Hojabrpour, Payman

Molecular Biology of the Cell
Vol. 19, 3212–3220, August 2008

An Essential Role for MCL-1 in ATR-mediated CHK1
Phosphorylation
Sarwat Jamil, Shadi Mojtabavi, Payman Hojabrpour, Stefanie Cheah,
and Vincent Duronio
Department of Medicine, University of British Columbia and Vancouver Coastal Health Research Institute,
Vancouver, BC, V6H 3Z6 Canada
Submitted November 26, 2007; Revised April 16, 2008; Accepted May 8, 2008
Monitoring Editor: William P. Tansey

Here we report a novel role for myeloid cell leukemia 1 (Mcl-1), a Bcl-2 family member, in regulating phosphorylation and
activation of DNA damage checkpoint kinase, Chk1. Increased expression of nuclear Mcl-1 and/or a previously reported
short nuclear form of Mcl-1, snMcl-1, was observed in response to treatment with low concentrations of etoposide or low
doses of UV irradiation. We showed that after etoposide treatment, Mcl-1 could coimmunoprecipitate with the regulatory
kinase, Chk1. Chk1 is a known regulator of DNA damage response, and its phosphorylation is associated with activation
of the kinase. Transient transfection with Mcl-1 resulted in an increase in the expression of phospho-Ser345 Chk1, in the
absence of any evidence of DNA damage, and accumulation of cells in G2. Importantly, knockdown of Mcl-1 expression
abolished Chk1 phosphorylation in response to DNA damage. Mcl-1 could induce Chk1 phosphorylation in ATMnegative (ataxia telangectasia mutated) cells, but this response was lost in ATR (AT mutated and Rad3 related)-defective
cells. Low levels of UV treatment also caused transient increases in Mcl-1 levels and an ATR-dependent phosphorylation
of Chk1. Together, our results strongly support an essential regulatory role for Mcl-1, perhaps acting as an adaptor protein,
in controlling the ATR-mediated regulation of Chk1 phosphorylation.

INTRODUCTION
Myeloid cell leukemia 1 (Mcl-1) was first identified as a gene
induced during myeloid cell differentiation (Kozopas et al.,
1993) and is a prosurvival member of the Bcl-2 family. The
activity of Bcl-2 family proteins involves several Bcl-2 homology (BH) domains (Danial and Korsmeyer, 2004) that
control protein–protein interactions. These proteins play an
important role in cancer by regulating cell death and survival. Compared with other prosurvival members such as
Bcl-2 or Bcl-xL, Mcl-1 has been shown to have more transient
survival effects (Zhou et al., 1997). Unlike other members of
the family, the half-life of Mcl-1 is very short and its expression is highly regulated by growth factors and a variety of
other agents. Moreover, Mcl-1 has an extended N-terminus
that is not conserved in any other Bcl-2 family protein. The
N-terminus contains two PEST domains, which are found in
proteins that are rapidly turned over, but deletion of the
PEST domains does not extend the short half-life of Mcl-1
(Akgul et al., 2000; Clohessy et al., 2004). When the Mcl-1
gene was knocked out in a mouse model, it resulted in
peri-implantation lethality (Rinkenberger et al., 2000), but
Mcl-1⫺/⫺ embryos showed no alterations in the extent of
apoptosis, indicating that Mcl-1 plays a role early in develThis article was published online ahead of print in MBC in Press
(http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E07–11–1171)
on May 21, 2008.
Address correspondence to: Vincent Duronio (vduronio@
interchange.ubc.ca).
Abbreviations used: ATM, ataxia telangectasia mutated; ATR, AT
mutated and Rad3 related; BH, Bcl-2 homology; Chk1, checkpoint
kinase 1; Mcl-1, myeloid cell leukemia-1.
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opment distinct from its prosurvival functions. We and others reported that Mcl-1 expression can lead to suppression of
cell proliferation and may have effects on cell cycle machinery (Fujise et al., 2000; Jamil et al., 2005). We showed that a
short nuclear form of Mcl-1 (snMcl-1) was associated primarily with the inactive form of Cdk-1 in the nucleus, most
prominently during the G2 phase of the cell cycle, which
suggested a potential means by which Mcl-1 mediates its
anti-proliferative effects (Jamil et al., 2005).
It is now well established that genome surveillance mechanisms function to cause a delay, or arrest, in progression of
the cell cycle at the boundary of G1/S or G2/M in response
to DNA damage (Zhou and Elledge, 2000; Abraham, 2001;
Kastan, 2001; Nyberg et al., 2002; Sancar et al., 2004). These
surveillance mechanisms are known to involve a network of
interacting proteins that recognize damage and elicit responses including cell cycle delay, DNA repair, and apoptosis (Elledge, 1996; Zhou and Elledge, 2000). Cdk’s, which
play a central role in cell cycle progression, are key targets of
these checkpoints (Iliakis et al., 2003). Yeast model systems
have revealed much about the mechanisms of the DNA
damage response, as well as the identity of the corresponding genes in higher eukaryotes. A role for a gene such as
Mcl-1 would not have been found from studies of more
primitive organisms, because Mcl-1 does not appear to
have any orthologues other than in vertebrates (e.g.,
search in OrthoMCL: http://orthomcl.cbil.upenn.edu/
cgi-bin/OrthoMclWeb.cgi?rm⫽index).
Because we had previously shown the interaction of Mcl-1
with Cdk-1 and we and others showed that Mcl-1 expression
causes a suppression of cell proliferation, we investigated
the potential change in Mcl-1 expression and whether Mcl-1
may possibly play a role when cells are treated with agents
that cause DNA damage. We found that Mcl-1 expression
© 2008 by The American Society for Cell Biology

Mcl-1 Functions in Chk1 Regulation
ICN Biomedicals (Costa Mesa, CA). RPMI 1640, fetal bovine serum (FBS),
Protein G agarose beads were purchased from Invitrogen (Carlsbad, CA).

Cell Treatments
Optimal concentrations of etoposide were determined for each cell line that
caused G2 arrest with the least amount of apoptosis. For HL-60 and FDC-P1
cells, 1.5 ␮M etoposide was used and for HeLa cells, 15 ␮M was required. For
UV treatments, cells were irradiated using a UVB source (5 J m⫺2 s⫺1) for 20 s
for a total of 100 J/m2, after which cells were washed twice with phosphatebuffered serum (PBS) and then allowed to incubate under normal conditions.

Subcellular Fractionation

Figure 1. Mcl-1 expression is increased, particularly in the nucleus,
in response to etoposide treatment. (A) HL-60 cells were either left
untreated (C) or treated with etoposide (Et) for 3 h. Cells were
fractionated into cytosolic and nuclear extracts. The blot was probed
with anti-Mcl-1 antibody, anti-vinculin antibody to indicate cytosol
purity, and anti-Oct1 antibody to indicate purity of nucleus fraction.
(B) HeLa cells were untreated (C) or treated with 15 ␮M of etoposide
(Et) for 3 h and prepared as in A. snMcl-1 refers to the protein
corresponding to the shorter nuclear form of Mcl-1 that we previously characterized. The samples from cytosol and nuclear extracts
represent an equivalent number of cells, resolved on the same gel
and blotted together and are thus a direct representation of the
relative amounts of Mcl-1. Results are representative of at least five
independent experiments.

increases, particularly in the nucleus, as a result of mild
DNA damage. More extensive damage had the expected
effect of decreasing expression of Mcl-1, as has been reported
previously (Nijhawan et al., 2003). After mild DNA damage,
the nuclear Mcl-1 was shown to be associated with active
Checkpoint kinase 1 (Chk1). Increased phosphorylation of
Chk1 at an activating site was observed after transient expression of Mcl-1, whereas no other evidence of a DNA
damage response was evident. Furthermore, knockdown of
Mcl-1 expression eliminated the Chk1 phosphorylation that
occurs after DNA damage. By comparison of ATM (ataxia
telangectasia mutated)-negative and ATR (AT mutated and
Rad3 related)-deficient cells, we could conclude that Mcl-1
plays a role in ATR-dependent activation of Chk1, revealing
a completely novel function for Mcl-1.
MATERIALS AND METHODS
Cell Lines
HeLa, HL-60, and FDC-P1 cells were obtained from ATCC (Manassas, VA).
ATM-deficient HT-144 cells were a kind gift from Dr. P. Olive (BC Cancer
Agency). ATR-defective fibroblasts, F02–98, were a kind gift from Dr. P.
Jeggo. Hs68, which are primary human foreskin fibroblasts (Wheaton and
Riabowol, 2004), maintained within 30 – 60 mean population doublings, were
kindly provided by Dr. K. Riabowol (University of Calgary). HL-60 cells were
cultured in RPMI1640 supplemented with 10% fetal bovine serum (FBS), 1
mM sodium pyruvate, 2 mM l-glutamine. FDC-P1 cells were grown in RPMI
supplemented with 10% fetal calf serum (FCS) and 2.5% WEHI-3-conditioned
medium as a source of IL-3. HeLa, Hs68, and F02–98 cells were grown in
DMEM and HT 144 cells were cultured in MEM, each with 10% FCS.

Subcellular fractionation was carried out as described (Shiio et al., 2003).
Briefly, cells were resuspended in buffer A (10 mM HEPES, pH 7.9, 10 mM
KCl, 1.5 mM MgCl2, 0.34 M sucrose, and 10% glycerol), containing protease
inhibitors and 1 mM dithiothreitol (DTT). To each extract, 0.1% Triton X-100
was added, and samples were incubated on ice for 7 min. The samples were
centrifuged for 5 min at 1300 ⫻ g. Supernatant containing cytosolic proteins
was centrifuged further for 10 min at 20,000 ⫻ g. The pellets containing the
nuclei were washed with buffer A, resuspended in buffer B (0.2 mM EGTA,
pH 8.0, and 3 mM EDTA, pH 8.0), and incubated on ice for 30 min. The
extracts were centrifuged at 1700 ⫻ g for 5 min. The pellets were washed
and centrifuged for 5 min at 1700 ⫻ g. The pellets were resuspended in
sample buffer and sonicated for 15 s. In experiments where nuclear proteins were extracted together with chromatin, 5 ␮g/ml each of DNase I
and RNase A were added to the buffer B and the nuclear preparations were
sonicated for 10 s.

Indirect Immunofluorescence Staining
Immunofluorescence staining was performed using the protocol as described
previously (Liu et al., 1999). Briefly, HeLa cells were seeded on glass coverslips, treated or not with either 15 ␮M etoposide, and fixed with fresh 4%
paraformaldehyde in PBS. Cells were blocked with 10% normal goat serum
and probed with 1:250 rabbit anti-Mcl-1 antibody or 1:150 mouse anti-Chk1.
Bound antibody was detected by goat anti-rabbit antibody conjugated to
Alexa fluor 488 or goat anti-mouse antibody conjugated to Alexa fluor 594.
The cell nuclei were stained with 1:10,000 dilution of Hoechst 33342. Stained
cells were analyzed using a Zeiss Axioplan 2 imaging microscope (Thornwood, NY).

Immunoprecipitation
Cells were washed with PBS before total cell lysates were obtained by lysing
cells in ice-cold solubilization buffer (20 mM Tris HCl, pH 8.0, 1% NP40, 10%
glycerol, 137 mM NaCl, and 10 mM NaF) with protease inhibitor cocktail, 200
␮M sodium vanadate, and 5 ␮g/ml each of DNase I and RNase A. Cells were
sonicated for 15 s and centrifuged at 32,000 ⫻ g for 10 min. Protein concentrations were determined by BCA protein assay. For immunoprecipitation,
cytosolic, nuclear or chromatin extracts were precleared with 20 ␮l of protein
G agarose beads for 30 min. Anti-Mcl-1 antibody at 1 ␮g/ml was added, and
after a 2-h incubation, the immunoprecipitates were collected by adding 50 ␮l
of protein G agarose beads. Beads were washed four times with solubilization
buffer.

Kinase Assays
For determination of Mcl-1–associated Chk1 activity, total cell lysates were
precleared by incubation with agarose G beads for 30 min. The samples were
then incubated with anti-Mcl-1 antibody for 2 h followed by addition of
agarose G beads for 1 h. After extensive washing, beads were resuspended in
assay dilution buffer (25 mM ␤-glycerophosphate, 20 mM MOPS, 5 mM
EGTA, 2 mM EDTA, 20 mM MgCl2, 250 ␮M DTT, 5 ␮M ␤-methyl aspartic
acid, pH 7.2). CHKtide at 5 ␮g/ml (Furnari et al., 1997), [␥-32P]ATP, and cold
ATP (25 ␮M) were added. The reaction was terminated after 20 min by
spotting 15 ␮l on p81 chromatography filter paper (Whatman, Clifton, NJ).
The filter papers were washed in 1% O-phosphoric acid, and the activity of
each sample was measured in a scintillation counter.

Flow Cytometry
For staining of cells to detect sub-G1 DNA levels and analysis of cell cycle
status, cells were fixed with 70% ethanol and subsequently stained with PBS
containing 50 ␮g/ml propidium iodide, 100 ␮g/ml RNase A, and 0.1%
glucose. Stained cells were analyzed using Epics XL flow cytometer (Coulter,
Hialeah, FL).

Antibodies and Reagents

Mcl-1 Small Interfering RNA Interference

Anti-Mcl-1 (Sc-19), Chk1 (FL-476), p85␣ (Z-8), and Oct I (C-21) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-Chk1
(Ser345) was from Cell Signaling (Beverly, MA). Monoclonal anti-human
vinculin was from Sigma (St. Louis, MO). Etoposide was purchased from
Calbiochem (La Jolla, CA). Isogranulatimide was a kind gift from Dr. M.
Roberge (University of British Columbia). [␥-32P]ATP was purchased from

For in vitro gene silencing cells were transfected with either Mcl-1 siRNA
sequence described by Zhang et al. (2002) or control siRNA (sense UUCUCCGAACGUGUCACGUdTdT, antisense ACGUGACACGUUCGGAGAAdTdT). The purified desalted and double-stranded Mcl-1 siRNA was ordered
from Dharmacon Research (Boulder, CO). Control siRNA was purchased
from Qiagen (Chatsworth, CA). HeLa cells were plated the day before being

Vol. 19, August 2008

3213

S. Jamil et al.

Figure 2. Mcl-1 translocation to nucleus after etoposide treatment. For intracellular localization of Mcl-1, HeLa cells were left untreated
(Control) or treated with 15 ␮M of etoposide for 2 h (Etoposide). Top, cells were stained with polyclonal anti-Mcl-1 antibody and detected
by goat anti rabbit antibody conjugated to Alexa fluor 488. Bottom, cell nuclei were stained with Hoechst 33342 and the combined staining.
Stained cells were visualized using a 40⫻ objective.

transfected with either 20 nM Mcl-1 or control siRNA using SILENTfect
(Bio-Rad, Richmond, CA) according to the manufacturer’s recommendations.
After 24 h the medium was replaced with fresh siRNA for another 24 h.

RESULTS
Mcl-1 Translocates to the Nucleus in Response to Etoposide
To induce DNA damage, cells were treated with etoposide,
which is a topoisomerase II inhibitor that results in doublestranded breaks and single-stranded gaps to trigger cell
cycle checkpoint activation (Cliby et al., 2002). To determine
whether etoposide treatment affects the cellular location of
Mcl-1, we performed subcellular fractionation followed by
immunoblotting with anti-Mcl-1 antibody. An increase in
Mcl-1 protein levels was observed after 3 h of etoposide
treatment in HL-60 cells; a similar increase in Mcl-1 expression was observed in HeLa cells (Figure 1). As we showed
previously in HL-60, some Mcl-1 is present in the nucleus
even in healthy asynchronous cells (Jamil et al., 2005). However, an increase in nuclear accumulation of Mcl-1 was
observed in both HL-60 and HeLa cells, upon etoposide
treatment. In both cell types, there is an increase in cytosolic
Mcl-1 (where the majority of Mcl-1 is normally present), but
there is a much greater increase in the amount of total Mcl-1
in the nucleus. In the nuclear extracts from HL-60 cells, the
predominant form was the previously reported short form,
snMcl-1 (Jamil et al., 2005), whereas in HeLa cells the predominant form was full-length Mcl-1. The significance of the
variable amounts of snMcl-1 in different cell types is not
clear at present. It is noteworthy that the concentrations of
etoposide used were optimized for each cell line to cause G2
arrest, with the least amount of apoptosis during the course
of the experiment. The three cell lines used in this study
were shown to arrest at G2 with either 1.5 ␮M (HL-60 and
FDC-P1) or 15 ␮M (HeLa) etoposide treatment overnight.
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We wanted to demonstrate, by an independent set of
analyses, the change in intracellular localization of Mcl-1
after DNA damage. We used immunocytochemistry to stain
endogenous Mcl-1 in HeLa cells. Our staining results
showed that Mcl-1 is present primarily in the cytosolic compartment in normally proliferating cells, as expected. However, after etoposide treatment, a substantial increase in the
nuclear accumulation of Mcl-1 was observed (Figure 2), with
the staining appearing as a punctate pattern.
Mcl-1 Associates with the Checkpoint Regulator Chk1
Because the increased amount of Mcl-1 protein and its accumulation in the nucleus were observed in response to a
DNA damaging agent, we sought to determine the potential
association of Mcl-1 with components of the DNA damage
response machinery. Chk1 plays an essential role in response to genotoxic stress by delaying cell cycle progression
through the S and G2 phases (Liu et al., 2000; Takai et al.,
2000). Potential physical association of Mcl-1 with Chk1 was
investigated in several ways.
Endogenous Chk1 was immunoprecipitated from the nuclear extracts of HeLa cells treated with or without etoposide
and immunoblotted for Mcl-1. Our results showed that some
Mcl-1 was coimmunoprecipitated with Chk1 from the nuclear extracts of untreated cells, and this association increased substantially in cells treated with etoposide (Figure
3A). It is interesting to note that in etoposide-treated cells,
the total level of nuclear Chk1 protein was also increased
after etoposide treatment, but increased association with
both full-length and short forms of Mcl-1 could be observed
in the nucleus. Chk1 has been previously shown to be
present in the nucleus in all phases of the cell cycle (Jiang et
al., 2003). We next sought to verify that reciprocal coimmunoprecipitation could be observed and at the same time
investigate the association of Mcl-1 with Chk1 kinase activMolecular Biology of the Cell

Mcl-1 Functions in Chk1 Regulation

Figure 3. Mcl-1 coimmunoprecipitates with active Chk1 after DNA damage. (A) HeLa cells were untreated (Con) or treated with etoposide
(Etop) for 16 h. Immunoprecipitation of endogenous Chk1 was performed from nuclear extracts (or antibody alone, with no extract, was used
as a control). Immunoprecipitates were probed for Chk1 and Mcl-1. (B) FDC-P1 cells expressing Mcl-1 or vector alone were untreated (Con)
or treated with etoposide (Etop) for 16 h. Anti-Mcl-1 immunoprecipitates were assayed for Chk1 activity. A parallel etoposide-treated sample
was incubated in vitro with 2.5 ␮M of isogranulatimide (Isogr.) for 30 min at 37°C before kinase assay (Etop ⫹ Isogr.). Results are the mean ⫾
SD from three independent experiments. (C) FDC-P1 cells with empty vector or overexpressing Mcl-1 were treated with etoposide (Etop.)
for 16 h as indicated. Pretreatment with either 2.5, 5, or 10 ␮M isogranulatimide (Isogr.) was for 10 min before the addition of etoposide. Cells
were analyzed by flow cytometry to determine the percentage of viable cells in G1 compared with S and G2. Numbers are averaged from
two independent experiments. (D) Representative flow cytometry profiles of empty vector control cells, compared with cells overexpressing
Mcl-1, after treatment with etoposide and 10 ␮M isogranulatimide. Apoptosis is indicated by cells having sub-G1 DNA content (indicated
by asterisk).

ity. Total lysates from the murine progenitor cell line, FDCP1, stably expressing human Mcl-1 or empty vector, were
immunoprecipitated with anti-human Mcl-1 (which does
not detect murine Mcl-1). These Mcl-1– expressing cells were
previously characterized and were shown to have a slower
rate of proliferation compared with parental cells (Jamil et
al., 2005). As shown in Figure 3B, Mcl-1 immunoprecipitates
from etoposide-treated cells contained kinase activity that
efficiently phosphorylated the Chk1 peptide substrate, CHKtide (Furnari et al., 1997). In untreated cells, there was no
activity detected above background levels. The elevated kinase activity was completely abolished by treatment with
the Chk1 inhibitor, isogranulatimide (Jiang et al., 2004).
However, this inhibitor has also been shown to inhibit
GSK-3␤ kinase activity, and thus we tested for the potential
involvement of this kinase. We found that levels of GSK-3␤
activity, assayed using its specific substrate, were in fact
decreased after etoposide treatment (data not shown). Furthermore, the CHKtide used as a substrate in the Chk1
kinase assay is unlikely to be phosphorylated by GSK-3
because it is not a phosphorylated peptide, which is a prerequisite for GSK-3 phosphorylation. Finally, we also confirmed that immunoprecipitation of endogenous murine
Mcl-1 from untransfected FDC-P1 cells also had increased
Vol. 19, August 2008

Chk1 activity after etoposide treatment, which was sensitive
to 1 ␮M isogranulatimide (data not shown).
After etoposide treatment, cells arrest at G2/M, which is
dependent on Chk1 activity (Jin et al., 2005), and the Chk1
inhibitor isogranulatimide can reverse the G2/M arrest that
is observed after DNA damage (Jiang et al., 2004). Thus, we
tested whether the overexpression of Mcl-1 altered the sensitivity of cells to the inhibitory effects of isogranulatimide.
Mcl-1– overexpressing and control FDC-P1 cells were used
for these experiments. Data in Figure 3C shows that both cell
populations have similar cell cycle profiles, as we reported
previously (Jamil et al., 2005), despite the fact that the Mcl1– expressing cells have a reduced proliferation rate. These
cells were exposed to etoposide, which caused G2 arrest
(Figure 3C). In control cells, 2.5 ␮M isogranulatimide was
able to completely reverse this G2 arrest. However, in cells
overexpressing Mcl-1, fourfold higher concentrations of
isogranulatimide were required to have a similar effect, indicating that the presence of increased Mcl-1 protein resulted in greater resistance to the Chk1 inhibitor. It should
be noted that, compared with control cells, the cells overexpressing Mcl-1 were much more resistant to the toxic effects
of the combined treatment with 10 ␮M isogranulatimide and
etoposide (Figure 3D).
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S. Jamil et al.

Figure 4. Mcl-1 and Chk1 colocalize after
DNA damage. (A) HeLa cells were untreated,
treated with 15 ␮M etoposide for 3 h, or
treated with etoposide for 3 h, followed by
washing and allowing cells to recover for 21 h.
Cells were stained using anti-Mcl-1 or antiChk1, or the figures were merged to show
colocalization. Additional staining with
Hoechst 33342 was used to visualize nuclei.
(B) Cell extracts from the same cells used in A
were used to immunoblot for the presence of
Mcl-1, phospho-Chk1, or actin as a loading
control.

Immunofluorescence staining was also used to monitor
potential colocalization of Mcl-1 and Chk1 after etoposide
treatment. As shown in Figure 4A, there was an increase in
both total and nuclear staining of Mcl-1 and Chk1 after
etoposide treatment, and these two proteins were colocalized in the nucleus. Interestingly, after cells were allowed to
recover for 21 h after washing out etoposide, there were still
elevated levels of both Mcl-1 as well as Chk1 phosphorylated at the activation site Ser345, as shown in Figure 4B.
However, the immunofluorescence staining showed that in
the majority of cells, little colocalization of the two proteins
could be detected. Together, all of these results indicated
that Mcl-1 can associate with active Chk1 in the nucleus after
DNA damage, although we have not yet determined
whether there is a direct association between the two proteins.
Overexpression of Mcl-1 Causes Ser345-Chk1 Phosphorylation in the Absence of DNA Damage
We next investigated the effect of transiently overexpressing
Mcl-1 on the events involved in DNA damage response. It is
well known that in response to DNA damage, Chk1 is
phosphorylated at two sites, Ser345 and Ser317, which are
required for kinase activation (Abraham, 2001; Zhao and
Piwnica-Worms, 2001; Shiloh, 2003). We found that Mcl-1
transiently overexpressed in HeLa cells resulted in phosphoSer345 Chk1 in the absence of exogenous DNA damage
(Figure 5A). Very little or no phospho-Ser345 Chk1 protein
was observed in control cells when empty vector was overexpressed. We next assessed the effect of transient Mcl-1
overexpression on cell cycle status. As shown in Figure 5B,
consistent with its enhancement of Chk1 phosphorylation, a
significant increase in the percentage of HeLa cells overexpressing Mcl-1 were found to accumulate in G2. This effect
of Mcl-1 is observed when high levels are expressed tran3216

siently, but it is important to point out that any cells selected
to stably express Mcl-1 (such as the FDC-P1 cells used
above) never show high levels of Mcl-1 expression, likely
due to the inhibitory effect of Mcl-1 on proliferation. We also
transiently transfected Mcl-1 in primary human fibroblasts
and again, the phosphorylation of Chk1 was observed (Figure 5C). These data suggest that increasing expression of
Mcl-1 alone can initiate the events leading to Chk1 phosphorylation.
To investigate whether Mcl-1-mediated phosphorylation
of Chk1 was associated with, or distinct from, the Bcl-2-like
prosurvival effects of Mcl-1, HeLa cells were transiently
transfected with mutant Mcl-1. Because all three BH domains of Mcl-1 are required for its complete antiapoptotic
effects (Clohessy et al., 2006), we generated a mutant Mcl-1
truncated at residue 304 (Mcl-1304), which deletes the BH2
and transmembrane domains. Transient expression of Mcl1304 in HeLa cells, detected as a band of lower molecular
weight than the endogenous Mcl-1, also caused increased
phosphorylation of Chk1 (Figure 5D).
In view of these results, we wanted to rule out the possibility that transient transfection with Mcl-1 may result in a
generalized stress response leading to Chk1 phosphorylation. We therefore investigated the phosphorylation of
H2Ax (␥-H2Ax), which is considered a hallmark of DNA
damage. In transiently transfected HeLa cells both empty
and Mcl-1 containing vectors showed a slight increase in the
␥-H2Ax (Figure 5E). This effect could be attributed to the
stress caused to the cells by transfection and was not due to
the overexpression of Mcl-1 protein. Compared with the
levels of ␥-H2Ax observed in transfected cells, a more robust
and dramatic increase in ␥-H2Ax was observed in cells
treated with etoposide. Similar results were observed with
Chk2 phosphorylation at Thr68 (an activating site on that
kinase), where no significant difference was observed beMolecular Biology of the Cell

Mcl-1 Functions in Chk1 Regulation
Figure 5. Transient overexpression of Mcl-1
increases phosphorylation of Chk1 at Ser345.
(A) HeLa cells were transiently transfected
with Mcl-1 and whole cell extracts immunoblotted for Mcl-1, phospho-Ser345 Chk1, or
vinculin as a loading control. (B) Cells overexpressing Mcl-1, or empty vector, as in A,
were analyzed for cell cycle status by flow
cytometry. Increased percentage of cells in G2
after Mcl-1 expression showed a significant
change (p ⫽ 0.05). (C) Hs68 primary fibroblasts were transiently transfected with empty
vector (Con) or with Mcl-1 and extracts
probed as in A, except for use of anti-Chk1 as
a loading control. (D) HeLa cells were transiently transfected with either Mcl-1 or Mcl1304 as indicated, and extracts were immunoblotted as in A, except for the additional antiChk1 blot, which also serves as a loading
control. (E) HeLa cells were untreated (C) or
treated with etoposide for 3 h (Et) or were
transiently transfected with vector alone (V)
or Mcl-1– containing vector (M). Chromatin
extracts were prepared, separated, and immunoblotted for the presence of phosphorylated
histone H2Ax (P-H2Ax) or for histone H4 (HH4) as a loading control. The pairs of samples separated by vertical lines were resolved on a
single gel.

tween the empty vector and Mcl-1– overexpressing cells
(data not shown). These results rule out the possibility that
expression of Mcl-1 itself may cause a generalized DNA
damage stress response.
Mcl-1–induced Chk1 Phosphorylation Requires ATR, but
not ATM
Chk1 is phosphorylated on Ser345 in response to DNA
damage primarily by two members of the phosphoinositide
3-kinase related kinase (PIKK) family of enzymes, ATM or
ATR (Abraham, 2001; Shiloh, 2003). We sought to determine

Figure 6. Mcl-1-induced phosphorylation of Chk1 is dependent on
ATR. ATM-deficient HT-144 cells (A) and ATR-defective fibroblasts,
F02–98 (B) were transiently transfected with empty vector (Con) or
Mcl-1, and extracts probed as indicated.

the kinase through which the effects of Mcl-1 were being
mediated. In HT-144 cells, which have a mutation in the
ATM gene (Ramsay et al., 1998), transient transfection of
Mcl-1 caused an increase in Chk1 phosphorylation similar to
that seen in other cell types (Figure 6A), ruling out an
essential role for ATM. Similarly, Mcl-1 overexpression
caused similar increases in Chk1 phosphorylation in cells
deficient for the related PIKK member, DNA-PK (data not
shown). ATR cannot be knocked out because it is an essential gene, and thus we investigated the involvement of ATR
by using fibroblasts derived from a Seckel syndrome patient,
F02–98 cells, which are known to have reduced ATR activity
(O’Driscoll et al., 2003). As shown in Figure 6B, transient
transfection of wild-type Mcl-1 in cells harboring the ATR
mutation was unable to induce Chk1 phosphorylation, suggesting that ATR is required for Mcl-1–mediated Chk1 phosphorylation.
Increase in Mcl-1 Expression with UV Irradiation Is ATR
Dependent
ATR is activated mainly by stalled replication forks and
agents that generate bulky lesions like UV irradiation (Guo
et al., 2000; Unsal-Kacmaz et al., 2002). We therefore examined the protein levels of Mcl-1 after exposure of cells to UV
radiation, which primarily activates ATR (Heffernan et al.,
2002). A low dose of UV resulted in the induction of Mcl-1
expression within 2 h in both HeLa cells and in primary

Figure 7. Low-level UV radiation induces
Mcl-1 expression and Chk1 phosphorylation.
HeLa cells (A), Hs68 cells (B), or F02–98 cells
(C) were untreated (Con) or irradiated with
UV, and extracts prepared after 2 h. Immunoblots of whole cell lysates were probed with
anti-Mcl-1, anti-phospho-Ser345 Chk1 (PChk1), or anti-p85 antibodies. (D) F02–98 cells
were untreated or treated with 2 mM hydroxyurea (HU) for 3 h. Whole cell lysates
were immunoblotted as indicated. For D, all panels were reblots of the same gel, and the pairs of lanes shown were at different portions of
the same gel and thus separated by a vertical line.
Vol. 19, August 2008

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S. Jamil et al.

human fibroblasts (Figure 7, A and B). In these cells in which
Mcl-1 was increased, a corresponding increase in phosphoChk1 was also observed. We and others have shown that
higher doses of UV cause Mcl-1 degradation (Nijhawan et
al., 2003; Germain and Duronio, 2007), but we consistently
observed increased Mcl-1 expression in response to lowlevel UV irradiation. In contrast to the increase in Mcl-1
observed in other cell lines, F02–98 cells showed little to no
increase in Mcl-1 levels in response to UV irradiation (Figure
7C). As expected, in these cells with low levels of ATR
activity, no increase in Chk1 phosphorylation was evident
after exposure to UV. However, when these same F02–98
cells were treated with a dose of hydroxyurea known to
induce DNA damage response in these cells (Alderton et al.,
2004), a significant increase in Chk1 phosphorylation could
be observed, with little or no change in the amount of Mcl-1.
These results demonstrate that the lack of Chk1 phosphorylation observed in response to low dose of UV was not due
to their general inability to phosphorylate Chk1.
Mcl-1 Is Required for Ser345 Chk1 Phosphorylation after
DNA Damage
The question of whether Mcl-1 plays an essential role in
Chk1 activation was tested in cells in which Mcl-1 expression was reduced using siRNA. HeLa cells were transfected
with Mcl-1–specific siRNA and cells were largely depleted
of Mcl-1 after 48 h (Figure 8A). The amount of total protein
in each cell extract was equivalent based on the immunoblots for the p85 subunit of PI 3-kinase. Furthermore, the
expression levels of the prosurvival protein Bcl-2 in Mcl-1–
depleted cells did not change over the time course of this
experiment. In control cells treated with an irrelevant
siRNA, treatment with etoposide resulted in phosphorylation of Chk1 at Ser345 as well as increases in Mcl-1 expression. However, in cells where Mcl-1 expression was knocked
down, phosphorylation of Chk1 was abolished after etoposide treatment (Figure 8A). Decreased Mcl-1 expression did
not have any effect on the endogenous level of Chk1 protein,
showing that the lack of Chk1 phosphorylation in the Mcl1– depleted cells could not be attributed to decreased levels
of Chk1. It is important to note that neither down-regulation
of Mcl-1 expression, nor treatment with etoposide, over this
short time period resulted in any noticeable cell death as
determined by trypan blue dye exclusion test (not shown) or
by propidium iodide (PI) staining for subdiploid DNA (Figure 8B).
DISCUSSION
This study has revealed an unexpected function of the Bcl-2
family protein, Mcl-1, in DNA damage responses. There is
increasing evidence from some recent studies of involvement of other members of the Bcl-2 family in DNA damage
response or repair (Kamer et al., 2005; Youn et al., 2005;
Zinkel et al., 2005; Wang et al., 2008), and thus several of
these proteins may have such alternate functions. Given that
overexpression of Mcl-1 alone is sufficient to direct the upstream kinase(s) to activate the key checkpoint signal transducer Chk1 and that suppression of Mcl-1 expression blocks
the ability of cells to respond to DNA damage by activating
Chk1, Mcl-1 must play a key role in this pathway. Our
results have ruled out an essential role for ATM or DNA-PK
in the Mcl-1–regulated phosphorylation of Chk1 and have
placed Mcl-1 upstream of Chk1 in the ATR-Chk1 pathway.
Interestingly, the functions of Mcl-1 appear to be very
similar to those that have been reported for Claspin. Downregulation of Claspin has been shown to inhibit Chk1 acti3218

Figure 8. Mcl-1 is required for Ser345 Chk1 phosphorylation after
DNA damage. (A) HeLa cells were transfected with control (left two
lanes) or Mcl-1 siRNA (right two lanes); 48 h after initial transfection, cells were left untreated (lanes 1 and 3) or were treated with
etoposide for 1 h (lanes 2 and 4). Total cell proteins were extracted
and immunoblotted with anti-Mcl-1, anti-phospho Chk1 (Ser 345),
anti-Chk1, anti-Bcl-2, and anti-p85 antibodies. (B) HeLa cells were
transfected with control siRNA or Mcl-1–specific siRNA for 48 h,
followed by treatment without or with etoposide for 1 h. Cells were
stained with PI and analyzed by flow cytometry for DNA content.

vation in response to DNA damage (Kumagai and Dunphy,
2000; Chini and Chen, 2003). However, unlike Claspin, Mcl1’s association with Chk1 does not depend on DNA damage
because we observed some association in untreated cells
(Figure 3B). It is possible that Mcl-1 plays a role in directing
the phosphorylating kinase to Chk1 during S and G2 phases
of cell cycle in unperturbed cells, and Claspin provides an
additional layer of control by further activating Chk1 and
consolidating the checkpoint response upon DNA damage.
It is noteworthy that Claspin was recently shown to be in
the same complex as proliferating cell nuclear antigen
(PCNA; Brondello et al., 2007), which is an important cell
cycle regulatory protein, suggesting Claspin may be a common link between DNA damage response and cell cycle
machineries. Interestingly, Mcl-1 is the only member of Bcl-2
family that has previously been shown to interact directly
with PCNA (Fujise et al., 2000) placing Mcl-1 at the interface
of apoptosis and cell cycle regulation. Based on these observations, a more relevant role of Mcl-1 in normal cell cycle
regulation may be suggested, because we have found Mcl-1
(or the shorter snMcl-1 form of the protein) in the nucleus in
several cell types (Jamil et al., 2005), and Mcl-1 has also been
shown to predominantly localize to the nucleus, where it
associates with PCNA, in U2OS cells (Fujise et al., 2000).
As we have mentioned earlier, our findings are based on
treatments with etoposide or UV radiation that are at lower
Molecular Biology of the Cell

Mcl-1 Functions in Chk1 Regulation

doses than those used in most studies. We were careful to
use doses that caused G2 arrest, but did not cause significant
apoptosis during the time course of the experiments. In fact,
it is clear that higher doses than we have used here, of either
etoposide or UV radiation, cause apoptosis together with
loss of Mcl-1 expression (Nijhawan et al., 2003; Germain and
Duronio, 2007). We cannot rule out that the increased Mcl-1
expression is also contributing to cell survival in cells treated
with lower levels of DNA-damaging agents. Thus, a dual
function for Mcl-1 can be suggested. It is possible that when
damage is sustained, up-regulated Mcl-1 is involved in mediating the checkpoint response that allows members of the
DNA damage response machinery to function. At the same
time as the DNA is being repaired, Mcl-1 at the mitochondria may also play a role in providing survival until the
repair has been completed.
Perhaps the most intriguing aspect of this study is that the
findings may offer an explanation for the essential function
of Mcl-1 that was demonstrated in studies of Mcl-1 knockout
mice (Rinkenberger et al., 2000), because those studies
showed that there was no change in the extent of apoptosis
in the Mcl-1⫺/⫺ embryos. Similar to Mcl-1 knockout mice,
knockout of either ATR or Chk1 are also embryonic lethal at
a preimplantation stage (de Klein et al., 2000; Liu et al., 2000;
Takai et al., 2000). Besides playing a key role in DNA damage response, the essential functions of these genes are also
likely to be to maintain normal DNA replication, as has been
suggested for Chk1 (Kaneko et al., 1999). As we and others
have reported previously, growth of cells overexpressing
Mcl-1 is dramatically inhibited, which is also consistent with
a proposed role for Mcl-1 in orchestrating a checkpoint
response. Indeed, we find that when high levels of Mcl-1 are
present after transient overexpression, we detected an accumulation of cells in G2, consistent with the expected effects
of increased Chk1 phosphorylation. During response to
DNA damage, Chk1 phosphorylation is enhanced, and as
we have also shown here, this can be independent of ATM
(Kaneko et al., 1999) and thus more dependent upon ATR.
The results of this study also show that regulation of Chk1
phosphorylation in response to DNA damage is dependent
on the presence of Mcl-1, because siRNA-mediated knockdown of Mcl-1 expression results in loss of DNA damage–
induced Chk1 phosphorylation.
To better understand the molecular events that are controlling Mcl-1 functions in the nucleus, future studies should
be aimed at finding the direct binding partners of Mcl-1. We
have established in this study the presence of Mcl-1 in a
complex that includes the checkpoint regulator Chk1, supporting the suggestion that a key function of Mcl-1 may be
in the coordination of events required to generate appropriate response to DNA damage and maintenance of chromosome integrity. These newly characterized properties of an
antiapoptotic member of the Bcl-2 family that has been
extensively studied in recent years may lead to important
new discoveries that expand our understanding of the important functions of Mcl-1.

ACKNOWLEDGMENTS
We thank Dr. Michel Roberge for helpful discussions regarding Chk1 and for
providing isogranulatimide, Dr. Karl Riabowol for providing primary human
fibroblasts, Dr. P. Olive for providing HT-144 cells and Dr. Susan Lees-Miller
(University of Calgary) for providing the ATR-defective cells that were obtained from Dr. P. Jeggo. We also thank Dr. Marco Garate for technical advice
and Dr. Andrew Sandford for critical review of the manuscript. This work
was supported by a grant to V.D. from the Canadian Institutes of Health
Research.

Vol. 19, August 2008

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