Molecular Cell, Vol. 9, 931–943, May, 2002, Copyright 2002 by Cell Press
The Anaphase-Promoting Complex:
Proteolysis in Mitosis and Beyond
Jan-Michael Peters1
Research Institute of Molecular Pathology
Dr.-Bohr Gasse 7
A-1030 Vienna
Austria
Summary
Key events in mitosis such as sister chromatid separation and subsequent inactivation of cyclin-dependent
kinase 1 are regulated by ubiquitin-dependent proteolysis. These events are mediated by the anaphase-promoting complex (APC), a cell cycle-regulated ubiquitin
ligase that assembles multiubiquitin chains on regulatory proteins such as securin and cyclins and thereby
targets them for destruction by the 26S proteasome.
Introduction
Cyclin and the “Cell Cycle Clock”
While working at Woods Hole in the summer of 1982,
Tim Hunt discovered a protein in rapidly dividing sea
urchin embryos that was synthesized during interphase
but suddenly destroyed during cell division. The cyclic
expression pattern of this protein, therefore called cyclin
(Evans et al., 1983), suggested immediately that proteolysis is key to its regulation. The observation that cyclin’s
disappearance coincided with blastomere cleavage raised
the interesting possibility that cyclin proteolysis might
control some important aspect of cell division. This hypothesis seemed so attractive because a proteolytic
mechanism could explain how a hypothetical mitotic
activator could be irreversibly inactivated, thereby resetting the “cell cycle clock” after completion of cell division.
In the two decades that have passed since then, these
ideas have turned into one of the central dogmas in our
understanding of the eukaryotic cell division cycle. We
refer to Hunt’s cyclin as cyclin B today and know that
it and several related “mitotic cyclins” are central regulatory elements of an enzymatic switch that triggers entry
into mitosis (Table 1). At the heart of this switch is cyclin
B’s binding partner, the cyclin-dependent kinase 1
(Cdk1). Cyclin B synthesis during interphase is essential
for activation of Cdk1 which, together with other mitotic
kinases, initiates many of the early events of mitosis.
The inactivation of Cdk1 during anaphase and telophase
is as important for exit from mitosis as Cdk1 activation
is for entry into mitosis. Without Cdk1 inactivation chromosomes do not decondense, nuclear envelopes do not
reassemble, and cells do not divide (reviewed by Peters
et al., 1998). Cdk1 inactivation is also essential for DNA
replication in the subsequent cell cycle by allowing the
formation of prereplicative complexes (Noton and Diffley, 2000), and partial Cdk1 inactivation might be required for sister chromatid separation in anaphase in
animal cells (Stemmann et al., 2001).
1
Correspondence: [email protected]
Review
In presumably all eukaryotes, cyclin proteolysis is the
major mechanism that is used to inactivate Cdk1. It is
now known that the enzymatic machinery that destroys
cyclin is unusually complex, both with respect to its
regulation and the number of components involved. This
machinery does much more than just reset the cell cycle
clock. It directly controls the chromosome cycle by initiating sister chromatid separation in anaphase, and it
regulates many other events inside and outside mitosis,
possibly even in postmitotic differentiated cells. In this
article, I discuss what we know today about the functions, regulation, and mechanisms of this pathway, with
particular emphasis on the anaphase-promoting complex (APC) or cyclosome.
The Anaphase-Promoting Complex
Cyclin is destroyed by ubiquitin-dependent proteolysis,
i.e., it is targeted for degradation by covalent attachment
of a multiubiquitin chain, allowing its recognition by the
26S proteasome (Glotzer et al., 1991; Hershko et al.,
1991). The 26S proteasome then degrades cyclin, leaving the catalytic Cdk1 subunit intact. Cyclin ubiquitination reactions depend on at least four components (reviewed in Morgan, 1999; Peters, 1999; Zachariae and
Nasmyth, 1999): the universal ubiquitin-activating enzyme E1, either one of two ubiquitin-conjugating enzymes (E2s), called UbcH5 and UbcH10 in human cells,
the APC, and one of several APC activator proteins such
as Cdc20 and Cdh1 (see Table 2 for names used in other
species). The E1 enzyme transfers activated ubiquitin
residues to UbcH5 or UbcH10, which then assemble
ubiquitin chains on cyclin with the help of the APC. The
APC was discovered as a mitosis and cyclin B specific
ubiquitin ligase in clam and Xenopus extracts (King et
al., 1995; Sudakin et al., 1995) and through the isolation
of budding yeast mutants that are defective in the degradation of mitotic cyclins (Irniger et al., 1995). The clam
enzyme was called cyclosome to illustrate the cyclic
activation and inactivation of the cyclin degradation
pathway (Sudakin et al., 1995), whereas the Xenopus
and yeast ubiquitin ligase was called anaphase-promoting complex to illustrate its essential role in anaphase
initiation (Irniger et al., 1995; King et al., 1995).
The APC is a high molecular mass complex composed
of at least 11 subunits, but it is only fully active as a
ubiquitin ligase once it has bound to Cdc20, Cdh1, or
related activators, resulting in distinct assemblies
called, for example, APCCdc20 or APCCdh1. Since Cdc20
and Cdh1 can bind to APC substrates, they may activate
ubiquitination reactions by recruiting substrates to the
APC (see below). APC-mediated ubiquitination depends
on either one of two rather poorly defined sequence
elements in the substrate, the destruction box (D box)
and the KEN box (Glotzer et al., 1991; Pfleger and
Kirschner, 2000). D boxes were first identified in the N
termini of mitotic cyclins and the KEN box in Cdc20,
but both these elements are found either singly or in
combination in all APC substrates known to date.
Not surprisingly, the ubiquitination of cyclin is regulated during the cell cycle, whereas proteasomal degradation of ubiquitinated cyclin does not appear to be.
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Table 1. Cyclin-Dependent Kinases in Budding Yeast and Animal Cells
Budding Yeast
Animal Cells
Cell Cycle Function
CDK
APC Substrate
CDK
APC Substrate
G1 phase
Cln1-Cdc28
Cln2-Cdc28
Cln3-Cdc28
Clb5-Cdc28
Clb6-Cdc28
Clb1-Cdc28
Clb2-Cdc28
Clb3-Cdc28
Clb4-Cdc28
no
no
no
yes
yes
yes
yes
yes
yes
CycD1-CDK4/6
CycD2-CDK4/6
CycD3-CDK4/6
CycE-CDK2
CycA-CDK2
CycA-CDK2
CycB1-CDK2
CycB2-CDK2
CycB3-CDK2
yesa
no
no
no
yesb
yes
yes
yes
yes
S phase
Mitosis
a
b
Cyclin D1 may be degraded in an APC-dependent manner in response to DNA damage (Agami and Bernards, 2000).
Cyclin A has S phase-promoting function in vertebrate cells, but in Drosophila it promotes mainly entry into mitosis.
The regulated step in this ubiquitination pathway is the
activation of the APC by its interaction with Cdc20 and
Cdh1 (Figure 1). In mitosis, APC is activated by binding
to Cdc20, and this is dependent on high Cdk1 activity. By
allowing Cdk1 activation in prophase, cyclin therefore
sows the seeds of its own destruction later in mitosis.
Cyclin B degradation begins in metaphase and continues while sister chromatids separate and then move
toward opposite spindle poles in anaphase (Clute and
Pines, 1999). In most species and cell types, Cdc20 is
also degraded during this period and replaced by Cdh1,
which keeps the APC active until the end of the subsequent G1 phase. In early mitosis, phosphorylation of
Cdh1 prevents its interaction with the APC. The binding
of Cdh1 to the APC during mitotic exit therefore depends
on Cdh1 dephosphorylation. At the G1-S transition,
Cdh1 is rephosphorylated and thereby dissociated from
the APC, allowing the reaccumulation of cyclins and
other APC substrates until APC binds Cdc20 in the subsequent mitosis.
Functions
Separase Activation and the Initiation of Anaphase
Expression of nondegradable cyclin mutants causes
fungal, plant, and animal cells to arrest in telophase, i.e.,
with their sister chromatids separated and transported
toward opposite spindle poles (reviewed in Peters et al.,
1998). However, genetic or biochemical inactivation of
the APC blocks mitosis before sister chromatids have
been separated in anaphase (Holloway et al., 1993; Irniger et al., 1995; Tugendreich et al., 1995). These observations demonstrated early on that APC activity is required for anaphase but that APC’s role in cyclin
proteolysis is not sufficient to explain its function in
initiating anaphase. Instead, the results implied that the
destruction of at least one other APC substrate is essen-
Table 2. Components of the APC Ubiquitination Pathway
Protein
Vertebrates
D.m.
C.e.
S.c.
S.p.
A.n.
Structural Motifs
E1
E2
E1
UbcH10/UbcX/E2-C
UbcH5/Ubc4
Apc1/Tsg24
Apc2
Apc3/Cdc27
Apc4
Apc5
Apc6/Cdc16
Apc7
Apc8/Cdc23
–
Apc10
Apc11
Cdc26
–
Cdc20/p55CDC
Cdh1 A, B, C, D
–
–
Mad2
Mad2B/Mad2L2
BubR1
Emi1
E1
–
–
–
–
–
–
Ida
–
–
–
–
–
–
–
–
–
–
MAT-2
–
MAT-1
EMB-30
–
EMB-27
–
MAT-3
–
–
–
–
–
FZR-1
E1
UbcP4
–
Cut4
–
Nuc2
Cut20/Lid1
–
Cut9
–
Apc8/Cdc23
–
Apc10
–
Hcn1
–
Slp1
Srw1/Ste9
Mrf1
–
Mad2
–
–
–
–
–
–
BIME
–
BIMA
–
–
BIMH
Fizzy
Fizzy-related
–
–
–
–
–
Rca1
Uba1
Ubc11
Ubc4/5
Apc1
Apc2
Cdc27
Apc4
Apc5
Cdc16
–
Cdc23
Apc9
Apc10/Doc1
APC11
Cdc26
Apc13
Cdc20
Hct1/Cdh1
–
Ama1
Mad2
–
Mad3 and Bub1
–
UBA signature
UBC domain
UBC domain
Rpn1/2 homology
Cullin homology
TPRs
WD40 repeats
TPRs
TPRs
TPRs
TRPs
–
DOC domain
RING-H2 finger
–
–
WD40 repeats
WD40 repeats
WD40 repeats
WD40 repeats
–
–
Protein kinase
F box, ZBR
APC subunits
APC activators
APC inhibitors
–
MDF-2
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
D.m., Drosophila melanogaster; C.e., Caenorhabditis elegans; S.c., Saccharomyces cerevisiae; S.p., Schizosaccharomyces cerevisiae; A.n.,
Aspergillus nidulans. TPRs, tetratrico peptide repeats; ZBR, Zn2⫹ binding region; Rpn1/2 are subunits of the 26S proteasome.
Review
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Figure 1. APC-Mediated Proteolysis during
the Cell Cycle
Temporal pattern of APCCdc20 and APCCdh1 activities (A) and of the destruction of some of
their substrates (B) during the cell cycle of
somatic animal cells. APCCdc20 is activated at
the onset of prometaphase (PM) when it initiates the degradation of cyclin A (CycA) and
Nek2A. Proteolysis of cyclin B (CycB) and
securin (Sec) also depends on APCCdc20 but is
delayed until metaphase (M) by the spindle
assembly checkpoint. During anaphase (A)
and telophase (T), APCCdh1 is activated and
mediates the destruction of additional substrates such as Plk1 and Cdc20. APCCdc20 is
inactivated during mitotic exit, whereas
APCCdh1 remains active until the onset of the
next S phase (S). G2, G2 phase; P, prophase.
tial for the initiation of anaphase. This substrate is known
as securin.
Expression of nondegradable securin mutants or overexpression of wild-type securin blocks sister chromatid separation (reviewed in Nasmyth, 2001), and, importantly, deletion of securin in budding yeast (which can
survive without it at low temperatures) allows sister
chromatid separation in the absence of APC activity
(Ciosk et al., 1998; Yamamoto et al., 1996). These results
imply that APC’s essential role in initiating anaphase
must be to destroy securin. Securin binds to a cysteine
protease, called separase (reviewed in Nasmyth, 2001).
Separase is essential for sister chromatid separation,
and its activation depends on securin proteolysis. APC’s
essential function in promoting anaphase therefore appears to be the activation of separase.
How then does separase initiate sister chromatid separation? Genetic screens have identified additional
genes whose mutation causes precocious sister separation (reviewed by Nasmyth, 2001). Several of these
genes encode the subunits of a chromosomal protein
complex that is required to hold sister chromatids together from S phase until their separation in anaphase.
This complex, called cohesin, is cleaved by separase at
the transition from metaphase to anaphase. In budding
yeast, this reaction is both required and sufficient to
induce sister chromatid separation (Uhlmann et al.,
1999, 2000), implying that cohesin cleavage directly dissolves cohesion between sisters, allowing their segregation toward opposite spindle poles in anaphase.
Similar although not identical mechanisms appear to
regulate sister chromatid separation in higher eukaryotes, including humans. In vertebrates two different
pathways dissociate cohesin from mitotic chromosomes (Waizenegger et al., 2000). The bulk of cohesin
is removed from chromosomes in prophase and prometaphase by a mechanism that depends on Polo-like
kinase but not on APC activation or on cohesin cleavage
(Hauf et al., 2001; Losada et al., 1998; Sumara et al.,
2002). Indeed, a small amount of cohesin remains on
chromosomes until the onset of anaphase, preferentially
in centromeric regions (Hauf et al., 2001; Waizenegger
et al., 2000). Once separase has been activated through
APC-dependent securin proteolysis, these cohesin
complexes are cleaved and thereby removed from chromosomes. It is not yet known why vertebrates and possibly other higher eukaryotes remove cohesin from chromosomes in two steps, but it is conceivable that this
process is required for chromosome condensation
(which is largely absent in budding yeast) or for the
separation of sister chromatid arms.
Although securin deletion in budding yeast allows sister separation in the absence of APC activity, recent
results suggest that in Xenopus APC’s role in activating
separase may not be restricted to securin destruction.
By carefully reinvestigating if cyclin B proteolysis is required for anaphase, Stemmann et al. (2001) discovered
that high Cdk1 activity prevents sister chromatid separation through inhibitory phosphorylation of separase.
This modification alone is sufficient to inhibit separase
in the absence of securin binding, possibly explaining
why securin-deficient human cells are viable and undergo anaphase with fairly normal timing (Jallepalli et al.,
2001). These results suggest that the APC may control
separase activation through two distinct mechanisms:
through partial cyclin B proteolysis to reduce Cdk1 activity and through securin destruction (Figure 2). This hypothesis could also explain why overexpression of nondegradable cyclin A delays anaphase both in Drosophila
and in human cells (reviewed in Su, 2001).
Promoting Anaphase—More Than Sister Separation
Once separase has been activated and sister chromatids have been separated, they are translocated toward
the spindle poles through a combination of microtubule
shrinkage and the activity of microtubule-dependent
motor proteins. Several observations in Xenopus and in
budding yeast suggest that APC activity is also critical
during this period of mitosis. By systematically screening for proteins that are associated with chromosomes
in metaphase but not with chromatids in anaphase, Funabiki and Murray (2000) discovered an APC substrate
that is required for chromosome alignment during meta-
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Figure 2. Regulation of Anaphase by APCCdc20
APCCdc20 initiates anaphase in Xenopus embryos through at least three distinct mechanisms. (1) APCCdc20 enables activation of the protease
separase by mediating the degradation of securin. Active separase separates sister chromatids from each other by cleaving cohesin complexes.
(2) APCCdc20 also helps to activate separase by initiating the degradation of cyclin B and other mitotic cyclins. Cyclin degradation inactivates
Cdk1 which in turn allows the removal of inhibitory phosphate residues from separase by protein phosphatases (PPases). (3) APCCdc20 also
initiates degradation of the kinesin Xkid on chromosome arms. Xkid is a microtubule plus end-directed motor that moves chromosomes away
from spindle poles in early mitosis. Xkid degradation, therefore, facilitates the segregation of chromatids toward spindle poles in anaphase.
phase in Xenopus egg extracts. This substrate, called
Xkid, is a kinesin-related motor protein that may be part
of the polar ejection force (“polar wind”) that moves
chromosomes away from spindle poles toward the spindle equator during prometaphase. Excess amounts or
nondegradable mutants of Xkid do not inhibit the separation of sister chromatids but prevent their movement
toward spindle poles in anaphase. In early Xenopus embryos, Xkid proteolysis may therefore be required to
inactivate polar ejection forces which might otherwise
prevent chromatid segregation in anaphase (Figure 2).
It is not yet clear if this mechanism is widespread because the bulk of Xkid is not degraded during anaphase
in somatic vertebrate cells. However, the microtubule
associated protein Ase1 and the kinesins Kip1 and Cin8
have also been identified as APC substrates in budding
yeast, suggesting that APC-dependent proteolysis is
more widely used to control spindle function (Gordon
and Roof, 2001; Hildebrandt and Hoyt, 2001; Juang et
al., 1997). During anaphase and telophase, the APC also
mediates the destruction of several other proteins, including regulators of DNA replication and several protein
kinases (Table 3).
APCCdh1 in G1 and G0—Keeping Cyclins under Control
In many species such as Drosophila and Xenopus, the
blastomeres of early embryos initiate DNA replication
immediately after exit from mitosis, allowing very rapid
cell divisions that lack gap phases. In these embryos
Cdh1 is not detectably expressed until a G1 phase is
established (Lorca et al., 1998; Sigrist and Lehner, 1997).
These observations suggest that APCCdc20 is sufficient
to allow cell proliferation, whereas APCCdh1 may have
evolved to perform G1-specific functions. In budding
and fission yeast Cdh1 is not essential for viability, however, implying that, at least in these organisms, APCCdh1
is not required for the establishment of a G1 phase.
What then is the function of APC during G1? Although
fission yeast cells lacking Cdh1 can proliferate, they fail
to arrest in G1 in response to nutrient starvation or mating factors which normally prevent entry into S phase
(Kitamura et al., 1998; Kominami et al., 1998). The same
effect is seen, both in fission and budding yeast, if APC
subunits are inactivated during G1 (Irniger and Nasmyth,
1997; Kumada et al., 1995). Such mutants inappropriately accumulate mitotic and S phase promoting cyclins
in G1 (Irniger and Nasmyth, 1997; Kominami et al., 1998),
and mutation of cyclin genes suppresses the G1 arrest
deficiency of APCCdh1 mutants in G1 (Kitamura et al.,
1998; Kominami et al., 1998). These observations suggest that in the presence of mating factors or under
starvation conditions APCCdh1 is required to prevent
cyclin accumulation and the activation of S phase promoting CDKs.
Numerous genetic observations suggest that the G1
function of APCCdh1 is partially redundant with the function of CDK inhibitor proteins, strongly supporting the
view that one of APCCdh1’s important functions in G1
is to keep CDKs inactive. For example, the viability of
budding yeast strains lacking Cdh1 depends on the CDK
inhibitor Sic1 (Schwab et al., 1997; Visintin et al., 1997),
and mutation of the fission yeast CDK inhibitor Rum1
causes phenotypes that are virtually identical to the phenotype of Cdh1 mutants (Kominami et al., 1998).
Cdh1 also has an important G1 function during Dro-
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Table 3. APC Substrates
Function of Substrate
Substrate
Species
Anaphase inhibitors
Securin/PTTG
Pimples
Pds1
Cut2
Plk1
Cdc5
Hsl1
Aurora A and B
Nek2A
NIMA
B-type cyclins
A-type cyclins
Dbf4
Cdc20
UbcH10/E2-C
Ase1
Kip1
Cin8
Xkid
Geminin
Cdc6
SnoN
B99
H. sapiens, X. laevis
D. melanogaster
S. cerevisiae
S. pombe
H. sapiens
S. cerevisiae
S. cerevisiae
H. sapiens
H. sapiens
A. nidulans
Several metazoans and unicellular fungi
vertebrates, D. melanogaster
S. cerevisiae
H. sapiens, S. cerevisiae
M. musculus, H. sapiens
S. cerevisiae
S. cerevisiae
S. cerevisiae
X. laevis
X. laevis, D. melanogaster
H. sapiens
H. sapiens
H. sapiens
Protein kinases
Regulatory subunits of protein kinases
APC cofactors
Motor, spindle, and kinetochore proteins
Regulators of DNA replication
Signaling molecules
Unknown function
sophila development. The Cdh1 homolog Fizzy-related
is expressed in fly embryos once cells become postmitotic (Sigrist and Lehner, 1997), and, similar to yeast,
Fizzy-related is required for arrest in G1. In addition,
Fizzy-related is needed to suppress mitosis in certain
cell types of the embryo so that these can amplify their
DNA content in endoreduplication cycles.
In vertebrate cells, the G1 function of APCCdh1 is less
well understood. Chicken cells whose Cdh1 alleles have
been deleted are viable and apparently proceed through
G1 in a normal manner (Sudo et al., 2001). However,
Cdh1’s deletion could possibly be compensated by one
of several Cdh1 homologs recently discovered in chickens (Wan and Kirschner, 2001). Taken together, the observations made in yeast, Drosophila embryos, and cultured chicken cells imply that APCCdh1 is not absolutely
essential for the establishment of G1 phases but that it
has critical roles in maintaining the G1 state under certain physiological conditions.
In vertebrates, APCCdh1 is also present and active in
quiescent cultured cells (Brandeis and Hunt, 1996), and
it is expressed in a variety of tissues that are predominantly composed of postmitotic cells, such as brain,
where it can be detected in the nuclei of differentiated
neurons (Gieffers et al., 1999). Although APCCdh1 could
simply be present in these cells to prevent the accumulation of cyclins and other S phase and mitotic activators,
it would be surprising if transcriptional regulation and
CDK inhibitor proteins were not sufficient to fulfill this
role. It is therefore possible that APCCdh1 has additional
roles in postmitotic cells that remain to be discovered.
More G1 and G0 Functions—TGF␤ Signaling
Support for the hypothesis that APC has additional functions in G1 and G0 comes from the recent finding that
APCCdh1 may regulate TGF␤ signaling in animal cells.
Signal transduction through the TGF␤ pathway has, depending on the cell type and developmental stage, been
shown to elicit a variety of biological responses, includ-
ing inhibition of cell proliferation, differentiation, and
apoptosis (reviewed by Massague et al., 2000). Once
activated through ligand binding, TGF␤ receptors at the
plasma membrane phosphorylate cytoplasmic Smad
proteins and thereby enable their transport into the nucleus where they control the transcription of specific
target genes. For example, TGF␤ signaling can activate
transcription of the CDK inhibitors p15 and p21 and
simultaneously inhibit expression of the growth-promoting transcription factor Myc, resulting in a G1 arrest.
TGF␤ signaling is antagonized both by regulated proteolysis of activated Smads and also by several corepressors that repress TGF␤ target genes. One of these
repressors, called SnoN, is rapidly degraded once TGF␤
signaling is activated. Furthermore, overexpression of
SnoN can decrease TGF␤ signaling, suggesting that
SnoN destruction is required for full activation of TGF␤
signaling. The ubiquitin ligase Smurf has been implicated in SnoN destruction, but recently it has been reported that SnoN can also be ubiquitinated by APCCdh1
and that the APC plays an important role in mediating
SnoN proteolysis in response to TGF␤ signaling
(Stroschein et al., 2001; Wan et al., 2001). These observations suggest that during G1 and G0, APCCdh1helps to
restrain cell cycle entry both directly by preventing the
accumulation of S phase and mitotic cyclins, and indirectly by allowing TGF␤ signaling, which would then
inhibit proliferation through trancriptional activation of
CDK inhibitor proteins.
Regulation
Mitotic Activation of APCCdc20
In yeast and in somatic animal cells, Cdc20 is degraded
during exit from mitosis in an APCCdh1-dependent manner. Cdc20 therefore has to be resynthesized during
S phase and G2 when APCCdh1 is inactive and Cdc20
transcription is upregulated (Fang et al., 1998; Kramer
et al., 2000; Prinz et al., 1998; Shirayama et al., 1998).
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Figure 3. Regulation of APCCdc20
In prophase or prometaphase, several subunits of the APC are phosphorylated by kinases such as Cdk1 or Plk1. APC phosphorylation
increases the binding of Cdc20 to the APC. Cdc20 may activate
ubiquitination reactions by recuiting substrates (S) to the APC. In
vertebrates, Emi1 inhibits APCCdc20, possibly by preventing the binding of substrates to Cdc20 until Emi1 itself is degraded early in
mitosis. The ability of APCCdc20 to ubiquitinate cyclin B and securin
is further delayed by Mad2 and BubR1 until all chromosomes have
attached to both poles of the mitotic spindle. During exit from mitosis, Cdc20 is degraded in an APCCdh1-dependent manner.
However, despite Cdc20 accumulation, APCCdc20 is not
activated until mitosis, implying that either additional
components or modifications are required for APCCdc20
activation or that inhibitors suppress its activity. There
is evidence supporting both of these possibilities, although their relative contribution is still poorly understood (Figure 3).
Binding of Cdc20 to the APC and APC activation in
mitosis correlates with the phosphorylation of several
APC subunits (Rudner and Murray, 2000), and biochemical experiments suggest that these phosphorylation reactions facilitate binding of Cdc20 to the APC (Kramer
et al., 2000; Rudner and Murray, 2000; Shteinberg et al.,
1999; Tang et al., 2001a). In budding yeast, mutation of
Cdk1 consensus sites in the APC subunits Cdc27,
Cdc16, and Cdc23 largely abolishes APC phosphorylation, reduces Cdc20 binding, and delays exit from mitosis, suggesting that APC phosphorylation by Cdk1 is
indeed essential for full activation of APCCdc20 (Rudner
and Murray, 2000).
In animal cells, numerous reports have implicated
Cdk1 and Plk1 in mitotic APC phosphorylation (reviewed
in Zachariae and Nasmyth, 1999), but it is still unclear if
this modification is essential for APC activation. Recent
work in Xenopus has identified an inhibitor of the APC,
called Emi1, whose depletion from Xenopus extracts
blocks entry into mitosis because mitotic cyclins fail to
accumulate (Reimann et al., 2001a). This observation
suggests that Emi1 is required to keep APCCdc20 inactive
in interphase and therefore implies that mitotic APC
phosphorylation may not be essential for activation of
APCCdc20. The identification of in vivo phosphorylation
sites and mutational analyses will therefore be required
to clarify if and how phosphorylation regulates APCCdc20
in animal cells.
Likewise, it will be important to understand how Emi1
regulates APCCdc20 and how widespread this mechanism
is. First experiments suggest that Emi1 can bind to
Cdc20 and prevent its interaction with APC substrates
but not the binding of Cdc20 to the APC (Reimann et
al., 2001b). Emi1 itself is degraded early in mitosis in an
APC-independent manner, consistent with its destruction being required for APCCdc20 activation (Reimann et
al., 2001a). In Drosophila, a homolog of Emi1, called
Rca1, has an important role in keeping APCCdh1 inactive
during G2 (see below), but surprisingly there is no evidence yet that Rca1 also regulates APCCdc20. It is therefore possible that mitotic APC phosphorylation and
Emi1/Rca1 destruction are of different importance for
APCCdc20 activation in different species or cell types or
that additional mechanisms of APCCdc20 regulation still
remain to be discovered.
Regulation of APCCdc20 by the Spindle
Assembly Checkpoint
The hypothesis that APCCdc20 is activated in early mitosis
either by phosphorylation or by Emi1/Rca1 degradation
can explain why APCCdc20 is inactive in interphase, but
it does not explain how the activation of APCCdc20 during
mitosis is timed in a way that enables the APC to precisely control late mitotic events such as anaphase and
exit from mitosis. It is well established that mitotic A-type
cyclins and the kinase Nek2A are degraded during prometaphase in an APC- and Cdc20-dependent manner,
indicating that APCCdc20 is already active early in mitosis
(Dawson et al., 1995; den Elzen and Pines, 2001; Geley
et al., 2001; Hames et al., 2001; Sigrist et al., 1995). In
contrast, other APC substrates such as securin and
cyclin B are stable until the beginning of metaphase.
Their degradation is initiated by APCCdc20 only once all
chromosomes have attached to both poles of the mitotic
spindle. This timing mechanism ensures that sister chromatid cohesion is not dissolved by separase before
spindle assembly has been completed, which could otherwise lead to chromatid missegregation and the formation of aneuploid daughter cells.
The biochemical pathway that delays securin and
cyclin B proteolysis until all chromosomes are bipolarly
attached is called the spindle assembly checkpoint (reviewed by Amon, 1999). Remarkably, a single unattached kinetochore is sufficient to delay the separation
of all the chromatids in a mitotic cell (Rieder et al., 1994).
The checkpoint is thought to measure tension that is
created at kinetochores once both sister chromatids are
attached to opposite poles (Li and Nicklas, 1995; Stern
and Murray, 2001), but there may also be mechanisms
that measure the attachment of spindle microtubules to
kinetochores directly, independent of tension (Skoufias
et al., 2001; Waters et al., 1998). How activation of the
checkpoint specifically inhibits the ability of APCCdc20 to
ubiquitinate securin and cyclin B is still poorly understood. Two known checkpoint proteins, Mad2 and
BubR1, can inhibit APCCdc20 in vitro, but it is unclear if
Mad2 and BubR1 collaborate to inhibit APCCdc20, or if
Mad2 and BubR1 are activated by different branches of
the spindle assembly checkpoint.
Mad2 can either bind to the checkpoint protein Mad1
or to Cdc20 but not simultaneously to both (Hwang et
al., 1998; Sironi et al., 2001). The binding of Mad2 to
Mad1 recruits Mad2 specifically to unattached kineto-
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937
chores, induces a major conformational change in
Mad2, and appears to facilitate the binding of Mad2 to
Cdc20 (Chen et al., 1996; Luo et al., 2002; Sironi et
al., 2001). These observations suggest that unattached
kinetochores recruit Mad1 which then can bind Mad2
and convert it into a form that can interact with Cdc20.
How Mad2-Cdc20 complexes then inhibit the APC is
not known. In vitro Mad2 can inhibit the association of
Cdc20 with the APC (Reimann et al., 2001b; Tang et
al., 2001a), suggesting that Mad2 could inhibit APC by
titrating out Cdc20. However, it is unclear if a single
unattached kinetochore, which is sufficient to delay anaphase, could inactivate enough Cdc20 to prevent cyclin
B and securin destruction. It is easier to envision that
Mad2-Cdc20 complexes inhibit APC function in a dominant manner. Consistent with this hypothesis, Mad2 can
indeed be specifically detected in mitotic APC immunoprecipitates (Kallio et al., 1998; Wassmann and Benezra,
1998). Mad2 can also slow down the dissociation of
Cdc20-substrate complexes in vitro (Pfleger et al.,
2001b), raising the possibility that Mad2 interferes with
some step during substrate ubiquitination.
Recent biochemical fractionation experiments showed
that only a complex containing Mad2, Cdc20, BubR1,
and Bub3 is able to efficiently inhibit APCCdc20 in vitro,
whereas fractions that contain Mad2 molecules which
are not part of this mitotic checkpoint complex (MCC)
have no inhibitory effect (Sudakin et al., 2001). Experiments with recombinant proteins suggest that BubR1
is largely responsible for the inhibitory activity of MCC
because this protein alone can bind to Cdc20, prevent
its interaction with the APC, and inhibit securin and
cyclin B ubiquitination in vitro (Tang et al., 2001a). In the
future it will be important to understand if MCC inhibits
Cdc20 via both its Mad2 and BubR1 subunits and if
active Mad2 exists exclusively as part of MCC, or if
independent checkpoint complexes exist. Likewise, it
will be crucial to understand how APCCdc20 is able to
ubiquitinate cyclin A but not B-type cyclins and securin
in the presence of an activated spindle assembly checkpoint.
Mitotic Exit—Switching from APCCdc20 to APCCdh1
During most of mitosis, Cdh1 is phosphorylated by
CDKs and thereby kept inactive. At the end of mitosis,
Cdh1 is dephosphorylated and thus able to bind and
activate the APC (Blanco et al., 2000; Jaspersen et al.,
1999; Kramer et al., 2000; Listovsky et al., 2000; Lukas
et al., 1999; Yamaguchi et al., 2000; Zachariae et al.,
1998a). APCCdh1 then ubiquitinates Cdc20 and thereby
inactivates APCCdc20 (Pfleger and Kirschner, 2000; Prinz
et al., 1998; Shirayama et al., 1998). Around the same
time, one of the E2 enzymes that collaborate with the
APC, UbcH10, is degraded (Yamanaka et al., 2000), but
the physiological consequence of this reaction is not yet
known. Neither Cdc20 proteolysis nor the degradation
of UbcH10 is essential for inactivation of APCCdc20 in
embryonic cell cycles because Cdc20 and orthologs
of UbcH10 are not degraded in early Drosophila and
Xenopus embryos that do not express Cdh1 (King et al.,
1995; Lorca et al., 1998; Sigrist and Lehner, 1997; Yu et
al., 1996). In these cell cycles, APC dephosphorylation,
subsequent dissociation of Cdc20, or binding to Emi1
may therefore be sufficient to inactivate APCCdc20.
APCCdh1 is activated once degradation of B-type
Figure 4. Regulation of APCCdh1
Cdh1 is phosphorylated during S, G2, and mitosis by S phase and
mitotic CDKs such as Cdk2 and Cdk1, respectively. Cdh1 phosphorylation prevents the association of Cdh1 with the APC, but it does
not inhibit the association of Cdh1 with APC substrates (Schwab et
al., 2001). Cdh1 is dephosphorylated at the end of mitosis, resulting
in APCCdh1 activation. In budding yeast, Cdh1 dephosphorylation is
mediated by the phosphatase Cdc14. In Drosophila and vertebrates,
APCCdh1 is, in addition, inhibited by Emi1/Rca1, which binds to Cdh1
and thereby might prevent the binding of substrates. Phosphorylation of Cdh1 also results in its destabilization. It is conceivable that
Emi1/Rca1 has a role in Cdh1 degradation because Emi1 is an F
box protein that interacts with SCF ubiquitin ligases.
cyclins has been initiated by APCCdc20, raising the simple
possibility that lowering Cdk1 activity may be sufficient
to allow Cdh1 dephosphorylation by constitutively active protein phosphatases. However, recent work has
revealed that regulation of Cdh1 dephosphorylation is
more complex (Figure 4). In budding yeast, APCCdc20 initiates mitotic cyclin degradation but is unable to complete
it (Baumer et al., 2000; Yeong et al., 2000). A fraction
of mitotic cyclins is protected from destruction by an
unknown mechanism until APCCdh1 is activated at the
end of mitosis. The activation of APCCdh1 in turn depends
on the prior activation of APCCdc20 and on the correct
positioning of the mitotic nucleus between the mother
and the future daughter cell (reviewed by Bardin and
Amon, 2001). Once these events have occurred, CDK
inactivation is completed by activation of APCCdh1 and
by the simultaneous accumulation of the CDK inhibitor
Sic1. This mechanism ensures that cells never exit from
mitosis before anaphase has been initiated or under
conditions where chromosomes would not be segregated correctly between mother and daughter cells.
In yeast, activation of both APCCdh1 and Sic1 depends
on the protein phosphatase Cdc14, which is kept in an
inactive form in the nucleolus through most of the cell
cycle. In anaphase and telophase, Cdc14 is released
from its nucleolar binding partner Cfi1/Net1. Once released, Cdc14 dephosphorylates at least three proteins,
Molecular Cell
938
Cdh1, Sic1, and the transcription factor Swi5, all of which
contribute to CDK inactivation (Shou et al., 1999; Visintin
et al., 1998). Cdh1 dephosphorylation activates APCCdh1,
whereas Sic1 dephosphorylation prevents its recognition by SCF ubiquitin ligases and therefore results in
Sic1 stabilization. Sic1 accumulation is also promoted
by Swi5 dephosphorylation which allows transport of
Swi5 into the nucleus where it stimulates Sic1 transcription. Once accumulated, Sic1 inactivates mitotic CDKs
through direct binding and might therefore collaborate
with APCCdh1 in promoting exit from mitosis.
Activation of the Cdc14 phosphatase is controlled
by at least two mechanisms. In anaphase, Cdc14 is
transiently released from the nucleolus by the FEAR
(Cdc fourteen early anaphase release) pathway (Stegmeier et al., 2002). This pathway depends on separase
activation, but what the critical separase substrates are
and how the FEAR pathway disscoiates Cdc14 from
Cfi1/Net1 are not yet known. Maintenance of Cdc14
activity and exit from mitosis depend on a second pathway, called the mitotic exit network (MEN). The MEN
represents a signaling cascade that consists of the
GTPase Tem1, two proteins that regulate Tem1 (a
GTPase activating protein and a gunanine-nucleotide
exchange factor), and several protein kinases (reviewed
in Bardin and Amon, 2001). Activation of the MEN is
controlled by multiple signals, including the correct positioning of the nucleus between the mother and the
future daughter cell, which brings the GTPase Tem1 into
close proximity to its GTPase activating protein, Lte1
(Bardin et al., 2000). Tem1 is concentrated at the spindle
pole that will move into the daughter cell, where Lte1 is
also enriched at the cortex. Tem1 is therefore only fully
activated once the Tem1-containing spindle pole has
moved into the daughter cell. Activation of Tem1 then
promotes release of Cdc14 from the nucleolus and
thereby initiates exit from mitosis.
Homologs of Cdc14 also exist in other eukaryotes,
but whether they are required for Cdh1 dephosphorylation and mitotic exit is unknown. In fission yeast, Cdc14
is regulated by a signaling cascade similar to the MEN
in budding yeast, called the septation initiation network
(SIN), but this pathway appears to control CDK inactivation by promoting inhibitory tyrosine phosphorylation
on Cdk1, not by activating cyclin proteolysis (reviewed
by Bardin and Amon, 2001). In higher eukaryotes, it has
been shown that a homolog of Cdc14 can dephosphorylate Cdh1 in vitro (Bembenek and Yu, 2001), but it is not
yet known if this enzyme is required for APC activation
in vivo.
Inactivation of APCCdh1 in S, G2, and Early Mitosis
APCCdh1 has to be inactivated at the G1-S transition to
allow the reaccumulation of APC substrates that are
needed for entry into S phase or later for entry into
mitosis. For example, to allow DNA replication, S phasepromoting cyclins have to accumulate, such as cyclin
A in vertebrates and Clb5 and Clb6 in budding yeast.
Phosphorylation of Cdh1 appears to be required for its
inactivation in all organisms studied so far. In budding
yeast, G1-CDKs, whose cyclin subunits are not APC
substrates, are thought to initiate Cdh1 phosphorylation,
but S phase-promoting CDKs complete and maintain
Cdh1 phosphorylation (Amon et al., 1994; Huang et al.,
2001). In Drosophila and mammalian cells, cyclin E-Cdk2
and cyclin A-Cdk2 have been reported as the major
Cdh1 kinases, respectively (Lukas et al., 1999; Sigrist
and Lehner, 1997).
Dephosphorylation of Cdh1 dissociates it from the
APC but also might target it for degradation. Cdh1 protein levels decrease during S phase in vertebrate cells
and fission yeast when Cdh1 is phosphorylated, and
nonphosphorylatable mutants of fission yeast Cdh1 are
stabilized in S phase (Blanco et al., 2000; Kramer et al.,
2000; Lukas et al., 1999; Yamaguchi et al., 2000). How
Cdh1 is degraded is unknown, but it is conceivable that
SCF complexes are involved because the recognition
of substrates by these ubiquitin ligases typically depends on substrate phosphorylation (reviewed by Deshaies, 1999).
Although Cdh1 phosphorylation is required for APCCdh1
inactivation, the recent identification of Rca1/Emi1 as
an APC inhibitor suggests that phosphorylation alone
is not sufficient to prevent APC-mediated proteolysis in
S and G2 phase. Drosophila Rca1 was initially characterized as a regulator of cyclin A whose mutation resulted
in a G2 arrest similar to the one caused by mutation of
cyclin A (Dong et al., 1997). More recently, Grosskortenhaus and Sprenger (2002) showed that mitotic cyclins
are prematurely degraded in Rca1 mutants and that
cyclin degradation mediated by the Drosophila Cdh1
ortholog Fizzy-related can be inhibited by Rca1. These
observations and the earlier finding that Rca1 mutants
arrest in G2 indicate that Rca1 function is required to
keep APCCdh1 inactive during G2, possibly because animal cells do not contain enough CDK activity in G2 to
inactivate Cdh1 by phosphorylation alone.
Similar although not identical results have been obtained with Emi1 in vertebrate cells. Emi1 was initially
found to inhibit APCCdc20 in Xenopus extracts (see above),
but more recently inhibition of APCCdh1 has also been
reported (Reimann et al., 2001b). Emi1 is able to inactivate purified APC, possibly by binding to Cdc20 and
Cdh1 and preventing their interaction with APC substrates. Since Emi1 interacts with the Skp1 subunit of
SCF ubiquitin ligases via an F box (Reimann et al.,
2001a), a sequence element that is typically found in
substrate adaptors of the SCF, it will be interesting to
test if Emi1 inhibits Cdh1 not only by direct binding but
also by targeting it for ubiquitination by SCF complexes.
Structure and Mechanism of the APC
Mechanism
Although it is clear that the APC is essential for the
multiubiquitination of its substrates, very little is known
about how the APC supports these reactions in a mechanistic sense. In operational terms, the APC fulfills the
role of a ubiquitin ligase, i.e., its presence is required
for the transfer of ubiquitin residues from E2 enzymes
to substrates. This reaction results in the formation of
an isopeptide bond between the C terminus of ubiquitin
and the epsilon amino group of a lysine residue in the
substrate. In subsequent reaction cycles, lysine residues within the attached ubiquitin residues can also
serve as acceptor sites, resulting in the assembly of a
multiubiquitin chain which is thought to function as a
recognition signal for the 26S proteasome.
Based on sequence homologies and biochemical
Review
939
properties, two types of ubiquitin ligases can be distinguished. HECT domain proteins form covalent ubiquitin
thioesters before they transfer ubiquitin residues to substrates. All other known ubiquitin ligases, including the
APC, are characterized by the presence of a RING finger,
a protein domain that is stabilized by the coordination
of two atoms of zinc. RING fingers are not known to
form ubiquitin thioester intermediates but have been
shown to directly bind to E2 enzymes (cited in Gmachl
et al., 2000). When mixed with E1 and E2 enzymes,
isolated purified RING finger proteins are able to support
ubiquitination reactions in vitro (Lorick et al., 1999). RING
finger proteins therefore appear to have an important
role in the catalysis of ubiquitination reactions, but it
remains unknown whether they directly participate in
catalysis or just facilitate it by generating proximity between E2 enzymes and substrates. Support for the first
possibility has been provided recently by Tang et al.
(2001b), who reported that high concentrations of Zn2⫹
ions alone are able to support ubiquitination reactions
in the absence of a RING finger protein.
Among RING finger-containing ubiquitin ligases, the
APC appears to be distantly related to SCF complexes
because its Apc2 subunit is partially homologous with
the cullin subunit of SCF (Yu et al., 1998; Zachariae et
al., 1998b). In SCF the C-terminal domain of the cullin
subunit binds to the RING finger subunit, whereas the
cullin N terminus interacts with two other proteins that
can bind to substrates (Deshaies, 1999; Zheng et al.,
2002). Cullins may therefore help to bring substrates,
RING finger proteins, and E2 enzymes into close proximity. Apc2 may have a similar function because it binds
to APC’s RING finger subunit, Apc11, which in turn is
able to bind E2 enzymes and to support ubiquitination
reactions in vitro (Gmachl et al., 2000; Leverson et al.,
2000; Tang et al., 2001b). However, it remains unknown
how substrates are recruited to Apc2-Apc11.
The APC is unusual among ubiquitin ligases with respect to its subunit complexity. Eleven to twelve core
subunits have been identified in the yeast and the human
APC (Gmachl et al., 2000; Grossberger et al., 1999; Yu
et al., 1998; Zachariae et al., 1998b). This complexity is
surprising because many other ubiquitin ligases are only
composed of one or a few subunits, implying that ubiquitin ligase activity does not necessarily depend on multiple subunits. It has been discussed that the multitude
of APC subunits could reflect the complexity of APC’s
regulation, but it is also not clear if a dozen subunits
would really be required to subject APC activity to tight
cell cycle control. It therefore remains a big mystery as
to why the APC is composed of so many subunits and
what their individual functions are.
Structure
Cryo-electron microscopy of purified human interphase
APC has yielded a low resolution model of the APC
that shows an asymmetrically shaped particle with an
internal cavity (Gieffers et al., 2001). This cavity would
be large enough to accommodate cofactors such as E2
enzymes or substrates of the APC. It will therefore be
interesting to analyze whether ubiquitination reactions
take place inside this cavity and whether the activity of
the APC can be regulated by spatially controlling the
access of substrates to catalytically important subunits
within this cavity. Higher resolution structures and the
mapping of individual subunits in the cryo model will be
important to test these ideas and to obtain insight into
how this complex may ubiquitinate substrates.
So far only the crystal structures of the small APC
subunit Apc10/Doc1 and of a fragment of Apc2 have
been reported (Au et al., 2002; Wendt et al., 2001; Zheng
et al., 2002). Apc10/Doc1 is characterized by a “Doc”
domain that is also found in several other putative ubiquitin ligases of both the HECT and the RING finger type
(Grossberger et al., 1999; Kominami et al., 1998). Apc10/
Doc1 may therefore have a function that is common to
some ubiquitin ligases. The structures of Apc10/Doc1
have revealed that the Doc domain is virtually identical
to the jellyroll domain previously found in a variety of
eukaryotic and bacterial proteins. In all known cases
the jellyroll domain binds ligands, such as sugars, nucleotides, phospholipids, nucleic acids, or proteins. By
analogy, Apc10/Doc1’s Doc domain may also bind an
unidentified ligand. This reaction could be important
for APC function because a mutation in budding yeast
Apc10/Doc1 that would be predicted to destabilize the
putative ligand binding pocket renders the yeast APC
temperature sensitive (Hwang and Murray, 1997).
Do Cdc20 and Cdh1 Recruit Substrates to the APC?
The role of Cdc20 and Cdh1 in regulating the APC was
first discovered in yeast and Drosophila where mutation
of these proteins stabilizes APC substrates, and Cdc20
and Cdh1 overexpression has the opposite effect (Dawson et al., 1995; Schwab et al., 1997; Sigrist et al., 1995;
Sigrist and Lehner, 1997; Visintin et al., 1997). Interestingly, budding yeast Cdc20 and Cdh1 appear to activate
the APC in a substrate specific manner; for example,
Cdc20 being required for degradation of the securin
Pds1 but not for complete proteolysis of Clb2 which
in turn depends on Cdh1 (Visintin et al., 1997). This
observation suggested early on that Cdc20 and Cdh1
may activate the APC by recruiting specific substrates
to the ubiquitin ligase, at least in budding yeast (in Drosophila and Xenopus embryos that lack Cdh1, APCCdc20
is clearly able to ubiquitinate both securin and mitotic
cyclins).
Recent support for the hypothesis that Cdc20 and
Cdh1 recruit substrates to the APC comes from two
observations. First, chicken cells were found to contain
four homologs of Cdh1 which in vitro all activated the
APC toward different substrates, consistent with the
possibility that these proteins recruit different substrates to the APC (Wan and Kirschner, 2001). Interestingly, these Cdh1 homologs are differentially expressed
in chicken tissues, also lending further support to the
hypothesis that the APC may have unidentified functions
in differentiating cells. Second, several groups found
that APC substrates can bind to Cdc20 and Cdh1 (Burton and Solomon, 2001; Hilioti et al., 2001; Ohtoshi et
al., 2000; Pfleger et al., 2001a; Schwab et al., 2001;
Sorensen et al., 2001). Importantly, Schwab et al. (2001)
observed that the ability of overexpressed Cdc20 and
Cdh1 to destabilize specific substrates in vivo corresponds to their ability to bind to these substrates in
vitro. Furthermore, Pfleger et al. (2001a) showed that
N-terminal fragments of Cdc20 and Cdh1 that can bind
APC substrates are also able to inhibit the degradation
of APC substrates in Xenopus extracts, suggesting that
Molecular Cell
940
the in vitro binding reactions are relevant for APC-mediated ubiquitination reactions.
Although these data suggest that Cdc20 and Cdh1
bind to APC substrates, there are also several conflicting
and unexplained observations. For example, Schwab et
al. (2001) observed that Cdh1 could bind to the mitotic
cyclin Clb2 even when Clb2’s D box had been deleted,
despite the fact that Cdh1-induced Clb2 degradation in
vivo is strictly dependent on the D box. This could imply
that in vitro binding is not always an accurate correlate
of the binding interaction that occurs in vivo or that the
D box is not actually required for binding of substrates to
Cdh1 but for their subsequent ubiquitination. Likewise, it
is not clear which parts of Cdc20/Cdh1 are required for
substrate binding. Sørensen et al. (2001) showed that
cyclin A can bind to a conserved sequence element in
the C-terminal WD40 domain of Cdh1, whereas Pfleger
et al. (2001a) proposed an important role for the N termini
of Cdc20 and Cdh1. However, if Cdc20’s and Cdh1’s
N-terminal domains were their only determinants of substrate specificity, it would be difficult to explain how the
recently identified homologs of Cdh1 in chicken cells
could confer substrate specificity to the APC, because
all of these homologs are almost identical in their
N-terminal domains (Wan and Kirschner, 2001).
Conclusions
APC’s mitotic functions and regulation are now understood in some detail, but several mysteries remain; for
example, how the APC is able to ubiquitinate cyclin A
early in mitosis without touching other substrates such
as cyclin B and securin. Also, the lists of APC’s cell cycle
substrates and of pathways that modulate its activity are
likely to grow in the future. Bigger surprises, however,
may come from areas other than cell cycle research,
which may uncover unexpected roles for the APC in
nonproliferating cells and tissues. What we are still missing almost entirely is a mechanistic understanding of
how the APC mediates ubiquitination reactions. Although this is also far from clear for other RING fingercontaining ubiquitin ligases, the APC represents a particularly puzzling example due to its unusual size and
still unexplained subunit complexity. The lack of mechanistic insight is in part due to the scarcity of this complex,
which makes its conventional purification in large scale
difficult, and to its subunit complexity, which has so
far prevented successful in vitro reconstitutions from
recombinant proteins. Therefore, many more years may
pass before we will fully understand how the APC works
and how exactly Hunt’s cyclin is degraded in mitosis.
Acknowledgments
I would like to thank Andrea Musacchio, Kim Nasmyth, and members
of my lab for comments on the manuscript, and Hannes Tkadletz and
Christian Gieffers for help with preparing the figures and referencing.
Work in my lab is supported by Boehringer Ingelheim, the Austrian
Science Fund, and the European Molecular Biology Organization.
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