BBRC
Biochemical and Biophysical Research Communications 301 (2003) 392–398
www.elsevier.com/locate/ybbrc

Nedd8-modiﬁcation of Cul1 is promoted by Roc1
as a Nedd8-E3 ligase and regulates its stabilityq
Mitsuru Morimoto,a Tamotsu Nishida,a Yudai Nagayama,a and Hideyo Yasudaa,b,*aDivision of Molecular Biology, School of Life Science, Tokyo University of Pharmacy and Life Science,
1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japanb
Department of Chemistry, School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Received 24 December 2002

Abstract
SCF is a ubiquitin ligase and is composed of Skp1, Cul1, F-box protein, and Roc1. The catalytic site of the SCF is the Cul1/Roc1
complex and RING-ﬁnger protein Roc1. It was shown earlier that when Cul1 was co-expressed with Roc1 in Sf-9 cells in a baculovirus protein expression system, Cul1 was highly neddylated in the cell, suggesting that Roc1 may function as a Nedd8-E3
ligase. However, there is no direct evidence that Roc1 is a Nedd8-E3 in an in vitro enzyme system. Here we have shown that Roc1
binds to Ubc12, E2 for Nedd8, but not to Ubc9, E2 for SUMO-1 and Roc1 RING-ﬁnger mutant, H77A, did not bind to Ubc12. Inin vitro neddylation system using puriﬁed Cul1/Roc1 complex expressed in bacteria, Roc1 promotes neddylation of Cul1. These
results demonstrate that Roc1 functions as a Nedd8-E3 ligase toward Cul1. Furthermore, Roc1 and Cul1 were ubiquitinylated in amanner dependent on the neddylation of Cul1 in vitro. In addition, Cul1 was degraded through the ubiquitin–proteasome pathway,
and a non-neddylated mutant Cul1, K720R, was more stable than wild-type in intact cells. Thus, neddylation of Cul1 might regulate
SCF function negatively via degradation of Cul1/Roc1 complex.
Ó 2003 Elsevier Science (USA). All rights reserved.
Keywords: Cul1; Nedd8; Roc1; SCF; Ubiquitin; Ubiquitin-like protein; Ligase; Ubc12; Cdc34; RING-ﬁnger

Protein degradation by the ubiquitin–proteasome
pathway plays an important role in many biologicalsteps including cell-cycle progression and immune responses. Proteasomes recognize the poly-ubiquitinylated
chain attached to the protein to be degraded. The
ubiquitinylation of proteins requires three enzymes: E1,
ubiquitin activating enzyme; E2, ubiquitin conjugating
enzyme; and E3, ubiquitin ligase [1,2]. The ubiquitin ligase recognizes the protein targeted to be degraded.
There are at least two types of ubiquitin ligases. One
type is ubiquitin ligase having a HECT-domain [3,4] andthe other is that having a RING-ﬁnger domain [5–8].
SCF is thought to be one of the RING-ﬁnger typeubiquitin ligases [9–11] and is composed of Skp1, Cul1
(or CDC53), F-box protein, and Roc1/Rbx1. The cataq
This work was supported in part by grants from the Ministry of
Education, Culture, Sports, Science and Technology of Japan.
*
Corresponding author. Fax: +81-3-3818-4621.
E-mail address: h-yasuda@yk9.so-net.ne.jp (H. Yasuda).

lytic site of the SCF ubiquitin ligase is the Cul1/Roc1
complex and Roc1 is a RING-ﬁnger domain-containing
protein [12–17]. This complex recruits the E2 andtransfers a ubiquitin moiety from E2 to the target protein, which is bound by the F-box protein. This F-box
protein recognizes the protein to be ubiquitinylated [18].
For example, Skp2 recognizes p27kip1 [19,20] in thepresence of cks1 [21,22] and b-TrCP recognizes IjBa
[23,24] and b-catenin [25].
Cul1 in SCF complex was found to be modiﬁed by aubiquitin-like protein Nedd8 [26,27]. Furthermore, Cul2
in VCB [28,29], another E3 complex, and all other cullinfamily members (Cul3, Cul4A, Cul4B, and Cul5) werealso modiﬁed by Nedd8 [30]. Nedd8 modiﬁcation(neddylation) of Cul1 was shown to be necessary for theactivity of SCF toward p27kip1 [31,32] or IjBa [33,34] invitro; and Rub1, a Nedd8 homologue in ﬁssion yeast,
was shown to be essential for cell viability [35]. Recently,
neddylation of Cul1 was shown to enhance the bindingof the ubiquitin-binding form of UbcH4 [36]. These

0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved.
doi:10.1016/S0006-291X(02)03051-6

M. Morimoto et al. / Biochemical and Biophysical Research Communications 301 (2003) 392–398

ﬁndings suggest that the modulation of neddylation
system is crucial for the regulation of SCF function.
Nedd8 modiﬁcation is catalyzed by APP-BP1/Uba3
acting as E1 and Ubc12 as E2 [28,37,38] and may beregulated by Nedd8-E3 ligase. When CDC53, the budding-yeast homologue of Cul1, was co-expressed with
Roc1 in insect cells in a baculovirus protein expression
system, CDC53 was modiﬁed markedly by Nedd8 in a
Roc1-dependent manner [39]. Therefore, Roc1 isthought to be a possible candidate for a Nedd8-E3 ligase, but the enhancement of neddylation by Roc1 invitro has not been shown so far.
Thus, it is an important issue to clarify whether Roc1
functions as a Nedd8-E3 ligase and what is a function of
the neddylation of Cul1. Here we show that Roc1 enhanced the neddylation of Cul1 and that this neddylation destabilized Cul1.

393

assay was performed essentially as described previously using biotinylated ubiquitin [40]. The ubiquitinylated proteins were detected byWestern blotting using peroxidase-conjugated avidin (Extravidin,
Sigma) and the ECL method (Amersham).
In vitro neddylation system. Substrate immobilized on glutathione–
Sepharose 4B was added to the ubiquitinylation reaction buﬀer containing neddylation enzymes (His-APP-BP1, His-Uba3, and Ubc12)
and Nedd8. Modiﬁcation of Cul1 by Nedd8 was done in vitro as described previously by using puriﬁed enzymes and Nedd8 [31]. The
neddylated proteins were detected by Western blotting using avidinperoxidase and the ECL method (Amersham Pharmacia). The Western
blotting of Nedd8 or 6xHis-tagged protein was done by use of antiNedd8 antibody (Alexis) or anti-6xHis antibody (Qiagen), respectively.
Anti-Cul1 antibody (Biosource) was used for detecting Cul1. The
secondary antibody used was peroxidase-conjugated anti-mouse rabbit
antibody (Dako).

Results
Roc1 is Nedd8-E3 ligase

Materials and methods
Cell culture and DNA transfection. Human U2OS cells and COS7
cells were maintained in DullbeccoÕs modiﬁed EagleÕs medium(DMEM) supplemented with 10% fetal bovine serum (FBS). Cells weretransfected with various kinds of plasmids by using FuGene 6 reagent(Roche) according to the manufacturerÕs instructions. When the stability of the Cul1 was monitored, the cells in several dishes were harvested 24 after transfection, mixed together, and then equal numbers ofcells were inoculated again into each dish. Cycloheximide (10 lg/ml)
was added to some dishes to inhibit protein synthesis.
Plasmid constructions and expression of recombinant proteins. To
amplify the His-Cul1 sequence with bacterial ribosomal binding site
(RBS-His-Cul1) by PCR, pTrcHis-Cul1 was used as a template. The
PCR-generated fragment was cloned into the pGEX-6P-2. Next, theresultant plasmids were inserted by the Roc1 cDNA to fuse in-frame
into a glutathione S-transferase (GST) sequence. The ﬁnal constructed
plasmid was designated as pGEX-6P-2-Roc1/RBS-His-Cul1. Recombinant GST- or His-Cul1, GST- or His-Roc1, GST-Skp1, GSTp53, His-Nedd8, His-APPBP1, and His-Uba3 proteins were expressedin a baculovirus expression system using pFastBac vector (Invitrogen)and Sf-9 cells as described previously [31]. GST-Roc1/His-Cul1, HisCdc34, UbcH5c, Ubc12, and Nedd8 were expressed in Escherichia coli
(BL21, LysS). The substitution mutants of Roc1 and Cul1 were prepared by the PCR ampliﬁcation method using speciﬁc mutation
primers.
Protein interaction assay. Sf-9 cells expressing GST-fusion proteins
were suspended in lysis buﬀer containing 50 mM Tris–HCl (pH 7.4),
0.4 M NaCl, 3 mM MgCl2 , 0.1% NP-40, and 1 mM PMSF, and disrupted by sonication. GST-fusion proteins were captured with glutathione–Sepharose 4B (Amersham). GST-fusion proteins immobilized
on beads and puriﬁed Ubc12 (5 lg) or Ubc9 (5 lg) were incubated inlysis buﬀer without NaCl at room temperature for 30 min. The beads
were then washed four times with the lysis buﬀer without NaCl, andthe bound proteins were eluted with SDS sample buﬀer, subjected to
SDS–PAGE, and immunoblotted with anti-T7 tag antibody (Novagen) or anti-His6 tag (Qiagen) and peroxidase-conjugated anti-mousesecondary antibody. The immunoblotted proteins were detected by the
ECL method (Amersham).
In vitro ubiquitinylation assay. Substrate immobilized on glutathione–Sepharose 4B (Amersham) was added to the reaction buﬀer(40 mM Tris–HCl [pH7.4], 5 mM MgCl2 , 2 mM ATP, and 1 mM DTT)
containing ubiquitinylation enzymes (mouse Uba1 and His-Cdc34 or
UbcH5c) and biotinylated ubiquitin. The in vitro ubiquitinylation

Since Nedd8 is a ubiquitin-like proteins, and themechanism of its conjugation to a target protein could
be analogous to that operating in ubiquitin-conjugation,
the Nedd8 conjugation system is thought to require
three enzymatic steps. However, Nedd8-E3 ligase has
not been identiﬁed so far; although it has been suggested
to be Roc1, based on the data that Rbx1/Roc1 enhanced
the neddylation of Cdc53, a yeast homologue of Cul1,
when Sf-9 cells were co-infected with these proteins [39].
To determine whether neddylation of Cul1 depends
on Roc1, we co-infected Sf-9 cells with baculoviruses
containing GST-Cul1 and His-Roc1. In that case, theCul1 was highly neddylated (Fig. 1A, lanes 3 and 4),
whereas when the cells were infected with viruses containing only GST-Cul1, the modiﬁcation was not detected (lane 1), as shown previously [39]. When the Cul1
expressed in Sf-9 cells was puriﬁed and then used in an
in vitro neddylation assay, only the Cul1/Roc1 heterocomplex enhanced the neddylation of Cul1 (Fig. 1A,
lane 4), but not neddylation-site mutant (K720R)/Roc1
heterocomplex or Cul1 alone (Fig. 1A, lanes 2 and 6).
These data suggest that Roc1 may function as a Nedd8E3 ligase. However, we cannot rule out the possibility
that some Roc1 associated protein originated from insect cells promotes the neddylation of Cul1.
To demonstrate that Roc1 functions as Nedd8-E3
ligase, indeed, an in vitro neddylation assay system isnecessary using recombinant proteins expressed in bacterial cells, which do not have a neddylation system. In
order to construct an appropriate in vitro neddylation
assay system, we co-expressed GST-Roc1 and His-Cul1
in bacterial cells. In this case, expressed proteins formed
complex in a cell. Nedd8 and Ubc12 were expressed in
E. coli and His-APP-BP1/His-Uba3 was expressed in
Sf-9 cells; and then each protein was puriﬁed. When HisCul1/GST-Roc1 was incubated in the presence or394

M. Morimoto et al. / Biochemical and Biophysical Research Communications 301 (2003) 392–398

Binding of Ubc12 to Roc1
The RING ﬁnger-type ubiquitin ligase (E3) binds E2
in a RING-ﬁnger domain-dependent manner as clearly
described in the case of c-Cbl and Ubc7 [41]. SUMO-E3
ligases, PIAS family proteins, also can bind Ubc9, an E2for SUMO, in a RING-ﬁnger-like domain-dependent
manner [42].
Since Roc1 has a RING-ﬁnger domain in its carboxyl
terminus, Ubc12 (Nedd8-E2) would be expected to bindto the RING-ﬁnger domain of Roc1. In an in vitro
binding assay using glutathione–Sepharose 4B, Ubc12
could bind to GST-Roc1 in either GST-Roc1 expressedalone or in GST-Roc1 expressed as a Cul1/Roc1 heterocomplex, but not to GST-Skp1, a component of SCF(Fig. 2A). However, Ubc9, an E2 for SUMO, could notbind to GST-Roc1 (Fig. 2B). p53 could bind to Ubc9 as acontrol. The same binding assay was then performed with
RING-ﬁnger mutant H77A. Ubc12 could bind to neither
GST-Roc1(H77A) nor GST-Roc1(H77A)/His-Cul1 (Fig.
2C). These data indicated that Roc1, not Cul1, binds
Ubc12 in a RING-ﬁnger domain-dependent manner.
Self-ubiquitinylation of the Cul1/Roc1 complex

Fig. 1. Roc1 enhances neddylation of Cul1 in a RING-ﬁnger-dependent manner in vitro. (A) GST-Cul1 (lanes 1 and 2), GST-Cul1/HisRoc1 (lanes 3 and 4), and GST-Cul1 (K720R)/His-Roc1 (lanes 5 and 6)
were expressed in Sf-9 cells in the baculovirus protein expression system. Total cell extracts were prepared and GST-Cul1 and its binding
protein (Roc1) were immobilized on glutathione–Sepharose 4B beads.
The proteins loaded into lanes 2, 4, and 6 had been subjected to in vitro
neddylation. Neddylated Cul1 was detected by Western blotting with
anti-Nedd8 antibody. (B) GST-Cul1, GST-Roc1, GST-Roc1/His-Cul1,
and GST-Roc1 (H77A)/His-Cul1 were co-expressed in E. coli. Total
cell extracts were prepared, and GST-Cul1, GST-Roc1, and GSTRoc1 bound His-Cul1 were pulled down using glutathione–Sepharose
4B beads. Then beads were subjected to neddylation in vitro, andneddylated Cul1 was detected by Western blotting with anti-Nedd8
antibody (upper panel) or anti-Cul1 antibody (middle panel). Coomassie staining of proteins is shown (lower two panels).

absence of His-APP-BP/His-Uba3 (Nedd8-E1), Ubc12(Nedd8-E2), or Nedd8, neddylated Cul1 was detected by
the anti-Nedd8 antibody or anti-Cul1 antibody (Fig.
1B). Cul1 was neddylated only in the presence of both
neddylation enzymes and Roc1 (Fig. 1B, lane 6). The
modiﬁcation of Cul1 was dependent on the RING-ﬁnger
domain of Roc1, because the H77A mutant, a RINGﬁnger domain mutant, did not enhance the modiﬁcation(Fig. 1B, lane 8). These data strongly suggest that Roc1
is a Nedd8-E3 ligase functioning in a RING-ﬁnger domain-dependent manner.

During the construction of in vitro neddylation assay
system, we noticed that Cul1 and Roc1 were self-ubiquitinylated. Since the neddylation of Cul1 was shown tobe necessary for ubiquitin ligase activity toward p27kip1
or IjBa, we examined whether this modiﬁcation wasnecessary for the self-ubiquitinylation of Roc1/Cul1
complex. Using Cdc34 as E2 enzyme, both Roc1 andCul1 in complex were self-ubiquitinylated in a neddylation system domain-dependent manner (Fig. 3A, lane6). However, in the presence of the Roc1 mutant, H77A,
Cul1/Roc1(H77A) did not show self-ubiquitinylation
activity (Fig. 3A, lane 8). In the case of using neddylation-site mutant Cul1 (K720R), neither Roc1 nor mutant Cul1 was self-ubiquitinylated (data not shown).
These ﬁndings indicated that neddylation of Cul1 wasnecessary for the self-ubiquitinylation activity of Roc1/
Cul1 complex. Roc1 expressed solely did not show the
self-ubiquitinylation activity (Fig. 3A, lanes 3 and 4).
When the same experiments were performed using
UbcH5c as E2 enzyme, both Roc1 expressed solely andCul1/Roc1 complex could catalyze the self-ubiquitinylation independent of the neddylation of Cul1 (Fig. 3B,
lanes 3 and 4). These results suggested that the neddylation of Cul1 enhances the ubiquitinylation activity of
Cul1/Roc1 complex through Cdc34 but not UbcH5c invitro.
Neddylated Cul1 is unstable
If Cul1/Roc1 complex is self-ubiquitinylated in acell, the ubiquitinylated complex will be degraded byM. Morimoto et al. / Biochemical and Biophysical Research Communications 301 (2003) 392–398

Fig. 2. Roc1 interacts with Ubc12, an E2 for neddylation in vitro. (A)GST-Roc1 (lane 1), GST-Roc1/His-Cul1 (lane 2), or GST-Skp1 (lane3) was expressed in Sf-9 cells in the baculovirus protein expression
system. Total cell extracts were prepared and GST-fusion proteins
were immobilized on glutathione–Sepharose 4B beads and then incubated with puriﬁed Ubc12. After extensive washing, Ubc12 bound toeach protein was detected by Western blotting with anti-T7tag antibody (upper panel). Coomassie staining of proteins is shown (lower
two panels). (B) Binding assay was done as shown in (A) by using HisUbc9, an E2 for sumoylation. (C) Binding assay was done as shown in(A) using GST-Roc1(H77A) mutant in place of GST-Roc1 wild-type.

proteasomes. To examine the involvement of proteasomes in Cul1 degradation, we estimated the eﬀects of aproteasome inhibitor, MG132, on the protein levels of
Flag-Cul1 expressed in U2OS cells. As shown in Fig.
4A, Cul1 was accumulated in the presence of MG132. If
neddylation of Cul1 was necessary for the self-ubiquitinylation, K720R mutant might be stable than wildtype in a cell. In order to assess the eﬀect of neddylationon the stability of Cul1, we evaluated the half life of395

Fig. 3. Self-ubiquitinylation of Cul1/Roc complex is dependent on the
neddylation of Cul1 in vitro. (A) GST-Cul1, GST-Roc1, GST-Roc1/
His-Cul1, or GST-Roc1 (H77A)/His-Cul1 was expressed in E. coli.
Total cell extracts were prepared and GST-fusion proteins were immobilized on glutathione–Sepharose 4B beads. After incubation with(lanes 2, 4, 6, and 8) or without the neddylation system (lanes 1, 3, 5,
and 7), beads were subjected to the in vitro ubiquitinylation reaction
using Cdc34 as E2 and biotinylated ubiquitin. Biotinylated proteins
were detected by peroxidase conjugate avidin (ExtrAvidin, upper panel). Coomassie staining of proteins is shown (lower two panels). (B)Experiments were done as shown in (A) except for using UbcH5c as E2
in the in vitro ubiquitinylation reaction.

396

M. Morimoto et al. / Biochemical and Biophysical Research Communications 301 (2003) 392–398

wild-type or K720R mutant in a cell. As expected, whenwild-type Flag-Cul1 was expressed in U2OS cells, its
half life was about 18 h, whereas that of the mutant one
was about 48 h (Figs. 4B and C). Similar data wereobtained by using COS7 cells. These ﬁndings suggestthat neddylation accelerates Cul1 degradation throughthe ubiquitin–proteasome pathway.
To detect the ubiquitinylated Cul1 in intact cells,
Flag-Cul1 (wild-type or K720R) was co-expressed with
HA-ubiquitin in U2OS cells. Wild-type Cul1 wasmarkedly ubiquitinylated (Fig. 4D, lanes 1 and 4), but
K720R mutant Cul1 was less ubiquitinylated than thewild-type one (Fig. 4D, lanes 2 and 5). The diﬀerence
between wild-type and K720R mutant in the ubiquitinylation in intact cells may reﬂect diﬀerences in theirstabilities.

Discussion
Roc1 functions as a Nedd8-E3 ligase toward Cul1

Fig. 4. K720R mutant Cul1 is more stable than wild-type Cul1. (A)U2OS cells were transfected with pFlag-Cul1 and treated with 20 lM
MG132 (lane 2) or DMSO (lane 1) as a vehicle control for 10 h. The
levels of Flag-Cul1 expression were determined by Western blottingwith anti-Flag antibody. (B) U2OS cells or COS7 cells were transfectedwith pFlag-Cul1 (wild-type or K720R). Twenty-four hours after
transfection, the cells were harvested and then re-inoculated into
dishes. After an additional 24-h incubation, the cells were harvested at
the times indicated. The levels of Flag-Cul1 expression were determined by Western blotting with anti-Flag antibody. (C) The relative
amounts of Cul1 were estimated by quantiﬁcation of band intensityfrom data in (B) using an imaging densitometer. (D) Cul1 is selfubiquitinylated in intact cells. U2OS cells were co-transfected withexpression plasmids encoding Flag-Cul1 (wild-type or K720R) and
HA-Ubiquitin. The cells were treated with 20 lM MG132 for 12 h.
Flag-Cul1 (wild-type or K720R) was immunoprecipitated (IP) fromtotal cell extracts with anti-Flag antibody. Bound proteins were subjected to Western-blotting assay (WB) using anti-HA antibody (leftpanel) or anti-Flag antibody (right panel).

The Cul1/Roc1 complex has been shown to be acatalytic site of SCF, a ubiquitin ligase. Furthermore,
Cul1 is not only self-ubiquitinylated but also neddylated. Nedd8 is one of the ubiquitin-like proteins andthe mechanism of its conjugation to a target protein
could be analogous to that of ubiquitin-conjugation.
The ubiquitin conjugation system requires three enzymes, E1, E2, and E3 or ubiquitin ligase. The Nedd8
conjugation system also seems to require three enzymes,
but Nedd8-E3 ligase has not been clearly identiﬁed sofar; although it might be Roc1, based on the data thatRoc1 enhanced the neddylation of Cul1 when Sf-9 cells
were co-infected with these proteins [39]. The ubiquitin
ligases bind E2 and the RING-ﬁnger-type ubiquitin ligase (E3) probably binds E2 mainly at that RING-ﬁnger domain [41]. If Roc1 is indeed a Nedd8 ligase
toward Cul1, since Roc1 has RING-H2 ﬁnger domain,
it should bind Ubc12 at the RING-ﬁnger domain. Also,a Roc1 mutant, in which this domain is disrupted,
should no longer bind Ubc12. The data described hereindemonstrated these characteristics of Roc1. To conﬁrm
that Roc1 was indeed acting as a Nedd8-E3 ligase, we
needed to prepare an appropriate in vitro neddylation
assay system. When Cul1/Roc1 complex was expressedin Sf-9 cells, Cul1 was highly neddylated. The nonneddylated Cul1 could be a better substrate than the
neddylated one for an in vitro neddylation assay system.
In addition, we were afraid that recombinant Roc1
puriﬁed from insect cells might bind some neddylationstimulation factors originated from the cells, because
the insect cells have a neddylation system. Therefore, we
puriﬁed recombinant Cul1/Roc1 complex expressedin bacterial cells, which does not have a neddylation

M. Morimoto et al. / Biochemical and Biophysical Research Communications 301 (2003) 392–398

system, and used it in vitro neddylation assay system. In
this assay system, Roc1 enhanced the neddylation of
Cul1in a RING-ﬁnger domain-dependent manner. Taken together, our data strongly suggest that Roc1 is a
Nedd8-E3 ligase toward Cul1. The RING-ﬁnger domain of Roc1 is thought to be the binding site ofubiquitin-E2 as proposed in the case of RING-ﬁngertype ubiquitin ligase. We can easily speculate that Roc2
could also be a Nedd8-E3 ligase. Further Roc1 in the
VBC complex could function as a Nedd8-E3 ligase toward Cul2.

Neddylated Cul1 has self-ubiquitinylation activity and isdegraded through the ubiquitin–proteasome pathway
We found in this study that Cul1/Roc1 complex has
self-ubiquitinylation activity through the Cdc34 or
UbcH5c. Cdc34-catalyzed self-ubiquitinylation activityof Cul1/Roc1 complex depends on the neddylation of
Cul1. In contrast, UbcH5c-catalyzed self-ubiquitinylation activity of Cul1/Roc1 complex is independent of theneddylation. Recently, other researchers also reported
similar data [43].
We also could ﬁnd that Cul1 is ubiquitinylated anddegraded through the ubiquitin–proteasome pathway ina cell. In addition, a non-neddylated mutant
Cul1(K720R) was less ubiquitinylated and more stable
than wild-type intact cells. These results suggest that theneddylation of Cul1 stimulates the degradation of Cul1/
Roc1 complex, leading to decreased SCF activity. Recent study shows that overexpression of Rbx1 stabilizes
the SCF substrate in Arabidopsis [44]. Although themechanism is unclear, hyper-neddylation of Cul1 seems
to regulate SCF function negatively. On the other handthe neddylation of Cul1 was shown to be necessary toenhance the ubiquitinylation of p27kip1 [31,32] and IjBa
[33,34] or for the polymerization of the ubiquitin moiety[43] by the SCF complex. We propose, therefore, thatthe neddylation of Cul1 regulates the SCF complex both
positively and negatively. The data described here further suggest that Cul1/Roc1 complex acts as a RINGH2 ﬁnger-type ubiquitin ligase, only when neddylationof Cul1 occurs. Possibly, F-box protein at ﬁrst binds tothe target protein and then the SCF complex forms
without neddylation of Cul1. The neddylation of Cul1,
through APP-BP1/Uba3 as E1 and Ubc12 as E2, would
activate the ubiquitin ligase activity of SCF. In thismodel, inactive SCF, being bound to the target protein,would be activated by neddylation of Cul1 to ubiquitinylate the target protein. In that time, Cul1/Roc1 complex in SCF would also be ubiquitinylated and could be
degraded by proteasomes after ubiquitinylation of thetarget protein. This model provides the auto-regulationof the activity of SCF. The ubiquitinylation of Skp2
could be included in this model.

397

Acknowledgments
We are grateful to Profs. Y. Kudo and H. Tanaka, Tokyo Univ.
Pharm. and Life Sci., for their helpful support throughout this study.

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