A Role for the Fizzy/Cdc20 Family of Proteins
in Activation of the APC/C
Distinct from Substrate Recruitment
Yuu Kimata,1Joanne E. Baxter,2Andrew M. Fry,2and Hiroyuki Yamano1,*
1Cell Cycle Control Laboratory, Marie Curie Research Institute, The Chart, Oxted, RH8 0TL Surrey, UK
2Department of Biochemistry, University of Leicester, LE1 9HN Leicester, UK
The Fizzy/Cdc20 family of proteins are essential acti-
vators of the anaphase-promoting complex/cyclo-
some (APC/C), a multisubunit E3 ubiquitin ligase.
However, apart from the well-established role of the
C-terminal WD40 domain in substrate recognition,
the precise roles of the activators remain elusive.
Here we show that Nek2A, which directly binds
the APC/C, can be ubiquitylated and destroyed
in Fizzy/Cdc20-depleted Xenopus egg extracts
when only the N-terminal domain of Fizzy/Cdc20
(N-Cdc20) is added. This activity is dependent upon
the C box and is conserved in the alternative activa-
tor, Fizzy-related/Cdh1. In contrast, canonical sub-
strates such as cyclin B and securin require both
the N-terminal and WD40 domains, unless N-Cdc20
is fused to substrates when the WD40 domain be-
cells, N-Cdc20 can facilitate Nek2A destruction in a
C box-dependent manner. Our results reveal a role
for the N-terminal domain of the Fizzy/Cdc20 family
of activators in triggering substrate ubiquitylation
by the APC/C.
Ubiquitin is a small protein and serves as molecular tag for pro-
tein degradation. This tagging process, ubiquitylation, is regu-
lated by a cascade of enzymes comprising a ubiquitin-activating
enzyme (E1), ubiquitin-conjugating enzymes (E2s), and ubiquitin
ligases (E3s) (Hershko and Ciechanover, 1998). The APC/C is an
E3 ubiquitin ligase that controls destruction of numerous
proteins at specific times in the cell cycle including the meta-
phase-to-anaphase transition and exit from mitosis by degrad-
ing securin/Cut2/Pds1 and cyclin B, respectively (Morgan,
2007; Peters, 2006; Thornton and Toczyski, 2006). However,
the molecular mechanisms by which the APC/C recognizes so
many different proteins at the correct time and place are still un-
clear. The primary candidate for such determinants is the Fizzy/
Cdc20 family of APC/C activator proteins, which have a charac-
teristic conserved WD40 repeat domain at their C termini (Yu,
2007). Although they are not stoichiometric components of the
APC/C, genetic and biochemical evidence suggests that these
activators are essential for the APC/C to function. First, Fizzy/
Cdc20/Slp1 is required for the APC/C activity in anaphase,
whereas Fizzy-related/Cdh1/Ste9 maintains activity during late
mitosis and G1 phase (Morgan, 2007; Peters, 2006; Thornton
and Toczyski, 2006). Second, the Fizzy/Cdc20 family of proteins
have been shown to bind several substrates (Burton and Solo-
mon, 2001; Hilioti et al., 2001; Pfleger et al., 2001; Schwab
et al., 2001), and recent photocrosslinking experiments have
demonstrated that APC/C substrates directly bind to the WD40
repeat domain of the Fizzy/Cdc20 family of activators (Kimata
et al., 2008; Kraft et al., 2005). Thus, an accepted view in the field
is that the APC/C activators achieve their essential function by
recruiting substrates to the APC/C, which would be equivalent
to the role of F box proteins in the SCF (Skp1-Cullin 1-F box pro-
tein) complex (Deshaies, 1999). Yet, compared to the number of
the F box proteins identified (Jin et al., 2004), so far only a few
Fizzy family activators have been identified (Morgan, 2007; Pe-
ters, 2006; Thornton and Toczyski, 2006). This suggests that it
is unlikely that substrate specificity is determined by individual
members of the Fizzy/Cdc20 family, since such a wide range
of substrates are degraded at different cell-cycle stages. In ad-
dition, core APC/C subunit or subunits have also been shown
to recognize substrates (Carroll et al., 2005; Hayes et al., 2006;
unclear how APC/C substrates are recognized and what are the
exact roles of the Fizzy family of proteins in APC/C-dependent
Nek2A is a vertebrate NIMA-related kinase involved in regula-
tion of the centrosome duplication cycle (Fry et al., 1998). We
have shown that Nek2A is an APC/C substrate that is strikingly
different from other canonical APC/C substrates. Nek2A stably
binds the APC/C core complex via its unique MR tail even in
the absence of the Fizzy family of activators in Xenopus egg ex-
tracts and is degraded in prometaphase when the spindle
checkpoint inhibits destruction of other APC/C substrates in
HeLa cells (Hames et al., 2001; Hayes et al., 2006). These unique
tified roles of the Fizzy/Cdc20 family.
Here we report that the activators are essential not only for
recognizing substrates but also for a postrecognition step, in
stimulating the ubiquitin ligase activity of the APC/C. This activ-
ity is dependent upon the C box, a short motif conserved
among members of the Fizzy/Cdc20 family, in the N-terminal
576 Molecular Cell 32, 576–583, November 21, 2008 ª2008 Elsevier Inc.
The N-Terminal Domain of Fizzy/Cdc20 Can Support
APC/C-Dependent Destruction of Nek2A,
but Not Cyclin B or Mes1
In the case of proteins such as cyclin B or securin, Fizzy/Cdc20
is thought to be required for substrate recognition and recruit-
ment, a prerequisite step for the subsequent ubiquitylation by
the APC/C. However, we wanted to establish whether or not
Fizzy/Cdc20 had additional roles in the control of substrate
degradation. We hypothesized that such a role(s) might only
be discovered by investigating a substrate, which directly binds
the APC/C independently of the Fizzy/Cdc20 activator, such as
Nek2A (Figure 1A) (Hayes et al., 2006). First, we examined
whether Fizzy/Cdc20 is indeed required for Nek2A destruction
in cell-free Xenopus egg extracts. Not only Nek2A but also
Cdc13/cyclin B and cyclin A were stabilized by Fizzy/Cdc20
depletion from egg extracts, whereas they are efficiently de-
stroyed in mock-treated extracts (Figure 1B). These results
indicate that Fizzy/Cdc20 is required for APC/C-dependent de-
struction even after a substrate such as Nek2A is recruited onto
the APC/C. We next sought to investigate this additional role by
first depleting Fizzy/Cdc20 from Xenopus egg extracts and add-
ing back in vitro-translated wild-type (WT) Cdc20 or mutated
versions of Fizzy/Cdc20 (Figure 1C). Three different APC/C
substrates, Nek2A, Cdc13/cyclin B, and Mes1, were used as
model substrates (Hames et al., 2001; Kimata et al., 2008; Ya-
mano et al., 1996). As expected, full-length Fizzy/Cdc20 (WT)
restored the destruction of all the substrates (Figure 1D, lanes
5–8). Surprisingly, destruction of Nek2A, but not Cdc13 or
Mes1, was supported by a fragment of Fizzy/Cdc20 that lacks
the entire WD40 domain (N159) (Figure 1D, lanes 25–28) as
well as by Fizzy/Cdc20 constructs with mutations in the C-ter-
minal IR motif (DIR) and the WD40 domain (WD2A and WD5A;
Figure 1D, lanes 13–24). In contrast, the C-terminal domain
alone (C348) did not rescue Nek2A destruction, suggesting
that only the N-terminal domain is essential for the destruction
of Nek2A. As predicted for substrates that require Fizzy/
Cdc20 for recruitment of the APC/C, neither Cdc13 nor Mes1
was destroyed when the truncated Fizzy/Cdc20 fragments
To further investigate this activity of the N-terminal domain,
we prepared bacterially purified GST-fused N159 (GST-N159)
and MBP-fused N159 (MBP-N159) and used these recombi-
nant proteins in place of WT Fizzy/Cdc20 (Figure 1E). Both
recombinant proteins were able to facilitate destruction of
Nek2A, but not that of Cdc13/cyclin B in Fizzy/Cdc20-
depleted egg extracts. Moreover, this destruction was depen-
dent upon the MR tail of Nek2A (data not shown). These
results indicate that the N-terminal 159 residues of Fizzy/
Cdc20 have an additional role besides substrate recruitment.
Consistent with this idea, the fragment containing the C-ter-
minal WD40 domain (C348), which is able to bind Mes1 (Ki-
mata et al., 2008), was unable to support Mes1 destruction
in these assays (Figure 1D, lanes 29–32), underscoring the
importance of the N-terminal domain of Fizzy/Cdc20 for a
postsubstrate recognition event in the APC/C-dependent
The Conserved C Box of Fizzy/Cdc20 Is Required
for Activation of the APC/C
In contrast to the conserved C-terminal WD40 repeat domain,
the N-terminal regions of the Fizzy/Cdc20 family are divergent
both in primary sequence and length. To identify the important
eral truncated versions of N159 and asked which were able to
activate Nek2A destruction in Fizzy/Cdc20-depleted Xenopus
egg extracts (see Figure S1 available online). We found that trun-
cation of the first 36 residues (37–159) still facilitated Nek2A
destruction, whereas further deletions of the N-terminal 82 resi-
dues (83–159) or the C-terminal 60 residues (N98) completely
abolished the activity. Within this N-terminal region there is a
C box. This is a highly conserved motif present in all members
of the Fizzy/Cdc20 family that is required for the association
with the APC/C (Schwab et al., 2001). We therefore investigated
whether the C box had a role in the activity of the N159 fragment.
We generated a version of GST-N159 with mutation of the C box
this protein to support destruction of Nek2A and Cdc13 in Fizzy/
Cdc20-depleted extracts. As shown in Figure 2A, this mutation
abolished the ability of N159 to activate Nek2A destruction. As
the C box is required for association with the APC/C, we inves-
tigated whether the C box in the N159 protein was responsible
for APC/C binding. When GST-N159 was added into Fizzy/
Cdc20-depleted Xenopus egg extracts, APC/C was copurified
with GST-N159 in a C box- and an N159-dose-dependent man-
ner (Figure 2B), suggesting that N159 indeed interacts with the
of GST-N159 fragments in the add-back experiments in Fizzy/
Cdc20-depleted extracts, we immunoprecipitated APC/C from
mock- or Fizzy/Cdc20-depleted extracts supplemented with
GST-N159 and quantified the amounts of Fizzy/Cdc20 associ-
ated with APC/C. The amount of recombinant N159 protein as-
sociated with the APC/C was equal to or slightly less than that
of endogenous Fizzy/Cdc20 (Figure S2).
As all members of the Fizzy/Cdc20 family of activators contain
a C box, we next sought to investigate whether this N-terminal
activity is conserved. We used bacterially purified N-terminal
Xenopus Fizzy-related/Cdh1 (GST-FzrN186) and human Cdc20
(GST-Hs Cdc20N151), both of which were capable of activating
the APC/C (Figure 2C). Interestingly, GST-FzrN186 could de-
stroy Nek2A in Fizzy/Cdc20-depleted interphase extracts in
a C box-dependent manner, but not Cdc13/cyclin B (Figure 2D).
The N-Terminal Domain of Fizzy/Cdc20 Facilitates
Ubiquitylation of Nek2A, but Not Cyclin B or Securin
We next examined whether the N-terminal domain of Fizzy/
Cdc20 could promote APC/C-dependent ubiquitylation in
a cell-free assay. We purified APC/C from Fizzy/Cdc20-depleted
extracts and analyzed Nek2A ubiquitylation supplemented with
Fizzy/Cdc20 or N159 protein. Consistent with the above finding
using cell-free destruction assays, N159 was able to support
ubiquitylation of Nek2A as efficiently as the full-length Fizzy/
Cdc20 (FzyWT) protein, whereas the N159 C box mutant
failed (Figure 3A, lanes 1–16). In contrast, N159 could not acti-
vate APC/C-dependent ubiquitylation of Cdc13 or securin
(Figure 3A, lanes 17–28, and Figure 3B). These results imply
C Box-Dependent Activation of APC/C
Molecular Cell 32, 576–583, November 21, 2008 ª2008 Elsevier Inc. 577
Figure 1. The Destruction of Nek2A, but Not Canonical APC/C Substrates, Is Activated by the N-Terminal Fragments of Fizzy/Cdc20
(A) Different modes of substrate recognition. (Left) Nek2A directly binds the APC/C through its MR motif. (Right) Canonical substrates normally require both
APC/C and Fizzy/Cdc20 for their efficient recruitment.
(B) Nek2A requires Fizzy/Cdc20 for its destruction in a cell-free destruction assay. Mock-depleted or Fizzy/Cdc20-depleted Xenopus egg extracts were used. As
substrates,35S-labeled in vitro-translated Nek2A, Cdc13 (fission yeast cyclin B), and cyclin A were used alongside a version of Cdc13 lacking the N-terminal 67
residues (D67, stable control). Samples were taken at indicated time points after addition of CaCl2.
(C) A schematic diagram of Fizzy/Cdc20 mutants used in this study.
(D) Addition of N-terminal 159 residues of Fizzy/Cdc20 (N159) is sufficient to trigger Nek2A destruction in Fizzy/Cdc20-depleted egg extracts, whereas the
full-length Fizzy/Cdc20 is required for Cdc13 and Mes1 destruction. Endogenous Fizzy/Cdc20 was depleted from CSF extracts and the destruction of APC/C
substrates was examined after in vitro-translated Fizzy/Cdc20 constructs were supplemented into the extracts. Samples were taken at the indicated time points
after addition of CaCl2. Asterisk indicates N159 protein used for this assay.
(E) Bacterially purified N-Cdc20 initiatesNek2A destruction, but not Cdc13.GST- and MBP-fused Fizzy/Cdc20 N159 alongside in vitro-translated (IVT) full-length
Fizzy and FzyN159 were used for destruction assay of Cdc13 and Nek2A in Fizzy/Cdc20-depleted extracts.
C Box-Dependent Activation of APC/C
578 Molecular Cell 32, 576–583, November 21, 2008 ª2008 Elsevier Inc.
that N159 directly interacts with the APC/C and promotes the
ubiquitin ligase activity of the APC/C.
We further asked whether the ubiquitylation of Nek2A bound
to the APC/C is processive. Using a specific anti-Apc3 antibody,
we first isolated APC/C prebound Nek2A from Fizzy/Cdc20-
andused it forthe ubiquitylation assaysupplemented with N159.
As shown in Figure 3C, more than 80% of Nek2A was converted
into ubiquitylated forms by addition of N159 (Figure 3C, lanes
5–8), suggesting that Nek2A bound to the APC/C via its MR tail
can be ubiquitylated in a processive manner in response to the
interaction between N159 and the APC/C.
was only required for substrate binding in the case of proteins
suchascyclin BandMes1,theWD40 domainwouldbedispens-
able when the N-terminal APC/C-activating domain (N159) was
directly fused to substrates, thereby bypassing the function of
substrate recruitment. To test this, we fused N159 to Mes1WT
(N159-Mes1) and a nondegradable version of Mes1 with muta-
tions in the D box and KEN box (N159-Mes1DK). Surprisingly,
not only N159-Mes1 but also N159-Mes1DK was strongly ubiq-
uitylated in a cell-free ubiquitylation assay without the addition of
either full-length or fragments of Fizzy/Cdc20 (Figure 3D, lanes
7–12), whereas the same fusion construct with the C box muta-
Figure 2. The Conserved C Box of the Fizzy/
Cdc20 Family of Proteins Is Required for
Nek2A Destruction and APC/C Interaction
(A) Mock, GST-fused WT, or C box mutant (DC
box) versions of N159 were added into Fizzy/
Cdc20-depleted egg extracts and examined for
activation of Nek2A and Cdc13 destruction.
(B) APC/C-binding assays with GST-N159. The in-
dicated amounts of purified GST-N159 WT, C box
mutant (DC), or GST alone (25 mg) were bound to
GSH-Sepharose and incubated with CSF extracts
at 23?C for 20 min. The amounts of bound APC/C
were analyzed by immunoblotting with anti-Apc3
(C) The C box-dependent activation of the APC/C
is conserved. GST-fused N-terminal 186 residues
minal 151 residues of human Cdc20 (Cdc20N151)
were added into Fizzy/Cdc20-depleted egg ex-
tracts and the destruction of Nek2A and Cdc13
examined. Adding back mock- and GST-N159
serves as a negative and a positive control,
(D) The C box is required for APC/C activation in
not only mitosis but also interphase. Mock, GST-
FzrN186 WT, or C box mutant (DC box) was added
into Fizzy/Cdc20-depleted CSF or interphase ex-
tracts and the destruction of Nek2A and Cdc13
tion (N159DC box-Mes1) was not ubiqui-
tylated (Figure 3D, lanes 4–6).
These data suggest that the N-terminal
domain of Fizzy/Cdc20 has an as-yet-
unidentified activity to stimulate APC/
sis. It should also be noted that the N159 protein itself was
not ubiquitylated, although it was loaded onto the APC/C
(Figure 3D, lanes 1–3; Figure S3).
The N-Terminal Domain of Cdc20 Is Sufficient
to Trigger Destruction of Nek2A In Vivo
Finally, we asked whether the N-terminal domain of Fizzy/Cdc20
(N-Cdc20) was also sufficient for degradation of Nek2A in vivo.
We first demonstrated that RNAi-mediated depletion of Cdc20
led to stabilization of GFP-Nek2A in nocodazole-arrested mitotic
cells (Figure 4A). Thus, Fizzy/Cdc20 is required for the destruc-
tion of Nek2A in prometaphase cells, despite not being required
to recruit Nek2A to the APC/C, in agreement with our previous
data (Hayes et al., 2006). We then generated Flag-tagged con-
structs expressing N-Cdc20 with or without an intact C box
and confirmed that the expression of both of these constructs
is equivalent in the Cdc20 knockdown cells (Figure 4B). We
then assayed Nek2A destruction in those cells by immunofluo-
rescence microscopy using anti-GFP (GFP-Nek2A) antibodies.
Following control depletion, only 15% of mitotic cells contained
detectable Nek2A. In contrast, 54% of cells which had been de-
pleted of Cdc20 showed a positive Nek2A signal during mitosis.
Importantly, in cells depleted of Cdc20, expression of N-Cdc20
triggered Nek2A destruction and reduced Nek2A-positive cells
C Box-Dependent Activation of APC/C
Molecular Cell 32, 576–583, November 21, 2008 ª2008 Elsevier Inc. 579
to 31%, whereas the percentage of cells expressing Nek2A did
not decrease when the N-Cdc20 protein with the C box mutation
was expressed (Figures 4C and 4D). This indicates that the WT
N-Cdc20, but not the construct lacking the C box, was capable
of rescuing the degradation of GFP-Nek2A.
Taken together, we conclude that the C box-dependent acti-
vation of the APC/C is an additional and essential role of the
Fizzy/Cdc20 family of proteins.
The Fizzy/Cdc20 family of APC/C activators are conserved pro-
teins that are required for the activation of the APC/C. However,
the APC/C through their C-terminal WD40 repeats domain (Yu,
2007). The results presented here demonstrate that the N-termi-
nal domain of the activators has an essential role to initiate ubiq-
uitylation of APC/C substrates in a C box-dependent manner.
is highly conserved among members of the Fizzy/Cdc20 family.
Itappears toberequiredforassociation oftheactivatorswiththe
APC/C (Schwab et al., 2001; Vodermaier et al., 2003). Yet, it re-
mains elusive whether it is required for recruitment of APC/C
substrates in conjunction with the C-terminal WD40 domain
and the IR (isoleucine-arginine) motif at the C terminus. Nek2A,
which does not need Fizzy/Cdc20 for its recruitment to the
APC/C, is ubiquitylated and destroyed in Fizzy/Cdc20-depleted
egg extracts or cultured cells when the N-terminal region with an
intact C box is present. Thus, the interaction between the C box
and the APC/C seems to allow activation of the ubiquitin ligase
activity by Fizzy/Cdc20. The N terminus may promote a confor-
mational change of the APC/C core complex, allowing move-
ment of recruited substrates relative to the catalytic center,
thereby triggering their ubiquitylation (see Figure 4E). This idea
is consistent with the recent finding that the main structural do-
main of the APC/C is rotated upon binding of Fizzy-related/Cdh1
Figure 3. The N-Terminal Domain of Fizzy/Cdc20 Is Required for APC/C Substrate Ubiquitylation
(A) APC/C was purified from Xenopus mitotic extracts from which Fizzy/Cdc20 had been predepleted and was used for in vitro ubiquitylation assays with buffer
(mock), IVT-full-length Fizzy (FzyWT), purified recombinant GST-FzyN159, or GST-FzyN159-DC box. Nek2A and Cdc13 were used as substrates. Samples were
taken at the indicated time points and analyzed by SDS-PAGE and fluorography.
(B) As for (A), except that Xenopus securin was used as a substrate.
(C) The APC/C-bound fraction of Nek2A is efficiently ubiquitylated by addition of FzyN159. In vitro-translated Nek2A was incubated in CSF extracts in the pres-
ence of the proteasome inhibitor MG-132. Nek2A-bound APC/C was immunopurified with anti-Apc3 antibody and used for ubiquitylation assay with buffer
(+mock) or GST-FzyN159.
(D) The WD40 repeat domain is dispensable if FzyN159 is fused to substrates. (Left) Schematic diagram of FzyN159 and the fusion constructs. Filled box in Mes1
indicates mutations in the D box and KEN box. (Right) Ubiquitylation assay was performed using APC/C purified from Fizzy/Cdc20-depleted mitotic extracts.
FzyN159 or the fusion constructs were added as substrates and activators to initiate the reaction. Samples were taken at the indicated time points and analyzed
by SDS-PAGE and fluorography.
C Box-Dependent Activation of APC/C
580 Molecular Cell 32, 576–583, November 21, 2008 ª2008 Elsevier Inc.
(Dube et al., 2005). It is also possible that the C box interaction
directly stimulates the intrinsic catalytic activity of the APC/C
ubiquitin ligase. Our data also highlight that the Fizzy/Cdc20
family of activators have at least two functionally distinct roles:
‘‘APC/C activation’’ and ‘‘substrate recognition’’ via the N- and
C-terminal domains, respectively. Importantly, the N-terminal
domain (e.g., the C box) seems to be vital for ubiquitylation of
all the substrates, but the C-terminal WD40 domain is dispens-
able once the substrates are recruited to the APC/C (Figures
3D and 4E).
In the context of our present study, it is intriguing to note that
theApc10/Doc1 subunitofthe APC/Chas aCbox-like (CL)motif
in the Doc domain (Figure S4) that is found in a variety of proteins
involved in ubiquitylation reactions (Au et al., 2002; Grossberger
et al., 1999; Kominami et al., 1998; Wendt et al., 2001). The
Apc10/Doc1 subunit is a well-conserved subunit from human
to the microsporidia Encephalitozoon cuniculi, and like the
Fizzy/Cdc20 family of activators, it has been reported to bind
Apc3/Cdc27 subunits via a C-terminal IR/LR tail region (Wendt
portant roles in promoting ubiquitylation as well as recognition of
substrates (Carroll et al., 2005; Carroll and Morgan, 2002; Pass-
more et al., 2003). Notably, the CL motif is located in the ligand-
binding interface where it may interact with other core APC/C
subunits and promote APC/C ubiquitin ligase activity. This idea
is in agreement with the recent report that mutations in the
b sheet (b 11), which is structurally adjacent to the CL motif,
prevented the Apc10/Doc1-dependent processing activity
(Carroll et al., 2005). Thus, the mechanism by which Apc10/
Doc1 stimulates processive ubiquitylation may underlie this
type of interaction.
In more general terms, the activity of the APC/C might be con-
trolled by an interaction between the APC/C and an individual
C box or a CL motif or a combination of the two, depending on
Figure 4. The N-Terminal Domain of Cdc20
Also Regulates Nek2A Destruction In Vivo
with 1 mg/ml doxycycline and treated with 100 nM
siRNA oligonucleotides directed against Cdc20 or
GAPDH. After 36 hr, cells were treated for a further
12 hr with 500 ng/ml nocodazole before mitotic
cells were collected by shake-off. Extracts were
prepared using RIPA buffer and analyzed by im-
munoblotting with antibodies against Cdc20,
GFP, or a-tubulin.
(B) U2OS:EGFP-Nek2A cells were induced and
nocodazole treatment. After 24 hr, cells were
either untransfected or transfected with Flag-
tagged N-terminal 151 residues of human Cdc20
(+N-Cdc20) or the same construct with C box mu-
tation (+ N-Cdc20 DC box) for a further 24 hr. Cell
extracts were prepared as above and analyzed by
immunoblotting with indicated antibodies.
against Flag (Flag-Cdc20) or GFP (GFP-Nek2A).
Images of mitotic cells are shown indicating that
both Flag-tagged N-Cdc20 proteins localize pre-
pears more intense than the N-Cdc20-DC box, as
the cell is more rounded up. Mergeimagesinclude
Flag-Cdc20 (red), GFP-Nek2A (green), and DNA
stained with Hoechst (blue). Scale bar, 10 mm.
(D) The histogram indicates the percentage of mi-
totic cells, treated as described in (B), that stained
positively for GFP-Nek2A. Fifty cells were counted
in three independent experiments, and error bars
show standard error.
of the Fizzy family of activators. In the absence
of activators, the APC/C is inactive and cannot
ubiquitylate any substrates (indicated by the color
blue). A substrate (e.g., Nek2A) that directly binds
the APC/C only requires the N-terminal region
(N-Cdc20),whereascanonical substrates (e.g., cyclinB and securin) needboth theN- and C-terminal regions (+FullCdc20)for theirubiquitylation. TheC-terminal
mutations, the fusion substrate (N-Cdc20-DK) can be ubiquitylated, indicating that the WD40 domain is dispensable once substrates are recruited to the APC/C.
C Box-Dependent Activation of APC/C
Molecular Cell 32, 576–583, November 21, 2008 ª2008 Elsevier Inc. 581
substrates or cell-cycle stages. Moreover, Fizzy/Cdc20 and
Fizzy-related/Cdh1 are both phosphorylated in mitosis, and sev-
eral phosphorylation sites lie within the N-terminal domain in
close proximity to the C box (Yu, 2007). Although the roles of
phosphorylation arepoorly understood, it ispossible that protein
kinases and phosphatases control the activity of the APC/C by
regulating the patterns of phosphorylation around the C box.
At present, we do not know the precise mechanism for the
C box-dependent activation of the APC/C. Equally, whether
to the C box in Fizzy/Cdc20 remains to be clarified, but our re-
sults certainly provide a paradigm for understanding protein
ubiquitylation by the APC/C ubiquitin ligase and the Fizzy/
Cdc20 family of activators.
See the Supplemental Data for additional experimental procedures.
Assays for APC/C-Dependent Ubiquitylation and Destruction
Destruction assays were performed essentially as described previously (Ya-
mano et al., 1996). Substrates were labeled with [35S]methionine (PerkinElmer)
in a coupled in vitro transcription-translation (IVT) system (Promega). IVT-
Fizzy/Cdc20 constructs or recombinant GST-FzyN159 proteins were added
to Fizzy/Cdc20-depleted egg extracts, and after 5 min incubation at 23?C,
the APC/C substrates were mixed. The samples were taken at the indicated
time points after adding CaCl2and were analyzed by SDS-PAGE and fluorog-
raphy. For ubiquitylation assays, Xenopus APC/C was immunoprecipitated
from 12.5 mlof Fizzy-depleted mitotic extracts using monoclonal anti-Apc3 an-
tibody (mAB, AF3.1) immobilized on Dynabeads Protein A (Invitrogen). Reac-
tions were performed at 23?C in 10 ml of the buffer (20 mM Tris-HCl [pH 7.5],
100 mM KCl, 2.5 mM MgCl2, 2 mM ATP, 0.3 mM DTT) containing 0.1 mg/ml
E1, 0.1 mg/ml UbcH5 and UbcH10, 1.5 mg/ml ubiquitin, 1 mM ubiquitin-alde-
hyde, 150 mM MG132, and 1 ml in vitro-translated (IVT) Fizzy or bacterially pu-
rified GST-FzyN159 protein. Reactions were stopped at the indicated time
points with SDS sample buffer, and mixtures were resolved by SDS-PAGE.
The Supplemental Data include Supplemental Experimental Procedures and
four figures and can be found with this article online at http://www.molecule.
The authors would like to thank T. Hunt, J. Gannon, and members of the Ya-
mano laboratory for helpful discussions and critical reading of the manuscript.
Research UK Clare Hall Laboratories Xenopus colony. This work was sup-
ported by Marie Curie Cancer Care and the Association for International Can-
cer Research (AICR).
Received: June 30, 2008
Revised: September 5, 2008
Accepted: September 25, 2008
Published: November 20, 2008
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