A role for the Fizzy/Cdc20 family of proteins in activation of the APC/C distinct from substrate recruitment.
ABSTRACT The Fizzy/Cdc20 family of proteins are essential activators of the anaphase-promoting complex/cyclosome (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 activator, Fizzy-related/Cdh1. In contrast, canonical substrates 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 becomes dispensable. Furthermore, in Cdc20-depleted 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.
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ABSTRACT: Polyubiquitination by E2 and E3 enzymes is a predominant mechanism regulating protein function. Some RING E3s, including anaphase-promoting complex/cyclosome (APC), catalyze polyubiquitination by sequential reactions with two different E2s. An initiating E2 ligates ubiquitin to an E3-bound substrate. Another E2 grows a polyubiquitin chain on the ubiquitin-primed substrate through poorly defined mechanisms. Here we show that human APC's RING domain is repurposed for dual functions in polyubiquitination. The canonical RING surface activates an initiating E2-ubiquitin intermediate for substrate modification. However, APC engages and activates its specialized ubiquitin chain-elongating E2 UBE2S in ways that differ from current paradigms. During chain assembly, a distinct APC11 RING surface helps deliver a substrate-linked ubiquitin to accept another ubiquitin from UBE2S. Our data define mechanisms of APC/UBE2S-mediated polyubiquitination, reveal diverse functions of RING E3s and E2s, and provide a framework for understanding distinctive RING E3 features specifying ubiquitin chain elongation.Molecular cell. 10/2014;
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ABSTRACT: Protein modification with ubiquitin chains is an essential signaling event catalyzed by E3 ubiquitin ligases. Most human E3s contain a signature RING domain that recruits a ubiquitin-charged E2 and a separate domain for substrate recognition. How RING-E3s can build polymeric ubiquitin chains while binding substrates and E2s at defined interfaces remains poorly understood. Here, we show that the RING-E3 APC/C catalyzes chain elongation by strongly increasing the affinity of its E2 for the distal acceptor ubiquitin in a growing conjugate. This function of the APC/C requires its coactivator as well as conserved residues of the E2 and ubiquitin. APC/C's ability to track the tip of an emerging conjugate is required for APC/C-substrate degradation and accurate cell division. Our results suggest that RING-E3s tether the distal ubiquitin of a growing chain in proximity to the active site of their E2s, allowing them to assemble polymeric conjugates without altering their binding to substrate or E2.Molecular cell. 10/2014;
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ABSTRACT: The Anaphase Promoting Complex or Cyclosome (APC/C) is critical to the control of mitosis. The APC/C is an ubiquitin ligase that targets specific mitotic regulators for proteolysis at distinct times in mitosis, but how this is achieved is not well understood. We have addressed this question by determining whether the same substrate, cyclin B1, is recognised in the same way by the APC/C at different times in mitosis. Unexpectedly, we find that distinct but overlapping motifs in cyclin B1 are recognised by the APC/C in metaphase compared with anaphase, and this does not depend on the exchange of Cdc20 for Cdh1. Thus, changes in APC/C substrate specificity in mitosis can potentially be conferred by altering interaction sites in addition to exchanging Cdc20 for Cdh1.Biology open. 09/2014;
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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