lentivirus-mediated small hairpin RNA delivery. Down-regulation of either subunit resulted in disruption of the CSN complex and in Cullin1 hyperneddylation. Functional consequences of CSN down-regulation were decreased protein levels of Skp2, the substrate recognition subunit of SCFSkp2, and stabilization of a Skp2 target, the cyclin-dependent kinase inhibitor p27Kip1. CSN down-regulation caused an impairment in cell proliferation, which could be partially reversed by suppression of p27Kip1. Moreover, restoring Skp2 levels in CSN-deficient cells recovered cell cycle progression, indicating that loss of Skp2 in these cells plays an important role in their proliferation defect. Our data indicate that the CSN is necessary to ensure the assembly of a functional SCFSkp2 complex and therefore contributes to cell cycle regulation of human cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The COP9 signalosome (CSN)4 is a highly conserved eight-subunit protein complex, first identified in Arabidopsis thaliana for its role in regulating light-dependent development (1). The subsequent characterization of the CSN in other eukaryotes, including Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, and mammalians (2–6), has confirmed the pleiotropic functions of the complex in regulating many developmental and signaling pathways. Several biochemical functions have been associated with the CSN: the complex may function as a platform that recruits protein kinases (inositol 1,3,4-triphosphate 5/6-kinase, protein kinase D and casein kinase II (7, 8)), and possibly some of their substrates, such as c-Jun or the p53 tumor suppressor protein (5). In addition, two different ubiquitin isopeptidase activities have been associated with the CSN, one consisting in cleavage of monoubiquitin from Cullin4 (9), the other in the depolymerization of polyubiquitin chains. This latter activity depends on the recruitment of a conventional deubiquitinating enzyme of the cystein protease family, called Ubp12 in S. pombe (10) or USP15 in mammalian cells (11). To date, the best characterized biochemical function of the CSN is an intrinsic NEDD8 isopeptidase activity that requires the Zinc protease motif (JAMM motif) of subunit 5 (CSN5) of the complex, as well as the integrity of the whole CSN complex (12, 13). The ubiquitin-like peptide NEDD8 is a post-translational modifier of cullins. Cullins assemble with a RING protein and other adaptor proteins to constitute a class of E3 ubiquitin ligases, the last components in the enzymatic cascade that leads to protein ubiquitination. Ubiquitin ligases are of particular importance in determining the specificity of the ubiquitination process, since they are responsible for direct substrate recognition. A well characterized cullin-based E3 is the SCF (Skp1, CDC53/cullin1, F-box) that marks several important cellular signaling proteins for destruction. The SCF can bind different substrate recognition subunits, such as the F-box protein Skp2, which recruits for ubiquitination a number of cell cycle regulators, among which the cyclin-dependent kinase inhibitors p27Kip1 and p21WAF or cyclins D1 and D3 (14).

NEDD8 conjugation is fundamental for cullin activity, as demonstrated by the severe impairment of cullin function upon loss of components of the neddylation pathway. Defects in the NEDD8/RUB conjugation process result in cell cycle arrest in S. pombe and developmental defects in C. elegans and mice (15–17). The molecular basis of the strict requirement for cullin neddylation is still debated. Neddylation is currently thought to regulate the assembly of a functional cullin complex by removing the inhibitor CAND1 and promoting interaction of Cullin1 both with the Skp1-F-box protein module (18, 19) and with the E2 ubiquitin-conjugating enzyme (20).

The CSN binds to a number of cullins, among which are Cullin1 (13) and Cullin4 (9), and promotes cullin deneddylation; however, the physiological consequences of CSN-mediated deneddylation are still unclear. In vitro studies have suggested that CSN-mediated removal of NEDD8 from cullin complexes reduces their ubiquitin ligase activity (9, 21). These data are in contrast with the observed accumulation of cullin substrates in CSN-deficient organisms (4, 10, 22–26), which would indicate that the CSN is in fact necessary for cullin function. To resolve this paradox, current models are based on the hypothesis that a cycle of NEDD8 attachment, and removal is necessary for the association of a functional cullin-based complex (27, 28). It has been further proposed that the CSN aids in stabilizing E3 complexes by counteracting proteolytic degradation of some of their components (29). In fission yeast csn-deletion mutants, a decrease in the levels of the Cullin3 adaptor Btb3p and of the Cullin1 adaptor Pop1p was observed (30). Similarly, disruption of csn-2 in Neurospora causes a reduction of the levels of the FWD-1 F-box (31), indicating that the CSN may promote cullin activity by maintaining adaptor stability.

Gene disruption of single CSN subunits has resulted in early embryonic lethality in the mouse, precluding further investigation of the physiological role of the complex in mammalian cells (26, 32, 33).

In the present work, we have used lentivirus-mediated RNA interference to down-regulate CSN subunits in human cell lines and to study the role of the CSN in regulating SCFSkp2 function and mammalian cell proliferation. Down-regulation of CSN subunits resulted in increased p27Kip1 half-life and impaired cell proliferation. Protein levels of the substrate adaptor Skp2 were suppressed in CSN-deficient cells but could be recovered by proteasome inhibition or by iRNA-mediated Cullin1 suppression. Our data indicate that the CSN is an important regulator of proliferation in human cells and that CSN activity promotes the assembly of a functional SCF complex by regulating the stability of its F-box subunit.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Transient Transfection—HEK293T and HeLa cells were grown at 37 °C in 5% CO2 and maintained in Dulbecco's modified Eagle's medium (Invitrogen), supplemented with 10% fetal bovine serum (HyClone). FLAG-tagged Skp2 (from M. Donzelli, IEO, Milan, Italy) was transiently transfected in HEK293T cells with the jetPEITM reagent according to the manufacturer's protocol (QBIOgene).

Plasmids—The lentivirus vector pTRIP-{Delta}U3-CMV was provided by P. Charneau (Institut Pasteur, Paris). The green fluorescent protein coding sequence was substituted with the extracellular sequence of the nerve growth factor receptor (NGFR), cloned into the BamHI-XhoI sites to generate the pTCN vector. The oligonucleotides used to generate small hairpin RNA sequences were the following: control, 5'-GATCCCCGAGCATGCGTATGGACGATTCAAGAGATCGTCCATACGCAATGCTCTTTTTGGAAA-3'; siCSN4, 5'-GATCCCCGCAGATCCAATCACTTTGTTTCAAGAGAACAAAGTGATTGGATCTGCTTTTTGGAAA-3'; siCSN5.1, 5'-GATCCCCGATGGTGATGCATGCCAGATTCAAGAGATCTGGCATGCATCACCATCTTTTTGGAAA-3'; siCSN5.2, 5'-GATCCCCGCTCAGAGTATCGATGAAATTCAAGAGATTTCATCGATACTCTGAGCTTTTTGGAAA-3'; sip27, 5'-GATCCCCGAGCCAACAGAACAGAAGATTCAAGAGATCTTCTGTTCTGTTGGCTCTTTTTGGAAA-3'. These oligonucleotides were cloned together with the H1 promoter from the pSuper vector (gift of R. Agami) in the EcoRI site of pTCN. All plasmid purifications were carried out using Qiagen plasmid kits.

Lentivirus Production and Transduction—2 x 106 HEK293T were plated on 100-mm plates. The following day 10 µgofthe small hairpin (shRNA) vector pTCN (control, iCSN4, or iCSN5), 10 µg of the packaging vector pCMV{Delta}8.9, and 5 µgof the vesicular stomatitis virus-glycoprotein envelope vector (from P. Charneau) were co-transfected by calcium phosphate precipitation. The medium was changed the next day and substituted with Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum, 10 mM Hepes (pH 7.4). After 24 h supernatants were collected and centrifuged at 2500 rpm for 10 min. For transduction, 3–5 x 105 HEK293T or HeLa cells were seeded in a 6-well plate and transduced with the viral supernatant by spinoculation (30 min at 2000 rpm). To evaluate the efficiency of viral transduction, cells were analyzed by fluorescence-activated cell sorter for NGFR expression 4 days after the infection (anti-NGFR murine monoclonal antibody clone 20.4). If less than 95% positive for NGFR expression, transduced cells were purified by positive selection using the CD271 (LNGFR) MicroBead kit (Miltenyi Biotec). All experiments were performed 5–10 days after transduction.

Cell Lysate and Western Blot Analysis—Cells were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris-Cl (pH 8), 1% Nonidet P-40, 0.5% deoxycholate, 0.05% SDS, 2 mM EDTA, and Pefabloc-SC (Roche Applied Science)). Protein content of the supernatant were measured by colorimetric reaction (Bradford, Bio-Rad), and equal amounts of proteins were resolved by SDS-PAGE, transferred to a membrane (Hybond-P, Amersham Biosciences), and hybridized with the appropriate antibodies, followed by detection by enhanced chemiluminescence (SuperSignal Western blotting, Pierce). For protein stability studies transduced cells were treated with 60 µg/ml of cycloheximide (Sigma) for the indicated times and lysed in RIPA buffer. Treatment with proteasome inhibitors was as follows: MG132 (Sigma), 20 µM for 8 h, lactacystin (Sigma), 10 µM for 4 h; or MG262 (Alexis), 1 µM for 4 h. The following antibodies were used for this study: anti-CSN4 (A300-013A) antibody was from Bethyl Laboratories; cyclin D1 (72–13G, sc-450 and M-20, sc-718) and p27 (C-19, sc-528) antibodies were from Santa Cruz Biotechnology; anti-CSN1 (PW 8285), CSN3 (PW 8235), CSN6 (PW 8295), and CSN8 (PW 8290) antibodies were from Affiniti; anti-Cullin1 (71–8700) and p45 SKP2 (32–3300) antibodies were from Zymed Laboratories; anti-NEDD8 (ALX-210-194) and anti-proteasome 20 S subunit C2 (210–295-C100) antibodies were from Alexis Biochemicals; anti beta-tubulin (clone TUB2.1, T 4026) antibody was from Sigma; and the anti-CSN5 monoclonal SP282 antibody was described previously (34).

Immunoprecipitation of Endogenous Cullin1—Transduced cells were lysed in 1% Nonidet P-40-Tris buffer plus CompleteTM protease inhibitors (Roche Applied Science) and N-ethylmaleimide (10 µM, Sigma). The lysates were cleared by incubation with protein A-Sepharose, then incubated with anti-Cullin1 antibody for 1 h at 4°C, followed by incubation with protein A-Sepharose. After washing, bound proteins were eluted with gel sample buffer and analyzed by Western blot.

Gel Filtration Chromatography—2 x 106 of transduced cells were lysed by mechanical shearing in hypotonic buffer (Hepes (pH 7.4) with protease inhibitors). Homogenates were spun at 14,000 rpm for 40 min and filtered with 0.45-µm filters (Sigma). 50 µg of proteins were loaded onto a Superose 6 column (SMART System, GE Healthcare) and eluted with Hepes buffer (20 mM Hepes, 150 mM NaCl, 1.5 mM MgCl2, protease inhibitors). Fractions 8–19 were resolved on a 10% SDS-PAGE, blotted onto a polyvinylidene difluoride membrane, and analyzed by Western blotting.

RNA Extraction and Quantitative Real-time PCR—RNA was extracted with the RNeasy kit (Qiagen) following manufacturer's instructions. 1 µg of total RNA was reverse-transcribed with the SUPERSCRIPTTM II enzyme (Invitrogen). Real-time fluorogenic RT-PCR was performed according to manufacturer's protocols (PE Applied Biosystem): 10–40 ng of cDNA were amplified in 25 µl final volume with 1.25 µl of the "Assays-on-Demand" oligonucleotides for CSN4, CSN5, SKP2, p27, CUL1, CUL4A, and 18S. Samples were analyzed with the ABI Prism 7300 real-time PCR machine. Relative mRNA levels were normalized to 18S RNA levels. Similar results were obtained with normalization to GAPDH or actin mRNA levels.

Proliferation Assay—Metabolic activity of viable cells was measured with the Cell Proliferation reagent WST-1 (Roche Applied Science), according to the manufacturer's protocol. Briefly, cells were plated into a flat bottom 96-well plate at a density of 7.5 x 103 cells/100 µl/well, 10 µl of WST-1 reagent was added at the indicated times after plating, and after 4 h of incubation at 37 °C, sample absorbance at 450 nm was measured using an ELISA reader. A colorimetric immunoassay (Cell Proliferation ELISA, Roche Applied Science) was used for the quantification of 5-bromo-2'-deoxyuridine (BrdUrd) incorporation during DNA synthesis. Briefly, 7.5 x 103 cells/100 µl/well were seeded in 96-well plates and cultured for 24 or 72 h. BrdUrd (10 µM final concentration) was added, and after 4 h cells were incubated with the anti-BrdUrd antibody, following the manufacturer's instructions. The reaction was stopped with 1 M H2SO4, and absorbance of the samples was measured in an ELISA reader at 450 nm.

Transient Transfection of Small Interference RNA Oligonucleotides—Lentivirus-transduced cells were transiently transfected with a non relevant siRNA (control) or with a Cullin1 siRNA oligonucleotide (control, no. 1022076; Cul1.5, no. SI02225657; Cul1.6, no. SI 02225664; Cul4A.1, no. SI00356937, Cul4A.2, no. SI00356944, Qiagen). Briefly, 2 x 105 cells were seeded in a 12-well plate and transfected with the oligonucleotide (10 nM final concentration) using 6µl of HiPerFect (Qiagen). 48 h later cells were lysed with RIPA buffer and analyzed by Western blotting.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Down-regulation of Either CSN4 or CSN5 Causes Cullin1 Hyperneddylation and Disruption of the COP9 Signalosome Complex—To inhibit CSN function in human cells, we have used lentivirus-mediated RNA interference to down-regulate the expression of subunit 4 (CSN4) or subunit 5 (CSN5) of the CSN. CSN5 was chosen because it harbors the deneddylase activity, while loss of CSN4 in Arabidopsis and in Drosophila was shown to be sufficient to disrupt the whole complex, with consequent destabilization of other CSN subunits (35–37).

293T or HeLa cells were transduced with a dual cassette lentivirus that co-expresses the CSN-targeting shRNA and the extracellular domain of the NGFR, as a marker for transduced cells. Cell populations used in our studies showed 80–95% suppression of the target mRNA and corresponding protein (Fig. 1, A and B). We tested whether down-regulation of CSN4 or CSN5 expression in our experimental system was sufficient to interfere with CSN function, by examining protein neddylation in transduced cells. Down-regulation of CSN4 and CSN5 was accompanied by an increase in neddylated proteins in total cell lysates, compared with cells treated with a control shRNA vector (Fig. 1C, right, and data not shown). Increased neddylation of Cullin1 was shown by the appearance of slower migrating forms in total lysates (Fig. 1C, right) and confirmed by immunoprecipitation of Cullin1 and hybridization with anti-NEDD8 antibodies (Fig. 1C, left).


Figure 1
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  		FIGURE 1.
Down-regulation of CSN4 or CSN5 by lentiviral vector-mediated shRNA delivery. 293T cells were transduced with lentiviral vectors expressing shRNAs for either CSN4 (iCSN4), CSN5 (iCSN5), or a control sequence and analyzed 4 days later. A, total RNA was extracted from transduced cells and the mRNA levels of CSN4 (left) and CSN5 (right) were analyzed by real-time RT-PCR, using Taqman probes. Results were normalized to 18 S amplification and represented relative to the control sample. Shown are average ± S.D. values of six independent experiments. B, transduced cells were lysed with RIPA buffer and analyzed by Western blotting with anti-CSN4 (left) or anti-CSN5 (right) antibodies. Total protein loading was controlled by hybridizing with anti-beta-tubulin antibodies. C, down-regulation of CSN5 results in increased Cullin1 neddylation. Cullin1 was immunoprecipitated from lysates of either control or CSN5kd cells (left panels), resolved by SDS-PAGE, and analyzed by Western blotting with anti-Cullin1 or anti-NEDD8 anti-bodies. Right panels show analysis of total lysates.

 
To study the effect of the down-regulation of CSN4 or CSN5 on the assembly of the CSN complex, we performed gel filtration chromatography of homogenates from cells in which we had down-regulated CSN4 (CSN4kd) or CSN5 (CSN5kd) and analyzed expression of CSN subunits in the elution fractions by Western blotting. Equal loading of total proteins on the gel filtration column was confirmed by Western blotting with an antibody directed against the C2 subunit of the 20 S proteasome (Fig. 2).


Figure 2
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  		FIGURE 2.
Down-regulation of either CSN4 or CSN5 reduces the levels of the entire CSN complex. 293T cells were transduced with control (left), iCSN4 (middle), or iCSN5 vectors (right). Cells were homogenized in a hypotonic buffer and proteins separated by gel filtration chromatography. Fractions 8–19 were resolved by SDS-PAGE, followed by Western blotting with antibodies against the indicated CSN subunits or against Cullin1. 20 S proteasome C2 subunit was used as loading control. ** indicates the fractions containing monomeric forms of the CSN5 subunit.

 


Figure 3
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  		FIGURE 3.
Down-regulation of either CSN4 or CSN5 causes accumulation of p27Kip1. A, 293T cells were transduced with the indicated shRNA vectors. p27Kip1, CSN5, and CSN4 protein levels were analyzed by SDS-PAGE and Western blot. beta-Tubulin was used as a loading control. iCSN5.1 and iCSN5.2 are two different shRNA constructs targeting CSN5. B, RNA was extracted from transduced cells and analyzed by quantitative RT-PCR for the p27 gene, using Taqman probes. Expression levels are normalized to 18 S amplification in each sample and represented relative to the expression levels of p27Kip1 in cells transduced with the control vector. Bars represent average ± S.D. values from seven independent experiments. C, 293T cells were transduced with the indicated shRNA vectors and treated with cycloheximide (60 µg/ml) for the indicated times. Lysates were resolved by SDS-PAGE and analyzed by Western blotting with anti-p27Kip1 antibody. Antibeta-tubulin was used as a control for protein loading. D, densitometric reading of the results shown in C. p27Kip1 values were normalized to beta-tubulin content in each sample and plotted relative to the values of untreated cells (time 0).

 
CSN subunits eluted in the same high molecular mass fractions, consistent with their association in the CSN complex (450–500 kDa, Fig. 2). As reported previously, CSN5 eluted also in lower molecular mass fractions corresponding to its predicted mass as a monomer (37.5 kDa, double asterisk in Fig. 2). ShRNA targeting of CSN4 caused decreased expression of the target CSN4 protein, as expected, but also of the other CSN subunits analyzed (Fig. 2, middle column), resulting in a net loss of the entire complex. Protein reduction of other CSN subunits not targeted by the shRNA occurred without changes in the mRNA levels, at least for subunits 5 and 3 (Fig. 1A and data not shown). Similarly, targeting of CSN5 caused a reduction of unrelated CSN subunits (Fig. 2, right column). The monomeric form of CSN5 completely disappeared upon treatment with CSN5 shRNA, but was unaffected by CSN4 down-regulation, indicating that CSN5 is stable when not associated with the CSN complex (see asterisks in Fig. 2). C2 protein expression and assembly into the 20 S proteasome were not affected by CSN down-regulation.

The analysis of Cullin1 expression in the elution fractions confirmed the enrichment of neddylated Cullin1 in the homogenates of CSN4kd and CSN5kd cells, compared with the control cells (Fig. 2, bottom panel). While a fraction of Cullin1 co-elutes with the CSN, the more neddylated forms of Cullin1 were enriched in fractions 13 and 14, which were devoid of CSN complex.

Down-regulation of CSN4 or CSN5 Increases p27Kip1 Protein Stability—Since the neddylation status of Cullin1 affects the ubiquitin ligase activity of the SCF complex, we asked what is the effect of CSN down-regulation on the protein levels of SCF ubiquitination substrates.

We observed an accumulation of the cyclin-dependent kinase inhibitor p27Kip1 in exponentially growing CSNkd cells (Fig. 3A). The observed increase in p27Kip1 did not result from increased p27Kip1 mRNA expression (Fig. 3B), indicating post-transcriptional regulation of protein levels.


Figure 4
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  		FIGURE 4.
Down-regulation of CSN4 or CSN5 impairs cell proliferation and DNA synthesis. A, down-regulation of CSN4 or CSN5 reduces cell proliferation. 293T cells were transduced with either control, iCSN4, or iCSN5 vectors. The metabolic activity of viable cells was measured at the indicated time points with a colorimetric assay: conversion of the tetrazolium salt WST-1 to formazan was determined by optical density reading at 450 nm. Each point represents the average value of three different samples. B, down-regulation of CSN4 or CSN5 impairs DNA synthesis. Cells were transduced as described for A. 24 or 72 h after plating, cells were incubated with BrdUrd for 4 h, and BrdUrd incorporation analyzed by ELISA. Bars represent the average values of three samples. C, shRNA-mediated down-regulation of p27Kip1. Control cells (left) or cells with suppressed CSN5 (right) were transduced with either a control or a p27Kip1 shRNA construct (ip27) and harvested 4 days later. Lysates were analyzed by Western blotting with anti-p27Kip1 antibody. beta-Tubulin was used as a loading control. D, the proliferation defect caused by CSN down-regulation is partially rescued by suppression of p27Kip1. Cells were transduced with the indicated combinations of shRNA vectors and tested in the WST-1 proliferation assay. Values represent the average of three samples. E, DNA synthesis is recovered by down-regulation of p27Kip1. Cells transduced as in D were analyzed for BrdUrd incorporation 24 or 72 h after plating. Bars represent average values ± S.D. of triplicate samples.

 
p27Kip1 levels during cell cycle progression are mainly regulated by ubiquitin-dependent proteolysis (38). To determine whether the accumulation of p27Kip1 in CSNkd cells is due to decreased turnover, we analyzed total lysates from control and CSNkd cells at different time points after addition of the protein synthesis inhibitor cycloheximide. While in control cells p27Kip1 was completely degraded in 5 h, 25–50% of the initial amount of the protein was still present in CSN4kd and CSN5kd cells, indicating that p27Kip1 degradation is impaired in these cells (Fig. 3, C and D).

Loss of CSN Function Causes a Defect in Cell Proliferation, Which Can Be Partially Reversed by Suppression of p27Kip1 Expression—p27Kip1 is a critical negative regulator of cell division and its abundance is strictly correlated to cell cycle progression (38). We therefore tested how CSN down-regulation and the consequent accumulation of p27Kip1 affect cell proliferation. 293T cells were transduced with lentiviral constructs targeting either CSN4 or CSN5. As a measure of cell number and proliferation rate, the metabolic activity of viable cells was determined at different times after plating, by measuring conversion of the tetrazolium salt WST-1 to formazan. As shown in Fig. 4A, CSN4kd and CSN5kd cells showed lower processing of the WST-1 reagent, compared with control transduced cells. Similar results were obtained with HeLa cells (data not shown).

Cell proliferation was also evaluated by measuring S phase entry by BrdUrd incorporation at two different times after plating the transduced cells. The fraction of CSN4kd or CSN5kd cells in S phase was reduced at all time points analyzed, compared with control cells (Fig. 4B).

To test whether the defect in proliferation observed in CSNkd cells is linked to the accumulation of p27Kip1, we suppressed p27Kip1 levels by lentivirus-mediated delivery of shRNA (Fig. 4C). p27Kip1 down-regulation alone does not affect proliferation of control cells, consistent with previous studies performed with embryonic fibroblasts from p27–/– mice (39). However, down-regulation of p27Kip1 partially recovered cell proliferation of CSN5kd cells, as measured by the WST-1 assay (Fig. 4D) or by BrdUrd incorporation (Fig. 4E), indicating that accumulation of p27Kip1 is important in determining the proliferation defect of CSNkd cells.

The CSN Controls the Levels of the F-box Protein Skp2—It has been proposed that the CSN may regulate cullin-based ubiquitin ligase activity by affecting the stability of selected components of cullin complexes, such as the substrate adaptor subunit (30, 31, 40) or cullin itself (41). We therefore asked whether the observed accumulation of p27Kip1 is due to altered levels of the Skp2 F-box adaptor protein in CSN4kd or CSN5kd cells. Skp2 protein levels were consistently reduced in total lysates obtained from CSNkd cells, compared with control cells (Fig. 5A), in the absence of obvious changes in Skp2 mRNA levels as measured by quantitative RT PCR (Fig. 5B). Treatment with proteasome inhibitors did not affect Skp2 levels in control cells; however, it recovered Skp2 levels in CSNkd cells, indicating that the decrease in Skp2 protein upon CSN down-regulation is due to increased proteasome-dependent degradation (Fig. 5C and supplemental Fig. 1). Analysis of Skp2 levels after cyclohexamide treatment demonstrated that Skp2 turnover is accelerated in CSN4kd or CSN5kd, compared with control cells (Fig. 5D).

A number of substrate-binding adaptors, including S. cerevisiae Cdc4, Grr1, Met30, and mammalian Skp2 and beta-TrcP, have been shown to be unstable when assembled into SCF complexes, raising the possibility that these F-box proteins are ubiquitylated through an autocatalytic mechanism (42–45). To investigate the role of Cullin1 ubiquitin ligase activity in the degradation of Skp2 in CSNkd cells, we transiently transfected control or CSN4kd cells with two different siRNA oligos directed against Cullin1 (iCul1.5 and iCul1.6) and analyzed Skp2 protein levels in total lysates. Fig. 6A shows that Skp2 levels were decreased in CSN4kd cells, as observed previously (compare lanes 1 and 4, transfected with control siRNA oligonucleotides). However, suppression of Cullin1 in CSN4kd cells recovered Skp2 protein expression (Fig. 6A, lanes 5 and 6), without affecting Skp2 mRNA levels (Fig. 6B, top panel), indicating a role for hyperneddylated Cullin1 in Skp2 down-regulation in these cells. Contrary to what previously observed for Drosophila cells (41), total Cullin1 levels were not affected by CSN down-regulation (see also Figs. 1 and 2).


Figure 5
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  		FIGURE 5.
Down-regulation of CSN4 or CSN5 reduces the expression of the F-box protein, Skp2. A, down-regulation of CSN4 or CSN5 results in a decrease of the Skp2 protein. 293T cells were transduced with the indicated vectors. Total lysates were harvested on day 5 and analyzed by Western blotting with antibodies against the indicated proteins (the arrow indicates the specific band for CSN4). Equal loading of lysates was controlled by blotting with anti-beta-tubulin antibody. B, Skp2 mRNA expression in CSN4 and CSN5 knock-down cells. Skp2 mRNA levels were measured by real-time RT-PCR and normalized to 18 S amplification levels. Results are calculated relative to the expression of skp2 in control cells; data show average values ± S.D. of six independent transductions. C, Skp2 levels are restored by proteasome inhibition. Transduced cells were treated with MG132 (20 µM) or Me2SO (0.1%) for 8 h. Total lysates were analyzed by SDS-PAGE and Western blot for Skp2 content. Actin was used as a loading control. D, 293T cells were transduced with the indicated shRNA vectors and treated with cycloheximide (60 µg/ml) for the indicated times. Lysates were resolved by SDS-PAGE and analyzed by Western blotting with anti-Skp2 or anti-actin antibodies. A longer exposure (60 min) of the Skp2 blots is also shown for CSN knock-down cells, to facilitate comparison with the control cells.

 
Recently, a Cul4A complex containing DDB1 and Skp2 has been implicated in the regulation of p27Kip1 levels (46, 47). Suppression of Cul4A expression by RNA interference increased Skp2 levels in control, as well as in CSNkd cells, suggesting that Cul4A is implicated in a similar autocatalytic mechanism of Skp2 regulation (Fig. 6C). Skp2 mRNA expression was not affected by Cul4A down-regulation (Fig. 6D, top panel).


Figure 6
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  		FIGURE 6.
Skp2 levels in CSN4 knockdown cells are rescued by Cullin1 or Cullin 4A suppression. A, cells transduced with the control or the iCSN4 vector were transfected with control siRNA oligos or with siRNA oligonucleotides targeting Cullin1 (iCul1.5 and iCul1.6). Total lysates were harvested after 48 h, and Cullin1 and Skp2 expression was analyzed by Western blotting. Actin was used as loading control. B, Skp2 and Cullin1 mRNA levels in iCul1.5 or iCul1.6 transfected cells were quantitated by real-time RT-PCR. C, cells transduced with the control or the iCSN4 vector were transfected with control siRNA oligonucleotides or with siRNA oligonucleotides targeting Cullin4A (iCul4.1 and iCul4.2). Total lysates were harvested after 48 h and Skp2, and actin expression was analyzed by Western blotting. D, Skp2 and Cullin4A mRNA levels in iCul4.1- or iCul4.2-transfected cells were quantitated by real-time RT-PCR.

 
Finally, we tested the importance of Skp2 suppression in the proliferation defect of CSNkd cells. Control, CSN4kd and CSN5kd were transfected with an expression vector encoding Skp2 or with an empty vector as a control (Fig. 7A). Equal number of cells was plated for each sample (white bars) and cell counts were obtained 48 h later for each population (gray bars). Transfection of Skp2 did not affect proliferation of control cells but largely recovered cell numbers of the CSNkd populations. Similarly, transfection of Skp2 in CSN5kd cells fully restored BrdUrd incorporation (Fig. 7B), indicating that suppression of Skp2 in these cells is a fundamental determinant of their proliferation defect. These findings demonstrate that CSN activity is necessary to preserve the stability of Skp2 and is therefore necessary for the assembly of a functional SCFSkp2 complex. Loss of the Skp2 adaptor plays an important role in the proliferation defect caused by CSN down-regulation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Our results show that down-regulation of the CSN complex leads to loss of the Skp2 F-box protein, with consequent impairment in the degradation of the cell cycle inhibitor p27Kip1 and a defect in cell proliferation. These findings support the interpretation that the CSN is necessary for maintenance of a functional SCFSkp2 complex.


Figure 7
View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 7.
Restoration of Skp2 levels recovers proliferation of CSN knockdown cells. A, control, CSN4, or CSN5 knockdown cells were transfected either with an empty vector or with a vector expressing Skp2. Equal number of cells were plated for each sample (white bars, t = 0), and cell counts were obtained for each population after 48 h (gray bars, t = 48). B, CSN5 knockdown cells were transfected with an expression vector encoding Skp2. BrdUrd incorporation was measured at two different time points after transfection (48 and 72 h). Shown are average values ± S.D. of three samples.

 
While the CSN is clearly implicated in regulating cullin-based E3 ligases, the consequences of CSN deneddylase activity have been somewhat controversial. Because the NEDD8 pathway is essential for cullin function, the CSN could be expected to act as a negative regulator of cullin-based complexes. This has been demonstrated to occur in vitro for Cullin4 and Cullin1 (9, 21). In particular, the CSN was shown to prevent p27Kip1 ubiquitination by SCFSkp2 in vitro and was therefore proposed to inhibit p27Kip1 degradation and to negatively regulate G1 to S phase progression (21). On the other hand, genetic evidence in several model organisms, including plants, yeast, and Drosophila, have shown that loss of the CSN is accompanied by accumulation of ubiquitination substrates of several cullin-based ligases, indicating that the CSN may indeed be necessary for cullin function (see Ref. 1 for a review). Consistently, in mammalians, increased cyclin E and p27Kip1 immunoreactivity was observed in CSN2 or CSN5 null blastocyts (26, 33). While no definite biochemical data are available at present to solve the discrepancy between the in vitro and in vivo studies, it has been proposed that the ability to cycle between a neddylated and deneddylated state is necessary for cullin activity in vivo (28). Based on their findings in S. Pombe, Wolf et al. (29) build on this assumption by proposing that neddylation cycles are necessary to stabilize and recruit different substrate adaptor proteins to the cullin complex. This model incorporates the notion that several F-box proteins are degraded through a proteasome-dependent pathway. Degradation is thought to occur in an autocatalytic fashion, since it requires association of the F-box protein to a cullin complex (42–44). The same mechanism may control the stability of several different substrate adaptors, such as the BTB/POZ proteins that recruit substrates to Cullin3 complexes (48), and may increase flexibility in cullin-mediated ubiquitination, by promoting exchange of the subunits that recognize the substrate. Our data showing that Skp2 levels are decreased and degradation of the SCFSkp2 target p27Kip1 is impaired in cells with diminished CSN activity are consistent with this model and support a positive role for the CSN in promoting F-box protein stability and ubiquitin ligase function of cullin complexes.

It has been proposed that the CSN may regulate p27Kip1 by mechanisms independent of cullin deneddylation. Overexpression of the single CSN5 subunit was reported to favor nuclear export of p27Kip1 and subsequent degradation in the cytoplasm (49, 50). Since ectopically expressed CSN5 only minimally incorporates in the CSN complex ((1, 51) and data not shown), this activity has been linked to the monomeric form of CSN5 or to smaller CSN5-containing complexes (49, 50). The biochemical and functional alterations observed in our studies occurred both when CSN5 or CSN4 were down-regulated. Our gel filtration analysis shows that down-regulation of CSN4 causes a decrease in the levels of the CSN complex but largely spares monomeric CSN5. We therefore believe that p27Kip1 accumulation and the consequent proliferation defect in our system are mainly due to a lack of CSN complex activity, rather than a defect in monomeric CSN5 function.

Size exclusion chromatography analysis of CSN4kd and CSN5kd homogenates shows that in both cases removal of a single subunit is sufficient to destabilize the complex and reduce the levels of other unrelated CSN subunits. In the case of CSN4, these findings are consistent with what described for Arabidopsis and Drosophila cells (35–37). On the contrary, the role of CSN5 in complex assembly and stability is unclear. Both Arabidopsis and Drosophila CSN5 mutants that fail to express the protein have been reported to still retain the complex (37, 52). However, a different study shows a loss of CSN complex in Drosophila CSN5 null cells (53), and immunohistochemical analysis could not detect the CSN1 subunit in CSN5 null mouse embryos (33), consistently with the results of our study. It is possible that loss of CSN5 still allows the assembly of an unstable complex, in particular experimental conditions.

Smaller CSN complexes, comprising only some of the CSN subunits in various combinations, have been detected by non-denaturing SDS-PAGE in a number of mammalian cell lines (50, 54), and their physiological significance is still investigated. In our gel filtration analysis some CSN subunits, notably CSN3 and CSN8, eluted in fractions compatible with smaller molecular mass complexes. However, since the amount of these smaller complexes increased upon shRNA-mediated disruption of the CSN, we favor the interpretation that they are a consequence of the destabilization induced by the loss of CSN4 or CSN5.

We found a clear proliferation defect in CSN4kd and CSN5kd cells. Suppression of p27Kip1 could partially recover proliferation, indicating that p27Kip1 accumulation indeed affects cell cycle progression in these cells. However, recovery was not complete, suggesting that multiple alterations contribute to aberrant cell cycle progression upon CSN loss. Consistently, in addition to p27Kip1, we could observe increased levels of other Skp2 targets, such as cyclin D1 and the inhibitor p21CIP/WAF (Fig. 5A and data not shown). Restoring Skp2 levels in CSNkd cells recovered cell cycle progression, providing evidence that loss of Skp2 is central to the proliferation defect in these cells. The CSN may also affect Skp2 independent pathways. In S. pombe, loss of CSN1 or CSN2 causes a delay in progression through S-phase, possibly because of failure to properly down-regulate the inhibitor Spd-1. In C. elegans, the CSN is necessary for degradation of the MEI-1/katanin microtubule-severing protein, which controls spindle formation during meiosis (4). Although a mitotic role for katanin has not been yet established, overexpression of katanin affects microtubule patterning during mitosis, and it is possible that abnormally high levels of katanin in CSNkd cells may affect mitotic progression (55, 56). Impaired proliferation of CSNkd cells is therefore likely the consequence of reduced degradation of several positive and negative regulators, resulting in a complex defect of cell cycle progression.

The broad activity of cullin-based ubiquitin ligases may provide an explanation to the pleiotropic function of the CSN. In addition to Skp2, other F-box proteins, such as Fbw7 or cyclin F, are down-regulated in CSN-deficient cells (40). Some cullin pathways, however, seem not to require the CSN to function. One example is the stimulus-dependent degradation of IkB{alpha}, which depends on the beta-TrcP F-box and occurs normally in CSNkd cells.5 Although the reason for this selectivity in CSN requirement is still unknown, a certain degree of specificity could be introduced by the different susceptibility of substrate adaptor modules to CSN-mediated stabilization. To improve our understanding of the cellular pathways controlled by CSN function, it will be important to identify which substrate adaptors are affected by the CSN and to understand the molecular mechanisms that make them susceptible to regulation by the CSN.


   FOOTNOTES
 
* * This work was supported in part by Association pour la Recherche sur le Cancer Grant 3402 (to L. R.) and by the Canceropole, Ile de France. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1. Back

1 These authors have contributed equally to this work. Back

2 Supported by a fellowship from Fondation de la Recherche Medicale. Back

3 To whom correspondence should be addressed. Tel.: 33-1-4061-3827; Fax: 33-1-4061-3204; E-mail: ebianchi{at}pasteur.fr.

4 The abbreviations used are: CSN, COP9 signalosome; NGFR, nerve growth factor receptor; shRNA, small hairpin RNA; ELISA, enzyme-linked immunosorbent assay; RT, reverse transcription; BrdUrd, 5-bromo-2'-deoxyuridine. Back

5 S. Denti and E. Bianchi, unpublished results. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Wei, N., and Deng, X. W. (2003) Annu. Rev. Cell Dev. Biol. 19, 261–286[CrossRef][Medline] [Order article via Infotrieve]
  2. Freilich, S., Oron, E., Kapp, Y., Nevo-Caspi, Y., Orgad, S., Segal, D., and Chamovitz, D. A. (1999) Curr. Biol. 9, 1187–1190[CrossRef][Medline] [Order article via Infotrieve]
  3. Mundt, K. E., Porte, J., Murray, J. M., Brikos, C., Christensen, P. U., Caspari, T., Hagan, I. M., Millar, J. B., Simanis, V., Hofmann, K., and Carr, A. M. (1999) Curr. Biol. 9, 1427–1430[CrossRef][Medline] [Order article via Infotrieve]
  4. Pintard, L., Kurz, T., Glaser, S., Willis, J. H., Peter, M., and Bowerman, B. (2003) Curr. Biol. 13, 911–921[CrossRef][Medline] [Order article via Infotrieve]
  5. Seeger, M., Kraft, R., Ferrell, K., Bech-Otschir, D., Dumdey, R., Schade, R., Gordon, C., Naumann, M., and Dubiel, W. (1998) FASEB J. 12, 469–478[Abstract/Free Full Text]
  6. Wei, N., Tsuge, T., Serino, G., Dohmae, N., Takio, K., Matsui, M., and Deng, X. W. (1998) Curr. Biol. 8, 919–922[CrossRef][Medline] [Order article via Infotrieve]
  7. Qian, X., Mitchell, J., Wei, S. J., Williams, J., Petrovich, R. M., and Shears, S. B. (2005) Biochem. J. 389, 389–395[CrossRef][Medline] [Order article via Infotrieve]
  8. Uhle, S., Medalia, O., Waldron, R., Dumdey, R., Henklein, P., Bech-Otschir, D., Huang, X., Berse, M., Sperling, J., Schade, R., and Dubiel, W. (2003) EMBO J. 22, 1302–1312[CrossRef][Medline] [Order article via Infotrieve]
  9. Groisman, R., Polanowska, J., Kuraoka, I., Sawada, J., Saijo, M., Drapkin, R., Kisselev, A. F., Tanaka, K., and Nakatani, Y. (2003) Cell 113, 357–367[CrossRef][Medline] [Order article via Infotrieve]
  10. Zhou, C., Wee, S., Rhee, E., Naumann, M., Dubiel, W., and Wolf, D. A. (2003) Mol. Cell 11, 927–938[CrossRef][Medline] [Order article via Infotrieve]
  11. Hetfeld, B. K., Helfrich, A., Kapelari, B., Scheel, H., Hofmann, K., Guterman, A., Glickman, M., Schade, R., Kloetzel, P. M., and Dubiel, W. (2005) Curr. Biol. 15, 1217–1221[CrossRef][Medline] [Order article via Infotrieve]
  12. Cope, G. A., Suh, G. S., Aravind, L., Schwarz, S. E., Zipursky, S. L., Koonin, E. V., and Deshaies, R. J. (2002) Science 298, 608–611[Abstract/Free Full Text]
  13. Lyapina, S., Cope, G., Shevchenko, A., Serino, G., Tsuge, T., Zhou, C., Wolf, D. A., Wei, N., and Deshaies, R. J. (2001) Science 292, 1382–1385[Abstract/Free Full Text]
  14. Nakayama, K. I., and Nakayama, K. (2005) Semin. Cell Dev. Biol. Cell Cycle and Development (Camb.) 16, 323–333
  15. Jones, D., and Candido, E. P. (2000) Dev. Biol. 226, 152–165[CrossRef][Medline] [Order article via Infotrieve]
  16. Osaka, F., Saeki, M., Katayama, S., Aida, N., Toh, E. A., Kominami, K., Toda, T., Suzuki, T., Chiba, T., Tanaka, K., and Kato, S. (2000) EMBO J. 19, 3475–3484[CrossRef][Medline] [Order article via Infotrieve]
  17. Tateishi, K., Omata, M., Tanaka, K., and Chiba, T. (2001) J. Cell Biol. 155, 571–579[Abstract/Free Full Text]
  18. Zheng, J., Yang, X., Harrell, J. M., Ryzhikov, S., Shim, E. H., Lykke-Andersen, K., Wei, N., Sun, H., Kobayashi, R., and Zhang, H. (2002) Mol. Cell 10, 1519–1526[CrossRef][Medline] [Order article via Infotrieve]
  19. Liu, J., Furukawa, M., Matsumoto, T., and Xiong, Y. (2002) Mol. Cell 10, 1511–1518[CrossRef][Medline] [Order article via Infotrieve]
  20. Kawakami, T., Chiba, T., Suzuki, T., Iwai, K., Yamanaka, K., Minato, N., Suzuki, H., Shimbara, N., Hidaka, Y., Osaka, F., Omata, M., and Tanaka, K. (2001) EMBO J. 20, 4003–4012[CrossRef][Medline] [Order article via Infotrieve]
  21. Yang, X., Menon, S., Lykke-Andersen, K., Tsuge, T., Di, X., Wang, X., Rodriguez-Suarez, R. J., Zhang, H., and Wei, N. (2002) Curr. Biol. 12, 667–672[CrossRef][Medline] [Order article via Infotrieve]
  22. Schwechheimer, C., Serino, G., Callis, J., Crosby, W. L., Lyapina, S., Deshaies, R. J., Gray, W. M., Estelle, M., and Deng, X.-W. (2001) Science 292, 1379–1382[Abstract/Free Full Text]
  23. Doronkin, S., Djagaeva, I., and Beckendorf, S. K. (2003) Dev. Cell 4, 699–710[CrossRef][Medline] [Order article via Infotrieve]
  24. Higa, L. A., Mihaylov, I. S., Banks, D. P., Zheng, J., and Zhang, H. (2003) Nat. Cell Biol. 5, 1008–1015[CrossRef][Medline] [Order article via Infotrieve]
  25. Bondar, T., Ponomarev, A., and Raychaudhuri, P. (2004) J. Biol. Chem. 279, 9937–9943[Abstract/Free Full Text]
  26. Lykke-Andersen, K., Schaefer, L., Menon, S., Deng, X. W., Miller, J. B., and Wei, N. (2003) Mol. Cell Biol. 23, 6790–6797[Abstract/Free Full Text]
  27. Cope, G. A., and Deshaies, R. J. (2003) Cell 114, 663–671[CrossRef][Medline] [Order article via Infotrieve]
  28. Schwechheimer, C., and Deng, X. W. (2001) Trends Cell Biol. 11, 420–426[CrossRef][Medline] [Order article via Infotrieve]
  29. Wolf, D. A., Zhou, C., and Wee, S. (2003) Nat. Cell Biol. 5, 1029–1033[CrossRef][Medline] [Order article via Infotrieve]
  30. Wee, S., Geyer, R. K., Toda, T., and Wolf, D. A. (2005) Nat. Cell Biol. 7, 387–391[CrossRef][Medline] [Order article via Infotrieve]
  31. He, Q., Cheng, P., and Liu, Y. (2005) Genes Dev. 19, 1518–1531[Abstract/Free Full Text]
  32. Yan, J., Walz, K., Nakamura, H., Carattini-Rivera, S., Zhao, Q., Vogel, H., Wei, N., Justice, M. J., Bradley, A., and Lupski, J. R. (2003) Mol. Cell Biol. 23, 6798–6808[Abstract/Free Full Text]
  33. Tomoda, K., Yoneda-Kato, N., Fukumoto, A., Yamanaka, S., and Kato, J. Y. (2004) J. Biol. Chem. 279, 43013–43018[Abstract/Free Full Text]
  34. Bianchi, E., Denti, S., Granata, A., Bossi, G., Geginat, J., Villa, A., Rogge, L., and Pardi, R. (2000) Nature 404, 617–621[CrossRef][Medline] [Order article via Infotrieve]
  35. Wei, N., Chamovitz, D. A., and Deng, X. W. (1994) Cell 78, 117–124[CrossRef][Medline] [Order article via Infotrieve]
  36. Staub, J. M., Wei, N., and Deng, X. W. (1996) Plant Cell 8, 2047–2056[Abstract]
  37. Oron, E., Mannervik, M., Rencus, S., Harari-Steinberg, O., Neuman-Silberberg, S., Segal, D., and Chamovitz, D. A. (2002) Development (Camb.) 129, 4399–4409[Abstract/Free Full Text]
  38. Bloom, J., and Pagano, M. (2003) Semin. Cancer Biol. 13, 41–47[CrossRef][Medline] [Order article via Infotrieve]
  39. Nakayama, K., Ishida, N., Shirane, M., Inomata, A., Inoue, T., Shishido, N., Horii, I., Loh, D. Y., and Nakayama, K.-I. (1996) Cell 85, 707–720[CrossRef][Medline] [Order article via Infotrieve]
  40. Cope, G. A., and Deshaies, R. J. (2006) BMC Biochem. 7, 1[CrossRef][Medline] [Order article via Infotrieve]
  41. Wu, J. T., Lin, H. C., Hu, Y. C., and Chien, C. T. (2005) Nat. Cell Biol. 7, 1014–1020[CrossRef][Medline] [Order article via Infotrieve]
  42. Zhou, P., and Howley, P. M. (1998) Mol. Cell 2, 571–580[CrossRef][Medline] [Order article via Infotrieve]
  43. Galan, J.-M., and Peter, M. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9124–9129[Abstract/Free Full Text]
  44. Wirbelauer, C., Sutterluty, H., Blondel, M., Gstaiger, M., Peter, M., Reymond, F., and Krek, W. (2000) EMBO J. 19, 5362–5375[CrossRef][Medline] [Order article via Infotrieve]
  45. Li, Y., Gazdoiu, S., Pan, Z. Q., and Fuchs, S. Y. (2004) J. Biol. Chem. 279, 11074–11080[Abstract/Free Full Text]
  46. Higa, L. A., Yang, X., Zheng, J., Banks, D., Wu, M., Ghosh, P., Sun, H., and Zhang, H. (2006) Cell Cycle 5, 71–77[Medline] [Order article via Infotrieve]
  47. Bondar, T., Kalinina, A., Khair, L., Kopanja, D., Nag, A., Bagchi, S., and Raychaudhuri, P. (2006) Mol. Cell Biol. 26, 2531–2539[Abstract/Free Full Text]
  48. Geyer, R., Wee, S., Anderson, S., Yates, J., and Wolf, D. A. (2003) Mol. Cell 12, 783–790[CrossRef][Medline] [Order article via Infotrieve]
  49. Tomoda, K., Kubota, Y., and Kato, J. (1999) Nature 398, 160–165[CrossRef][Medline] [Order article via Infotrieve]
  50. Tomoda, K., Kubota, Y., Arata, Y., Mori, S., Maeda, M., Tanaka, T., Yoshida, M., Yoneda-Kato, N., and Kato, J. Y. (2002) J. Biol. Chem. 277, 2302–2310[Abstract/Free Full Text]
  51. Naumann, M., Bech-Otschir, D., Huang, X., Ferrell, K., and Dubiel, W. (1999) J. Biol. Chem. 274, 35297–35300[Abstract/Free Full Text]
  52. Dohmann, E. M., Kuhnle, C., and Schwechheimer, C. (2005) Plant Cell 17, 1967–1978[Abstract/Free Full Text]
  53. Gemmill, R. M., Lee, J. P., Chamovitz, D. A., Segal, D., Hooper, J. E., and Drabkin, H. A. (2005) Oncogene 24, 3503–3511[CrossRef][Medline] [Order article via Infotrieve]
  54. Fukumoto, A., Tomoda, K., Kubota, M., Kato, J. Y., and Yoneda-Kato, N. (2005) FEBS Lett. 579, 1047–1054[CrossRef][Medline] [Order article via Infotrieve]
  55. Clandinin, T. R., and Mains, P. E. (1993) Genetics 134, 199–210[Abstract]
  56. Srayko, M., Buster, D. W., Bazirgan, O. A., McNally, F. J., and Mains, P. E. (2000) Genes Dev. 14, 1072–1084[Abstract/Free Full Text]

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