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On again – off again: COP9 signalosome turns the
key on protein degradation
Albrecht G von Arnim
The COP9 signalosome is an eight-subunit protein complex that
regulates protein ubiquitination and protein turnover in a variety
of plant developmental and physiological contexts, including
light-regulated development, hormone signaling, and defense
against pathogens. In all eukaryotes tested, the COP9
signalosome is able to posttranslationally modify the cullin
subunit of E3-ubiquitin-ligase complexes by cleaving off the
covalently coupled peptide, Nedd8. Two contrasting models
ascribe stimulatory or inhibitory roles to the modification of cullin/
E3 that is mediated by the COP9 signalosome. There is
considerable disagreement as to whether Nedd8 cleavage
underlies all of the COP9 signalosome’s numerous cellular and
phenotypic effects. This is because macroscopic phenotypes do
not always correlate with biochemical defects in COP9
signalosome mutants. Additional biochemical activities,
including protein interactions with the cellular machineries for
protein phosphorylation, protein turnover, and protein
translation, have been proposed to account for the role of the
COP9 signalosome in development and disease.
Addresses
Department of Botany, The University of Tennessee, Knoxville,
Tennessee 37996-1100, USA
e-mail: [email protected]
Current Opinion in Plant Biology 2003, 6:520–529
This review comes from a themed issue on
Cell Biology
Edited by Takashi Hashimoto and Dirk Inzé
1369-5266/$ – see front matter
ß 2003 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.pbi.2003.09.006
Abbreviations
AUX
AUXIN
axr1
auxin resistant1
COP9
CONSTITUTIVELY PHOTOMORPHOGENIC9
CSN
COP9 signalosome
DDB1
DAMAGED DNA-BINDING1
E1
activating enzyme for ubiquitin or Nedd8
E2
conjugating enzyme for ubiquitin or Nedd8
E3
ligase enzyme for ubiquitin or Nedd8
eIF3
eukaryotic translation initiation factor 3
IAA
INDOLEACETIC ACID
JAMM Jab1/MPN domain metalloenzyme
Kip1
Kinase inhibitor protein1
MPN
Mpr1-Pad1-amino-terminal
PCI
proteasome–COP9 complex–elF3
Pcu4
Schizosaccharomyces pombe cullin4
RAR1
REQUIRED FOR DISEASE RESISTANCE1
Rbx1
Ring-box protein1
Rub
Related to ubiquitin
SCF
SKP1–CDC53/cullin–F-box protein
Current Opinion in Plant Biology 2003, 6:520–529
SGT1
Spd1
TIR1
UFO
Suppressor of the G2 allele of skp1-4
S-phase delayed1
TRANSPORT INHIBITOR RESPONSE1
UNUSUAL FLORAL ORGANS
Introduction
The COP9 signalosome (CSN) is a complex of about
500 kDa that was first discovered through loss-of-function
mutations that repressed photomorphogenetic development in Arabidopsis [1,2]. Mutations in all six of the eight
CSN subunits tested (CSN1, CSN2, CSN3, CSN4, CSN7
and CSN8) destabilize the entire complex and therefore
cause almost identical seedling phenotypes. These phenotypes include a failure to etiolate upon germination in
darkness, concomitant derepression of normally lightinducible gene transcription and seedling lethality
(reviewed in [3,4]). The first CSN gene to be identified,
COP9/CSN8, was originally named for its constitutively
photomorphogenetic mutant phenotype. A compelling
array of experimental evidence, which is briefly summarized below, indicates that the CSN functions in the
regulation of protein turnover by E3 ubiquitin ligases
and the 26S proteasome. This review focuses on recent
experiments on the CSN of green plants, fungi, and
metazoans that have illuminated the biochemical activity
of the CSN.
The 26S proteasome is a 2 MDa macro-molecular
machine that consists of a cylindrical catalytic core
(20S) and up to two regulatory particles on either side
(19S) (Figure 1). The regulatory particles are composed of
a base and an eight-subunit lid complex. Substrate proteins, which are normally polyubiquitinated, are deubiquitinated by a proteolytic activity that is most likely
associated with the regulatory particle non-ATPase11
(Rpn11) lid subunit [5,6] and then transferred to the
base. Substrate proteins are unfolded by the base complex in a process involving ATP hydrolysis and fed into
the proteolytic core (reviewed in [7]).
Proteins are fated for 26S-proteasome-mediated turnover
by tagging with ubiquitin, a 76-residue protein. After
activation by a generic ubiquitin-activating enzyme
(E1), ubiquitin is transferred to a member of the family
of ubiquitin-conjugating enzymes (E2). Much of the
specificity that allows the identification of the correct
target protein (i.e. ‘substrate’) is provided by the E3
ubiquitin ligases, which are thought to number in the
hundreds in Arabidopsis [7]. These E3 ubiquitin ligases
complex with an E2 and guide the ubiquitination of the
substrate (Figures 1 and 2). The SCF (SKP1–CDC53/
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COP9 signalosome and protein degradation von Arnim 521
Figure 1
are the consequence of the CSN’s role in protein turnover
or whether the CSN has functions that are independent of
protein turnover.
26S Proteasome
Structure of the COP9 signalosome
Ub
Lid
Ub
Ub
E2
E3
Base
E1
Core
19S
20S
Ub
Ub
Substrate
Ub
Current Opinion in Plant Biology
Ubiquitination and degradation by the 26S proteasome. Ubiquitin (Ub) is
passed over a bucket brigade of three enzymes, an E1 activating
enzyme, an E2 ubiquitin conjugase and an E3 ubiquitin ligase. E3
ubiquitin ligases catalyze the transfer of ubiquitin onto a specific target
protein (i.e. proteasome ‘substrate’), which may be tagged for protein
turnover by yet other modifications (e.g. phosphorylation). Degradation
of a ubiquitinated substrate in the 20S core of the proteasome is
contingent on deubiquitination by the lid and unfolding by the base.
The COP9 signalosome is essentially conserved in all
eukaryotes. Following the purification of the CSN from
cauliflower [2], orthologous CSN complexes have been
characterized in mammals [14,15], Drosophila [16],
fission yeast [17,18] and more recently Caenorhabditis
elegans [19] and Aspergillus [20]. Mutant defects in yeast
and Arabidopsis CSN subunits have been rescued with
orthologs from Drosophila, C. elegans and mammals
[12,19,21,22], indicating substantial functional conservation. In budding yeast, a CSN-like complex that
includes the CSN5/Rri1 protein is responsible for cullin
deneddylation, although this complex is smaller and not
well-conserved in comparison with the CSN from other
organisms [12,13,23,24].
The COP9 signalosome consists of eight subunits, ordered
CSN1 to CSN8 by decreasing molecular mass [25]. Two of
these subunits, CSN5 and CSN6, contain the conserved
MPN domain. The remaining six are characterized by a
PCI domain (proteasome–COP9 complex–eukaryotic
translation initiation factor 3 [eIF3] domain). Both of these
domains have primarily been found in two other protein
complexes, the regulatory lid subunit of the 26S proteasome and eIF3 [26]. The PCI and MPN domains are
thought to be important in subunit–subunit interactions
that hold the CSN together [16,27–29]. No high-resolution
three-dimensional structure is available for the CSN as
yet, but electron microscopy and comprehensive pairwise
yeast-two-hybrid experiments with mammalian and Arabidopsis CSN have resulted in fairly consistent interaction
maps of the subunits. These maps suggest that the CSN
has an asymmetric and compact, rather than an extended
or ring-like, architecture [27,29,30].
cullin–F-box protein) complexes are a major subclass of
E3s. SCF complexes comprise the Ring-box protein1
(Rbx1, which has a central role in ubiquitin transfer), a
large scaffold protein (cullin), the SKP1 adaptor protein
and, as a determinant for substrate recognition, one of
numerous proteins carrying the SKP1-interacting ‘F-box’
domain. In all of the eukaryotes studied, the cullin subunit is subject to an idiosyncratic posttranslational modification, that is, the covalent attachment of the small
ubiquitin-like protein Nedd8 (Related to ubiquitin
[Rub]; Table 1) on a specific lysine residue. Neddylation
stimulates the E3 ligase activity of the corresponding
SCF-complex in vitro by facilitating the interaction
between the cullin and an E2 conjugating enzyme [8]
or by preventing the binding of an inhibitor of SCF
complex assembly [9]. As detailed below, the only known
enzymatic activity that is indigenous to the CSN complex
is the proteolytic cleavage of Nedd8 from the cullin
(‘deneddylation’) [10,11]. A putative zinc-binding motif
known as the Mpr1-Pad1-amino-terminalþ (MPNþ) or
c-Jun activation domain binding protein1 (Jab1)/MPN
domain metalloenzyme (JAMM) motif within the
CSN5 subunit is thought to form part of the active site
for the deneddylation reaction ([12,13]; Figure 2).
In biochemical size fractionation experiments, a few of
the CSN subunits appear in monomeric or otherwise
CSN-independent forms [16,31,32,33,34]. Mutations
in CSN5 preferentially destabilize the non-CSN forms
of CSN4 and CSN7 [33], suggesting the existence of a
pared down version of the CSN (‘mini-CSN’) [34]. CSN5
that is outside the CSN may be preferentially cytoplasmic
rather than nuclear-localized [31,34], and this may reflect
a nuclear export activity of overexpressed CSN5 [34].
Overall, however, the functional significance of these
‘monomeric’ or ‘mini-CSN’ subunits is unclear.
Although originally described as a repressor of lightregulated gene expression, and more recently as a regulator of protein turnover, the CSN appears to regulate
numerous cellular activities, including protein kinases,
nuclear protein localization and cell cycle progression
[3,4]. One open question is whether all of these activities
Aside from the eight core subunits of the CSN, numerous
other proteins copurify with the CSN, depending on
species and isolation conditions [26,35,36]. An interaction
between the CSN and subunits of its distant cousin, the
eIF3 complex, has been observed in several species. Up to
three subunits of eIF3, most consistently eIF3e but also
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Current Opinion in Plant Biology 2003, 6:520–529
522 Cell biology
Figure 2
Neddylation
AXR1/
ECR1
E1
Deneddylation
3
Nedd8
1
5
Nedd8
4
7
COP9
Signalosome
2
6
EXnHXHX10D
JAMM-motif
RCE1
E2
8
Nedd8
AUX/IAA
TIR1
Nedd8
E3
ASK1
RBX1
Cullin1
F-boxprotein
SKP1-homolog
SCF-type
Conventional E3 ubiquitin ligase
DDB2
Nedd8
RBX1
DDB1
Cullin4
Unconventional E3 ligase
Current Opinion in Plant Biology
Neddylation and deneddylation pathways. The Nedd8 peptide is transferred onto cullin via a pathway that biochemically resembles ubiquitination but
is genetically distinct. Cullins are scaffold subunits of a subset of E3 ubiquitin ligases. The figure shows the core structure of a conventional
Arabidopsis cullin complex, SCFTIR1, in complex with a substrate (i.e. AUX/IAA protein). A newly recognized E3 complex, which is active in the repair
of mammalian DNA damage [59], is shown at the bottom of the figure. It appears as if the function of DDB1 is analogous that of the adaptor protein
SKP1/ASK1, whereas DDB2 might function as specificity determinant, analogous to an F-box protein. The Arabidopsis gene products that are
responsible for Nedd8 activation (E1) and Nedd8-conjugation (E2) are shown. The E3 ligase for Nedd8 is probably RBX1 [64]. The deneddylation
activity of the CSN requires a conserved motif (JAMM motif) that is found in the CSN5 subunit. The Nedd8 peptide is known as Rub1 in yeast
and Arabidopsis, it is referred to as Nedd8 in this review for simplicity.
Table 1
Dictionary of proteins involved in protein turnover and related processes.
Name used in this
review (species)
Function
Other names
Nedd8
AXR1/ECR1 (At)
RCE1 (At)
RBX1
SKP1
Cullin
CSN1
CSN5
eIF3e
Modifying peptide for cullins
Heterodimeric E1 (Nedd8-activating) enzyme
E2 (Nedd8-conjugating) enzyme
Subunit of SCF complexes, also functions as the E3 for Nedd8
Subunit of SCF complexes, binds F-box protein
Subunit of SCF complexes
CSN subunit with PCI domain
CSN subunit with MPNþ/JAMM motif
eIF3 subunit with PCI domain
Rub1 (Sc, At)
APP-BP1/Uba3 (Mm)
Ubc12
ROC1 (Mm), Hrt1 (Sc)
ASK1 (At)
Cdc53 (Sc), Pcu1 (Sp)
Caa1 (Sp), FUS6 (At), COP11 (At)
Jab1 (Mm), AJH1 (At), Sgn5 (Hs), Rri1 (Sc)
Int-6, Int6 (Mm), Yin6 (Sp), Pci8p (Sc)
Abbreviations: AJH1, Arabidopsis JAB1 HOMOLOG1; APP-BP1, AMYLOID PRECURSOR BINDING PROTEIN1; ASK1, Arabidopsis SKP1
HOMOLOG1; At, Arabidopsis thaliana; Caa1, Check1 arrest attenuator1; ECR1, E1 carboxyl terminus related1; FUS6, FUSCA6;
Hs, Homo sapiens; Int-6/Int6, mouse mammary tumor virus integration site number6; Mm, mammalian; ROC1, REGULATOR OF CULLINS1;
Sc, budding yeast; Sgn5, Signalosome subunit5; Sp, fission yeast.
Current Opinion in Plant Biology 2003, 6:520–529
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COP9 signalosome and protein degradation von Arnim 523
eIF3c and eIF3h, associate with the CSN in Arabidopsis
and mammalian cells [37–39]. Curiously, the closest budding yeast homolog of eIF3e [40], named Pci8p, cooperates with other putative CSN subunits in cullin
deneddylation [13,23,24]. Moreover, Pci8p interacts biochemically with other yeast CSN subunits and contributed to the efficient nuclear localization of CSN5/Rri1
[13,23,24]. In light of the overwhelming evidence that
the CSN is a regulator of protein turnover (see below),
these data insinuate a novel functional, as opposed to
just an evolutionary, relationship between the regulation
of translation initiation by eIF3 and protein turnover by
the CSN.
In mammalian cells, the signalosome copurifies with at
least three protein kinases [41,42], which originally
prompted the name ‘signalosome’. These findings may
reflect the CSN’s spatial sequestering of generic protein
kinases for specific signaling applications; for example,
for labeling proteins for ubiquitination and turnover
(reviewed in [36]).
The COP9 signalosome is involved in diverse
plant signaling pathways
Although the CSN was originally identified as a repressor
of photomorphogenesis, genetic manipulations that
reduced CSN activity while avoiding early loss of function proved that the CSN supports plant growth and
development more broadly. For example, RNA silencing
of the Arabidopsis CSN5 subunit, for which no mutation
has become available because of genetic redundancy,
caused reduced auxin signaling [31,43]. Silencing of
CSN3 and CSN6 gave rise to floral developmental problems that were reminiscent of those caused by mutations
in the F-box protein UNUSUAL FLORAL ORGANS
(UFO) [44,45]. Phenotypes produced by the deliberately
incomplete transgenic rescue of a csn1-null mutation
revealed that the floral ‘B domain’ transcription factor
APETALA3 remains underexpressed when CSN1 is
compromised [21,46,47]. The floral phenotype can
be suppressed by overexpressing UFO, and the CSN
associates with SCFUFO [47]. A similar scenario, unfolding in the arena of plant pathogen interactions, suggests
that the CSN also regulates resistance to tobacco mosaic
virus downstream of the N gene [48] and to powdery
mildew in barley [49]. In these cases, the resistance
depends on two other interaction partners of SCF-type
E3 ligases, SGT1 and REQUIRED FOR DISEASE
RESISTANCE1 (RAR1) [48,49], implying that protein
degradation and the CSN have a role in disease resistance.
Furthermore, the CSN supports systemic plant defense
responses, as exemplified by the jasmonate response
downstream of the SCF complex that contains the
CORONATINE INSENSITIVE1 (COI1) F-box protein
(i.e. SCFCOI1) [50]. Together, these data demonstrate
that the CSN regulates various developmental pathways
by interacting with multiple SCF complexes. Yet, given
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the pleiotropic phenotypes of csn mutants in postembryonic development, it is striking that no early embryodevelopment functions whatsoever have been ascribed
to the Arabidopsis CSN.
CSN as a regulator of protein turnover by
the 26S proteasome
A number of early or circumstantial pieces of evidence
suggested that the CSN is a regulator of protein turnover.
First, mutations in CSN genes inhibit the turnover of the
Arabidopsis LONG HYPOCOTYL5 (HY5) and HY5
HOMOLOG (HYH) basic leucine zipper (bZip) transcription factors [51,52]. Second, null mutations in the
CSN cause a phenotype that is all but identical to that
caused by mutations in COP1 and COP10. COP1 is
believed to be part of a non-SCF E3 ubiquitin ligase
[53] and COP10 resembles the E2 ubiquitin-conjugating
enzymes [54]. COP10 can physically interact with both
CSN and COP1, although it is not known how the three
function together. Perhaps COP1 and COP10 are part of a
non-SCF E2/E3 ligase complex that plays a key role in
the regulation of early seedling development by light.
The COP1 and CSN null phenotypes would be expected
to resemble each other if COP1 were the first strictly
CSN-dependent E3 ligase to be active during plant
development. Third, the CSN is related evolutionarily
and structurally to the proteasome lid [27,29]. Fourth, the
CSN may be able to interact with the proteasome directly
in fungi and plants [24,55,56]. Fifth, a lid-like complex
appears to detach from the proteasome in csn mutants
[57]. Sixth, csn mutants accumulate elevated amounts of
ubiquitinated proteins [44,45]. And finally, mammalian
CSN was first discovered as a contaminant of 26S proteasome preparations [14].
These observations suggested, but by no means proved,
that CSN’s immediate function is in protein turnover. In
fact, other studies have suggested alternative scenarios
that are encapsulated by the terms ‘protein kinase platform’ [41], ‘transcriptional coregulator’, ‘nucleocytoplasmic trafficking’, and ‘translational control’ (reviewed in
[4,26,36]). However, each of these alternative models is
awaiting a combination of strong biochemical and genetic
support. What, then, is the irrefutable evidence in favor of
‘protein turnover’? A number of key discoveries were
published in a pair of articles in 2001 [10,43]. First, the
CSN was found to co-purify with mammalian, fission
yeast and Arabidopsis SCF-type E3 ligase complexes.
Second, the cullin subunits of fission yeast, budding yeast,
and Arabidopsis SCF complexes are conjugated with the
small peptide Nedd8 to an abnormally high level upon
mutation or RNA silencing of CSN. And third, CSN
purified from mammalian cells is capable of cleaving
off Nedd8 in a fission yeast csn cell extract [10,43].
Meanwhile, the key role of the CSN in protein turnover
activity in Arabidopsis came into focus when it was
shown that a substrate of the SCFTIR1 complex, the
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524 Cell biology
Table 2
Proteins whose abundance is regulated by the CSN.
(a) Destabilized by CSN (or stabilized in csn mutants)
Arabidopsis:
HY5, HYH [51,52], PSIAA6 [43]
Mammalian:
P53, p27Kip1[34]
Fission yeast:
Spd1 [61]
C. elegans:
Mei-1/Katanin [19]
Drosophila:
Cyclin E [22]
(b) Stabilized by CSN
Mammalian:
c-Jun, p27Kip1[66]
Abbreviation: Mei, Meiosis1.
auxin-sensitive transcriptional coregulator Pisum sativum
INDOLEACETIC-ACID-INDUCIBLE PROTEIN6
(PSIAA6) [58], was stabilized in CSN5-silenced plants.
The molecular defects in the deneddylation and stabilization of INDOLEACETIC ACID (IAA)-inducible proteins were accompanied by auxin insensitivity in TIR1dependent as well as TIR1-independent auxin signaling
pathways, such as those leading to auxin-insensitive root
growth and reduced apical dominance [43]. Since then,
the hyperneddylation of cullins and the stabilization of
SCF substrate proteins (Table 2) has been confirmed
as a typical phenotype of csn loss-of-function mutants
in all of the eukaryotes examined [18,19,22,24,59].
Thus, the biochemical and genetic data dovetail to assign
an immediate role for the CSN in modifying a cellular
protein, cullin, whose only known role is in the specification of protein turnover events.
The conundrum — biochemical
deneddylation activity does not correlate
with phenotypic effects
The data discussed above do not prove that all the
physiological roles of the CSN are mediated by its
deneddylation activity. If the ‘deneddylation hypothesis’
were the exclusive law of ‘zome-land’, there would be
complete genetic correlation between the deneddylation
activity of the CSN and all aspects of the CSN’s phenotypic spectrum. That is, a quantitative increase (or
decrease) in neddylation caused by a mutation in the
CSN would be accompanied by a corresponding loss(or gain-) of-function at the phenotypic level. As we will
see, this correlation does not hold up, suggesting that the
CSN must have additional molecular activities.
Predictions of the deneddylation hypothesis became
experimentally testable after the exciting discovery that
site-directed amino-acid substitutions in a predicted protease catalytic site of the fission yeast CSN5 subunit and
its budding yeast homolog Rri1p disrupt CSN deneddylation activity without destabilizing the CSN complex
([12]; Figure 2). Corresponding mutations in this socalled MPNþ/JAMM motif of CSN5 from Drosophila,
C. elegans and human cause deneddylation defects in
yeast [19,21,59]. As an aside, although it is quite clear
Current Opinion in Plant Biology 2003, 6:520–529
that the CSN can cleave a single Nedd8 from cullins,
other proteins may contribute to the deneddylation of
cullins by shortening multi-Nedd8 chains down to a
single Nedd8 [60].
Does the JAMM motif control all of the cellular activities
of the CSN? This question can be addressed in two
steps. First, how well do mutations in the JAMM motif
mimic the csn5 loss-of-function phenotype? Second, do
CSN5DJAMM phenotypes mimic those of plants with null
alleles for other CSN subunits? In Arabidopsis, the presumed genetic redundancy between two CSN5 genes
has thwarted the facile testing of this question, but
CSN5DJAMM mutations created in Drosophila, fission
yeast and budding yeast mimicked csn5 null alleles
[12,22], suggesting that the main task of at least the
CSN5 subunit is its isopeptidase activity using the JAMM
motif. In answer to the second question, deletion of
CSN5 is not generally equivalent to deletion of other
CSN subunits, such as CSN4. In Drosophila, the csn5-null
phenotype generally resembles a csn4-null. Both phenotypes involve a failure of oogenesis and arrested development during the third larval instar [16,22,33].
Conspicuous differences between the csn4-null and
csn5-null phenotypes were also seen, such as molting
problems specifically in csn4, melanotic tumors in csn5,
and differences in the spatial expression pattern of the
marker gene oskar during oogenesis [33]. Even considering that such phenotypic differences may be conditioned
in part by differential turnover rates of the maternally
contributed CSN4 and CSN5 subunits, these results
suggest that at least some CSN subunits have roles
beyond chaperoning CSN5’s JAMM motif.
In fission yeast, csn5 mutants have a rather subtle and
conditional phenotype, that is, hyperneddylation of cullins and concomitant enhancement of temperaturesensitive SCF-complex mutations. In comparison, csn1
and csn2 mutants are much more striking as they lack the
G2-DNA-damage checkpoint and display S-phase delay
[17,18,61]. Therefore, the deneddylation of cullins by
CSN5 cannot be the sole function of the CSN, a conclusion confirmed by recent data from Arabidopsis. Informative hypomorphic csn1 transgenic lines were constructed
by partial complementation of a csn1-null with CSN1
deletion transgenes [46,47]. The three different
CSN1-mutant alleles tested each incorporated into the
CSN complex and all rescued the deneddylation defect of
the csn1-null. However, each of these mutants displayed
profound defects during floral development or before.
Given that these defects are though to be due to the
misregulation of the E3 ubiquitin ligase SCFUFO [47],
and together with the fission yeast data, these results shed
serious doubts on the hypothesis that cullin deneddylation is the sole molecular output of the CSN. Deneddylation appears to be necessary but not sufficient for full
CSN activity.
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COP9 signalosome and protein degradation von Arnim 525
Does the COP9 signalosome regulate protein
turnover in more than one way?
It appears, then, that the CSN has multiple biochemical
activities, one of which is deneddylation. Three recent
reports have given us intriguing hints as to the nature of
these activities. Fission yeast CSN1 and CSN2, but not
CSN5, are needed for proteasome-mediated turnover of
the ribonucleotide reductase inhibitor Spd1 (S-phase
delayed1) [61]. Failure to degrade Spd1 inhibits the
nuclear export of ribonucleotide reductase, which in turn
leads to misregulation of deoxynucleotide synthesis and
S-phase delay. Biochemical data suggest that Spd1 is
degraded by an unconventional E3 ligase that is built
around the Schizosaccharomyces pombe cullin4 (Pcu4) and a
yeast homolog of the DDB1 subunit of the DAMAGED
DNA-BINDING protein, but that lacks the conventional
SKP1 and F-box protein subunits [61].
Both mammalian and fission yeast CSN also appear to be
associated with deubiquitinating activities. Data obtained
with mammalian CSN suggest that the CSN5-JAMM
motif supports the cleavage of not only Nedd8 but also
mono-ubiquitin [59]. Moreover, the CSN copurifies with
yet another strong deubiquitinating activity [59] that is
not CSN5-JAMM dependent; in fission yeast this activity
is encoded by Ubp12 [62]. The deubiquitinating activity might counteract the ubiquitin ligase activity of CSNassociated E3 complexes [59], or it may serve to reverse
accidental mis-ubiquitination of F-box substrate adaptor
proteins in the SCF-type E3 complexes [62].
What would be the purpose of cullin
neddylation and deneddylation?
Biochemical data suggest that neddylation increases the
affinity of the cullin for the E2 enzyme [8] and genetic
data provide evidence that neddylation stimulates E3
activity [63]. It has been suggested that cycles of neddylation and deneddylation are needed for cullin/E3
function (‘cycling model’) [10,64]. Perhaps deneddylation
is needed to exchange a ‘spent’ E2 protein for a ubiquitincharged E2 [19], although it cannot be ruled out that
the E2 is recharged with ubiquitin without dissociating
from the SCF complex. The alternative model posits
that a specific steady-state level of occupancy with
Nedd8 is needed for optimal cullin activity (‘static model’;
Figure 3).
The best argument put forward in favor of the cycling
model is genetic; if the static model were correct, then
mutations in the neddylation pathway and the deneddylating CSN should cause opposing and mutually suppressing phenotypes. To test this prediction, it is necessary to
measure whether SCF-complex substrate proteins such as
AUX/IAA proteins are stabilized not only in csn mutants
[43] but also in neddylation pathway mutants. The test is
not easy to perform because knockout of neddylation
causes early embryo defects in Arabidopsis [63], whereas
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knockout of deneddylation (i.e. the CSN) does not. Mild
mutant alleles in the Arabidopsis neddylation pathway
have, however, resulted in the subtle stabilization of
AUX/IAA substrate proteins, consistent with the cycling
model [64,65]. Meanwhile, the epistatic interaction
between a mutation in the neddylation pathway, auxin
resistant1 (axr1), and silencing of CSN5 has been interpreted as additive, consistent with the cycling model
[43]. Additional genetic interactions within the neddylation pathway [64] and between axr1 and the CSN-interacting COP10 gene add weight to the evidence [65],
notwithstanding that the precise relation between
COP10 and the CSN is not known. Similar, rather than
opposing, phenotypes of neddylation and deneddylation
mutants have also been observed in budding yeast (slow
growth; [10,12]) and C. elegans (mitotic defects; [19]).
Together, these data are explained elegantly by the
cycling model, although they do not rule out a static
model, because the final phenotypes being measured,
growth rate or auxin response or the like, may be determined by the ubiquitination of both positive and negative pathway intermediates.
Additional biochemical evidence that Nedd8 cycles on
and off the cullin E3 subunit during ubiquitination has
led to a more elaborate version of the cycling model
[62]. Many F-box substrate adaptor proteins in E3
complexes are themselves subject to ubiquitination and
rapid turnover, presumably a form of collateral damage
sustained during their exposure to E2 ubiquitin conjugase
as ubiquitin can be attached to almost any surface-exposed
lysine residue. According to this model (Figure 3), the
CSN serves as a safe harbor for the reassembly of (F-box)
substrate adaptor proteins into (SCF type) E3 complexes.
E2 activity is kept at a low level by the CSN because cullin
is deneddylated and accidental ubiquitination is counteracted by the CSN-associated Ubp12 deubiquitinating
enzyme [62]. One prediction of the model, that the
half-life of F-box proteins will be reduced in a csn mutant
even as the half-life of regular substrate proteins increases,
is borne out by initial experimental data. The model also
predicts that non-neddylated cullin is preferentially associated with the CSN, whereas neddylated cullin is not.
However, neddylated cullin is preferentially found in
higher-molecular-weight complexes, bona fide CSN–SCF
supercomplexes, contrary to expectations [50,66]. Thus,
this model will presumably be fine-tuned after additional
experimental testing.
Despite the beauty and popularity of the ‘cycling model’,
certain data are more consistent with a static model. CSN
inhibits SCF E3 ligase activity in vitro [11], perhaps
because deneddylated cullin preferentially binds an inhibitor of SCF complex assembly [9]. Second, in C. elegans
neddylation and deneddylation mutants can suppress
each others’ phenotypes [19]. This is exactly what is
expected according to the static model, even though the
Current Opinion in Plant Biology 2003, 6:520–529
526 Cell biology
Figure 3
Static model
F
R
Deneddylation
S
Cullin
Inactive
Neddylation
COP9
Signalosome
E2 -Ub
F
N
R
S
Cullin
SCF-complex
active
Ub- Substrate
Substrate
Ubiquitination
Cycling model
Deneddylation
F
E2
Inactive
N
R
Cullin
R
S
COP9
Signalosome
Ub- Substrate
N
R
E2 -Ub
Ub- F
S
Cullin
DUB
Neddylation
E2 -Ub
Substrate
F
Cullin
S
Ubiquitination
F
N
R
Cullin
S
SCF-complex
Active
Current Opinion in Plant Biology
Two alternative models to explain the biological function of cullin neddylation and deneddylation. The static model proposes that neddylation
activates E3 ligase activity whereas deneddylation inhibits it, perhaps by regulating the binding of E2 ubiquitin-conjugase. The cycling model proposes
that both neddylation and deneddylation are needed for sustained E3 ligase activity. The figure illustrates two mutually compatible rationales for
cycling. Cycling may be needed to replace a spent E2 enzyme with a ubiquitin-charged E2 [19] or to replace accidentally ubiquitinated F box proteins
[62]. F, F-box protein; N, Nedd8; R, RBX1; S, SKP1.
authors interpreted the sum of their data as favoring a
cycling model [19]. Third, in mammalian cells, p27Kip1,
an inhibitor of the G1!S cell cycle transition and a
SCFSKP2 substrate protein, is stabilized by the CSN in
an in vitro cell lysate assay. This is consistent with
inhibition of protein turnover by CSN-mediated deneddylation, as predicted by the static model. Furthermore,
carefully controlled microinjection of the CSN caused
blockage of the G1!S transition in vivo in mammalian
cells, consistent with the stabilization of p27Kip1 [66].
Tests to distinguish between the cycling model and the
static model should become easier once more examples of
partnerships between F-box proteins (e.g. TIR1) and
their direct targets (e.g. AUX/IAA proteins) are precisely
Current Opinion in Plant Biology 2003, 6:520–529
defined. We must also take into account that the F-box
proteins may themselves be targets of ubiquitination and
turnover, or that additional negative regulatory proteins
(e.g. cyclin E upstream of p27Kip1) that tag specific substrate proteins for turnover by phosphorylating them are
subject to regulation by protein turnover.
Conclusions
The CSN, which was initially characterized as a repressor
of photomorphogenesis, is involved in numerous additional plant processes. These include, but are not
limited to, auxin signaling, flowering, and plant defense
responses. The CSN regulates protein turnover by interacting with E3 ubiquitin ligases. The CSN deneddylates
www.current-opinion.com
COP9 signalosome and protein degradation von Arnim 527
the cullin subunit of a subset of E3 ligases through the
JAMM motif of the CSN5 subunit; however, the CSN
also has additional biochemical activities whose full spectrum remains to be resolved. Research on the CSN will
illuminate the role of neddylation in the function of
cullin-containing protein complexes. Currently, a static
model and a cycling model are being considered. Curiously, biochemical experiments appear to favor the static
model, whereas genetic studies tend to favor the cycling
model. How CSN activity is itself regulated by developmental or external signals remains poorly understood.
Acknowledgements
I regret that work published before 2001 could not be cited comprehensively
because of space constraints. Research in my laboratory is supported by
grants from the National Science Foundation, the US Department of
Energy, and the US–Israel Binational Science Foundation. Thanks to
Byung-Hoon Kim and Daniel Chamovitz for discussion and critical
comments on the manuscript.
References and recommended reading
Papers of particular interest, published within the annual period of
review, have been highlighted as:
of special interest
of outstanding interest
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Current Opinion in Plant Biology 2003, 6:520–529
528 Cell biology
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Current Opinion in Plant Biology 2003, 6:520–529
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COP9 signalosome and protein degradation von Arnim 529
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PROTEIN1, which function in DNA repair (Figure 2). Repression of the E3
involves a strong CSN-associated deubiquitinating (ubiquitin isopeptidase) activity. Knockdown of CSN5 by RNAi does not crumble the rest of
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downstream of the DNA damage checkpoint pathway [17], control DNA
replication by degradation of the ribonucleotide reductase inhibitor Spd1.
They might achieve this through an association with an unconventional
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The authors report correlations between cullin deneddylation, p27Kip1
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Their data suggest that deneddylation inhibits SCF activity (i.e. the ‘static
model’).
62. Zhou C, Wee S, Rhee E, Naumann M, Dubiel W, Wolf DA:
Fission yeast COP9/signalosome suppresses cullin activity
through recruitment of the deubiquitylating enzyme Ubp12p.
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67. Doronkin S, Djagaeva I, Beckendorf SK: CSN5/Jab1
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by activating a meiotic checkpoint. Development 2002,
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Current Opinion in Plant Biology 2003, 6:520–529