[Cell Cycle 8:13, 2041-2049; 1 July 2009]; ©2009 Landes Bioscience
The COP9 complex (signalosome) is a known regulator of
the proteasome/ubiquitin pathway. Furthermore it regulates the
activity of the cullin-RING ligase (CRL) families of ubiquitin
E3-complexes. Besides the CRL family, the anaphase-promoting
complex (APC/C) is a major regulator of the cell cycle. To inves-
tigate a possible connection between both complexes we assessed
interacting partners of COP9 using an in vivo protein-protein
interaction assay. Hereby, we were able to show for the first
time that CSN2, a subunit of the COP9 signalosome, interacts
physically with APC/C. Furthermore, we detected a functional
influence of the COP9 complex regarding the stability of several
targets of the APC/C. Consistent with these data we showed a
genetic instability of cells overexpressing CSN2.
The temporal regulation of degradation of damaged or
misfolded as well as short-lived proteins in a specific manner is an
essential feature of eukaryotic growth and development. Hereby,
the SCF (Skp1/Cullin/F-box) complex, an E3 ubiquitin ligase,
marks specific proteins with ubiquitin (Ub) for destruction by the
26S proteasome.1 As a protein complex, that possesses structural
similarities to a subunit of the 26S proteasome, the COP9 signalo-
some was discovered.2 The COP9 signalosome (also named CSN)
was first identified in Arabidopsis and comprises eight conserved
subunits, CSN1-CSN8, in all eukaryotic cells.3 These subunits
bear remarkable homologies to the 19S lid of the 26S proteasome
as well as to the translational initiation complex eIF3 and are
postulated recently to possess an as yet undetermined function
in protein degradation.4,5 Further, there are suggestions that the
COP9 complex is able to substitute the 19S lid functionally.6
The COP9 complex is able to interact with the 19S regula-
tory complex, replace or interact with the 19S lid resulting in a
supercomplex containing 26S proteasome and COP9 signalsosome
and E3 ligases.7 There are data available indicating that the CSN
subunits interact directly with the 26S proteasome. In Arabidopsis
as well as in Drosophila, CSN1 interacts with Rpn6, a subunit
of the 19S lid.8,9 Furthermore, the COP9 signalosome competes
with the 19S lid for binding to the 26S proteasome in vitro.10
Additionally, it has been shown that subunits of COP9 interact
with different subunits of cullin dependent E3 ligases. Hereby, the
CSN5 subunit which possesses a metalloproteinase activity is able
to remove Nedd8 from cullins.11 Cycles of neddylation and dened-
dylation of cullins seem to regulate the ubiquitinating activity
of the cullin-based Ub ligases.12 As yet, there are no data avail-
able showing an interaction of the COP9 signalosome with the
cullin like protein APC2. Besides the SCF complex, the anaphase
promoting complex (APC/C) is the second major E3 ligase medi-
ating degradation of cyclins during cell cycle.13 Since APC/C
especially regulates the mitosis and the segregation of centrosomes
it has a great impact on the genomic stability.14-16 Recently, it
was revealed that COP9 is essential for cell cycle progression and
genomic stability in Arabidopsis.17
In a former study, we identified interacting proteins of the
co-repressor Alien, an isoform of the CSN2 subunit of COP9
signalosome, under in vivo conditions.18 The aim of the present
study was to detect and analyze functional interactions between
the COP9 complex and proteins involved in cell cycle as well as
maintenance of genomic stability to get a detailed insight in cell
cycle regulation and cell proliferation.
CSN2 interacts with the anaphase-promoting complex in
vivo. CSN2 as a subunit of the COP9 signalosome was shown to
interact with several factors of the ubiquitin-proteasome pathway.7
Recently, it was shown that the signalosome binds to cullin 1–4
and affects their function.19-23 To extend our knowledge about
this network, it is necessary to detect further interaction partners
of COP9. For this reason, we performed a protein-protein interac-
tion assay. Therefore, we precipitated an endogenously expressed
subunit of COP9, CSN2, from crude U2OS cell extract using
a specific antibody against CSN2. In order to recognize unspe-
cific precipitated proteins we used as a negative control a rabbit
*Correspondence to: Christian Melle; Core Unit Chip Application (CUCA);
Institut für Humangenetik und Anthropologie; Universitätsklinikum Jena; 07740
Jena, Germany; Tel.: +49.0.3641.935529; Fax: +49.0.3641.935518;
Submitted: 02/18/09; Revised: 04/24/09; Accepted: 04/27/09
Previously published online as a Cell Cycle E-publication:
Regulation of the anaphase-promoting complex by the COP9
Robert Kob,1 Juliane Kelm,1 Nicole Posorski,1 Aria Baniahmad,2 Ferdinand von Eggeling,1 Christian Melle1,*
1Core Unit Chip Application (CUCA); 2Molecular Genetics; Institute of Human Genetics and Anthropology; Jena University Hospital; Jena, Germany
Key words: 26S proteasome, COP9 signalosome, CSN2, anaphase-promoting complex/cyclosome, Alien, protein-protein interactions
2042 Cell Cycle2009; Vol. 8 Issue 13
homolog (Z-score: 0.73; supplemental Fig. 3; theoretical MW:
71,656 Da) as the best candidates for the three detected interac-
tion partners. APC1, APC4 as well as APC6 are subunits of the
anaphase-promoting complex (APC/C). The APC/C is involved
in protein degradation of cell cycle regulating factors. To confirm
the presence of a protein complex containing CSN2 and APC1
we performed a co-immunoprecipitation experiment (CoIP).
In line with the previous results, we were able to co-precipitate
CSN2 using a specific APC1 antibody from U2OS cells (Fig. 1C).
Additionally, we were able to coprecipitate APC1 by a specific
antibody against CSN2 in a reciprocal CoIP (Fig. 2, lanes 1
and 2). The CSN2/APC1 complex appeared in a cell cycle depen-
dent manner (Fig. 2). These data suggest that endogenous CSN2
exists together with endogenous APC/C, at least transiently, in one
and the same stable protein complex.
CSN2 binding to APC/C but not to the base of the 19S
proteasome is independent by interaction to COP9. The base,
a sub-complex of the 19S proteasome, seems to interact with
different subunits of the COP9 signalosome.9,10 In order to
uncover the biological function of the interaction of COP9 with
pre-immune serum. Captured proteins were eluted and analyzed
using SELDI-MS (Fig. 1A). A specific precipitated signal possessed
an m/z of 218,752 which roughly corresponds to the molecular
weight of the anaphase-promoting complex subunit APC1. This
signal was absent in the negative control. To identify the 219 kDa
signal we subjected the eluted proteins to SDS-PAGE and detected
a specific band in the range of ~220 kDa. Beside this specific band
we detected further specific bands at ~100 kDa and at ~70 kDa
(Fig. 1B). The negative control using a rabbit pre-immune serum
did not reveal bands at these positions. Thus, we confirmed the
presence of specific CSN2-interacting proteins. These specific
bands were excised from the gel and subsequently subjected to an
in-gel digestion by trypsin and protein identification. As control,
an empty gel piece underwent the same treatment. The digest
yielded solution was spotted on a Au array and the peptide mass
fingerprints (PMF) were determined by SELDI-MS. Database
searches (Profound; prowl.rockefeller.edu/prowl-cgi/profound.exe)
revealed APC1 (Z-score: 2.39; supplemental Fig. 1; theoretical
MW: 216,500 Da), APC4 (Z-score: 0.76; supplemental Fig. 2;
theoretical MW: 92,116 Da), and APC6, also named CDC16
Regulation of APC/C by COP9
Figure 1. Endogenous CSN2 interacts with several subunits of the APC/C in vivo. (A) In protein-protein complex detection assays, a specific anti-CSN2
antibody was coupled on IDM beads and incubated with crude U2OS cell extract. Bound protein complexes were eluted and analyzed by SELDI-MS.
A number of specific signals were detectable in the assay using the specific CSN2 antibody compared to an unspecific antibody. Among other specific
signals two signals possessing 218.7 kDa and 94.5 kDa, respectively, were detected which correspond well to the molecular mass of APC1 and APC4,
respectively. (B) The eluate was separated using an SDS-PAGE. Three specific protein bands (labeled with arrows) were excised from the coomassie
stained gel and subjected to tryptic in-gel digestion. The generated peptide mass fingerprints (PMF) were analyzed by SELDI-MS and compared to an
online database. Hereby, APC1 (at approx. 220 kDa), APC4 (at ~100 kDa) and APC6 (at ~70 kDa) were obtained as the best candidates. (C) The
CSN2–APC1 protein interaction was confirmed by reverse CoIP experiments. CSN2 (labeled with an arrow) was co-immunoprecipitated (IP) from crude
U2OS cell extract by a specific APC1 antibody as detected by an immunoblot (IB) (lane 2) compared to a negative control using an unspecific anti-
body (lane 1). A faint band in the negative control (lane 1) was unspecific precipitated by the non-specific antibody. Using a densitometrical analysis,
this unspecific band represents only 19% of the protein band corresponding to CSN2 (lane 2) which was specific co-precipitated by the anti-APC1
www.landesbioscience.com Cell Cycle2043
SUG1-specific antibody, treatment of the cells with NEM led to a
strong reduction of the co-precipitated CSN2 signal compared to
untreated control cells as quantified by a densitrometric assessment
(Fig. 3A; compare lane 7 with lane 3). Hereby, the co-precipitated
signal from cells treated with NEM corresponding to CSN2
was reduced for approx. 75% compared to the CSN2 signal
co-precipitated from control cells. This loss of interaction between
CSN2 and the 19S subunit SUG1 after treatment of U2OS cells
with NEM which results in the disruption of COP9 signalosome
is further confirmed in similar intensity by a reverse CoIP with
a specific CSN2 antibody used for co-precipitation of SUG1
(Fig. 3B). In contrast, there was only a little effect on the binding
of APC1 to CSN2 or SUG1, respectively (compare Fig. 3A,
lane 6 with Fig. 3B, lane 6). In summary, the binding of CSN2 to
the 19S base sub-complex of the proteasome seems to be depen-
dent on its integration in the COP9 complex. In contrast, the
interaction between APC1 and CSN2 is NEM resistant which has
the notion that CSN2 could bind directly to APC/C. As APC1
interacts with the base of the 19S proteasome even in the presence
of NEM, we concluded that the COP9 complex is not exclusively
necessary to recruit the APC/C to the proteasome.
The COP9 signalosome specifically affects stability of APC/C
target proteins. In the past few years there were several reports
available that COP9 influences the stability of many different
targets of the ubiquitin/proteasome pathway.21-23 This activity
is mediated by different factors bound to the complex. For
the proteasome as well as APC/C, we performed additional protein-
protein interaction assays. Hereby, we found that endogenously
expressed CSN2 interacts with at least four subunits of the base
of the 19S proteasome namely SUG1 [proteasome subunit p54/
SUG; also named thyroid hormone receptor-interacting protein 1
(TRIP1)], TBP1 (TATA-box binding protein), 26S proteasome
subunit S5B and S4. Additionally, an interaction between the
COP9 subunit CSN2 and the 20S proteasome subunit alpha 6 was
also detectable using the protein-protein complex identification
assay (Suppl. Figs. 4–9). To further confirm these interactions, we
performed a number of CoIP experiments using several antibodies
that recognized different subunits of the proteasome (Suppl. Fig.
10). These results strongly suggest that endogenously expressed
CSN2 is integrated in vivo in a protein complex containing the
19S sub-complex base.
Since CSN2 interacts with both the 26S proteasome and the
APC/C we asked whether the COP9 signalosome is necessary to
connect these two protein complexes resulting in degradation of
APC/C in the proteasome. N-ethylmaleimide (NEM) is able to
completely disrupt the COP9 complex.20 In this case, a complete
disruption of the COP9 complex using NEM would prevent the
APC/C from binding to the proteasome. For that purpose, U2OS
cell extract was preincubated with either 5 mM NEM or an equal
volume ethanol as the solvent control for 1 hour followed by
capturing the COP9 and 19S base containing protein complexes
using specific antibodies (Fig. 3). In CoIP experiments with a
Regulation of APC/C by COP9
Figure 2. Interaction between CSN2 and APC1 is cell cycle dependent. U2OS cells were synchronized at different cell cycle stages. Synchronization
in specific phases was achieved by following treatments. G1 phase: 0.5 mM mimosine 24 h. G1/S phase: 2 mM hydroxy urea over night. S phase/
G2 phase: U2OS cells were blocked in G1 by serum starvation for 24 h following addition of fresh medium containing FBS supplemented with
2.5 mM thymidine over night. M phase: cells were blocked in mitosis by incubation with 100 ng/ml nocodazol for 16–18 h. Cells were lysed using
lysis buffer and cleared by centrifugation (15 min; 15,000 rpm) at 4°C. The supernatants corresponding to protein extracts of different cell cycle stages
were immediately used for CoIP experiment using a specific antibody against CSN2 for precipitation. APC1 was detected in an immunoblot using a
specific anti-APC1 antibody (lanes 2–7), or as negative control, an unspecific antibody (lane 1). As a control for equal protein loading corresponding
actin levels were shown.
2044 Cell Cycle2009; Vol. 8 Issue 13
Regulation of APC/C by COP9
Figure 3. The interaction of CSN2 with APC1 and SUG1, respectively, is
differentially affected by NEM. (A) Left panel: In CoIP experiments using
crude U2OS cell extract, specific antibodies against SUG1 and APC1,
respectively, co-precipitate (IP) CSN2 as detected in an immunoblot (IB)
(lane 2 and 3). Right panel: The APC1 antibody was capable to co-precip-
itate CSN2 (lane 6) after preincubation of the U2OS cells with 5 mM NEM
for one hour. In contrast, the SUG1 antibody failed to precipitate CSN2
after the mentioned treatment with NEM (lane 7). (B) Left panel: Antibodies
against APC1 as well as CSN2 were both able to co-precipitate SUG1
from crude U2OS lysate (lane 2 and 3) as shown by immunoblot with
a specific SUG1 antibody. right panel: A specific APC1 antibody was
able to co-precipitate SUG1 from crude U2OS lysate preincubated with
5 mM NEM for one hour (lane 6). A specific CSN2 antibody failed
to co-precipitate SUG1 from U2OS cells treated as described above
(lane 7). An unspecific antibody which was used as negative control was
not able to precipitate neither CSN2 nor SUG1 from untreated U2OS cells
or treated U2OS cells (A, lane 1 and 5; B, lane 1 and 5).
example, the attached deubiquitinase USP15 mediates degradation
of marked proteins.20 For this reason, we hypothesized that the
signalosome affects protein stability of APC/C targets. It was
shown that overexpression of CSN2 leads to a de novo assembly of
the COP9 complex.10 Therefore, we transfected U2OS cells with
an expression plasmid coding for CSN2 or, as control, the empty
vector. Cells were harvested, lysed and subjected to SDS-PAGE.
Thereafter, known APC/C target proteins including cyclin A,
cyclin B, CDC6 and SnoN were analyzed in the differentially
transfected U2OS cells by immunoblotting. In three independent
experiments we detected decreased protein signals for CDC6
and SnoN in cells transfected with the CSN2 expression plasmid
compared to mock-transfected control cells. In contrast, an
increased signal corresponding to cyclin A was detected in cells
overexpressing CSN2 due to the specific expression plasmid. No
differences were found regarding cyclin B signal in specific trans-
fected cells and control cells (Fig. 4A).
In order to proof these findings, we knocked down CSN5
by RNA interference using a specific CSN5 siRNA. As control,
an unspecific non-silencing siRNA (nsc) was used. In contrast
to CSN2 when a downregulation of this subunit is lethal, the
CSN5 subunit of the COP9 is dispensable for complex formation
but recruits several enzymes, e.g, a deubiquitinase to the signalo-
some.24-26 We manipulated the activity of CSN2 and CSN5 as
both subunits are located in different sub-complexes of the COP9
signalosome.27 The COP9 is active if the complex is complete.
Cells were harvested 72 h after siRNA transfection and proteins
were separated on a SDS gel and analyzed by immunoblotting
using specific antibodies. Signal intensities of immunoblots corre-
sponding to CDC6, cyclin A, SnoN and cyclin B were compared
between U2OS cells treated with the specific CSN5 siRNA
and control cells treated with unspecific, non-silencing siRNA
(Fig. 4B). Thereby, we found an increase of both SnoN and CDC6
in CSN5 downregulated U2OS cells. By contrast, there was a
lower signal corresponding to cyclin A detectable in cells treated
with specific CSN5 siRNA. No difference in signal intensity of
cyclin B was detectable between CSN5 downregulated cells and
controls. It is obvious that both CDC6 and SnoN were downregu-
lated if a subunit of COP9 was overexpressed. In line with this
both CDC6 and SnoN were up-regulated when a COP9 subunit
was depleted. The results regarding cyclin A were completely
opposite. The overexpression of a COP9 subunit correlates well
with cyclin A upregulation well as the depletion of COP9 subunit
downregulates the cyclin A expression.
Thereafter, we were interested whether the half life of the
proteins was altered by transfection with a vector encoding CSN2
or, as a control, an empty vector. Cycloheximide was applied 48 h
after transfection and the cells were harvested 1, 4, and 8 hours,
respectively, after incubation. The protein stability of APC/C
targets is influenced by CSN2 as shown in Figure 5. Both, SnoN
and CDC6 were more rapidly degraded in cells over-expressing
CSN2 due to a specific vector encoding CSN2 compare to control
cells as densitometrical determined. A significant decreased of
SnoN and CDC6, respectively, was detectable even four hours
after cycloheximide induction. The half life of cyclin B was not
affected by CSN2 overexpression at all. Surprisingly, we detected
a decreased kinetic of cyclin A degradation in cells over-expressing
CSN2 compared to control cells. These results confirm our find-
ings regarding to the alteration of protein levels of APC/C targets
after over-expression of CSN2 or down-regulation of CSN5 as
www.landesbioscience.com Cell Cycle2045
CSN2 overexpression leads to genomic instability. Both, the
COP9 signalosome and the APC/C are well characterized regula-
tors of the cell cycle. For this reason, we asked if overexpression of
CSN2 causes an altered cell cycle distribution. Fluorescence acti-
vated cell sorting (FACS)-technique was applied using U2OS cells
fixed 48 h after transient transfection with the CSN2 encoding
vector or, as a control, with the empty vector followed by staining
of the DNA with propidium iodide and analysis of DNA content.
Thereby, we did not detect any significant changes of cell cycle
transition of cells expressing higher levels of CSN2 (Fig. 6A).
Consistently, there were only very slight differences in cell
viability determined by colony forming assay (data not shown).
Recently, it was shown that APC/C is involved in the segregation
of chromatides and is necessary for accurate DNA replication.16,28
The temporally regulated degradation of its targets seems suffi-
cient to enable genetic stability. Since our data suggest that CDC6
protein level is altered by CSN2 overexpression, we hypothesized
that this could lead to genetic instability. Using a 50K microarray
analysis from Affymetrix to investigate single nucleotide poly-
morphism (SNP), we detected a higher rate of both deletions and
duplications of genes in a pool of U2OS cells stably transfected
with a vector coding CSN2 compared to a pool of control cells
stably transfected with the empty vector (Fig. 6B). DNA instability
was not detectable at specific sites in the genome, but they were
randomly distributed over the whole genome (data not shown).
These results suggest that overexpression of CSN2 leads to genetic
instability and indicate that the proper regulation of the APC/C
dependent protein degradation by the signalosome seems impor-
tant for genetic stability. Interestingly, we did not detect an effect
on the cell cycle phase distribution, although cells revealed an
increase in genome instability. A possible explanation could be the
lack of functional p53 in these cells. The rate of apoptosis of CSN2
over-expressing cells was analyzed by monitoring the caspase 3/7
activity. Hereby, we found only a slight reduction of the apoptotic
cell fraction of CSN2 overexpressing cells compared to cells treated
with the empty vector (data not shown).
Taken together, the data suggest that CSN2 interacts with the
APC/C and influenced functionally the stability of APC/C targets.
Further, in cells overexpressing CSN2 the genomic stability of
these cells massively perturbed.
In the past few years many publications discussed about the
influence of the COP9 signalosome on the ubiquitin/proteasome
pathway. It could be shown the direct interaction of Flag-CSN2
with the 26S proteasome in mouse B8 fibroblasts.10 In the present
study, we extended the panel of interacting partners of COP9
in vivo by the detection of several protein interactions between
CSN2, a subunit of COP9, and subunits of the base of the 19S
proteasome as well as of the 20S proteasome. These protein inter-
actions seemingly depend on COP9 as CSN2 did not bind to the
base of the 19S proteasome after disruption of the signalosome in
vitro by NEM. Furthermore, we found that CSN2 binds at least
three proteins of the anaphase promoting complex/cyclosome
(APC/C). Beside the SKP1 Cullin F-box (SCF) complex, APC/C
Regulation of APC/C by COP9
Figure 4: Overexpression of CSN2 as well as downregulation of CSN5
influenced expression of targets of the APC/C. (A) CSN2 was transiently
overexpressed in U2OS cells by transfection with a pcDNA3-CSN2 plas-
mid (lane 2) and compared with cells transfected with the empty vector
(lane 1). Transfected cells were lysed and several targets of the APC/C
including cyclin A, CDC6, SnoN, and cyclin B were analyzed by immuno-
blot using specific antibodies. (B) Protein extracts of U2OS cells transfected
with either a specific CSN5 siRNA (lane 2) or an unspecific non-silencing
siRNA (nsc; lane 1) were subjected to immunoblotting against cyclin A,
CDC6, SnoN, and cyclin B using specific antibodies. (C) The results of
three independent experiments of CSN5 knock down and over-expression
of CSN2 were densitometrically determined and summarized. The Y-axes
shows the fold expression compared to controls transfected with the empty
vector which were set to 1.
well as disintegration of the COP9 signalosome. Based on these
findings, it seems that the COP9 signalosome influences stability
of several APC/C targets on protein level.
2046 Cell Cycle2009; Vol. 8 Issue 13
by APC/C. Additionally, it seems that COP9 is involved in regula-
tion of the cell cycle. Taken together, CSN2 overexpression seems
to lead to genomic instability. On the other hand, these cells pass
cell cycle checkpoints without being arrested and are prevented
from apoptosis leading to further accumulation of genomic altera-
Materials and Methods
Cell culture and cell cycle synchronization. Human U2OS
osteosarcoma cells were cultured in DMEM supplemented with
10% fetal bovine serum. Cells were harvested at 70–90% conflu-
ence using PBS with 0.05% EDTA and trypsin. In order to
synchronize U2OS cells, cells were submitted to different treat-
ments as described elsewhere.35 Mitosis: Cells were blocked
in mitosis by incubation with 100 ng/ml nocodazol (Sigma)
for 16–18 h. Mitotic cells were detached by mitotic shake-off
and cleared from the medium by centrifugation for 5 min on
1500 rpm at room temperature. G1 phase: Synchronization in G1
was achieved by mimosine treatment (0.5 mM, for 24 h, Sigma).
G1/S phase: to accumulate cells at the G1/S transition hydroxy urea
(Sigma; 2 mM, overnight) was used. S phase/G2 phase: In a first
step, U2OS cells were blocked in G1 by serum starvation for 24 h.
Afterwards fresh medium with FBS supplemented with thymidine
(Sigma; 2.5 mM over night) was added. The cell cycle block was
released by washing cells twice with PBS and normal medium was
applied. Cells were harvested after 4 h reflecting to S phase or after
8 h corresponding to G2 phase, respectively.
Cells were lysed in a buffer containing 100 mM sodium
phosphate pH 7.5, 5 mM EDTA, 2 mM MgCl2, 0.1% CHAPS,
500 µM leupeptin, and 0.1 mM PMSF. After centrifugation
(15 min; 15,000 rpm; 4°C) the supernatant was immediately
Protein-protein complex identification assay. The protein-
protein interaction assay was performed as described before.36 In
short, 4 µl protein A (Sigma) was bound to 20 µl of Interaction
Discovery Mapping (IDM) beads (Bio-Rad) overnight at 4°C.
After discarding the supernatant the pellet was washed twice with
a buffer containing 50 mM sodium acetate pH 5.0. Afterwards,
unspecific binding sites were blocked by incubation with a buffer
is responsible for the temporally degradation of cyclins during cell
cycle.13 As CSN2 is known to interact with cullins we suggested
that it interacts directly with the cullin-like protein APC2.24,25
Consistent with that, we found CSN2 to be bound to the APC/C
even in the absence of an intact signalosome. Whether the COP9
complex recruits the APC/C to the proteasome or if these interac-
tions are independent remains unknown. Our data clearly show
that APC/C is able to bind to the proteasome even if the COP9
complex was disrupted. In our opinion, there are two possible
explanations for this. On the one hand, the signalosome and the
lid of the 19S proteasome compete for the APC/C mediating
substrate specifity. Such a competitive binding of both complexes
was shown for the binding to the base of the 19S proteasome.10
On the other hand, it might be possible that the APC/C directly
binds to the base. In this case, the COP9 would be able to influ-
ence the APC/C independently of proteasome binding.
The specificity of the APC/C for its multiple substrates is
regulated by the competitive binding of Cdh1 and Cdc20 to the
E3 ligase dependent on the cell cycle phase.29-32 We found that
degradation of cyclin A, SnoN and CDC6 was regulated by the
COP9 complex while protein levels of cyclin B which is degraded
at the end of mitosis was not affected.33 Consistent with these data,
we found that CSN2 did not bind to the APC/C during mitosis.
Because of these reasons, we speculate that the regulation of APC/C
by the COP9 is both high substrate and cell cycle specific.
Because of its role as a regulator of DNA duplication and chro-
matide separation, the APC/C was shown to mediate genomic
stability.16 Otherwise, a stabilization of APC/C targets may initiate
a perturbation of these processes causing a p53 response by deregu-
lating G1 phase.30 Consistently, we showed that overexpression of
CSN2 and the resulting influence on the APC/C targets led to
genomic alterations. This instability seems not to affect cell cycle
distribution or cell viability. An explanation for unaffected cell
cycle despite deregulation of APC/C targets stability might be as
U2OS cell possess an inactivated form of p53. In a large genetic
screen in yeast, the COP9 was shown to have a great impact on
cell cycle without complete explanation for the results.34 Our
presented data show, that the signalosome regulates both the SCF
and also the second important pathway for degradation of cyclins
Regulation of APC/C by COP9
Figure 5. CSN2 deregulates stability of APC/C targets. U2OS cells were transiently transfected either with pcDNA3-CSN2 or the empty vector. 48 hours
after transfection fresh medium with 50 µg cycloheximide/ml was applied and the cells were harvested after the indicated time points (1 hour: lane 1
and 4; 4 hours: lane 2 and 5; 8 hours: lane 3 and 6) and lysed using a lysis buffer. Lysates of the transfected cells treated with cycloheximide were
analyzed by western blotting with antibodies specific for SnoN, Cdc6, Cyclin A and Cyclin B, respectively.
www.landesbioscience.com Cell Cycle2047
Regulation of APC/C by COP9
SDS-PAGE for separation of containing proteins followed by
staining with Simply Blue Safe Stain (Enhanced Coomassie,
Invitrogen). Specific gel bands were excised, destained, and dried
followed by rehydration and digestion with 10 µl of a trypsin solu-
tion (0.02 µg/µl; Promega) at 37°C overnight. The supernatants
of the in-gel digestions were applied directly to a gold (Au) arrays
(Bio-Rad). After addition of the matrix (CHCA), peptide fragment
masses were analyzed by SELDI-MS. A standard protein mix (all-
in-1 peptide standard mix; Bio-Rad), including Arg8-vasopressin
(1082.2 Da), somatostatin (1637.9 Da), dynorphin (2147.5 Da),
ACTH (2933.5 Da), and insulin beta-chain (3495.94 Da) was
used for calibration. Proteins were identified using the fragment
masses searching in a publicly available database (Profound; prowl.
Co-immunoprecipitation. Specific antibodies which recognise
CSN2 (rabbit polyclonal), S4 (rabbit polyclonal; Abcam), SUG1
(25D5, mouse monoclonal; Abcam), p42 (p42-23, mouse mono-
clonal; Calbiochem), TBP1 (TBP1-19, mouse monoclonal), TBP7
(TBP7-27, mouse monoclonal), proteasome α 6 subunit (MCP20,
mouse monoclonal; Abcam), APC1 (H-300, rabbit polyclonal;
Santa Cruz) or, as negative control, normal rabbit IgG (Pepro Tech
Inc.) were bound on protein A-agarose beads. The antibody loaded
beads were incubated with 150 µl of crude U2OS cells extract
for 1 hour at 4°C. Unspecific bound proteins were removed by
three washes with CoIP buffer containing 20 mM HEPES/KOH
pH 8.0, 50 mM KCl, 0.1 mM EDTA and 0.05% CHAPS.
containing 0.5 M Tris/HCl pH 9.0 and 0.1 % Triton X-100 for
1 hour at room temperature. The beads were washed three times
with 1x PBS. Thereafter, a specific antibody which recognized
human CSN2 (rabbit polyclonal), or normal rabbit IgG (Pepro
Tech Inc.; Rocky Hill, NJ) as negative control, in 50 mM sodium
acetate pH 5.0 was applied to the beads and allowed to bind at
room temperature for 1 hour in an end-over-end mixer. The specific
anti-CSN2 antibody was described before.37 Unbound antibodies
were removed by washing in 50 mM sodium acetate. Following
two washes with 1 x PBS the beads were incubated with at least
100 µl of crude U2OS cell extract for two hours at 4°C in an end-
over-end mixer. Unbound proteins were removed by sequential
washes in 0.5 M sodium chloride, 0.1 % Triton X-100 and PBS.
Afterwards proteins were eluted from the IDM beads by 25 µl
50% acetonitrile/0.5% trifluoroacetic acid and gently vortexed for
30 minutes. Five microliters of the eluted samples were subjected
on an activated H50 ProteinChip Array (Ciphergen Biosystem,
Inc., Fremont, CA) and the array was analyzed in a ProteinChip
Reader (series 4000; Ciphergen, Bio-Rad) according to an auto-
mated data collection protocol by SELDI-MS. This includes an
average of 265 laser shots to each spot with a laser intensity of
2300 nJ and 3500 nJ (20–200 kDa), respectively, dependent on
the measured region (low = 2.5–20 kDa and high = 20–200 kDa,
respectively) and an automatically adapted detector sensitivity.
The volume of the eluted samples was reduced to a maximum
of 10 µl using a speed-vac (ThermoServant) and subjected to
Figure 6. CSN2 overexpression promotes genetic instability. (A) U2OS cells were transfected with pcDNA3-CSN2 or the empty vector and ethanol
fixation following staining of DNA with propidium iodide and measuring cell cycle distribution by FACS analysis. (B) Stably transfected U2OS cells
with pcDNA3-CSN2 or the empty vector, respectively, were analyzed with a 50K microarray (Affymetrix), which shows the copy number of genomic
sequences. CSN2 overexpressing cells exhibit loss as well as well as gain of allels compared to cells transfected with the empty vector control. The DNA
changes in the control cells transfected with the empty vector were normalized to a copy number of 2 chromosomes. Dots above the black line imply
DNA amplifications in cells transfected with the pcDNA3-CSN2 vector (copy number of 3; black arrow); dots below the black line shows DNA losses
in CSN2 over-expressing cells (copy number 1; white arrow). The experiment was done twice. (C) Percentage DNA loss or DNA gain, respectively, of
the whole genome of the CSN2 over-expressing cells.
2048 Cell Cycle2009; Vol. 8 Issue 13
Regulation of APC/C by COP9
Harvested transiently transfected cells and medium were collected
together to obtain all cell cycle phases and apoptotic cell popula-
tion. After centrifugation (5 min, 1500 rpm, RT) the resulting
pellet was washed twice in PBS. For fixation, 1 ml of ice-cold
70% ethanol was added slowly to the cells. Afterwards, the cells
were resuspended carefully and incubated for 1 h on ice. After
centrifugation and washing in PBS, the cells were resuspended
in 300 µl PBS with 1 mg/ml RNase A (Fermentas). The samples
were incubated for 10 min at room temperature before staining
the DNA with 50 µg propidium iodide (Sigma) for 10 min at RT.
Fluorescent labeling was measured with a FAC-Scan using Cell
Quest Software (Becton Dickinson).
Apoptosis assay. Changes of the apoptotic population of
U2OS cells transiently transfected with either pcDNA3-CSN2 or
the empty vector, respectively, were analyzed with the Apo-ONE
Homogeneous Caspase-3/7 Assay (Promega) according to
manufacturer´s instructions. Hereby, the amount of total protein
of cell lysates was measured with a NanoDrop device (ND-1000
spectrometer; Peqlab, Erlangen, Germany) and samples were
diluted to same concentrations. Afterwards, samples were measured
on a fluorescence plate reader with excitation maximum at 498 nm
and emission maximum of 521 nm.
Genetic stability. The gene chip copy number analysis was
based on the detection of single nucleotide polymorphisms (SNP).
Therefore, a 50K microarray from Affymetrix was used to detect
differences of chromosomal imbalance between U2OS cells
overexpressing CSN2 and untreated U2OS cells. The procedure
was performed according to the Mapping 100K Assay manual
from Affymetrix (www.affymetrix.com). First, total DNA was
isolated from U2OS cells using a Qiagen Mini Kit following
by digestion with XbaI restriction enzyme and amplification by
one-primer PCR. After amplification the genomic DNA was puri-
fied, fragmented and labeled. Finally, the DNA fragments were
hybridized to the 50.000 SNPs on the XbaI microarray surface.
After 16 h hybridization, the DNA was stained with streptavidin
phycoerythrin (SAPE), washed and scanned. The difference in
fluorescence intensity was caused by variation in concentration of
bound DNA. This difference indicates a loss or gain of chromosomal
material. Following primers were used. XbaI adaptor sequence:
primer, 001: 5'-ATTATGAGCACGACAGACGCCTGATCT-3'.
This work was supported by a grant of the Interdisciplinary
Center for Clinical Research (IZKF), Jena to C.M., DFG
BA1457/3 to A.B. and of the German Federal Ministry of
Education and Research (BMBF).
Supplementary materials can be found at:
Afterwards beads were boiled in 4 x SDS loading buffer (200 mM
Tris-Cl pH 6.8, 4% SDS, 30% Glycerol, 10% β-mercaptoethanol,
0.002% Bromophenol blue) and bound proteins were subjected to
a 10% SDS–PAGE and analyzed by immunoblotting. For detec-
tion in immunoblots following antibodies apart from the ones
mentioned above were used: CSN2 (goat polyclonal; Abcam),
β-actin (A266, rabbit polyclonal; Sigma), cyclin A (C-19, rabbit
polyclonal; Santa Cruz), cyclin B1 (M-20, rabbit polyclonal; Santa
Cruz), CDC6 (0.T.17, mouse monoclonal; Santa Cruz), SnoN
(H-317, rabbit polyclonal; Santa Cruz) and CSN5 (FL-334, rabbit
For CoIP experiments using crude U2OS cell extract prein-
cubated with N-ethylmaleimide (NEM), lysates were split into
two aliquots. The sample was treated either with 5 mM NEM or
with an equal volume of ethanol at 4°C for 1h in an end-over-end
Small interfering RNA-mediated knockdown of CSN5. For
knockdown by RNA interference the following small inter-
fering RNA (siRNA) duplex oligonucleotides was used in
this study that was based on the human cDNA encoding
CSN5. CSN5: 5'-GCAAUCGGGUGGUAUCAUAdTdT-
3'(sense), 5'-UAUGAUACCCGAUUGCdAdT-3' (antisense)
(QIAGEN GmbH, Hilden, Germany); nonsilencing control
siRNA: 5'-UUCUCCGAACGUGUCACGUdTdT-3' (sense),
(QIAGEN GmbH, Hilden, Germany). U2OS cells (3 x 105) were
seeded in a six-well plate 24–48 h before transfection and were
50% confluent when siRNA was added. The amount of siRNA
duplexes applied was 1.5 µg/well for CSN5. Transfection was
performed using the amphiphilic delivery system SAINT-RED
(Synvolux Therapeutics B.V., Groningen, The Netherlands) as
described.38 Briefly, siRNA was complexed with 15 nmol of trans-
fection reagent and added to the cells for 4 hours. Subsequently,
2 ml of culture medium was added and incubation proceeded for
Transfection. U2OS cells were seeded into 6-well plates at a
density of 2.5 x 105 cells per well 24 h prior to transfection. Fresh
DMEM supplemented with 10% FCS was added four hours
before transfection. Afterwards, transfection was performed with
CaPO4 as described.22 Hereby, one microgram of pcDNA3-Linker
or pcDNA3-CSN2 were used per well. Cells were washed three
times with 2 ml PBS and new medium was applied after 24 h.
In case of transient transfection, cells were harvested 48 h after
initial transfection. In case of cycloheximide chase, 50 µg cyclo-
heximide per ml medium were applied and cells were collected
after 1, 4, and 8 hours, respectively. For stable transfection, cells
were selected with 0.5 mg/ml geneticin sulphate (G418). Medium
and hygromycin were replaced every 2–3 days until all cells were
died in a transfection control experiment. All experiments were
performed at least three times.
Densitometrical assessment. Signal intensities of corresponding
proteins were densitrometrical assessed using the Lab Image 1D
program (Kapelan Bio-Imaging, Leipzig, Germany) according to
the manufacturer’s instructions.
FACS. Cell cycle distribution of U2OS cells were shown
by fluorescence activated cell sorting (FACS) as described.39
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