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Journal of Cell Science 113, 1929-1938 (2000)
Printed in Great Britain © The Company of Biologists Limited 2000
JCS1193
Chromatin-bound Cdc6 persists in S and G2 phases in human cells, while
soluble Cdc6 is destroyed in a cyclin A-cdk2 dependent process
Dawn Coverley*, Cristina Pelizon, Sarah Trewick‡ and Ronald A. Laskey
Wellcome/CRC Institute, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR and Department of Zoology,
University of Cambridge, Cambridge, UK
*Author for correspondence (e-mail: [email protected])
‡Present address: ICRF, Clare Hall Laboratories, South Mimms, Herts, EN6 3LD, UK
Accepted 8 March; published on WWW 10 May 2000
SUMMARY
Cdc6 is essential for the initiation of DNA replication in all
organisms in which it has been studied. In addition,
recombinant Cdc6 can stimulate initiation in G1 nuclei in
vitro. We have analysed the behaviour of recombinant
Cdc6 in mammalian cell extracts under in vitro replication
conditions. We find that Cdc6 is imported into the nucleus
in G1 phase, where it binds to chromatin and remains
relatively stable. In S phase, exogenous Cdc6 is destroyed
in a process that requires import into the nucleus and
phosphorylation by a chromatin-bound protein kinase.
Recombinant cyclin A-cdk2 can completely substitute for
the nucleus in promoting destruction of soluble Xenopus
and human Cdc6.
Despite this regulated destruction, endogenous Cdc6
persists in the nucleus after initiation, although the
amount falls. Cdc6 levels remain constant in G2 then fall
again before mitosis. We propose that cyclin A-cdk2
phosphorylation results in destruction of any Cdc6 not
assembled into replication complexes, but that assembled
proteins remain, in the phosphorylated state, in the
nucleus. This process could contribute to the prevention
of reinitiation in human cells by making free Cdc6
unavailable for re-assembly into replication complexes
after G1 phase.
INTRODUCTION
Romanowski and Madine, 1996, 1997; Rowles and Blow,
1997; Stillman, 1996; Wuarin and Nurse, 1996). Assembly
takes place sequentially. ORC, which occupies predetermined
sites of initiation in yeast, recruits Cdc6, which in turn directs
loading of the Mcm complex (Cocker et al., 1996; Coleman et
al., 1996; Donovan et al., 1997; Romanowski et al., 1996;
Rowles et al., 1996; Tanaka et al., 1997). Together they form
a pre-replication complex (pre-RC), which is activated at the
start of S phase.
Analysis of the proteins associated with yeast replication
origins in cyclin-dependent kinase (cdk) mutants, and of the
behaviour of the Mcm proteins in the presence of protein
kinase inhibitors in mammalian cells (Coverley et al., 1998;
Dahmann et al., 1995; Piatti et al., 1996), suggest that
phosphorylation events at the start of S phase are required for
activation of the pre-RC and that continued kinase activity
throughout S and G2 prevents reassembly of new complexes.
The proteins of the pre-RC generate a specific DNase I
protection profile in G1, which becomes less extensive in S and
G2 phases (reviewed in Diffley, 1996). This is consistent with
destruction or displacement of some of the proteins involved
in initiation. In Xenopus, yeast and mammalian cells, Mcm
proteins are displaced from chromatin as cells proceed through
S phase (Chong et al., 1995; Krude et al., 1996; Kubota et al.,
1995; Madine et al., 1995b; Todorov et al., 1995) and in yeast,
Cdc6 protein levels decrease (Cocker et al., 1996; Jallepalli et
Duplication of nuclear DNA is strictly coordinated with the rest
of the cell cycle so that it occurs only once before the cycle is
reset at mitosis. Regulated initiation of mammalian DNA
replication can now be achieved entirely in vitro using soluble
protein extracts made from human cells synchronised in S
phase. A single round of DNA synthesis can be induced in
nuclei from HeLa cells chemically synchronised in late G1
(Krude et al., 1997) or nuclei from mouse cells released from
quiescence into G1 (Stoeber et al., 1998). In the latter case the
proportion of nuclei which begin DNA synthesis is greatly
increased by supplementing S-phase extracts with recombinant
Cdc6 protein.
Cdc6 was first identified in yeast (Zhou et al., 1989), but
homologues have been found in higher eukaryotes including
Xenopus (Carpenter et al., 1996) and humans (Hateboer et al.,
1998; Saha et al., 1998; Williams et al., 1997). All evidence
suggests that it is a key regulatory protein, which is essential
for initiation of DNA replication (Carpenter et al., 1996;
Cocker et al., 1996; Coleman et al., 1996; Hateboer et al., 1998;
Kelly et al., 1993; Yan et al., 1998). Cdc6, the origin
recognition complex (ORC) and the Mcm family of proteins
assemble at sites of future initiations in G1 in a regulated
process known as replication licensing (reviewed in Botchan,
1996; Diffley, 1996; Muzi-Falconi et al., 1996; Newlon, 1997;
Key words: Cdc6, Proteolysis, S phase, Cyclin-dependent kinase
1930 D. Coverley and others
al., 1997; Nishitani and Nurse, 1995; Piatti et al., 1996, 1995).
In higher eukaryotes the fate of Cdc6 after initiation is still
controversial.
There is confusion in the literature about the behaviour of
endogenous human Cdc6 in S phase. A number of reports
looked at the total level of endogenous Cdc6 in the cell cycle
by western blot analysis in Wi38 cells (Williams et al., 1997),
CV1 cells (Petersen et al., 1999), 3T3 cells (Saha et al., 1998)
and U2OS cells (Jiang et al., 1999). They found that apart from
a drop in level during quiescence and during mitosis the total
amount of Cdc6 protein remains relatively stable in late G1, S
and G2 phases. These observations are not in question.
However, analysis of the subcellular distribution of Cdc6 upon
transition from G1 to S phase has yielded conflicting data.
Immunofluorescence data showing the subcellular distribution
of overexpressed Cdc6 in U2OS cells (Jiang et al., 1999;
Petersen et al., 1999; Saha et al., 1998) or endogenous Cdc6
in CV1 cells (Petersen et al., 1999) find that nuclear staining
is present in G1 but absent in S phase. Total levels of
endogenous protein remain fairly constant, so loss of S-phase
staining has been linked to increased cytoplasmic staining.
There is some evidence in favour of increased cytoplasmic
Cdc6 in S phase (Jiang et al., 1999; Petersen et al., 1999; Saha
et al., 1998). Contradictory to these observations, several
studies report persistent Cdc6 in the S-phase nucleus. In nuclei
from 3T3 cells, Cdc6 remains present beyond G1 (Stoeber et
al., 1998) and in HeLa cells the level of nuclear bound Cdc6
was unchanged between G1, S phase and G2 (Fujita et al.,
1999) although cytoplasmic Cdc6 became apparent in S phase.
In addition, nuclear Cdc6 has been detected by
immunofluorescence in S phase in newborn human fibroblasts
and in 90% of nuclei in premalignant human cervix tissue
(Williams et al., 1998).
Our analysis shows conclusively that Cdc6 persists in human
nuclei beyond S phase and also demonstrates the existence of
two isoforms, one of which is unstable in isolated nuclei. The
relevance of these isoforms to the apparent conflict in the
literature is discussed.
In addition, by monitoring the fate of exogenous free Cdc6
in vitro, we extend the results obtained in overexpression
experiments (Jiang et al., 1999; Petersen et al., 1999; Saha et
al., 1998). Free Cdc6, not assembled into replication
complexes, is destroyed by proteolysis in a reaction triggered
specifically by cyclin A-cdk2 in the S-phase nucleus. Thus
reassembly of replication complexes in S phase is prevented,
allowing only one round of replication within a single cell
cycle.
MATERIALS AND METHODS
Cell culture and synchronisation
HeLa cells, NIH 3T3 cells and Wi38 cells were cultured in Dulbecco’s
modified Eagle’s medium supplemented with 10% foetal calf serum,
10 i.u./ml penicillin and 0.1 mg/ml streptomycin at 37°C. Sf9 insect
cells were grown in Grace’s medium containing 10% foetal calf serum
and 5 µg/ml gentamycin (all reagents from Gibco-BRL). HeLa cells
were synchronised in S phase by double thymidine block (0.4 mg/ml)
of 17 hours and 15 hours separated by a 9-hour release. To generate
a G1 population, pre-synchronised cells were arrested in mitosis by
40 ng/ml nocodazole (Sigma) and released into G1 by mitotic shake
off. NIH 3T3 and Wi38 cells were synchronised in G0 by contact
inhibition as previously described (Stoeber et al., 1998). G1 phase
populations were achieved by releasing confluent cells from
quiescence by subculturing and replating 1 in 6 into standard medium
for 16-18 hours. Synchronies were checked by flow cytometry and by
in vitro elongation assays, which report on the proportion of nuclei in
S phase.
Preparation of nuclei and cytosolic extracts
Nuclei were prepared as previously described (Krude et al., 1997).
Briefly cells were washed and swollen in hypotonic buffer then
scraped from the plate and disrupted by 10 light strokes in a dounce
homogeniser, not 25 as previously described. In some experiments
homogenisation was done in the presence of 1 µM okadaic acid
(Calbiochem). Nuclei were pelleted and the supernatants used to
produce cytosolic extract. Nuclei were washed and stored as
previously described (Stoeber et al., 1998). Supernatants or
homogenised mitotic cells were spun at 14,000 rpm for 20 minutes to
remove residual insoluble material and frozen in aliquots in liquid
nitrogen. All materials were frozen and thawed only once.
Production and purification of Cdc6
Sf9 cells were infected with a 6×His-tagged Xenopus Cdc6
recombinant baculovirus (Coleman et al., 1996) or a derivative in
which the serine residue in all five Cdk consensus phosphorylation
sites were changed for alanines. This mutant is designated M9 (C.
Pelizon et al., manuscript in preparation). A baculovirus expression
construct coding for 6×His-tagged human Cdc6 was produced by
inserting the coding sequence for human Cdc6 (from GenBank) into
the BamHI site of vector pVL-6His. Recombinant protein was purified
using a nickel affinity column according to published procedures
(Kunagai and Dunphy, 1995). Column fractions containing Cdc6 were
pooled and dialysed against 20 mM Tris, pH 7.4, 150 mM NaCl, 5%
glycerol, 1 mM DTT then stored at −80°C.
Antibodies
Anti-Xenopus Cdc6 polyclonal antibody was raised in rabbits using
full-length recombinant Xenopus Cdc6 as antigen and purified by
standard procedures (Harlow and Lane, 1988). Purified anti-Xenopus
Cdc6 antibody was used at 1:1200 dilution for immunofluorescence
studies, and under these conditions did not cross-react with human or
mouse Cdc6.
The anti-human Cdc6 polyclonal antibody has been published
previously by this laboratory and was produced and purified as
described (Stoeber et al., 1998).
Actin in isolated nuclei was detected on western blots using
affinity-purified rabbit anti-peptide actin antibody (Sigma).
Nuclear import, protein degradation and phosphorylation
reactions
Cdc6 (100 ng) was incubated with or without intact nuclei (1-5×104)
in 10 µl of cytosol, for analysis by western blot. Reactions were scaled
up 2-3 times for immunofluorescence studies. Cytosols were prepared
and supplemented as for replication reactions (Krude et al., 1997) with
1.3 mM MgCl2, 20 mg/ml creatine phosphokinase and a 10× frozen
reaction pre-mix (to give 40 mM Hepes, pH 7.8, 7 mM MgCl2, 3 mM
ATP, 0.1 mM GTP, 0.1 mM CTP, 0.1 mM UTP, 0.1 mM dATP, 0.1
mM dGTP, 0.1 mM dCTP, 0.5 mM DTT, 40 mM creatine phosphate)
just before use. Reactions were incubated for 30 minutes, or as
specified in figure legends, at 37°C. Reactions were variously
supplemented with 0.5 mM olomoucine (Sigma), importin β dominant
negative mutant (Kutay et al., 1997), 100 nM leptomycin B (Nishi et
al., 1994), 0.05 mM MG132 (Sigma), p21 (Furuno et al., 1999) or sf9
cell lysates containing human cyclin-cdk complexes. Protein kinase
activity levels in unpurified sf9 lysates made from cells infected with
human cyclin A and cdk2 or cyclin E and cdk2, or cyclin D and cdk6
baculovirus constructs, were normalised by their ability to
phosphorylate the retinoblastoma protein in standard kinase assays.
Cdc6 in S phase in human cells 1931
5×105 Rb phosphorylating units were added per 20 µl reaction.
Reaction products were analysed in one of two ways.
For immunofluorescence studies, reactions were diluted 50-fold in
phosphate-buffered saline (PBS) or PBS/0.5% Triton X-100 and then
fixed with an equal volume of 8% paraformaldehyde. Nuclei were
spun through 30% sucrose in PBS onto polylysine-coated coverslips
as described (Mills et al., 1989). Exogenous Cdc6 was detected using
Xenopus specific purified polyclonal antibody in ‘antibody buffer’
(0.1% Triton X-100, 0.02% SDS, 10 mg/ml BSA in PBS) and
visualised using anti-rabbit-FITC (Vector labs). DNA was
counterstained with propidium iodide for visualisation by
fluorescence confocal microscopy and digital images were merged
using Adobe photoshop.
For immunoblotting, whole reactions were stopped by the addition
of SDS-PAGE loading buffer and heated to 95°C for 10 minutes.
Reaction products were separated by electrophoresis through 7 or 8%
polyacrylamide gels and transferred to nitrocellulose. Blots were
developed with polyclonal anti-Xcdc6 or polyclonal anti-human Cdc6
and anti-rabbit conjugated to horse radish peroxidase (Sigma), using
standard protocols. Blocking and antibody incubations were in 0.4%
Tween 20, 10% low-fat milk powder in PBS. Bands were visualised
using Enhanced ChemiLuminesence (ECL, Amersham) and preflashed Kodak film. Quantification of band intensities was done using
NIH image software and short ECL exposures.
Replication initiation and elongation assays
Nuclei (approximately 1×104 per 10 µl reaction) were incubated at
37°C for 2 hours in 75 mM NaCl, 250 mM sucrose, 0.5 mM spermine,
0.15 mM spermidine, 3% BSA for elongation assays or S-phase
cytosolic extract for initiation assays supplemented with ATP and
ATP-regenerating system and 10× nucleotide mix as above. In
addition reactions were supplemented with biotinylated dUTP, which
was detected after fixation in paraformaldehyde, using streptavidinFITC (Mills et al., 1989). Replication is expressed as the percentage
of nuclei in a population that show fluorescence.
RESULTS
Exogenous Cdc6 accumulates in intact G1 nuclei
We first asked whether Cdc6 enters the nucleus under in vitro
replication conditions. We chose to use purified recombinant
Xenopus Cdc6 protein in these studies for three reasons. Firstly,
because we know it stimulates replication in mammalian nuclei
in vitro (Stoeber et al., 1998), secondly because we have a
mutant form in which all five consensus cdk phosphorylation
sites are mutated and thirdly, the ease with which Xenopus
Cdc6 is detected in immunofluorescence assays or by western
blot using species-specific polyclonal antibodies that do not
recognise either the endogenous human or mouse proteins.
Using G1-phase cytosol prepared from 3T3 cells 16.5 hours
after release from quiescence we monitored the accumulation
of exogenous Cdc6 in nuclei from the same cells. In
immunofluorescence assays (Fig. 1A) we found that Cdc6
efficiently accumulated in G1 in vitro. In order to prove that
accumulation is not the result of diffusion through a damaged
nuclear membrane, we included a dominant-negative mutant of
the import factor importin β (Kutay et al., 1997). The mutant
protein completely abolished accumulation of Cdc6 in the
nucleus (+ import inhibitor). This provides an important control
for nuclear membrane integrity. In addition, this data is strong
evidence that Cdc6 is actively transported into G1 nuclei and is
consistent with reports of a functional nuclear localisation
signal (NLS) (Petersen et al., 1999; Takei et al., 1999).
We then asked whether imported Cdc6 binds to chromatin.
Accumulated Cdc6 resisted extraction with detergent (+TX100
wash) showing that it binds to structures within the nucleus,
probably chromatin. Furthermore, detergent extraction did not
significantly decrease Cdc6 staining intensity, suggesting that
all of the Cdc6 in the nucleus is chromatin bound. The
functional significance of this binding has not yet been
demonstrated.
We looked for evidence that nuclear protein export might
regulate Cdc6 levels using leptomycin B, a potent inhibitor of
Crm1-dependent export (Nishi et al., 1994). In several
immunofluorescence experiments with this drug, we detected
no increase in protein in the nucleus (Fig. 1A, +leptomycin B)
implying that in late G1, nuclear protein export does not control
nuclear Cdc6 levels. However, as a number of soluble export
factors can be lost from isolated nuclei (Kehlenbach et al.,
1998) it is likely that nuclear protein export does not function
optimally in our system. This has allowed identification of
another clear level of regulation (see later).
Exogenous Cdc6 does not accumulate in the
nucleus in S phase
We examined the behaviour of exogenous Cdc6 using nuclei
and cytosols from different phases of the cell cycle. We used
isolated nuclei and the equivalent soluble extracts from
quiescent 3T3 cells (G0), 3T3 cells released from quiescence
for 16.5 hours (late G1) or 21 hours (S) and from HeLa cells
released for 4 hours from a mitotic nocodazole block (early
G1), or released for 2 hours (S) or 8 hours (G2) from thymidine
arrest. Exogenous Cdc6 protein accumulated in the nucleus in
G1 and G0 3T3 nuclei and to a lesser extent in early G1 HeLa
nuclei, but failed to accumulate in the nucleus in S phase and
G2 (Fig. 1B). This is consistent with reports which look at the
localisation of overexpressed Cdc6 in intact cells (Jiang et al.,
1999; Petersen et al., 1999; Saha et al., 1998). Absence of Sphase nuclear staining in our experiments suggests that
exogenous recombinant Cdc6 is subject to controls similar to
overexpressed Cdc6 in intact cells. We next asked why
exogenous protein fails to accumulate in S phase.
Neither S-phase nuclei nor S-phase cytosol allow
nuclear accumulation of Cdc6
We dissected the G1-specific nuclear accumulation of Cdc6 by
mixing nuclei and cytosol from different phases of the cell
cycle and assaying for the presence of Cdc6 in the nucleus by
immunofluorescence (Fig. 2A). G1, S, G2, M and G0 cytosols
were assayed for the ability to support accumulation of
exogenous Cdc6 in early G1 HeLa cell nuclei. Only G1 and G0
cytosols supported accumulation of Cdc6. These observations
show that factors in the soluble fraction from S and to a lesser
extent G2 cells can prevent Cdc6 accumulation in G1 nuclei.
Fig. 2A also compares the effect of S phase and G1 cytosolic
environments on import into G1, S and G2 nuclei. Neither S
phase nor G2 nuclei accumulated Cdc6, even in a G1 cytosolic
environment. Therefore, contributions of either S/G2 nuclei or
S/G2 cytosol prevent accumulation of Cdc6 in the nucleus.
Fig. 2B shows a control reaction in which the behaviour of
Xenopus Cdc6 is compared to that of Xenopus nucleoplasmin.
Incubation with either S phase or G1 cytosol produced the usual
S-phase-dependent exclusion of Cdc6 from G1 nuclei while
nucleoplasmin efficiently accumulated in both. This shows that
1932 D. Coverley and others
Fig. 1. Exogenous Cdc6 accumulates in G1 nuclei but not S-phase nuclei. (A) Recombinant Xenopus Cdc6 accumulates inside intact nuclei
from NIH 3T3cells, when incubated in G1 cytosol (Cdc6 only). Accumulation in the nucleus in G1 is completely blocked by inclusion of a
dominant negative importin β fragment (+ import inhibitor) but is unaffected by a nuclear protein export inhibitor (+ leptomycin B).
Accumulated protein is resistant to extraction with detergent (+ TX100 wash), indicating that imported Cdc6 is bound to structures within the
nucleus. (B) Cdc6 accumulates inside intact nuclei from G1 or G0 NIH 3T3 cells or G1 HeLa cells, but not in nuclei from S-phase 3T3 cells or
S-phase or G2 HeLa cells, during incubation in matching cytosols. A clear transition is seen between G1 and S phase. Nuclei are stained red
with the DNA stain propidium iodide. Cdc6 is detected using a species-specific affinity purified polyclonal antibody raised against recombinant
Xenopus cdc6, and a secondary antibody conjugated to FITC, generating a yellow image in positive nuclei when images of the two
fluorochromes are merged.
S-phase-dependent exclusion from the nucleus does not apply
to all proteins, but is selective for Cdc6.
S-phase nuclei promote destruction of Cdc6
Recombinant Cdc6 does not accumulate in the nucleus in S
phase, so we looked at the total level of Cdc6 protein, which
survives under the conditions used in the immunofluorescence
experiments shown in Figs 1 and 2. Complete nuclear import
reactions containing nuclei, cytosol and 100 ng recombinant
Cdc6 were analysed by western blot after separation by SDSPAGE. When incubated with S-phase nuclei and S-phase
cytosol the majority of Cdc6 was destroyed after 30 minutes
(Fig. 3A, lanes 3 and 5). Cdc6 destruction is absolutely
dependent on the presence of nuclei, as incubation with Sphase cytosol alone (lanes 1 and 4) does not affect Cdc6
survival compared to the amount of protein present at the start
Fig. 2. S-phase nuclei and Sphase cytosol both block
accumulation of Cdc6.
(A) Nuclei from
synchronized cells were
mixed with cytosols from
different cell-cycle phases
and assayed for their ability
to accumulate exogenous
Cdc6. S-phase nuclei do not
support accumulation in any
cytosol and S-phase cytosol
does not support
accumulation in any nuclei,
therefore both S cytosol and
S nuclei contain regulators of
Cdc6 subcellular distribution.
G2 nuclei behaved the same
as S nuclei, while G2 cytosol
and M-phase cytosol were
similar, but slightly less
efficient than S-phase
cytosol, at blocking
accumulation into G1 nuclei. G0 cytosol behaved like G1 cytosol and supported accumulation into G1 nuclei. Nuclei are stained red with
propidium iodide; Cdc6 staining as in Fig. 1, generates a yellow image in positive nuclei. (B) Xnucleoplasmin accumulates in the nucleus in S
phase. To show that the differing ability to accumulate Cdc6 in the nucleus between G1 and S phase is not a general phenomenon we monitored
the behaviour of another protein under identical conditions. FITC-conjugated Xnucleoplasmin accumulated efficiently in G1 nuclei when
incubated in both G1 and S-phase cytosols, while in the same experiment Cdc6 did not. Cdc6 is detected using species-specific anti-Cdc6
antibody and FITC-conjugated secondary antibody. Nuclei are not counterstained.
Cdc6 in S phase in human cells 1933
Fig. 3. Phosphorylation inside the nucleus in S
phase promotes destruction of free Cdc6.
(A) Destruction of Cdc6 in S phase is dependent
upon the presence of nuclei. Incubation of 100 ng
of recombinant Xenopus Cdc6 in S-phase cytosol
in the absence of nuclei (lanes 1 and 4) does not
result in any loss of protein compared to the
amount of Cdc6 present at the beginning of a
reaction (lane 2). Inclusion of S-phase nuclei
(lanes 3 and 5) or G1 nuclei (lane 6) resulted in
substantial loss of exogenous Cdc6 within 30
minutes at 37°C. (B) S-phase nuclei also promote
destruction of Cdc6 in G1 cytosol (lane 3), but G1
nuclei do not (lane 2). The slight increase in
survival of Cdc6 seen with G1 nuclei compared to
the control reactions that contain no nuclei (lanes
1 and 4) is abolished when nuclear import is
inhibited (lane 5). This suggests that the G1
nuclear environment can protect Cdc6 from slow
degradation by cytosolic extract. (C) The
recombinant Cdc6 that survives in S phase
fractionates entirely with the nucleus after
permeabilization with detergent. (D) Rapid Sphase destruction of Cdc6 (lane 2) is dependent
upon nuclear import. Inclusion of importin β
dominant-negative mutant protein to an S-phase
nuclei/S-phase cytosol incubation (lane 3)
resulted in Cdc6 survival, compared to survival in
the control reaction. (E) Cdc6 is protected from
destruction during incubation with S-phase
nuclei/S-phase cytosol by either olomoucine
(OM) or MG132. This is consistent with a model
in which cdk phosphorylation activates proteosome-mediated destruction of Cdc6. (F) Mutant Xenopus Cdc6 protein, in which the serine
residues in all five consensus Cdk phosphorylation sites were mutated to alanines, survives incubation with S-phase nuclei/S-phase cytosol,
arguing for direct phosphorylation of Cdc6 as the trigger for destruction. Destruction assays ran for 30 minutes and were stopped by addition of
SDS-PAGE loading buffer. Total products of each reaction were analysed by western blot for survival of the exogenous protein using Xenopus
Cdc6-specific polyclonal antibody. In C, reactions were first mixed with an equal volume of 0.5% Triton X-100 then separated into soluble and
insoluble fractions by centrifugation to assess whether surviving Cdc6 is present in the nucleus or cytosol.
of the reaction (lane 2). When incubated in S-phase cytosol,
G1 nuclei also promote destruction of Cdc6 (lane 6). Since Sphase cytosol alone does not catalyse destruction of Cdc6 it is
the combination of S-cytosol and G1 nuclei that is responsible
for loss of exogenous protein in this experiment. This suggests
that G1 nuclei can be induced to perform S-phase functions by
soluble S-phase factors, and parallels the induction of DNA
replication in G1 nuclei by S-phase cytosol (Krude et al., 1997;
Stoeber et al., 1998). Furthermore, transient exposure to Sphase cytosol will activate G1 nuclei to perform both Cdc6
destruction and initiation of DNA replication even in G1
cytosol (not shown), suggesting that the switch to S-phase
function is irreversible.
G1 nuclei do not promote destruction when assayed in G1
cytosol (Fig. 3B). In fact, by monitoring survival of the whole
population of exogenous Cdc6 (rather than just the nuclear
protein by immunofluorescence), G1 nuclei were found to
promote overall survival of Cdc6, as slightly more Cdc6 is
observed at the end of an incubation if G1 nuclei are included
(compare lane 2 to lanes 1 and 4). During similar reactions
containing only cytosol and Cdc6 some loss of protein (91%
survival) was observed after 30 minutes while significant loss
(56% survival) was not seen until after 60 minutes (not shown).
G1 nuclei could protect exogenous Cdc6 from this ‘slow mode’
cytosolic degradation by sequestering protein within the
nucleus. To ask whether improved survival is dependent on
nuclear import we included importin β dominant-negative
mutant protein. When import is blocked, protection by G1
nuclei is abolished (compare lanes 2 and 5). This is consistent
with a model in which chromatin binding protects against Cdc6
instability.
Similar to the protection by G1 nuclei, a small amount of
Cdc6 always survives proteolysis in S-phase nuclei in vitro,
even when reactions are incubated for several hours rather than
the usual 30 minutes. Furthermore, when increasing numbers
of nuclei are added the amount of protein that survives
increases (not shown), suggesting that nuclei contain a limited
number of binding sites capable of protecting Cdc6. Consistent
with this, surviving Cdc6 protein fractionates with the nucleus
after permeabilization with detergent (Fig. 3C).
Slow degradation in G1 cytosol differs from the rapid
destruction catalysed by S-phase nuclei in several ways. Most
significantly, when nuclear import is blocked by the mutant
importin β protein, S-phase destruction of Cdc6 is reduced
(Fig. 3D). The rapid destruction of Cdc6 by S-phase nuclei is
in agreement with the absence of immunofluorescence signal
in S-phase nuclei (Figs 1 and 2), and demonstrates a role for
regulated proteolysis of Cdc6 in the mammalian cell cycle.
1934 D. Coverley and others
Other workers have proposed that the lack of
immunofluorescence signal in S-phase nuclei is caused by
export of Cdc6 from the nucleus, as overexpressed Cdc6 was
found to remain in the nucleus (and not accumulate in the
cytoplasm) when transfected human tumour cells were treated
with leptomycin B and cycloheximide (Jiang et al., 1999). In
those experiments cytoplasmic absence could reflect cessation
of synthesis of new Cdc6, because of cycloheximide rather
than blocked nuclear export. In addition they do not report
quantification of the Cdc6 levels remaining in the nucleus and
therefore do not rule out a contribution of nuclear proteolysis.
Our experiments, which follow the fate of a defined amount of
protein, are consistent with these previous reports but reveal an
additional level of regulation.
S-phase destruction of Cdc6 is regulated by cyclindependent kinases
It has been reported that loss of Cdc6 immunofluorescence
signal from the nucleus in S-phase mammalian cells is
regulated via phosphorylation of conserved N-terminal CDK
consensus sites (Jiang et al., 1999; Petersen et al., 1999; Takei
et al., 1999). Phosphorylation drives Cdc6 from the nucleus.
We asked whether S-phase-specific degradation of Cdc6 is
also regulated by cdk-dependent phosphorylation. Cdc6 was
incubated with S-phase cytosol and nuclei in the presence of
the cdk inhibitor olomoucine or MG132, a peptide inhibitor
of the proteosome (Lee and Goldberg, 1998). Destruction of
exogenous Cdc6 was efficiently blocked by either drug (Fig.
3E), consistent with a model in which cdk phosphorylation
targets Cdc6 for destruction. More conclusively, a nonphosphorylatable mutant version of Xenopus Cdc6 remains
unaffected during incubation with S-phase nuclei/S-phase
cytosol (Fig. 3F), demonstrating directly that phosphorylation
of Cdc6 is an essential regulatory event in the pathway to
destruction.
We also looked for any indication that S-phase-specific
destruction of Cdc6 might be linked to ongoing DNA synthesis
or transcription, but we saw no effect on Cdc6 survival when
aphidicolin or actinomycin D were included (not shown).
Activation of Cdc6 destruction by recombinant
cyclin A-cdk2
Cdc6 has been shown conclusively to interact specifically with
active cyclin A-cdk2 complexes (Petersen et al., 1999) and to
disappear from the nucleus when cyclin A-cdk2 is activated at
the start of S phase. We asked whether destruction of Cdc6
could be promoted in G1 cytosol by recombinant cyclin A-cdk2
(Fig. 4A). Compared to the control reactions with no additions,
cyclin A-cdk2, but not equivalent amounts of cyclin E-cdk2 or
cyclin D-cdk6 activity, caused efficient destruction of
recombinant Xenopus Cdc6, in the absence of any nuclei.
Importantly, when human Cdc6 was incubated in G1 cytosol,
supplemented with cyclin A-cdk2 it behaved in exactly the
same way as Xenopus Cdc6 (Fig. 4B), eliminating the
possibility that the regulated destruction seen here is specific
to the Xenopus protein.
Although the in vivo phosphorylation event that marks Cdc6
for destruction is mediated by a nuclear-associated protein
kinase, this experiment shows that when cyclin A-cdk2 is
added the proteolysis event can occur in the cytosolic fraction.
Additional analysis in which Cdc6 was incubated with S-phase
Fig. 4. Cyclin A-cdk2 activated destruction of Cdc6. (A) Equivalent
amounts of recombinant human cyclin A-cdk2, cyclin E-cdk2 or
cyclin D1-cdk6 activity were added to G1 cytosol in the presence of
exogenous Cdc6. Cdk2 or cdk6 uncomplexed to cyclin were included
as controls. Cyclin A-cdk2 promoted destruction of Cdc6 in the
absence of any nuclei. (B) Like Xenopus Cdc6, destruction of human
Cdc6 is regulated by cyclin A-cdk2. Cytosols from G1 HeLa cells or
G1 Wi38 cells were supplemented with cyclin A-cdk2 as in A.
(C) Destruction of Cdc6 by S-phase nuclei/S-phase cytosol is
efficiently inhibited by the specific cdk inhibitor p21. Reactions were
processed and analysed as for Fig. 3, except that recombinant human
Cdc6 was detected with anti-human Cdc6 polyclonal antibody.
nuclei in buffer, in the absence of cytosol (not shown),
indicated that proteolysis is not dependent on the cytosolic
fraction. These observations are consistent with presence of the
proteosome in both the nuclear and cytoplasmic compartments
(reviewed in Lee and Goldberg, 1998).
When S-phase nuclei are incubated in, but then removed
from, S-phase cytosol the activating kinase does not leak out
(not shown), consistent with the kinase being chromatin bound.
The experiments shown here argue that this kinase is
chromatin-bound cyclin A-cdk2.
Finally, to confirm that destruction of Cdc6 is activated by
a cdk2-dependent kinase we have shown that the G1 cyclindependent kinase inhibitor p21 (Furuno et al., 1999) blocks Sphase destruction of Cdc6 (Fig. 4C). Consistent with this, other
workers have reported that all cells in an asynchronous
Cdc6 in S phase in human cells 1935
Fig. 5. Human Cdc6 protein
persists in the nucleus in S
phase. (A) HeLa cells
arrested in mitosis (M),
early G1, S phase (S) or G2
were separated into soluble
and nuclear associated
fractions by dounce
homogenization and
centrifugation. 1×105 cell
equivalents were loaded for
each sample and separated
by SDS-PAGE. Human
Cdc6 was detected using
affinity-purified polyclonal
antibody raised against
recombinant human Cdc6.
Two Cdc6 isoforms are
present in S and G2 nuclei.
(B) The slower migrating
isoform of nuclear Cdc6 is
unstable. In a separate experiment nuclei were prepared from S-phase cells at room temperature in the presence and absence of the phosphatase
inhibitor okadaic acid. In both preparations the fast migrating form is the most abundent while the slower migrating form is only apparent when
prepared in the presence of inhibitor. (C) Modification of exogenous Xenopus Cdc6 protein by cytosolic extracts from synchronised HeLa cells.
S- and G2-phase cytosols, but not G1-phase cytosol, modify exogenous Cdc6 to a slower migrating form. This modification is prevented by
inclusion of the cdk inhibitor olomoucine. Soluble extract from nocodazole-arrested M-phase cells produced a greater degree of retardation,
which may be due to more extensive phosphorylation. (D) HeLa cells arrested at the beginning of S phase by thymidine block were released
into medium containing nocodazole. Nuclei were prepared at 0, 3, 6, 9 and 11 hours. Flow cytometry profiles for the different populations are
overlayed to demonstrate progression through S phase. By 11 hours approximately 30% of the population was arrested in mitosis; these cells
were discarded to produce a late G2 population. (E) Approximately 1×105 nuclei from the 0, 3, 6, 9 and 11 hour populations shown in D were
denatured, seperated by SDS-PAGE and blotted for Cdc6 and actin. (F) Cdc6 and actin band intensities were quantified using NIH image
software. Cdc6 levels were normalised against actin and plotted (open circles) on the same axes as the percentage of elongating nuclei for each
population (closed circles). Nuclei that were actively replicating in vivo at the time of isolation continue to elongate their DNA in vitro, when
incubated in a buffered mix of nucleotides and energy regenerating system. This provides an independent measure of progression through S
phase. Cdc6 levels fell by half upon entry into S phase and then remained almost constant. By 9 hours S phase was completely finished yet
Cdc6 protein levels remained high, until just before mitosis.
population that stain positively for nuclear p21 are also positive
for epitope-tagged human Cdc6 (Saha et al., 1998). These data
suggest that one of the roles of p21 in vivo in G1 phase is to
protect Cdc6 from modification and subsequent destruction, so
facilitating assembly of replication complexes.
Chromatin-bound endogenous Cdc6 persists in S
phase
We have monitored the level of endogenous human Cdc6
protein in HeLa cell cytosols and nuclei prepared under in vitro
replication conditions, using a previously characterised
affinity-purified polyclonal antibody raised against
recombinant human Cdc6 (Stoeber et al., 1998). We see
persistent Cdc6 in S-phase nuclei and also in G2 phase nuclei,
which typically include around 30% late S-phase cells (Fig.
5A). Quantification of nuclear Cdc6 band intensity (Fig. 5E)
in synchronised populations taken 0, 3, 6, 9 and 11 hours after
release from thymidine arrest reveals that Cdc6 levels fall to
approximately one half (relative to actin) by 3 hours into S
phase, and then remain constant until just before mitosis. At
11 hours after release a large proportion of cells were arrested
in mitosis due to the presence of nocodazole. Mitotic cells were
removed by vigorous shaking, leaving a late G2 population in
which Cdc6 levels had fallen again. Cdc6 band intensities were
normalised against actin levels and plotted using arbitrary units
on the same axes as the number of elongating nuclei in each
population (Fig. 5F). In vitro elongation assays and flow
cytometry profiles (Fig. 5D) provide independent measures of
progression through S phase, allowing the firm conclusion that
at 9 hours after release from thymidine arrest DNA replication
has completely finished, yet Cdc6 remains in the nucleus.
Consistent with expression of Cdc6 at G1-S (Saha et al.,
1998), the level of Cdc6 in post-G1 nuclei is higher than that
in early G1 nuclei (Fig. 5A). Our analysis also reveals cell cycle
changes in Cdc6 isoforms. We see a single fast migrating form
in G1 and two equally abundant isoforms in S phase and G2.
The upper form is unstable in isolated S-phase nuclei at room
temperature, but recovery is improved in the presence of the
phophatase inhibitor okadaic acid, suggesting that the retarded
form is phosphorylated (Fig. 5B). Consistent with this
instability, human Cdc6 has recently been reported to interact
with a regulatory subunit of protein phosphatase 2A (Yan et
al., 2000).
S-phase phosphorylation of Cdc6 is observed directly when
exogenous Xenopus Cdc6 protein is incubated with soluble
extracts prepared from synchronised cells, in the absence of
nuclei. S cytosol, but not G1 cytosol, modifies Cdc6 so that it
migrates more slowly (Fig. 5C). Furthermore, this modification
is prevented by olomoucine, suggesting that cdk-dependent
phosphorylation causes the reduced mobility. G2 cytosol also
generates a reduced mobility form of Cdc6, and M-phase
extract causes an even greater degree of retardation, which
probably reflects phosphorylation at more of the ten potential
phosphorylation sites in Xenopus Cdc6 (Coleman et al., 1996).
1936 D. Coverley and others
DISCUSSION
Regulated destruction of Cdc6
In human cells the behaviour of Cdc6 is regulated in the cell
cycle by several different mechanisms. Cdk consensus sites
near the N-terminal nuclear localisation signal (NLS) are
believed to influence nuclear protein import (Takei et al.,
1999), while inhibition of nuclear protein export suggests that
overexpressed Cdc6 migrates from the nucleus in S phase
(Jiang et al., 1999). By using a finite amount of exogenous
reporter protein we provide evidence for another level of
control, regulated proteolysis, which rapidly destroys free
Cdc6 in S phase.
In yeast, analysis of cdc18/CDC6 mRNA and protein levels
has produced a coherent picture of its regulation in the cell
cycle. Transcription begins in mitosis and ends in S phase, and
protein levels are regulated by proteolysis. Cdc18 protein is
targeted for destruction in mitosis by cdc2-mediated
phosphorylation and only begins to accumulate when mitotic
kinase activity falls. Later, the rise of S-phase cdk activity
again phosphorylates cdc18/CDC6, resulting in rapid
destruction (Baum et al., 1998; Jallepalli et al., 1997; Nishitani
and Nurse, 1995). Phosphorylation of cdc18/CDC6 in S phase
seems to be central to the mechanism that restricts DNA
replication to one round per cell cycle. Expression of a mutant
form which lacks consensus cdk phosphorylation sites or
overexpression of the wild-type protein results in unregulated
initiation (Baum et al., 1998; Jallepalli et al., 1997; Liang and
Stillman, 1997; Nishitani and Nurse, 1995). Our results on
regulated proteolysis of Cdc6 are partly similar to the picture
that has emerged from studies in yeasts, but instead of seeing
degradation of all Cdc6 in S phase, we see degradation of
only the free pool. Endogenous, chromatin-bound Cdc6 is
conserved but, we believe, modified in a way that some
antibodies fail to recognise (see later).
Rapid proteolysis of free Cdc6 in S phase is dependent upon
import into the nucleus and upon a nuclear-associated, p21sensitive protein kinase. Cyclin A-cdk2 can completely
substitute for the nucleus activating Cdc6 destruction in G1
cytosol. This suggests that nuclear cyclin A-cdk2 is the major
trigger for Cdc6 destruction in vivo. Cyclin A-cdk2 is known
to be activated at G1-S phase (Carbonaro-Hall et al., 1993;
Girard et al., 1991; Pagano et al., 1992; Pines and Hunter,
1990) and has been found at sub-nuclear sites of DNA
replication (Cardoso et al., 1993). It is possible therefore that
destruction of Cdc6 in vivo is initiated in S phase by
phosphorylation of Cdc6 at sites of replication. Whether or not
phosphorylation of Cdc6 is coupled to replication, the
consequence is elimination of excess protein from the nucleus.
Our data is consistent with this being a negative regulatory
event that contributes to the prevention of re-initiation by
preventing Cdc6 from rebinding to chromatin after initiation.
Nuclear membrane permeabilization and the
stimulation of DNA replication
Extensive experimental evidence argues that the nuclear
membrane plays an important role in regulating the formation
and activation of replication complexes, by partitioning key
proteins to the cytoplasm or the nucleus (Blow and Laskey,
1988; Coverley et al., 1993; Hua et al., 1997; Leno et al., 1992;
Leno and Munshi, 1994; Madine et al., 1995a; Walter et al.,
1998). In one model it serves to concentrate factors that
simultaneously block replication complex assembly and
activate initiation, while in another model it restricts access of
essential initiation factors to chromatin. These models are not
mutually exclusive.
Recent work with an in vitro mammalian initiating system,
from this laboratory, adds to the current body of evidence about
the regulatory role of the nuclear membrane. Stimulation of in
vitro initiation by recombinant Cdc6 protein was found to be
dependent on artificial permeabilization of the nuclear
membrane (Stoeber et al., 1998), adding weight to the theory
that Cdc6 might be the Xenopus Mcm ‘loading factor’ whose
access to the nucleus is restricted when the nuclear membrane
is intact (Madine et al., 1995a).
Early reports suggested that Cdc6 does not posses an NLS
as mutations in the conserved putative bipartite NLS of the
human protein resulted in no detectable alteration in nuclear
localisation (Saha et al., 1998). This implied that access to the
nucleus might occur by association with another protein or
experimentally through a permeabilized nuclear membrane.
However, recent analysis has shown that the amino acids
altered in these experiments overlap with the cyclin binding
domain (Petersen et al., 1999). Total deletion of this domain
results in increased nuclear localisation of Cdc6 in S phase
rather than the reduced nuclear localisation that might be
expected from mutation of an NLS. More recently, mutations
in another N-terminal conserved putative NLS abolished
nuclear localisation of Cdc6 (Takei et al., 1999), and the two
serine residues that flank the site were found to be important
for regulating its function (Petersen et al., 1999; Takei et al.,
1999). We present strong evidence that Cdc6 is actively
transported into G1 nuclei, consistent with reports of a
functional NLS. This would argue against Cdc6 as the Xenopus
‘loading factor’.
Our current observations about regulated proteolysis of
Cdc6 in S phase provide a trivial explanation for why nuclear
membrane permeabilization might allow Cdc6 to stimulate in
vitro replication. Most of the high level of Cdc6 protein used
by Stoeber et al. (1998) to supplement S-phase cytosol would
have been destroyed upon addition of nuclei. Permeabilization
could allow a proportion of the input protein to bind to G1
chromatin and escape immediate destruction. Consistent with
this, detergent-resistant Cdc6 was detected only if the nuclear
membrane was permeabilized (Stoeber et al., 1998). This
explanation is analogous to the proposal that the nuclear
membrane serves to generate an intranuclear environment
which inhibits replication complex assembly (Hua et al., 1997).
However, our work does not exclude the possibility that
permeabilization allows additional replication factors to gain
access to the nucleus. In fact efficient pre-loading of Cdc6 into
intact G1 nuclei in vitro did not increase the number of
initiating nuclei, upon transfer to S-phase cytosol (D. C. and
S. T., unpublished observations). This suggests that nuclear
membrane permeabilization is required for productive binding
of Cdc6 in this system.
Endogenous human Cdc6
Finally and importantly, this study looks at the status of
endogenous human Cdc6 in cytosolic replication type extracts
and isolated nuclei from HeLa cells. Consistent with three of
the reports in the literature (Fujita et al., 1999; Stoeber et al.,
Cdc6 in S phase in human cells 1937
1998; Williams et al., 1998), endogenous nuclear Cdc6 persists
in S phase. Quantification of protein levels reveals a distinct
drop in nuclear Cdc6 after initiation, followed by almost
constant levels in S and G2, and a sharp fall just before mitosis.
Early analysis of yeast mutants showed that cdc18/Cdc6 is part
of the checkpoint control that prevents mitosis from occurring
until DNA synthesis is complete (Kelly et al., 1993). A full
investigation of the nuclear associated Cdc6 fraction in human
G2 nuclei will be of interest with regard to this function.
The immunofluorescence experiments, in which
overexpressed Cdc6 disappeared from the nucleus in S phase,
suggested that Cdc6 is displaced from chromatin in a manner
similar to the Mcm proteins, and then exported to the
cytoplasm (or degraded) under the control of cyclin-dependent
kinases (Jiang et al., 1999; Petersen et al., 1999; Saha et al.,
1998; Takei et al., 1999). While we believe that free Cdc6 is
regulated in this way, our data clearly show that some Cdc6
remains associated with the nucleus long after replication has
finished.
After G1, nuclear Cdc6 exists as two distinct isoforms, of
which the slow migrating, phosphorylated form is unstable in
isolated nuclei. This form is also antigenically different to the
faster migrating form as it is not recognised by a second antihuman Cdc6 polyclonal antibody that was raised against amino
acids 145-360 (not shown). If, as our data suggests, chromatinbound Cdc6 exists in S phase as the phosphorylated form, its
altered antigenicity is one possible reason why some studies
fail to detect it.
We observed abundant free, undegraded protein in S-phase
cytosols, although levels varied between different preparations
(not shown). Given the very high capacity of all our S-phase
nuclei/S-phase cytosol preparations to degrade exogenous
Cdc6, this is slightly surprising. High endogenous Cdc6 levels
might arise in S-phase cells as a consequence of the
synchronisation procedure, as suggested previously (Petersen
et al., 1999). Our cells were arrested and held for two
consecutive cell cycles at the beginning of S phase, the point
at which mRNA levels are known to peak in the unaltered cell
cycle (Saha et al., 1998). This could produce unphysiological
Cdc6 levels which, our data shows, could only be degraded
after nuclear protein import and phosphorylation inside the
nucleus.
Our aim has been to better understand the regulation of Cdc6
as a step toward a broader goal of dissecting the mammalian
cellular initiation process. In this study we present strong
evidence that human Cdc6 persists in the nucleus beyond S
phase. In addition we have analysed the regulatory forces
acting upon free Cdc6 at the transition from G1 to S phase.
Exogenous Cdc6 is eliminated from the nucleus at the start of
S phase, by cyclin A-cdk2 regulated proteolysis. This
mechanism could function in vivo to remove any Cdc6 not
assembled into replication complexes, as well as the Cdc6,
which is lost from chromatin after initiation. This could
contribute to the prevention of reinitiation by blocking
formation of new replication complexes.
We are pleased to thank the following people for kindly providing
reagents: P. Romanowski for anti-human Cdc6 antibody, K. Stoeber
and G. Williams for isoform-specific anti-human Cdc6 polyclonal
antibody, H. Laman for cyclin D-cdk6 and cyclin E-cdk2, W. Krek
and C. Wirbelauer for cyclin A-cdk2, D. Gorlich’s laboratory for
purified importin β dominant-negative mutant, Nobuo Furuno for
purified p21 protein, Minoru Yoshida for leptomycin B, and P.
Carpenter, T. Coleman, W. Dunphy for the baculovirus Xcdc6
expression construct. We are also grateful to M. Jackman for helpful
discussions. This work was supported by the Cancer Research
Campaign except for C.P., who was supported by a post-doctoral
Human Frontiers Science Program fellowship.
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