Biosci. Rep. (2010) / 30 / 243–255 (Printed in Great Britain) / doi 10.1042/BSR20090171
Control of cell cycle progression by
phosphorylation of cyclin-dependent kinase (CDK)
substrates
Randy SURYADINATA*, Martin SADOWSKI* and Boris SARCEVIC*†1
*Cell Cycle and Cancer Unit, St Vincent’s Institute of Medical Research, The University of Melbourne, Fitzroy, Melbourne, Victoria 3065,
Australia, and †Department of Medicine, St Vincent’s Hospital, The University of Melbourne, Fitzroy, Melbourne, Victoria 3065, Australia
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Synopsis
The eukaryotic cell cycle is a fundamental evolutionarily conserved process that regulates cell division from simple
unicellular organisms, such as yeast, through to higher multicellular organisms, such as humans. The cell cycle
comprises several phases, including the S-phase (DNA synthesis phase) and M-phase (mitotic phase). During S-phase,
the genetic material is replicated, and is then segregated into two identical daughter cells following mitotic M-phase
and cytokinesis. The S- and M-phases are separated by two gap phases (G1 and G2 ) that govern the readiness of cells
to enter S- or M-phase. Genetic and biochemical studies demonstrate that cell division in eukaryotes is mediated by
CDKs (cyclin-dependent kinases). Active CDKs comprise a protein kinase subunit whose catalytic activity is dependent
on association with a regulatory cyclin subunit. Cell-cycle-stage-dependent accumulation and proteolytic degradation
of different cyclin subunits regulates their association with CDKs to control different stages of cell division. CDKs
promote cell cycle progression by phosphorylating critical downstream substrates to alter their activity. Here, we will
review some of the well-characterized CDK substrates to provide mechanistic insights into how these kinases control
different stages of cell division.
Key words: cell cycle, cyclin-dependent kinase (CDK), phosphorylation, protein kinase, substrate specificity, target
substrate
&
INTRODUCTION
The phosphorylation of proteins on serine, threonine and tyrosine
residues by protein kinases is a fundamental post-translational
modification that is important for regulating protein function in
nearly all eukaryotic pathways, such as cell proliferation and
differentiation, cell mobility and motility, metabolism and apoptosis. The human genome encodes for over 500 protein kinases,
which represents approx. 2 % of all human genes, exemplifying
the central role of protein phosphorylation in regulating cellular function [1]. Deregulation of protein kinase activity plays a
central role in many human diseases (reviewed in [2–6]).
Ultimately, protein kinases mediate their effects by phosphorylation of specific downstream substrate(s) to alter their biochemical and biological function(s) and elicit specific cellular
responses. Considerable effort has been directed at identifying
and characterizing protein kinase substrates to understand how
%
protein kinases mediate their biological effects. Substrates need
to satisfy several criteria to validate that they are bona fide substrates of a particular protein kinase. These include establishing
that the substrate is phosphorylated by the protein kinase on the
same sites in vitro and in vivo. The substrate must be localized in
the same cellular compartment as the protein kinase during the
period in which it is active. Finally, phosphorylation should be
of sufficient stoichiometry to induce biochemical changes in the
substrate to mediate biological effects.
CDKs: KEY REGULATORS OF THE
CELL CYCLE
The critical role of protein kinases in central biological processes is exemplified by the CDKs (cyclin-dependent kinases),
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Abbreviations used: APC/C, anaphase-promoting complex/cyclosome; BRCT, BRCA1 (breast cancer early-onset 1) C-terminus; CDC, cell division cycle; CDH1, CDC20 homologue 1;
CDK, cyclin-dependent kinase; CDT1, chromatin licencing and DNA replication factor 1; CP110, centrosomal protein of 110 kDa; cy motif, cyclin-binding motif; E16.5, etc., embryonic
day 16.5, etc.; Emi1, early mitotic inhibitor 1; G1 , Gap1 phase; G2 , Gap2 phase; GINS, from the Japanese go-ichi-ni-san meaning 5-1-2-3, after the four related subunits of the complex
Sld5, Psf1, Psf2 and Psf3; HDAC, histone deacetylase; MCM, minichromosome maintenance; M-phase, mitosis; NPAT, nuclear protein mapped to the AT locus; NPM, nucleophosmin;
ORC, origin of recognition complex; pRb, retinoblastoma protein; SCC1, sister chromatid cohesion protein 1; SCF, Skp1/cullin/F-box; Skp, S-phase kinase-associated protein; S-phase,
DNA synthesis phase.
1 To whom correspondence should be addressed ([email protected]).
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Figure 1 Cyclin–CDK regulation of the mammalian cell cycle
The cell cycle consists of a DNA synthesis (S) phase and a mitotic (M)
phase, separated by two gap (G1 and G2 ) phases. In mammalian cells
different cyclin–CDK complexes regulate progression of cells through
the different phases of the cell cycle.
which control cell cycle progression in eukaryotic organisms.
This evolutionarily conserved process is fundamental in regulating cell division from unicellular organisms, such as yeast,
through to higher organisms, such as mammals. The cell division cycle is characterized with four distinct phases, including
two gap phases (G1 and G2 ) that prepare and assess the readiness of cells to enter the S-phase (DNA synthesis phase) or the
M-phase (mitotic phase) [7] (Figure 1). Strict control of S-phase
is important to ensure that cells only undergo a single round of
chromosomal DNA replication, while M-phase governs cytokinesis, where the duplicated genetic material is segregated into two
identical daughter cells [7]. In mammalian cells, commitment to
cell division is regulated by mitogenic factors that act in the G1 phase of the cell cycle to activate CDKs and transverse a point,
termed the restriction point (R), after which they are committed
to a round of cell division independent of growth factors [8].
Progression of cells through the cell cycle is tightly regulated
by cyclin–CDK dimeric kinase complexes, where the CDK is
the catalytic kinase subunit and the cyclin is the activating subunit. Schizosaccharomyces pombe and Saccharomyces cerevisiae
CDCs (cell division cycles) are regulated by a single CDK protein, p34cdc2 and Cdc28 respectively, coupled with several different cyclins that are expressed at different cell cycle phases
[9,10]. In contrast, higher organisms possess different CDKs that
associate with the different cyclins and are important for progression through different cell cycle phases. Therefore, mammalian
cell culture studies have demonstrated that CDK4 and CDK6 in
association with D-type cyclins are activated and important for
progression during the G1 cell cycle phase [11,12]. The cyclin
E–CDK2 complex is important during transition from G1 - into Sphase [13]. Cyclin A–CDK2 and cyclin A–CDK1 are important
during S- and G2 -phase progression respectively [14–17]. Finally, progression of cells through mitosis is dependent on cyclin
B–CDK1 [18] (Figure 1). Given the importance of cyclin–CDKs
in mediating cell cycle progression, these kinases are tightly regulated at several levels to ensure that cell cycle progression occurs only under appropriate circumstances when cell division is
required. Therefore, in addition to the binding of activating cyclins, CDKs are regulated by the binding of inactivating proteins
(cyclin-dependent kinase inhibitory proteins) and activating and
inactivating phosphorylation events on critical residues of the
CDK subunit (reviewed in [3,19–22]).
The fundamental roles of cyclin–CDK complexes for cell cycle
progression and development in mammals has been extensively
studied in mouse models, through knockout or overexpression
of genes expressing particular cyclin or CDK subunits. These
studies have unveiled significant complexity in terms of developmental importance and tissue-specific action and redundancy
of CDK action. For example, mice lacking cyclin D1 display abnormalities associated with retinal development and mammary
gland development during pregnancy [23,24]. In addition, these
mice have reduced body size when compared with the wild-type
litter-mates [23,24]. In contrast, cyclin-D2-deficient mice exhibit
impaired proliferation of peripheral B-lymphocytes [25,26]. Furthermore, cyclin-D2-deficient female mice are sterile, whereas
the males retain their fertility, but exhibit smaller testes [27].
Conversely, cyclin-D3-deficient mice display impairment in the
thymus-derived T-lymphocyte development [28]. These studies
demonstrate tissue-specific action of D-type cyclins. However,
mice lacking all three D-type cyclins die by E16.5 (embryonic
day 16.5) [29], indicating that the presence of at least one D-type
is essential for viability. Similarly, mice lacking either cyclin E1
or E2 do not show a significant impact on their development;
however, double knockout of the E-type cyclins is embryonically lethal by E11.5 due to defects in endoreplication in placental
trophoblast cells [30]. Mice lacking either cyclin B1 or cyclin B2
expression demonstrated that, although cyclin-B2-deficient mice
display normal developmental patterns, no pups carrying homozygous cyclin B1 deletion survived [31]. Interestingly, cyclin B1
expression compensates the loss-of-function of cyclin B2-null
mice, but not vice versa [31]. Mice lacking either CDK4 or CDK6
remain viable, despite displaying defects in the proliferation in
specific cell types. CDK4 is important for lactotroph-derived
prolactin production and secretion [32]. Secretion of prolactin
is important for the female reproductive system as it maintains
the luteal phase (late phase of the menstrual cycle), which, in
turn, is important for pregnancy. CDK4-deficient female mice
are sterile [32]. In addition, CDK4-null mice develop diabetes
due to impaired pancreatic β-cell proliferation [33]. In contrast,
CDK6 plays a role in the expansion of differentiated haemopoietic population [34]. Deletion of both CDK4 and CDK6 results
in lethality during late embryogenesis, although fibroblasts derived from early embryos proliferate in vitro, suggesting that
other CDKs can compensate for their loss during G1 cell cycle
progression [34]. CDK2-null mice are viable for up to 2 years,
indicating that CDK2 is one of several cell cycle genes that appear to be dispensable [35,36]. However, both male and female
CDK2-deficient mice are sterile, indicating that CDK2 plays a
crucial role in germ cell development [35,36]. Unlike CDK2,
CDK4 and CDK6, CDK1 is important for early stage embryonic
development [37]. Interestingly, knockout studies demonstrate
that CDK1 can interact with cyclins D and E to form active kinase
complexes to compensate for the loss of CDK4 and CDK2 [37].
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CDK substrates
This study [37] challenges the absolute neccessity of CDKs 2,
4 and 6 in regulating cell cycle progression. However, although
CDK1 is able to partially compensate the role of CDKs 2, 4 and
6 in general, their presence is necessary to phosphorylate specific target substrates important for proliferation of specialized
cell types [37]. Altogether, the whole-animal studies with knockout mice demonstrate tissue-specific roles for different cyclins
and CDKs, and significant functional redundancy of the different
family members.
APPROACHES FOR IDENTIFYING
PROTEIN KINASE SUBSTRATES
The identification of protein kinase substrates has represented
a significant challenge, with technical limitations to the various
approaches adopted by different laboratories. Indeed, as yet there
is no single method that can simply identify the cellular substrate
complement of a protein kinase. Irrespective of the approach
used, further studies are required to validate that a protein isolated
by screening methods is a bona fide substrate of a particular
protein kinase. This is further complicated by the possibility of
multiple phosphorylation sites, as well as the same sites being
targeted by multiple protein kinases.
(i) Radioisotope labelling in vivo
Earlier approaches to identify in vivo protein kinase substrates involved incubating cells with [32 P]Pi radioisotope to radioactively
label intracellular ATP pools. Following activation of the kinase,
phosphoproteins can be separated on one- and two-dimensional
polyacrylamide gels and analysed to identify potentially phosphorylated substrates [38–40]. A major limitation of this approach is the poor signal-to-noise ratio as many cellular proteins
are phosphorylated, leading to high background phosphorylation and difficulty in identifying specifically phosphorylated substrates, particularly low-abundance protein substrates.
(ii) Radioisotope labelling in vitro
Due to the limitations of these in vivo-based approaches, most
laboratories have utilized in vitro-based phosphorylation approaches, where kinases are incubated with proteins or cellular extracts under appropriate phosphorylation conditions to
identify putative substrates. These approaches also suffer from
background phosphorylation issues, since cell lysates contain
numerous endogenous kinases and substrates, making it difficult
to identify specific substrates of the exogenous kinase. Different strategies have been adopted to circumvent these limitations.
For example, several groups have employed chromatographic
techniques to separate cellular extracts into different fractions
to reduce the background phosphorylation and enable detection
of substrates phosphorylated by added kinase [41–43] (reviewed
in [44]). An alternative in vitro approach involves engineering a
mutant protein kinase in the ATP-binding domain that specifically utilizes an analogue of ATP that cannot be utilized by other
cellular kinases [45–47]. Incubation of cellular extract with the
engineered kinase and the ATP analogue thus results in specific
phosphorylation of substrate proteins by the engineered kinase
with no background phosphorylation from other kinases. Phosphorylated proteins can then be separated on polyacrylamide
gels, isolated and identified by MS. These techniques allow for
phosphorylation of substrates in their native conformation, but a
potential limitation is that low-abundance proteins may be harder
to detect or identify by MS. This approach has successfully been
used to identify substrates of Raf1, JNK (c-Jun N-terminal kinase)
and CDKs [47–49].
(iii) Phosphorylation of phage-display or cDNA
expression libraries
In addition to phosphorylation of proteins from cell extracts, other
approaches have involved phosphorylation screening of recombinant proteins expressed from λ-phage cDNA expression libraries [50,51]. This approach involves expression of recombinant
proteins from phage cDNA expression libraries that are immobilized on nitrocellulose filters and incubated with recombinant
protein kinase in the presence of [γ-32 P]ATP. Phosphorylated
proteins are visualized by autoradiography and identified by sequencing of the cDNA of the corresponding phage clone. In
principle, this method allows for the equivalent expression of
different recombinant proteins and thus is not biased towards the
detection of highly abundant proteins. However, a caveat of this
technique is that bacterial expression may result in partially expressed protein and incorrect folding, leading to false positives.
This approach has been used to identify substrates of MAPK
(mitogen-activated protein kinase) [52] and CDKs [53]. In addition to phosphorylation of bacterially expressed proteins, other
groups have phosphorylated proteins following in vitro transcription and translation from a cDNA library in a reticulocyte lysate
system [54]. Protein expression in this system avoids the issues
of incorrect protein folding that may occur with bacterial expression. Expressed proteins were mixed with mitotic cell extracts,
and phosphorylated substrates were identified using a mitosisspecific anti-MPM-2 (mitotic protein monoclonal 2) phospho-specific antibody or by reduced mobility on SDS/PAGE.
(iv) Phosphorylation of peptides
Apart from phosphorylation of proteins, a number of laboratories have utilized peptide-based phosphorylation screening
approaches to identify the optimal phosphorylation motif of
a particular protein kinase. Protein kinases phosphorylate
residues in substrates within a defined consensus sequence context [55,56]. Initially, these were established using synthetic
peptides corresponding to local phosphorylation site sequences
of native substrates [55], but were subsequently superseded by
combinatorial peptide libraries to determine the consensus phosphorylation sequences for PKA (protein kinase A), CDKs, SLK1
(STE20-like kinase 1), protein kinases CK1 and CK2, ERK1
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(extracellular-signal-regulated kinase 1), calmodulin-dependent
kinase, phosphorylase kinase and NIMA (never in mitosis gene A
of Aspergillus nidulans) protein kinase has been defined [57–59].
By determining the consensus phosphorylation motif for a protein kinase, bioinformatic approaches have been used to identify
substrates of IKKβ and IKKε (IκB kinases β and ε) [60,61]. The
potential limitations associated with this method is that, in addition to substrate consensus phosphorylation motifs, kinases often
require other structural aspects to recognize substrates, such as
binding motifs [62–64]. In addition, if a consensus motif for a
particular kinase is not highly specific or stringent, bioinformatic searches are difficult as the motif will be present in many
proteins, necessitating the use of additional selection criteria.
(v) Yeast two-hybrid screen
Another approach that has been used to identify protein kinase
substrates is the yeast two-hybrid interaction screen, which relies
on interaction between the kinase and substrate [65–68]. However, this technique is prone to isolation of interacting proteins
that may not be substrates of a protein kinase, but binding proteins
or regulatory subunits.
(vi) Structural basis for substrate predicition:
Predikin
In addition to direct experimental approaches, an in silico
predictive approach termed ‘Predikin’ has been developed to
identify putative protein kinase substrates [69,70]. This approach
uses a set of rules based on either the known crystal structure of a protein kinase or defining the substrate determining
residues in the catalytic domain of a protein kinase based on its
primary sequence. This information is then used to predict which
heptapeptide sequences are the best substrate for a particular
protein kinase, which can then be used to search sequence databases. A limitation of this approach is that some kinases utilize
substrate-specificity determinants in addition to the sequences
proximal to the phosophorylated residue, such as anchoring or
adaptor proteins, scaffolds or subunits. Residues within the consensus site may be phosphorylated which may affect specificity.
In addition this approach does not take into account if a specific
protein kinase and putative substrate are co-localized to ensure
catalysis.
SUBSTRATES OF CDKS
Since CDKs promote cell division, defining their substrates is
critical to understanding the mechanistic basis of their action.
Although numerous CDK substrates have been described in the
literature to date, a detailed understanding of how CDK-mediated
phosphorylation controls their function and cell cycle progression at a mechanistic level has been well characterized for only
some of these. It is not within the scope of this review to discuss
all CDK substrates reported to date. A relatively comprehensive
catalogue of CDK substrates described to date can be found at
a public database (www.phosphosite.org). Rather, we will describe some of the key substrates whose CDK-mediated phosphorylation has been well characterized to provide mechanistic
insights into the key role of these kinases in controlling cell cycle
progression.
Analysis of the CDK phosphorylation sites in proteins and
peptides has revealed that these enzymes are proline-directed
serine/threonine kinases. Hence, CDKs require a proline immediately C-terminal to the phosphorylated serine or threonine residue
(S/T-P) [57–59]. Although this recognition motif is the minimal
requirement for CDKs to phosphorylate substrates, in many substrates the optimal phosphorylation motif generally consists of a
positively charged lysine or arginine three positions downstream
of the phosphorylated site (S/T-P-X-R/K) [57–59]. Although
the consensus phosphorylation motif for different CDKs is conserved, CDKs can be specific to particular substrates. A major
mechanism for regulating substrate specificity of CDKs is via the
bound cyclin subunit. For example, cyclins E and A possess a hydrophobic patch, Met-Arg-Ala-Ile-Leu (M-R-A-I-L), that recognizes and interacts with an Arg-X-Leu (R-X-L) cy motif (cyclinbinding motif) within substrates [62–64]. Mutation of either the
cy motif in the substrate or the hydrophobic patch in the cyclin impairs cyclin E–CDK2 or cyclin A–CDK2-mediated phosphorylation of particular substrates [63,64,71]. Although cyclin
E–CDK2 and cyclin A–CDK2 have overlapping substrates, each
can also target unique substrates, indicating that other distinct
features of cyclins E and A can control substrate specificity [42].
In addition to regulating phosphorylation of different substrates,
different CDKs can phosphorylate the same substrate on different sites to regulate different aspects of substrate function. For
example, cyclin D1–CDK4 and cyclin E–CDK2 phosphorylate
different sites on pRb (retinoblastoma protein) to regulate different aspects of its transcriptional inhibitory activity [72]. The
mechanism underpinning this selectivity is unclear, but may be
related to the cyclin subunits binding to different regions of pRb.
Another level of CDK-mediated substrate specificity involves differential subcellular localization. Both cyclins E and A localize
and are active with their respective CDKs in the nucleus [73–75].
Conversely, cyclin B1 exhibits dynamic subcellular trafficking
between the cytoplasm and nucleus during the cell cycle. Synthesis of cyclins B1 and B2 begins during interphase and their
localization is restricted to the cytoplasm. As the cells progress
into prophase, cyclin B1 is abruptly translocated into the nucleus,
whereas cyclin B2 remains in the cytoplasm [76,77]. The ability
of cyclins B1 and B2 to maintain their cytoplasmic localization
during interphase is dependent on an N-terminal region of 42
amino acid residues, namely the CRS (cytoplasmic-retention sequence) [77]. The dynamic cell-cycle-dependent cytoplasmic and
nuclear localization of cyclin B1 is related to the diverse range
of substrate targets of cyclin B–CDK1 complexes, whose phosphorylation is required for the major cytoskeletal and nuclear
alterations that occur during the mitotic transition. For example,
phosphorylation of the nuclear lamins is thought to contribute to
the breakdown of the nuclear envelope during mitosis [78,79].
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Other cytoskeletal proteins include the centrosomal protein of
55 kDa, Cep55 [80], and caldesmon [81–83].
(i) CDK substrates during G1 –S-phase
pRb
A major substrate of cyclin D–CDK4/6 in G1 phase is pRb, a
tumour suppressor protein of 928 amino acids [84]. pRb inhibits
G1 –S-phase cell cycle progression, and this inhibitory effect is
reversed by CDK-mediated phosphorylation (reviewed in [85]).
pRb inhibits cell cycle progression largely through binding and
inhibition of the E2F transcription factors whose activities are
required for cell cycle progression [86,87]). Transcriptionally
active E2F induces the expression of numerous genes important
for cell cycle progression, such as CDC6 [88], DHFR (dihydrofolate reductase) [89], TK (thymidine kinase) [90] and DNA
polymerase α [91]. The synthesis of cyclin E, which occurs during late G1 , is also regulated by the E2F family of transcription factors [92–95]. Initial phosphorylation of pRb by cyclin
D–CDK4/6 complexes leads to partial activation of E2F, which
allows the transcription of cyclin E gene by E2F family members.
Cyclin E then interacts with CDK2, forming an active complex
that further phosphorylates pRb, resulting in full activation of
E2F. This provides a positive feedback loop mechanism, whereby
phosphorylation of pRb by cyclin E–CDK2 induces further transcription of the cyclin E gene (reviewed in [96]).
pRb contains two functional domains, consisting of a so-called
pocket region and a C-terminal region [97,98]. The pocket of pRb
is approx. 45 kDa, comprising two domains, A and B, that are
separated by a spacer region. pRb binding inhibits E2F transcriptional activity by two mechanisms. One mechanism is through
direct inhibition of the E2F transactivation domain, where the pRb
A–B interface provides docking for E2F [97]. The C-terminal
pRb domain also binds E2F to repress activity [99]. In addition to inhibition of transactivation activity, pRb inhibits E2Fs
through active repression by recruiting members of the HDAC
(histone deacetylase) family to E2F promoters [100,101]. HDACmediated deacetylation of the core histones leads to a tighter association between the core histones and DNA on chromatin, thus
impairing access of transcriptional co-activators and leading to
transcriptional inhibition [102]. pRb contains 16 consensus CDK
phosphorylation sites, and several studies have demonstrated
that cumulative phosphorylation on multiple sites is required to
achieve full inactivation of pRb-mediated E2F inhibitory function
to allow G1 –S-phase cell cycle progression [72,103,104]. Both
cyclin D–CDK4/6 and cyclin E–CDK2 inactivate pRb through sequential phosphorylation of different sites, leading to progressive
loss of pRb-mediated E2F inhibitory function. Therefore, initial
phosphorylation by cyclin D–CDK4/6 on the pRb C-terminal
serine residues at positions 788 and 795 destabilizes its interaction with E2F [98] and induces dissociation of HDACs [72].
Subsequent phosphorylation during late G1 by cyclin E-CDK2
on threonine residues 821 and 826 and residues in the pocket
region results in an intramolecular interaction between the pRb
C-terminal and pocket regions, leading to dissociation from E2F
to allow transcription [72,98] (Figure 2).
NPAT (nuclear protein mapped to the AT locus)
In addition to regulating the transcription of genes important
for G1 –S-phase cell cycle progression through phosphorylation
of pRb, CDK-mediated phosphorylation of the NPAT transcriptional regulator is important for increased expression of histones,
required for assembling with newly synthesized DNA into chromatin prior to mitosis. NPAT was initially identified as a cyclin
E–CDK2 substrate using an in vitro phosphorylation screen of
λ-phage cDNA expression libraries [51]. Overexpression of active cyclin E–CDK2 increases NPAT phosphorylation in U2OS
osteosarcoma cells, and co-expression of NPAT with cyclin E–
CDK2 co-operates in promoting S-phase cell cycle entry. This
effect is not observed with cyclin A–CDK2 co-expression, indicating that NPAT is specifically targeted by cyclin E–CDK2 to
promote S-phase entry [51,105]. NPAT is associated with histone
gene clusters and activates histone gene transcription [105,106].
Mutation of the NPAT cyclin E–CDK2 phosphorylation sites at
Ser-775, -779 and -1100 and Thr-1270 and -1350 suppresses
its transcription from the histone H2B promoter, underlying the
critical role of cyclin E–CDK2 phosphorylation in regulating
NPAT-dependent histone gene transcription [105,106].
Centrosome duplication
Chromosomal segregation during mitosis is dependent on the
formation of bipolar mitotic spindle [107,108]. Microtubules
which are the major component of the mitotic spindle attach
to kinetochores of sister chromatids at the metaphase plate and to
centrosomes at the spindle poles. During mitosis, sister chromatids are separated and retracted by microtubules from the
metaphase plate towards the centrosomes at the spindle poles
to segregate the genetic material prior to cytokinesis to generate
two identical daughter cells. Centrosomes thus play a pivotal role
in separating sister chomatids during mitosis. Following cytokinesis, each daughter cell inherits one centrosome, and therefore it
is necessary to duplicate the centrosome prior to the next round
of mitosis to establish a bipolar mitotic spindle. The centrosome
consists of a pair of core centrioles, surrounded by an amorphous
mass of proteins termed PCMs (pericentriolar materials; providing support to the core centrioles [109]). Cyclin E–CDK2 promotes centrosome duplication [110,111] by phosphorylation of
the centrosomal proteins NPM (nucleophosmin)/B23 [108,112]
and CP110 (centrosomael protein of 110 kDa) [107]. Cyclin
E–CDK2-mediated phosphorylation of NPM/B23 results in its
dissociation from the centrosome to trigger centriole duplication. Microinjection of an anti-NPM/B23 antibody inhibits cyclin E–CDK2-mediated NPM/B23 phosphorylation and centriole duplication. The importance of cyclin E–CDK2-mediated
NPM phosphorylation in centrosome duplication is exemplified
by studies demonstrating that expression of a NPM/B23 T199A
phosphosite mutant remains associated with centrosomes and inhibits their duplication [112]. Duplicated centrosomes remain
free of NPM/B23 until mitosis. When the nuclear membrane
breaks down during mitosis, NPM/B23 relocalizes to centrosomes, and upon cytokinesis each daughter cell receives one
centrosome bound by NPM/B23. Therefore NPM/B23 is thought
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Figure 2
Cumulative phosphorylation of the pRb tumour suppressor by cyclin D–CDK4/6 and cyclin E–CDK2 alleviates
its inhibitory activity towards the E2F transcription factor
The inhibitory activity of pRb on E2F involves two mechanisms. pRb binds directly to the transactivation domain of E2F
and, in addition, recruits HDAC. Phosphorylation (P) by cyclin D–CDK4/6 in early-mid G1 phase destabilizes the C-terminal
(C-term) portion of pRb, resulting in the dissociation of HDAC. Further phosphorylation by cyclin E–CDK2 results in
conformational changes to pRb, liberating E2F which activates transcription of genes necessary for progression of cells
into S-phase.
to constitute a ‘licencing system’ for centrosome duplication. By
phosphorylation of NPM/B23 and other substrates important for
G1 –S-phase cell cycle progression, cyclin E–CDK2 co-ordinates
centrosome and DNA duplication in preparation for mitosis. Another cyclin E–CDK2 centrosomal substrate is CP110. CP110
is expressed during G1 phase, peaks at S-phase and its levels
diminish as cells enter mitosis [107]. Depletion of CP110 suppresses centrosome reduplication, whereas expression of a CP110
CDK phosphosite alanine mutant to mimic a constitutively nonphosphorylated form leads to impaired centrosome separation
and polyploidy [107].
(ii) CDK substrates during S-phase
During S-phase, CDKs play important roles in both promoting
DNA replication and ensuring that only one round of replication occurs per cell cycle. As cells enter S-phase, chromosomal
DNA replication is initiated at numerous origins concurrently
and a conserved mechanism termed the licencing system ensures
that DNA replication occurs only once per cell cycle. The DNA
replication origins consist of initiator proteins that interact with
replicator elements within the DNA [113]. The initiator protein
complex, which is collectively referred to as the ORC (origin
recognition complex), consists of six polypeptides that serve as
a foundation for protein–protein interactions important for DNA
replication, including the licencing protein complex, consisting
of the MCM (mini-chromosome maintenance) proteins 2–7 [114]
that are evolutionarily conserved. Their C-terminus display sequence homology to hexameric DNA helicases and function to
unwind the DNA for replication. All six MCM proteins are required for DNA replication, with MCM 4, 6 and 7 involved in
ATP binding and catalysis, whereas MCM 2, 3 and 5 possess
regulatory functions [115]. Studies in yeast show that during
DNA replication initiation, CDC6 and CDT1 (chromatin licencing and DNA replication factor 1) proteins mediate the interaction between the MCM proteins and the ORC, thereby creating
the pre-RC (pre-initiation replicative complex) [116,117]. In the
absence of CDC6 cells enter a pseudo-mitotic state in the absence
of replicated DNA [118]. DNA polymerase α, δ and ε are also
recruited to the origins by CDC45, which initiates DNA synthesis
(reviewed in [119]). The ORC is regulated by CDKs to prevent
re-licencing of replicated DNA prior to mitosis to prevent polyploidy. The phosphorylation status of the ORC varies during the
cell cycle with the ORC being hypophosphorylated during early
G1 , increased phosphorylation during the G1 –S transition and
being hyperphosphorylated when cells are in S-phase through to
mitosis [120–123]. Several proteins are phosphorylated by CDKs
to regulate the ORC. Hence, cyclin A–CDK1 phosphorylates
the ORC subunit Orc1 during mitosis, preventing its interaction with chromatin [123]. Another major target of CDKs in
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Figure 3 CDK regulation of CDC6 and CDT1 prevents re-replication
during DNA synthesis
During S-phase, DNA replication initiation occurs at ORCs at multiple
sites, which serve as a foundation for protein – protein interactions. The
interaction between the ORC and the licencing complex is mediated by
CDC6 and CDT1. CDK-mediated phosphorylation (P) of the ORC, as
well as the licencing complex, including CDC6 and CDT1, prevents re-licencing and DNA re-replication prior to mitosis. CDK-mediated phosphorylation of CDT1 leads to its ubiquitination (Ub) and proteosomal
degradation, whereas phosphorylation of CDC6 results in its transport
out of the nucleus.
preventing DNA re-replication in budding yeast is CDC6 (the
S. pombe homologue is cdc18). CDK-mediated phosphorylation
of CDC6 results in its ubiquitin-mediated degradation at the G1 –
S-phase transition [124]. In higher eukaryotes, CDK-mediated
phosphorylation controls nuclear transport of excess CDC6 during S-phase [125] (Figure 3). Another licencing protein which
is regulated by CDK complexes is CDT1. In higher eukaryotes,
CDK2 and/or CDK4 complexes and cyclin A–CDK1-mediated
phosphorylation of CDT1 results in its interaction with the F-box
protein Skp2 (S-phase kinase-associated protein 2) of the SCF
(Skp1/cullin/F-box) ubiquitin ligase complex, which mediates
ubiquitin-mediated proteolysis of CDT1 [126,127] (Figure 3).
Furthermore, in vitro phosphorylation of CDT1 reduces its capacity to bind to DNA, whereas inactivation of CDK1 in murine FT210 cells resulted in de-phosphorylation and re-binding
of CDT1 to chromatin [127]. In contrast, CDK-mediated phosphorylation of S. cerevisiae CDT1 resulted in its nuclear export
[128]. The MCM proteins are also targeted by the S. cerevisiae
CDK complex during S, G2 and early mitosis, which lead to
their export from the nucleus to the cytoplasm, ensuring that rereplication of the chromosomal DNA does not occur in a single
round of cell division [129].
Apart from governing DNA replication licencing, CDKs are
also crucial for regulating DNA synthesis. In S. cerevisiae, CDKmediated phosphorylation of Sld2 is necessary for initiating DNA
replication. Phosphorylated Sld2 interacts with DNA replication
protein Dpb11 to promote DNA replication [130]. A phosphosite
mutant of Sld2 where all six CDK consensus phosphorylation
sites are mutated to alanine interferes with Sld2–Dpb11 complex
formation and prevents DNA replication [130]. The ability of
Sld2 to interact with Dpb11 is due to the presence of the BRCT
[BRCA1 (breast cancer early-onset 1) C-terminus] domains located at the C-terminus of Dpb11 [131]. In addition, Dpb11 also
possesses N-terminal BRCT domains which interact with another
CDK substrate, Sld3 [132]. The primary amino acid sequence of
Sld3 contains 12 potential CDK phosphorylation sites, two of
which (Thr-600 and Ser-622) are essential for cell survival [132].
Furthermore, both Thr-600 and Ser-622 have been demonstrated
to be phosphorylated during the G1 –S-phase transition and during
S-phase at the same time as ORC6 [132]. The ability of Sld2 and
Sld3 and the effect of their phosphorylation by CDKs in regulating DNA replication remain unclear. However, it is thought that
they may target downstream replication proteins such as CDC45
and the GINS (from the Japanese go-ichi-ni-san meaning 5-1-23, after the four related subunits of the complex Sld5, Psf1, Psf2
and Psf3) complex. CDC45 interacts with Sld3 and together with
GINS forms a complex with the MCM proteins at the replication
fork (reviewed in [133]). In addition, CDC45 is also involved in
the recruitment of DNA polymerases (reviewed in [119]).
(iii) CDK substrates during M-phase
APC/C (anaphase-promoting complex/cyclosome)
Progression through mitosis is primarily governed by cyclin B–
CDK1 and inactivation of this kinase during late mitosis is necessary for cell cycle exit. A major target of cyclin B–CDK1
in regulating mitotic progression is the APC/C ubiquitin ligase.
Cyclin B–CDK1 and APC/C interplay in feedback regulation
to control mitotic progression and exit. The APC/C, which is
an evolutionarily conserved ubiquitin ligase of the RING family
consisting of at least 12 core subunits, is critical for mitosis [134].
Activation of the APC/C requires the binding of one of two
activator proteins, CDC20 or CDH1 (CDC20 homologue 1).
Both proteins are active during different phases of mitosis [135].
CDC20 binding activates APC/C in early mitosis, whereas CDH1
activates APC/C in late mitosis through to G1 phase [135]. During
early mitosis, cyclin B–CDK1-mediated phosphorylation on the
APC/C core subunits CDC27, CDC16, CDC23 and APC7 plays
a major part in activation of the APC/C and binding of CDC20
to APC/C [136]. In addition to phosphorylation of the APC/C
core subunits facilitating CDC20 binding, concurrent cyclin B–
CDK1-mediated phosphorylation of CDH1 prevents its interaction with the APC/C [137], thus maintaining APC/CCDC20 as the
major ubiquitin ligase complex during mitosis. Once activated,
APC/CCDC20 initiates the ubiquitination and proteasomal degradation of the securin proteins and mitotic B-type cyclins during the metaphase–anaphase transition. Securin is an anaphase
inhibitor protein that blocks sister chromatid separation [138–
140]. Following ubiquitin-mediated degradation of securin, the
separase enzyme is activated, which in turn cleaves the SCC1
(sister chromatid cohesion protein 1) subunit of the cohesion
complex, thus allowing the separation of the two sister chromatids [141–144]. In addition, APC/CCDC20 also ubiquitinates
cyclin B, leading to its degradation and inactivation of cyclin
B–CDK1 during late anaphase [145–147]. This in turn relieves
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R. Suryadinata, M. Sadowski and B. Sarcevic
Figure 4
Interplay of cyclin B–CDK1 and the APC/C in controlling mitosis
Early mitosis: Cyclin B–CDK1 becomes active following increased synthesis of cyclin B in late G2 phase. During early mitosis, cyclin B–CDK1-mediated phosphorylation (P) of core APC/C subunits stimulates the binding of CDC20. Concurrently,
cyclin B–CDK1 phosphorylates CDH1, preventing its interaction with the APC/C. CDC20 or CDH1 binding to the APC/C during mitosis differentially regulates the substrate preference of this ubiquitin ligase. The APC/CCDC20 complex ubiquitinates
(Ub) several substrates, including the anaphase inhibitor protein securin, which is an inhibitor of the separase protease.
Ubiquitination and proteasomal degradation of securin leads to activation of the separase enzyme which proteolyses
SCC1, allowing the separation of sister chromatids. Late mitosis: through feedback regulation, the APC/CCDC20 complex
also ubiquitinates cyclin B as cells progress through mitosis, reducing the activity of cyclin B–CDK1. Reduced cyclin
B–CDK1 activity allows CDH1 interaction with the APC/C during late mitosis. The APCCDH1 targets numerous substrates,
including CDC20 and cyclin B, to abolish CDK1 activity and mediate mitotic exit.
the phosphorylation of CDH1, thus allowing CDH1 interaction
with APC/C. As cells exit mitosis, active APC/CCDH1 targets substrates such as the CDC20 protein [148,149]. Upon re-entering
the G1 phase, the APC/CCDH1 remains active to prevent the early
activation of mitotic APC/CCDC20 complex, which can then be reactivated following increased cyclin B synthesis prior to the next
round of mitosis. The interplay of cyclin B–CDK1 and the APC/C
in controlling mitotis represents an important illustration of how
CDK-mediated phosphorylation and ubiquitin-mediated proteolysis control cell cycle progression (Figure 4).
Emi1 interacts with CDC20 and inhibits the APC/C to induce
mitotic arrest and stabilization of cyclin B [150]. Emi1 levels
oscillate through the cell cycle where its levels are increased during S-phase and depleted in M-phase [150]. Emi1 destruction at
early mitosis is mediated by the SCF ubiquitin ligase complex
[151,152]. Synergistic phosphorylation by Plk1 (polo-like kinase
1) and cyclin B–CDK1 is required for SCF-mediated ubiquitination of Emi1 in vitro [153].
Emi1 (early mitotic inhibitor 1)
CONCLUDING REMARKS
In addition to CDC20 and CDH1, an inhibitor to the APC/C,
Emi1, is a CDK substrate. Emi1 contains an F-box domain, a ZBR
(zinc-binding domain), five potential CDK phosphorylation sites
and two potential NLSs (nuclear localization sequences) [150].
Genetic and biochemical studies over the last three decades have
provided significant insights into the molecular mechanisms of
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CDK substrates
cell cycle progression and revealed that CDKs are pivotal regulators of this fundamental process. These kinases phosphorylate
numerous substrates to orchestrate the dynamic changes in cell
behaviour during cell division, such as DNA replication, mitosis and cytokinesis. How CDKs control these processes at a
mechanistic level has been well described for a few substrates
reported to date. The characterization of the full repertoire of
CDK substrates and the understanding of how their phosphorylation controls cell division awaits further study. Given the central
role of de-regulated CDK activity in many human cancers, these
studies will provide a better understanding of mechanisms of
oncogenesis.
12
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ACKNOWLEDGEMENT
We thank Bruce E. Kemp for critical reading of the manuscript prior
to submission.
18
19
FUNDING
Work in the laboratory on CDK substrates is supported by The
Cancer Council Victoria [grant number 502619]; The Australian Research Council [grant number DP0771366]; The National Breast
Cancer Foundation [grant number KCF7]; and The Breast Cancer Research Program from The Department of Defence Congressionally
Directed Medical Research Programs [grant number BC020800].
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Received 23 December 2009/29 January 2010; accepted 5 February 2010
Published on the Internet 17 March 2010, doi 10.1042/BSR20090171
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