Flag-CSN2 was permanently expressed in mouse B8 fibroblasts and Flag pull-down experiments revealed the forma-tion of an intact Flag-CSN complex, which is associated with the 26S proteaso
Trang 1Xiaohua Huang1, Bettina K J Hetfeld1, Ulrike Seifert2, Thilo Ka¨hne3, Peter-Michael Kloetzel2, Michael Naumann3, Dawadschargal Bech-Otschir4and Wolfgang Dubiel1
1 Division of Molecular Biology, Department of Surgery, Charite´, Universita¨tsmedizin Berlin, Germany
2 Institute of Biochemistry, Charite´, Universita¨tsmedizin Berlin, Germany
3 Institut fu¨r Experimentelle Innere Medizin, Universita¨t Magdeburg, Germany
4 MRC Human Genetics Unit, Western General Hospital, Edinburgh, UK
The COP9 signalosome (CSN) has been discovered in
plant cells as a negative regulator of
photomorphogen-esis [1] It occurs in all eukaryotic cells and consists of
eight core subunits, CSN1–CSN8 [2] Six of the CSN
subunits contain PCI (proteasome, COP9 signalosome,
initiation factor 3) domains and two contain MPN
(Mpr-Pad1-N-terminal) domains [3] These two
charac-teristic domains have been found in three protein
com-plexes: the CSN, the 26S proteasome lid complex (lid)
and the eukaryotic translation initiation factor 3
(eIF3) complex The two domains are composed of
about 150–200 amino acids at the N- or C-terminus of the CSN subunits The PCI domain has been demon-strated to be important for interactions between CSN subunits Thus, it might have scaffolding function [4,5] The MPN+ or JAMM domain of CSN5 is responsible for an intrinsic metalloprotease activity of the complex [6] The function of the MPN domain of CSN6 is unknown
The CSN is associated with a large number of pro-teins [7], most of which are substrates or regulators
of the ubiquitin (Ub) system Analysis of associated
Keywords
COP9 signalosome; lid; p53; PCI domain;
26S proteasome
Correspondence
Division of Molecular Biology, Department
of Surgery, Charite´, Universita¨tsmedizin
Berlin, Monbijoustr 2, 10117 Berlin,
Germany
Fax: +49 30 450522928
Tel: +49 30 450522305
e-mail: wolfgang.dubiel@charite.de
(Received 9 May 2005, accepted 6 June
2005)
doi:10.1111/j.1742-4658.2005.04807.x
The COP9 signalosome (CSN) occurs in all eukaryotic cells It is a regula-tory particle of the ubiquitin (Ub)⁄ 26S proteasome system The eight sub-units of the CSN possess sequence homologies with the polypeptides of the 26S proteasome lid complex and just like the lid, the CSN consists of six subunits with PCI (proteasome, COP9 signalosome, initiation factor 3) domains and two components with MPN (Mpr-Pad1-N-terminal) domains Here we show that the CSN directly interacts with the 26S proteasome and competes with the lid, which has consequences for the peptidase activity
of the 26S proteasome in vitro Flag-CSN2 was permanently expressed in mouse B8 fibroblasts and Flag pull-down experiments revealed the forma-tion of an intact Flag-CSN complex, which is associated with the 26S proteasome In addition, the Flag pull-downs also precipitated cullins indi-cating the existence of super-complexes consisting of the CSN, the 26S pro-teasome and cullin-based Ub ligases Permanent expression of a chimerical subunit (Flag-CSN2-Rpn6) consisting of the N-terminal 343 amino acids
of CSN2 and of the PCI domain of S9⁄ Rpn6, the paralog of CSN2 in the lid complex, did not lead to the assembly of an intact complex showing that the PCI domain of CSN2 is important for complex formation The consequence of permanent Flag-CSN2 overexpression was de-novo assem-bly of the CSN complex connected with an accelerated degradation of p53 and stabilization of c-Jun in B8 cells The possible role of super-complexes composed of the CSN, the 26S proteasome and of Ub ligases in the regula-tion of protein stability is discussed
Abbreviations
CSN, COP9 signalosome; PCI, proteasome-COP9 signalosome-initiation factor 3; MPN, Mpr-Pad1-N-terminal; Ub, ubiquitin.
Trang 2enzymatic activities implies that the CSN is a
compo-nent of the Ub pathway Originally CSN associated
kinase activity has been described [8] Recently a
num-ber of kinases associated with the complex have been
identified [9,10] CSN associated kinases phosphorylate
important cellular proteins such as p53 and regulate
the stability of the tumour suppressor towards the Ub
system [11] It has been demonstrated that subunits of
the eIF3 complex interact with components of the CSN
[12,13] The exact function of this interaction is
unknown Recently many groups found that the CSN
is associated with Ub ligases in particular with
cullin-based ubiquitinating enzyme complexes Cullins 1–7 are
scaffolding proteins forming a family of highly diverse
Ub ligase complexes, which are responsible for the
ubiq-uitination of important cell cycle regulators and
tran-scription factors So far it has been shown that the CSN
interacts with cullin 1 to cullin 4 [14–18] An important
reason for the relationship between the CSN and the
cullin-based Ub ligase complexes seems to be the
intrin-sic metalloprotease activity of the CSN, which removes
the Ub-like protein Nedd8 from cullins [6] Cycles of
neddylation and deneddylation of cullins seem to
regu-late the ubiquitinating activity of the cullin-based Ub
ligases [19] The metalloprotease activity of CSN5 is also
able to deubiquitinate proteins [15] In addition, the
CSN is associated with a deubiquitinating enzyme called
Ubp12 in the fission yeast Schizosaccharomyces pombe
[20], which counteracts autocatalytic degradation of
components of cullin-based Ub ligases [20,21]
All described CSN interactions clearly indicate that
the complex is a component of the Ub system
More-over, there are data on a direct interaction of the CSN
with the proteolytic machinery of the Ub system, the
26S proteasome Several years ago it was shown that the
CSN cofractionates with the 26S proteasome from
human cells [8] The yeast two-hybrid screen revealed
that the C-terminal domain of the Arabidopsis at CSN1
subunit interacts with at Rpn6 of the 26S proteasome
lid [22] Recently gel filtration size-fractionation of
material from cauliflower in the presence of ATP and
phosphatase inhibitors indicated that the CSN1 and
CSN6 subunits coelute in the same fractions as subunits
of the 26S regulatory complex Moreover,
coimmuno-precipitations revealed the existence of super-complexes
consisting of the CSN, the 26S proteasome and
cullin-based Ub ligase complexes [23] Although these results
indicate an association of the CSN and the 26S
pro-teasome, the exact mode of this interaction, the role of
PCI domains, and its consequences for the stability of
cellular proteins is not known
Here we show for the first time that the CSN
directly interacts with the 26S proteasome and that the
purified human CSN has impact on 26S proteasome activity The CSN seems to compete with the 26S pro-teasome lid In cells permanently overexpressing CSN2 the amount of the CSN complex increases, which has consequences for the stability of p53 and c-Jun
Results
Flag pull-downs with lysates from B8 fibroblasts permanently expressing Flag-CSN2 contain subunits of the 26S proteasome
To study the interaction between the CSN and the 26S proteasome the human CSN2 cDNA was cloned into
an eukaryotic expression vector coding for an N-ter-minal Flag-tag The construct was permanently expressed in mouse B8 fibroblasts Human and mouse CSN2 are identical on the amino acid level and there-fore we expected the integration into the mouse CSN Interestingly, permanent expression of the Flag-CSN2 construct in HeLa cells was not successful, because cells died (data not shown)
First it was tested whether the Flag-CSN2 was integ-rated into large protein complexes Glycerol gradient centrifugation and subsequent western blots revealed that the Flag-CSN2 sediments into the same fractions as the CSN In Fig 1A, middle panel, two bands are seen with the anti-CSN2 Ig The upper band corresponds to Flag-CSN2 and the lower one is endogenous CSN2, which occurred in a ratio of approximately 1 : 1 Both Flag-CSN2 and endogenous CSN2 were efficiently integrated into complexes Due to de novo assembly the total amount of the CSN complex increased in B8 cells permanently expressing Flag-CSN2 (see below)
To study CSN associated proteins, lysate of B8 cells expressing Flag-CSN2 was incubated with Flag-beads After washing, bound proteins were specifically eluted with the Flag-peptide The SDS⁄ PAGE and subse-quent Coomassie stain of the Flag pull-down is shown
in Fig 1B Selected bands were cut out and analyzed
by mass spectrometry revealing the presence of all core CSN subunits The eluted proteins were analyzed under nondenaturing conditions In a nondenaturing gel followed by western blotting the complex migrates exactly to the position of the CSN It can be detected
by the anti-Flag as well as by the anti-CSN3 Ig A smear was detected in the region of the 20S⁄ 26S pro-teasome with the anti-Flag Ig (Fig 1C)
Previously it has been shown that the purified CSN phosphorylates c-Jun and p53 by CSN associated kin-ases [10,24] To test whether eluted proteins were able
to phosphorylate c-Jun and p53, kinase assays were performed As shown in Fig 1D, the two proteins as
Trang 3well as recombinant CSN2, another substrate of CSN
associated kinases [25], were phosphorylated in a
curcumin-sensitive manner; curcumin being a typical
inhibitor of CSN associated kinases [10,26]
To study association of the CSN with the 26S
pro-teasome western blots were performed The data are
summarized in Fig 1E First proteins of the Flag
pull-downs were probed with antibodies against subunits of
the CSN (Fig 1E, left panel) All subunits of the CSN
tested were detected supporting our data obtained by
mass spectrometry (Fig 1B) Figure 1E (right panel)
shows the western blots with antibodies against
sub-units of the 26S proteasome In all Flag pull-downs
the components of the 26S proteasome base S1⁄ Rpn2, S4⁄ Rpt2, S6b ⁄ Rpt3 and S6a ⁄ Rpt5 and the 20S protea-some were clearly identified Under our conditions the subunits of the lid S10a⁄ Rpn7 and S12 ⁄ Rpn8, but not S13⁄ Rpn11, were detected There were no unspecific proteins eluted from the Flag-beads as demonstrated
by control pull-downs with lysate from B8 cells
The CSN binds directly to the 26S proteasome and most likely competes with the lid
It has been shown that the CSN forms super-com-plexes with the 26S proteasome and with cullin-based
E
Fig 1 Flag-CSN2 permanently expressed in
B8 cells is integrated into an intact CSN
complex, which interacts with the 26S
pro-teasome (A) Glycerol gradient centrifugation
was performed with lysates of B8 cells
expressing Flag-CSN2 Subsequent western
blotting with glycerol gradient fractions
using antibodies against Flag, CSN2 and the
20S proteasome (20S) revealed
sedimenta-tion of the Flag-CSN2 into the same
frac-tions as the CSN complex The asterisk
indicates that the anti-CSN2 Ig interacts
with the Flag-CSN2 (upper band) as well as
with endogenous CSN2 (lower band) (B)
Mass spectrometry of selected bands from
SDS ⁄ PAGE of Flag-CSN2 pull-downs (C)
Flag-CSN2 pull downs were analyzed by
nondenaturing gel electrophoresis
Subse-quently proteins were blotted to
nitrocellu-lose and tested with anti-Flag and anti-CSN3
Igs Positions of the CSN, the 20S and the
26S proteasome were determined with
spe-cific antibodies (D) Flag-CSN2 pull-downs
were used as a source of kinase activity in
kinase assays Recombinant CSN2, p53 and
c-Jun were used as substrates The reaction
was inhibited by the kinase inhibitor
curcu-min (E) Western blot analyses with
antibod-ies against CSN subunits (left panel) and
antibodies against 26S proteasome subunits
(right panel) were performed with lysate
from B8 cells (controls) and lysate from B8
cells permanently expressing Flag-CSN2.
Trang 4Ub ligases [23] However, direct interaction between
the CSN and the 26S enzyme had not been
demonstra-ted so far Therefore, we performed in vitro
immuno-precipitations with isolated human CSN and 26S
proteasome The two purified particles were
preincu-bated with a molar ration of 1 : 1 for 30 min in the
presence of ATP Then immunoprecipitation was
per-formed with the anti-CSN7 Ig or with preimmune
serum as a control The data are shown in Fig 2A
The western blot of the precipitate revealed the
coim-munoprecipitation of the CSN and S1⁄ Rpn2 of the
26S proteasome indicating a direct interaction of the
two complexes
To test whether the CSN competes with the 26S
pro-teasome lid complex purified CSN and 26S propro-teasome
were incubated as in Fig 2A using different molar rations of the two complexes After incubation immuno-precipitations with a monoclonal antibody against the 20S proteasome subunit a6⁄ C2 were performed The data in Fig 2B demonstrate that CSN1 and CSN5 can
be well detected in immunoprecipitates after incuba-tion with a 20-fold molar excess of the CSN Of note, after long-term exposure CSN1 and CSN5 were also seen in samples with 1 : 1 ratios (data not shown) In contrast, the lid subunit S10a⁄ Rpn7 was well seen in the absence of the CSN, but was not detectable at a molar ratio of 1 : 20 The base subunit S4⁄ Rpt2 and the 20S proteasome did not change depending on the molar ratios of 26S⁄ CSN (Fig 2B)
Based on these data we speculated that the CSN might replace the lid Possible competition between the CSN and the lid complexes might be also reflected by changed proteasome activity To see whether the direct interaction of the CSN with the 26S proteasome has
an effect on proteasome peptidase activity, assays with purified CSN and 26S proteasome and with succinyl-Leu-Leu-Val-Tyr-AMC as substrate were carried out
As shown in Fig 2C, measured fluorescence revealed that 26S proteasome peptidase activity in the presence
of ATP is slightly inhibited by a molar excess of the CSN There was no effect detected without ATP Ubiquitinated proteins are the physiological substrates
of the 26S proteasome Unfortunately, because of the high deubiquitinating activity associated with the CSN [20], the impact of the CSN on the degradation of model ubiquitinated substrates by the 26S proteasome was difficult to estimate under in vitro condition Sub-strates were quickly deubiquitinated before the 26S proteasome had a chance to degrade them (data not shown)
The PCI domain of CSN2 is essential for CSN complex assembly and its interaction with the 26S proteasome
The paralog subunit of CSN2 in the lid complex is Rpn6 We were interested to see whether substitution
of the PCI domain of CSN2 by the PCI domain of Rpn6 has consequences for the complex integration of the chimerical CSN2-Rpn6 protein The cDNA enco-ding the first 343 amino acids of human CSN2 and the PCI domain of human Rpn6 (Rpn6 amino acids 291– 422) were linked together and cloned into an eukaryotic expression vector possessing a N-terminal Flag-tag (Fig 3A) The construct was permanently expressed in B8 cells In glycerol gradients the Flag-chimerical pro-tein sedimented into similar fractions as the CSN, which is shown by western blotting in Fig 3B Again,
C
Fig 2 Direct interaction between the CSN and the 26S
protea-some in vitro (A) Co-immunoprecipitation of the CSN and the 26S
proteasome in vitro using the anti-CSN7 Ig or preimmune serum
(control) Purified CSN and 26S proteasome were incubated at a
molar ratio of 1 : 1 in the presence of ATP The precipitate was
analyzed by western blotting with the anti-S1 ⁄ Rpn2 Ig (B)
Co-im-munoprecipitations of the CSN and the 26S proteasome using the
monoclonal anti-a6 ⁄ C2 Ig Isolated 26S proteasome and different
amounts of the purified CSN were incubated for 30 min in the
pres-ence of ATP After incubation immunoprecipitations were
per-formed and precipitates were analyzed by western blotting using
anti-CSN1, anti-CSN5, anti-S10a ⁄ Rpn7, anti-S4 ⁄ Rpt2 and anti-20S
proteasome Igs (C) Fluorescence was measured with isolated 26S
proteasome (0.15 pmol per sample) in the presence of
succinyl-Leu-Leu-Val-Tyr-MCA as substrate with or without ATP Purified
CSN was added in molar rations indicated.
Trang 5the anti-CSN2 Ig revealed two bands, the
Flag-CSN2-Rpn6 (upper band) and the endogenous CSN2 (lower
band), which were integrated into complexes In this
case the expression of the chimerical protein was
signi-ficantly less than that of the endogenous CSN2
Flag pull-downs were performed as described above
to determine: (a) if the chimerical CSN2-Rpn6 protein
was integrated into an intact CSN or into the 26S
proteasome lid complex; (b) if there was an interaction with the 26S proteasome? Proteins specifically eluted with the Flag-peptide were analyzed with antibodies against subunits of the CSN as well as the 26S protea-some According to the data shown in Fig 3C the Flag-CSN2-Rpn6 protein was not integrated into either an intact CSN or lid complex The Flag pull-downs contained significant amounts of CSN1 protein, but only S1⁄ Rpn2 and traces of S4 ⁄ Rpt2 indicating that there is no interaction with the 26S proteasome complex
Are there CSN-26S proteasome super-complexes?
We were interested to see whether the Flag pull-downs contain additional B8 cell proteins besides the CSN and the 26S proteasome Therefore western blots were carried out after Flag pull-downs using antibodies against p53, c-Jun, cullin 1, cullin 3, Ub, associated kinase CK2a subunit and a subunit of the eIF3 com-plex, INT6 The large number of blots is not shown and the data are summarized in Table 1 Positive reac-tions are indicated Again, controls with B8 cell lysate alone showed that no unspecific proteins were eluted from the Flag-beads In pull-downs with the wild-type Flag-CSN2, p53 and c-Jun, two typical substrates of the CSN [24], were detected In addition, cullin 1 and cullin 3 were found, suggesting an association with cullin-based Ub ligase complexes These results confirm earlier observations on the existence of super-complexes consisting of the CSN, the 26S proteasome and cullin-based Ub ligases [23] The anti-Ub Ig reacted with high-molecular weight material indicating the binding
of Ub conjugates The CSN interaction with CK2 [10] and with INT6 [12] has been published before Table 1 shows that basically none of the tested proteins, except p53, interacted with the Flag-CSN2-Rpn6 chimerical
A
B
C
Fig 3 The PCI domain of CSN2 is essential for CSN complex
for-mation (A) The Flag-CSN2-Rpn6 construct codes for the first 343
amino acids of CSN2 and for the PCI domain of its lid paralog Rpn6
(amino acids 291–422) (B) The Flag-CSN2-Rpn6 chimera was stably
expressed in B8 cells and the cell lysate was analyzed by glycerol
gradients and subsequent western blotting The asterisk indicates
that the anti-CSN2 Ig interacts with both the upper
Flag-CSN2-Rpn6 protein and the lower endogenous CSN2 (C) Western blot
analyses with antibodies against CSN subunits (left panel) and
anti-bodies against 26S proteasome subunits (right panel) were
per-formed with lysate from B8 cells (controls) and lysate from B8 cells
permanently expressing the Flag-CSN2-Rpn6 chimera.
Table 1 Proteins detected in Flag pull-downs by western blotting Flag pull-downs were performed as described (Experimental proce-dures) Eluted proteins were separated by SDS ⁄ PAGE, blotted to nitrocellulose and probed with the antibodies indicated in the table The + symbol indicates a positive antibody reaction; ND, not deter-mined.
Trang 6protein demonstrating that the intact CSN is essential
for most bindings including the interaction with the
26S proteasome and with cullin-based Ub ligases
Changes of the CSN and of CSN substrates in
B8 cells permanently expressing Flag-CSN2 or
Flag-CSN2-Rpn6 chimera
To test whether the CSN was modified in fibroblast
per-manently expressing Flag-CSN2 or Flag-CSN2-Rpn6
western blots with cell lysates were performed using
antibodies against CSN subunits As demonstrated in
Fig 4A, permanent expression of wild-type Flag-CSN2
led to elevated levels of CSN3 and CSN5 subunits in B8
cells suggesting a de novo assembly of the CSN complex
In contrast, expression of the Flag-CSN2-Rpn6 chimera
did not change the amount of CSN subunits in B8 cells
as compared with control cells
Typical CSN substrates p53, c-Jun, p27 and IjBa
are phosphorylated by the associated kinases, which
regulate the stability of the proteins [24] Therefore the
influence of Flag-CSN2 or Flag-CSN2-Rpn6
expres-sion on the stability of these proteins in B8 cells was
studied by western blotting As shown in Fig 4B,
sig-nificant changes of p53 and c-Jun levels were detected
in B8 cells permanently expressing Flag-CSN2 as
compared to control cells While p53 in Flag-CSN2 B8 cells almost completely disappeared, c-Jun was clearly stabilized The impact on p27 and IjBa steady state levels in B8 cells is less pronounced as compared to p53 and c-Jun
Discussion
Here we show for the first time that the CSN directly interacts with the 26S proteasome It can compete with the lid, which has consequences for 26S proteasome peptidase activity In addition, we demonstrate the essential role of the PCI domain of CSN2 for complex formation and consequences of permanently overex-pressed CSN2 for the stability of p53 and c-Jun
The CSN interacts directly with the 26S proteasome and influences proteasome cleavage activity
Although the exact mode of CSN⁄ 26S proteasome inter-action is still obscure, it has been speculated that the CSN might be an alternative lid [27] It has been known for many years that the CSN copurifies with subunits of the 26S proteasome [8] and analyses by mass spectrome-try revealed components of the 26S base in our CSN preparation from red blood cells (our unpublished data)
To study CSN⁄ 26S interaction, Flag-CSN2 was perma-nently expressed in mouse B8 cells Data show that human CSN2 with a Flag-tag at its N-terminus was integrated into a complete mouse CSN complex This is not surprising, as mouse and human CSN2 protein are 100% identical No unspecific proteins were detected in our control pull downs with B8 cell lysate and using the Flag-peptide for specific elution Western blot analysis
of Flag-CSN2 pull-downs revealed the presence of 20S core particle and 26S proteasome base subunits Under our conditions, it was difficult to detect components of the lid (Fig 1E, right panel) The direct interaction between the CSN and the 26S proteasome is shown by
in vitro coimmunoprecipitation (Fig 2A) A possible competition between the CSN and the lid is demonstra-ted by immunoprecipitations after incubations of the 26S proteasome and the CSN at different molar ratios The data shown in Fig 2B demonstrate that a molar excess of the CSN most likely replaces the lid Because
of significant sequence homologies between the compo-nents of the CSN and the lid, it is likely that the two complexes can be substituted by and compete with each other Competition is also indicated by measuring pepti-dase activity with a fluorogenic peptide in the presence
of purified human 26S proteasome and purified human CSN Increasing amounts of the CSN slightly
Fig 4 Permanent expression of Flag-CSN2 causes de novo
assem-bly of the CSN complex in B8 cells connected with degradation of
endogenous p53 and stabilization of c-Jun (A) Lysates of B8 cells
(controls), B8 cells permanently expressing Flag-CSN2-Rpn6 and B8
cells permanently expressing Flag-CSN2 were tested by western
blotting using antibodies against CSN3 and CSN5 (B) Lysates of
B8 cells (controls), B8 cells permanently expressing
Flag-CSN2-Rpn6 and B8 cells permanently expressing Flag-CSN2 were tested
by western blotting using antibodies against Flag, p53, c-Jun, p27
and IjBa The anti-actin Ig was used as an internal control
demon-strating equal loading of proteins.
Trang 7pressed 26S proteasome peptidase activity indicating a
direct interaction with the 26S complex The effect of
the CSN on 26S enzyme activity can be explained by a
conformational change caused through the replacement
of the lid by the CSN At the moment it is unclear
whe-ther this effect of the CSN on 26S proteasome peptidase
activity has physiological relevance
The fraction of CSN particles associated with the
26S proteasome and vice versa is small As seen in the
nondenaturing gel in Fig 1C, most of Flag-CSN2
integrated into the free CSN complex and only small
amounts migrated into regions of the 26S proteasome
This is also reflected by the fact that
immunoprecipita-tions in cell lysates using antibodies against CSN or
26S components sometimes failed to detect
coimmuno-precipitation of the two complexes With phosphatase
inhibitors and ATP included in buffers a fraction of
5–10% of CSN was estimated to be associated with
the 26S proteasome in plant cells [23] Under our
con-ditions without adding ATP or inhibitors of
phospha-tases this fraction seems to be below 5%
The role of the PCI domain of CSN2 for complex
formation
It has been shown before that PCI domains are
important for CSN complex formation [4,28]
How-ever, the exact role of PCI domains in the complex
assembly process or in subunit targeting to the right
complex is still obscure Here we demonstrate that the
PCI domain of CSN2 is essential for the formation of
an intact CSN particle and subsequently for the
forma-tion of super-complexes consisting of the CSN, the 26S
proteasome and Ub ligases
The rationale for generating a chimerical protein
consisting of the N-terminal part of CSN2 and the
PCI domain of its paralog lid subunit, Rpn6, was to
test, whether the PCI domain has the information for
targeting it to the right complex Our data revealed
that the PCI domain of Rpn6 is not sufficient to serve
as an address for the lid complex The chimerical
pro-tein did not show any interaction with lid subunits
In contrast, the CSN2-Rpn6 chimera interacted with
CSN1 obviously independently of the CSN2-PCI
domain The formation of a CSN1–CSN2 subcomplex
with specific function in cell cycle would explain the
exclusive phenotypes obtained with csn1 and csn2
dele-tions in S pombe Knockouts of the two subunits, but
not of other CSN subunits, cause cell cycle delay in
S-phase [14,29] In csn1 and csn2 deletion mutants the
cell cycle inhibitor Spd1 accumulates causing
Suc22-dependent suppression of ribonucleotide
reduc-tase connected with S-phase delay and DNA damage
sensitivity [14] The existence of a CSN1–CSN2 sub-complex has to be verified in the future
Possible functions of CSN-based super-complexes The presented data confirm our earlier findings that overexpression of Flag-CSN2 leads to de novo assembly
of the CSN complex followed by the stabilization of c-Jun transcription factor [30] In addition, here we show that an increase of the CSN in B8 cells signifi-cantly accelerated the degradation of the tumour sup-pressor p53 (Fig 4B) This is not surprising, as the CSN targets p53 to degradation by the Ub system [11] and increased amounts of the CSN accelerate the degrada-tion Currently the question whether this process is mediated by super-complexes consisting of the CSN, the 26S proteasome and Ub ligases cannot be answered At the moment two hypothesis on the function of the complexes can be distinguished First, the super-complexes are proteolytic machineries for the degrada-tion of a certain set of substrates, which are channelled from substrate labelling by phosphorylation and ubiqui-tination to complete proteolytic cleavage In this model the CSN would act as an alternative lid or a platform bringing together specific Ub ligases and the 26S protea-some Second, the CSN is a platform that allows Ub ligase re-assembly This hypothesis is based on an idea
by Wolf and coworkers assuming that the CSN blocks cullin-based complex activity including auto-ubiquiti-nation and provides an environment necessary for the assembly of new cullin-based complexes [21,31] Accord-ing to the second hypothesis one would expect that association of the 26S proteasome to the super-complex might also cause inhibition of the protease to protect cullins and other components from degradation How-ever, our data indicate that the CSN does not efficiently inhibit the 26S proteasome activity in vitro In addition, elevated CSN amounts in B8 cells did not cause a gen-eral inhibition of Ub-dependent proteolysis Therefore
we favour the first model in which super-complexes are large proteolytic machines that carry out specific proteo-lysis Future work is necessary to fully understand the function of CSN⁄ 26S proteasome interaction and of the super-complexes
Experimental procedures
Materials The kinase inhibitor curcumin was obtained from Sigma Antibodies against CSN5 (a gift from B Christy), Ub (Dako, Glostrup, Denmark), Flag (Sigma, St Louis, Missouri, USA), 20S proteasome (Affiniti⁄ Biomol, Hamburg,
Trang 8Germany), p53 (BD Biosciences, San Jose, CA, USA), c-Jun
as well as CK2a (Calbiochem, Schwalbach, Germany), p27
as well as IjBa (Santa Cruz, CA, USA) and cullin 1
(Onco-gene, Schwalbach, Germany) were used in western blots The
anti-S1⁄ Rpn2 Ig was a gift from K Hendil, August Krogh
Institute, Copenhagen, Denmark and the anti-INT6 Ig was a
gift from C Norbury, University of Oxford, UK
Preparations, assays and immunoprecipitation
Preparation procedure for the 26S proteasome as well as
the CSN from human red blood cells and kinase assays
were described before [8] Peptidase assays with the purified
proteasome and with succinyl-Leu-Leu-Val-Tyr-AMC as
substrate were outlined earlier [32] Mass spectrometry was
performed as described [8] In vitro immunoprecipitation
was carried out with 2.3 pmol of the CSN as well as the
26S proteasome First the two particles were incubated in
the presence of 2 mm ATP at 37C for 30 min The
immuno-precipitation with the anti-CSN7 Ig or with the preimmune
serum was carried out as before [10]
Cell culture, Flag pull-downs
B8 mouse fibroblast cells were cultured using Iscove’s
MEM (Biochrom, Berlin, Germany) with 125 lgÆmL)1
G418 Stable transfected B8 cells were established using
cal-cium phosphate precipitation and selected with 1 lgÆmL)1
puromycin Human CSN2 and human CSN2-Rpn6 chimera
cDNAs were cloned into pcDNA3.1 vector (Invitrogen,
Carlsbad, CA, USA) coding for an N-terminal Flag-tag
Expression of Flag-CSN2 or Flag-CSN2-Rpn6 protein was
tested by western blots with an anti-Flag Ig
Flag pull-downs with B8 cells were performed as
recom-mended by the manufacturer (Sigma) Briefly, stably
trans-fected cells were rinsed twice with ice-cold 1· NaCl ⁄ Piand
collected Ice-cold lysis buffer (50 mm Tris⁄ HCl pH 7.4,
150 mm NaCl, 1 mm EDTA, 1% Triton X-100) with
freshly added phenylmethylsulfonyl fluoride (1 mgÆmL)1)
was added to the cells on ice After centrifugation at 15 000 g
for 10 min at 4C supernatants were loaded onto the
pre-pared ANTI-FLAG M2 affinity column After washing
with 20 column volumes of 1· TBS (50 mm Tris ⁄ HCl
pH 7.4, 150 mm NaCl, 1 mm EDTA), proteins were eluted
by competition with the Flag peptide (100 lgÆmL)1)
Eluted proteins were used for western blots, nondenaturing
electrophoresis and kinase assay
Glycerol gradients, nondenaturing electrophoresis
and western blots
Glycerol gradient centrifugation was performed as outlined
before [32] For nondenaturing electrophoresis 2 lL of Flag
pull-downs were separated on a 4–15% (w⁄ v) Phast-gel
(Pharmacia Biotech., Inc.) at 300 VÆh)1 Proteins were blot-ted onto nitrocellulose and probed with an anti-Flag or anti-CSN3 Ig All western blots were developed by ECL technique (Amersham, Buckinghamshire, UK)
Acknowledgements
This work was supported by a grant from the G.I.F., the German-Israeli Foundation for Scientific Research and Development, and grant DU 229⁄ 6–2 from the Deutsche Forschungsgemeinschaft to W D
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