Loss of ΔNp63α promotes mitotic exit in epithelial cells.
ABSTRACT Protein p63 is a key regulator in cell proliferation and cell differentiation in stratified squamous epithelium. ΔNp63α is the most commonly expressed p63 isoform, which is often overexpressed in human tumor. In the present work we report the potential involvement of ΔNp63α in cell cycle regulation. ΔNp63α accumulated in mitotic cells but its expression decreased during mitotic exit. Moreover, ΔNp63α knockdown promoted mitotic exit. ΔNp63α shares a conserved destruction box (D-box) motif with other potential targets of the Anaphase-Promoting Complex/Cyclosome (APC/C). Overexpression of APC/C coactivator Cdh1 destabilized ΔNp63α. Our results suggest that ΔNp63α level is cell cycle-regulated and may play a role in the regulation of mitotic exit.
- SourceAvailable from: Cindy M Bukach[Show abstract] [Hide abstract]
ABSTRACT: Studies of perceptual expertise typically ask whether the mechanisms underlying face recognition are domain specific or domain general. This debate has so dominated the literature that it has masked the more general usefulness of the expertise framework for studying the phenomenon of category specialization. Here we argue that the value of an expertise framework is not solely dependent on its relevance to face recognition. Beyond offering an alternative to domain-specific accounts of face specialization in terms of interactions between experience, task demands, and neural biases, expertise studies reveal principles of perceptual learning that apply to many different domains and forms of expertise. As such the expertise framework provides a unique window onto the functional plasticity of the mind and brain.Trends in Cognitive Sciences 05/2006; 10(4):159-66. · 16.01 Impact Factor
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ABSTRACT: We used a colour Mondrian--an abstract scene with no recognizable objects--and its achromatic version to image the change in blood oxygenation in the brains of 12 human subjects, with the aim of learning more about the position and variability of the colour centre in the human brain. The results showed a consistent association of colour stimulation with activation of an area that is distinct from the primary visual areas, and lies in the ventral occipitotemporal cortex; we refer to it as human V4. The position of human V4, as defined on functional grounds, varies between individuals in absolute terms but is invariably found on the lateral aspect of the collateral sulcus on the fusiform gyrus. There was no indication of lingual gyral activation. In further studies designed to reveal the topographic map within V4, we stimulated the superior and inferior visual fields separately, using the same stimuli. We found that human V4 contains a representation of both the superior and inferior visual fields. In addition, there appears to be retinotopic organization of V4 with the superior visual field being represented more medially on the fusiform gyrus and the inferior field more laterally, the two areas abutting on one another. We find no evidence that suggests the existence of a separate representation of the inferior hemifield for colour in more dorsolateral regions of the occipital lobe.Brain 01/1998; 120 ( Pt 12):2229-42. · 9.92 Impact Factor
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ABSTRACT: Expertise with unfamiliar objects ('greebles') recruits face-selective areas in the fusiform gyrus (FFA) and occipital lobe (OFA). Here we extend this finding to other homogeneous categories. Bird and car experts were tested with functional magnetic resonance imaging during tasks with faces, familiar objects, cars and birds. Homogeneous categories activated the FFA more than familiar objects. Moreover, the right FFA and OFA showed significant expertise effects. An independent behavioral test of expertise predicted relative activation in the right FFA for birds versus cars within each group. The results suggest that level of categorization and expertise, rather than superficial properties of objects, determine the specialization of the FFA.Nature Neuroscience 03/2000; · 15.25 Impact Factor
Loss of DNp63a promotes mitotic exit in epithelial cells
Pok Man Haua, Yim Ling Yipa, Michael S.Y. Huenb, Sai Wah Tsaoa,⇑
aDepartment of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region
bGenome Stability Research Laboratory, Department of Anatomy and Centre for Cancer Research, The University of Hong Kong, Hong Kong Special Administrative Region
a r t i c l ei n f o
Received 16 January 2011
Revised 23 July 2011
Accepted 26 July 2011
Available online 3 August 2011
Edited by Angel Nebreda
a b s t r a c t
Protein p63 is a key regulator in cell proliferation and cell differentiation in stratified squamous epi-
thelium. DNp63a is the most commonly expressed p63 isoform, which is often overexpressed in
human tumor. In the present work we report the potential involvement of DNp63a in cell cycle reg-
ulation. DNp63a accumulated in mitotic cells but its expression decreased during mitotic exit.
Moreover, DNp63a knockdown promoted mitotic exit. DNp63a shares a conserved destruction
box (D-box) motif with other potential targets of the Anaphase-Promoting Complex/Cyclosome
(APC/C). Overexpression of APC/C coactivator Cdh1 destabilized DNp63a. Our results suggest that
DNp63a level is cell cycle-regulated and may play a role in the regulation of mitotic exit.
? ? 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Unlike p53 which is commonly mutated in over 50% of human
cancers, its homolog p63 is rarely mutated [1,2]. Deregulation of
p63 expression is commonly associated with carcinogenesis. The
TP63 locus is located within a frequently amplified region in squa-
mous cell carcinoma which may account for its high level of p63
expression in cancer . Due to alternative promoter usage, two
classes of p63 arise: TA and DN (truncated TA domain) isoforms.
Each form is further subdivided into a, b and c isoforms due to
alternative splicing at the C-terminus. Recently, two more C-termi-
nus alternative splicing variants, d and e, were identified and be-
lieved to be involved in cell differentiation . The identification
of new p63 isoforms reveals the complexity of p63 functions in
various biological processes which warrants further investigations.
As shown by many studies, DNp63a is the most abundant isoform
in cancer cells . Upregulation of DNp63 isoform is commonly
associated with carcinogenesis. Furthermore, DNp63a had been
reported to modulate apoptotic response in cells by upregulating
the anti-apoptotic heat shock protein 70 (HSP70)  and repress-
ing expression of pro-apoptotic gene, IGFBP-3 .
In addition, DNp63a also interferes with cellular senescence by
inhibiting p53 transcriptional activity and many of its downstream
target genes involved in regulation of senescence. In fact, p63 defi-
ciency in mice causes premature aging. Senescence was induced in
somatic and germline cells after p63 knock-down with RNAi .
There are many reports showing involvement of p63 in cell prolif-
eration. Recent reports showed that p63 are modulated during cell
cycle and bound to the p53-responsive elements in the regulatory
region of cell cycle progression genes [8,9]. Furthermore, the p63
maintains cell cycle progression by directly repressing miRNA mol-
ecules that suppress the expression of G1 cell cycle regulators .
These studies support a role of p63 in controlling cell proliferation
and cell cycle progression.
In this report, we provide evidence that DNp63a expression is
modulated during cell cycle. The DNp63a is expressed at inter-
phase, accumulated in early mitosis and decreased during mitotic
exit. Knockdown of DNp63a by shRNA promoted mitotic exit.
The DNp63a may be a target for APC/C during metaphase-ana-
2. Materials and methods
All reagents were obtained from Sigma–Aldrich (St. Louis, MO,
USA) unless stated otherwise.
2.2. Cell culture and transfection
HaCaT, HeLa and HEK293T cells were cultured in DMEM media
supplemented with 10% fetal bovine serum (FBS). All cell lines
were maintained in 5% CO2at 37 ?C. Cell transfection was per-
formed using Lipofectamine 2000 (Invitrogen, CA, USA) following
the manufacturers’ protocols.
0014-5793/$36.00 ? 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
⇑Corresponding author. Fax: +852 2817 0857.
E-mail address: email@example.com (S.W. Tsao).
FEBS Letters 585 (2011) 2720–2726
journal homepage: www.FEBSLetters.org
The expression plasmid, DNp63a in pcDNA3.1/myc-His, was a
gift from Dr. Kurt Engeland (University of Leipzig, Germany).
H2B-GFP was a kind gift from Professor Randy Poon (Hong Kong
University of Science and Technology, Hong Kong). HA-Cdc20 and
HA-Cdh1 in pCR2 were kindly provided by Dr. Patrick Ling
(Queensland University of Technology, Australia). The short hair-
pin RNA targeted specifically against p63 DNA binding domain
(p63 shRNA) and the empty vector (pSUPER) were kindly provided
by Dr. James DiRenzo (Dartmouth-Hitchcock Medical Center, USA).
Short hairpin RNA shCdc20 and shCdh1 are provided by Dr. Serigo
Moreno (University of Salamanca, Spain) and Dr. Ralph Wäsch (Al-
bert-Ludwigs University Medical Center, Freiburg, Germany)
respectively. Gene silencing was performed by transient transfec-
tion into cell lines using Lipofectamine 2000 (Invitrogen, CA,
USA) according to manufacturer’s protocol.
2.4. Generation of DNp63a mutant
Based on the construct DNp63a in pcDNA3.1/myc-His, the
DNp63a D-box mutant (R225A, L228A) was generated by overlap-
ping extension PCR using two complementary primers (50-CAGA-
and flanking primers (50-GGATCCATGTTGTACCTGGAAAAC-30and
50-TCTAGATCACTCCCCCTCCTCTTTG-30). The PCR fragments were
cut with BamHI and XbaI and religated into pcDNA3.1/myc-His.
2.5. Antibodies and immunological methods
Immunoblotting was performed as described previously .
Primary antibody for p63 (4A4), p63a (H-109), b-actin (I-19), cy-
clin B1 (V152), phosphor-histone 3 (Ser10) and Aurora A were pur-
chased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The
Aurora B, Cdc20 and Cdh1 antibodies were purchased from Abcam
(Cambridge, UK). The HA (12CA5) antibody was obtained from
Roche (Roche, Germany). The Phospho-Cdc2 (Tyr15) and HRP-con-
jugated secondary antibodies were purchased from Cell Signaling
Technology (CST, MA, USA). The a-tubulin antibody was purchased
from LabVision (Fremont, CA, USA). The Alexa-Fluor 488 and 555
conjugated secondary antibodies were purchased from Invitrogen
(Invitrogen, CA, USA).
For immunostaining studies, cells were grown on glass-cover
slips, washed in PBS and further incubated in 4% paraformalde-
hyde for 15 min. Cells were then permeabilized and blocked in
solution with 0.1% triton X-100 and 3% BSA for 30 min at room
temperature. Cells were washed once with PBS, incubated with
primary antibodies for 1 h, and then washed three times with
PBS before incubating with secondary antibodies for 1 h. Cells
were counterstained with Hoechst 33258 to visualize their nu-
clei. The coverslips were mounted on glass slides with fluores-
imaged using a Carl Zeiss Epi-fluorescence microscope at 400?
magnification and captured by SPOT camera (Diagnostic Instru-
ments, Inc, MI, USA).
2.6. Time-lapse microscopy
Live cells transfected with plasmid constructs were grown in 6
wells plate. The cultures were transferred to an incubator chamber
(37 ?C, 5% CO2) and imaged for over 16 h with the Carl Zeiss LSM
510 microscope, equipped with a 40? Plan-Neo DIC 1.3 numerical
aperture objective. Images were captured at time intervals of 8 min
over 16 h using the Axiocam software (Carl Zeiss).
3.1. DNp63a knockdown promotes mitotic exit
To examine if DNp63a may play any role in cell cycle progres-
sion, we first examined if silencing p63 may have any effects on
mitotic exit in cells. Time lapse imaging experiment of HaCaT, an
immortalized human keratinocyte cell line, undergoing mitosis
was performed. The predominant isoform of p63 expressed in Ha-
CaT cells was shown previously to be DNp63a . We knocked
down DNp63a in HaCaT cells by transfectiing a short hairpin
RNA expression vector targeted specifically against p63 DNA bind-
ing domain (p63 shRNA) (kindly provided by Dr. James DiRenzo,
Dartmouth-Hitchcock Medical Center, USA). . The p63 shRNA
was cotransfected with the plasmid expressing the histone H2B-
GFP at a ratio of 10 to 1 for visualization of chromosome segrega-
tions in mitotic cells using time-lapse live cell imaging microscopy.
Mitotic cells in p63-knockdown cells and control cells, transfected
with control empty vector (pSUPER) were examined simulta-
neously for the length of time required to exit mitosis. Interest-
ingly, a shorter time span was observed for DNp63a-knockdown
cells to enter anaphase after nuclear envelope breakdown (NEB)
(41.9±9.0 min; n = 30) compared to control cells (63.5±11.2 min;
n = 30) (Fig. 1A). We then conducted cell synchronization experi-
ments to confirm the time-lapse live cell imaging results by Wes-
tern blotting analysis using specific markers for mitotic cells. We
first synchronized the DNp63a-knockdown cells and control cells
with nocodazole, a spindle-disrupting agent that inhibits the for-
mation of spindles, to arrest cells at pro-metaphase. Cells were
then released by washing out the drug and harvested at various
time points for Western blotting analysis. The mitotic marker,
phospho-Histone H3 (Histone 3-P), was used to monitor cells exit-
ing mitosis after release of nocodazole. The levels of Histone 3-P in
control cells decreased at 2–3 h after release of nocodazole while
that of DNp63a-knockdown cells diminished at 1–2 h after noco-
dazole release (Fig. 1B). Hence both live cell imaging and cell syn-
chronization experiment showed that knock down of DNp63a
promoted mitotic exit and support a role of DNp63a in regulation
of cell cycle progression.
3.2. DNp63a expression is cell cycle-dependent
The DNp63a is highly expressed in the basal layer of stratified
epithelium and decreased abruptly when cells undergo differenti-
ation . Complementation study in p63 ?/? mice with DNp63a
showed that expression of DNp63a is important for formation of
the basal layer in stratified epithelium . The p63 is expressed
in proliferating cells and is modulated during cell cycle progression
. Based on this premise, we synchronized HaCaT cells at early S
phase by double thymidine block and examined the levels of
DNp63a expression during mitosis transition during cell cycle pro-
gression (Fig. 2A). Entry and exit of mitosis was indicated by accu-
mulation and decrease of Cyclin B1 protein, Cdc2-P (Y15) and
Histone 3-P. In general, all these mitotic markers accumulated at
around 8–9 h after thymidine block release. The expression of His-
tone 3-P and other mitotic markers decreased at 11–12 h after re-
lease indicating that most cells had exited mitosis. Interestingly,
the protein levels of DNp63a correlated well with the expression
of these markers which also decreased at mitotic exit.
To further confirm the decrease of DNp63a level at mitotic exit,
we synchronized cells at pro-metaphase by nocodazole and exam-
ine its expression profile after release. Again, we confirmed that
the protein level of DNp63a decreased as cells exited mitosis
(Fig. 2B). The decrease of DNp63a lagged slightly behind than
the decrease of Cyclin B1, but ahead of Aurora A and Aurora B.
P.M. Hau et al./FEBS Letters 585 (2011) 2720–2726
To validate the close association of DNp63a expression with mito-
tic exit, we also expressed DNp63a in a p63-negative cell line,
HeLa cells, by transfecting a DNp63a expression plasmid and
examined for the levels of DNp63a after nocodazole synchroniza-
tion and release (Fig. 2C). The levels of exogenously expressed
DNp63a was also observed to decrease when cells exiting mitosis
as indicated by the downregulation of Histone 3-P and other mito-
tic markers (Fig. 2C). The decrease in levels of DNp63a protein at
mitotic exit resembles the behavior of other cell cycle regulators
which are involved to suppress premature mitotic exit.
Fig. 1. DNp63a knockdown promotes mitotic exit in HaCaT cells. (A) Live cell imaging of p63 knockdown cells. HaCaT cells were cotransfected with H2B-GFP plasmid and
either control shRNA or p63 shRNA cells in ratio 10:1 respectively. Cells were captured as described in Materials and Methods. The time required for cells to progress from
nuclear envelope breakdown (NEB) to anaphase was estimated. More than 30 cells were counted in each group. Representative time-lapse photos captured for each group
were shown. The upper panel is the brightfield image and the lower panel is the H2B-GFP signal captured from the same cells. The images were captured in 8 min intervals.
(B) HaCaT cells were transfected with either control shRNA or p63 shRNA together with H2B-GFP expression plasmid containing blasticidin-resistant gene. Cells were selected
with blasticidin for 24 h. After 16 h post-transfection, cells were then synchronized in prometaphase with nocodazole (0.1 lg/ml) for 16 h before washing the arrested cells
with PBS. Cells were re-plated and harvested at the indicated time. Cells were harvested and subjected to Western Blot using anti-p63 (4A4) and phospho-Histone H3 (S10)
P.M. Hau et al./FEBS Letters 585 (2011) 2720–2726
3.3. Decrease of DNp63a protein is associated with mitotic exit
Various mitotic regulators are involved in the timely co-ordina-
tion of events involved in mitosis. Cells that harbor defects in these
mitotic regulators are prone to have mitotic deregulation resulting
in genome instability. Progression to late mitosis is tightly regu-
lated by the destruction of APC/C-targeted substrates. Failure to
degrade these substrates halts the cells at different stages in mito-
sis and often leads to various mitotic defects including lagging and
breakage of chromosomes as well as cytokinesis failure .
After all the chromosomes have achieved bipolar attachment to
spindles and aligned at metaphase plate, two important events
take place. First, the Cyclin B1 is destroyed by the APC/Ccdc20lead-
ing to decrease in CDK1 kinase activity. Second, the Securin is
degraded freeing the Separase to cleave the Cohesin to facilitate
segregation of sister chromatids. The inhibition of the CDK1 kinase
activity allows cells to proceed from metaphase to anaphase. The
Cdc20 is a substrate of APC/Ccdh1which is degraded at anaphase.
The APC/Ccdh1is responsible for the degradation of substrates at
late mitosis and cytokinesis. Aurora A and Aurora B are substrates
of APC/Ccdh1targeted for destruction at telophase and cytokinesis.
We have compared the expression levels of DNp63a expression
with these key mitotic regulators including Aurora A, Aurora B,
Cdc20 and Cdh1 in HaCaT cells synchronized by double thymidine
block (Fig. 3A). After releasing HaCaT cells from double thymidine
block, we observed that decrease in DNp63a is associated with the
destruction of cyclin B1 and cdc20. As expected, cdh1 degradation
lagged behind cdc20 degradation during mitotic exit.
Fig. 2. DNp63a expression is cell cycle-dependent. (A) Synchronization of HaCaT cells in S phase. Cells were synchronized with double thymidine block. In brief, cells were
treated with 2 mM thymidine for 16 h. Cells were then washed with PBS for 3 times to remove the drug and allowed to recover for 8 h. Cells were again treated with 2 mM
thymidine for 16 h to arrest the cells at early S-phase. Arrested cells were released from drug by washing with PBS and harvested according to the time points stated. Western
blotting were performed with antibodies against p63 (4A4), Cyclin B1 (V152), p-Histone H3 (S10) and Cdc2-P (Y15). (B) Synchronization of HaCaT cells in prometaphase.
HaCaT cells were achieved by treating cells with nocodazole 0.2 lg/ml for 16 h, then washed with PBS and re-plated. Immunoblotting were performed using antibodies
against p63, Cyclin B1, Cdc2-P (Y15), p-Histone H3 (S10), Aurora B, Cdc20 and Cdh1. (C) HeLa cells were transfected with DNp63a expression construct. Cells were harvested
and subjected to Western Blot using anti-p63 (4A4), Cyclin B1 (V152), Cdc2-P (Y15) and p-Histone H3 (S10) antibodies described above, together with anti-Aurora B, anti-
Cdc20 and anti-Cdh1 antibodies (D).
P.M. Hau et al./FEBS Letters 585 (2011) 2720–2726
We further performed immunofluorescence studies of DNp63a
in HaCaT cells during mitosis. The expression of DNp63a could be
detected in the nuclei of interphase cells. The expression of
DNp63a appears to accumulate in mitotic cells beginning pro-
phase (Fig. 3, the first panel). However, in metaphase cells, we ob-
served two different patterns of DNp63a expressions. Half of the
metaphase retained high DNp63a expression, while others had re-
duced level of DNp63a expression (Fig. 3, the second and third
panels). The expression of DNp63a was lowest in anaphase cells
(Fig. 3, the forth panel). The DNp63a level remained low at telo-
phase (Fig. 3, the fifth panel) and begins to increase in interphase
cells. Together, our results indicate that DNp63a is a target for deg-
radation during metaphase to anaphase transition.
3.4. DNp63a contains a potential destruction box (D-box) and may be
a target of APC/C
Two coactivators are required for APC/C to recognize and target
specific substrates to ubiquitin-mediated proteolysis. They are
Cdc20 (cell-division cycle protein 20) and Cdh1 (Cdc20-homologue
1). Both Cdc20 and Cdh1 contain the protein domain WD40
repeats. The WD40 repeats domain recognizes specific motifs
present in APC/C substrates and accounts for substrate specificity.
Most studies focus on two regions: (1) destruction box (D-Box) and
(2) the KEN box. These coactivators confer substrate specificity to
APC/C ligase activity. While APC/Ccdc20recognizes D-box-contain-
ing substrates, the APC/Ccdh1recognizes both D-box and KEN box
substrates . D-Box motif is also present in Cyclin B1 and Secu-
rin while both D-Box and KEN Box motif are present in Aurora A,
Aurora B, as well as Cdc20. Recently, it was suggested that the rec-
ognition of substrate with different motifs is not mutually exclu-
sive [18,19]. Different motifs were suggested to coordinate with
each others to promote substrate recognition by APC/C.
We have explored if APC/C activation may be involved in the
reduction of levels of DNp63a at mitotic exit. To begin with, the
DNp63a protein sequence was scanned to determine the presence
of potential targets of either Cdc20 or Cdh1. A consensus D-
Box (RxxLxxxxN) motif is present in DNp63a which is highly con-
served among closed species (Fig. 4A). The potential D-Box is a tar-
get of both Cdc20 and Cdh1 of the APC/C.
HA-tagged Cdh1 in 293 cells and investigated the stability of
tein synthesis. Fig. 4B shows that overexpressing Cdh1 was more
effective than Cdc20 to reduce the protein stability of expressed
be rescued by treating cells with MG132 which is a proteasome
Fig. 3. Decreased in DNp63a during mitotic exit. HaCaT cells were synchronized in early S phase with double thymidine block as in Fig. 1A. Cells were harvested and cell
extracts were prepared. Protein was transferred to PVDF membrane and immunoblotted with anti-p63 (4A4), Cyclin B1 (V152), Aurora A, Aurora B, Cdc20 and Cdh1
antibodies. HaCaT cells were immunostained with anti-p63a antibody (H-129, 1:500) and/or a-tubulin antibody (Ab-4, 1:500) for 1 h. Cells were further washed 3 times with
PBS before incubated with secondary antibody (Alexa 555 Goat anti-rabbit or Alexa 488 Rabbit anti-mouse, 1:1000) for 1 h. The nuclei were counterstained with Hoechst
stain (1:8000) for 5 min before mounting the slides. In the figure, DNp63a was labeled in red, tubulin was labeled in green and nuclei were stained with Hoechst (Blue).
P.M. Hau et al./FEBS Letters 585 (2011) 2720–2726
inhibitor. Furthermore, mutation of the putative D-box motif (R225-
XX-L228to A225-XX-A228) of DNp63a abolished its destabilization in
Cdh1 knock-down experiments using shRNA. As shown in Fig. 4C
icant changes in DNp63a levels, Cdh1 knockdown suppressed
DNp63a degradation. These data indicate that DNp63a could be a
potential target of APC/CCdh1at mitosis. Further experiments are
warrant to delineate the interaction of DNp63a with APC/CCdh1.
There are many studies suggesting that the isoform of p63,
DNp63a, promotes cell proliferation. By applying gene profiling
study and chromatin immunoprecipitation on chip assay in human
keratinocytes, Testoni (2006) reported that many p63 target genes
are actually cell cycle regulated including p21, p57, 14-3-3 r and
adenosine deaminase (ADA) . Furthermore, the loss of strati-
fied epithelia skin in p63-knockout mice is attributed to the loss
of epidermal stem cells [21,22]. Therefore, p63 isoforms may regu-
late transcription of cell cycle genes and promotes cell cycle pro-
gression in basal cells. In this study, we have observed that the
DNp63a is a cell cycle-regulated protein and may function during
mitosis to delay mitotic exit in epithelial cells.
We have utilized shRNA to knock down DNp63a expression in
HaCaT cells and observed a shortened time interval for mitotic cells
to progress from NEB to anaphase using live cell imaging micros-
copy (Fig. 1A). This is the first report suggesting that DNp63a
may regulate timing of mitotic exit in cells. Immunoblotting also
confirmed that knockdown of p63 in HaCaT cells reduced the time
required to exit mitosis as indicated by the expression level of His-
tone 3-P in synchronized cell populations (Fig. 1B). To further
examine the role of DNp63a during cell cycle progression, we have
compared the levels of DNp63 expression at different phases of cell
cycle with other mitotic markers and regulators including Histone
Fig. 4. DNp63a as a potential target of APC/Ccdh1. (A) The diagram shows the presence of a potential Destruction box (D-box) in DNp63a. The potential D-box locates at
amino acid 225-234 in the DNA-binding domain. It is conserved in all p63 isoforms. D-box motif in the other APC/C targets, Cyclin B1 and p21 which contain the common
motif are also shown here for comparison. (B) 293 cells were cotransfected with DNp63a and either with vector, HA-cdc20 (upper panel) or HA-cdh1 (lower panel) expressing
vectors. The cells were treated with 10 lg/ml cycloheximide (CHX) and harvested according to the time specified. For the rescue experiment, 10 lM MG132 was added 1 h
before addition of cycloheximide. Cell extract were prepared and subjected to immunoblot with anti-HA (12CA5) and anti-p63 (4A4) antibodies. (C) (Left panel) 293 cells
were cotransfected with HA-cdh1 and wild type DNp63a or DNp63a D-box mutant. The cells were treated and cycloheximide as in (b) and harvested according to the time
specified. (Right panel) HaCaT cells were co-transfected plasmid containing blasticidin-resistant gene together with scramble, shCdc20 or shCdh1. Cells were selected with
blasticidin for 36 h before subjected to cell lysis. Cell extract were prepared and subjected to immunoblot with anti-p63 (4A4) (Santa Cruz), Cdc20 (Abcam) and Cdh1 (Abcam)
P.M. Hau et al./FEBS Letters 585 (2011) 2720–2726
3-P, cyclin B1, cdc2-P, cdc20 and cdh1 (Fig. 2A–C). Histone H3
phosphorylation is commonly used as marker for mitotic exit. De-
crease of Histone 3-P indicates cells exiting mitosis. In all our Wes-
tern blotting experiments, degradation of DNp63a was always
observed during mitotic exit. Immunofluorescence study reveals
the presence of DNp63a in interphase cells (Fig. 3). The DNp63a
accumulates from prophase to metaphase and degraded during
mitotic exit. Expression of DNp63a reappeared in interphase cells
after mitosis, albeit at a lower level compared to mitotic cells. Our
results indicate a novel role of DNp63a in regulating cell cycle pro-
gression at mitotic exit.
At this stage, the underlying mechanism of degradation of
DNp63a in mitotic cells is not defined. Mitotic regulators are tar-
geted for ubiquitin-mediated proteolysis during mitosis passage.
This is an intricate process during which mitotic regulators are
sequentially degraded as cells transit through mitosis to cytokine-
sis. The timely degradation of these mitotic regulators is crucial for
successful cell division. Various mitotic defects have been reported
in cells that fail in degrading these regulators . It is well docu-
mented that these mitotic regulators are targeted by APC/C. The
APC/C recognizes potential substrates for degradation via two
coactivators Cdc20 and Cdh1. By recognizing specific motifs on
the substrate, the Cdc20 and Cdh1 direct their substrates to APC/
C ubiquitin ligase for ubiquitin-conjugation and subsequent degra-
dation by proteosome. At present, the D-Box and the KEN box mo-
tif are present in many substrates involved in cell cycle regulation
which include various types of cyclins, polo-like kinases, Aurora ki-
nases, and even the APC/C coactivators themselves . Our re-
sults showed that DNp63a resembles the behaviors of other
mitotic regulators which are targets of APC/C for degradation dur-
ing mitosis. Our results suggest that DNp63a itself may also be a
substrate of APC/C. Analysis of the amino acids sequence of
DNp63a reveals a putative D-Box motif that can be recognized
by either Cdc20 or Cdh1 (Fig. 4A). The D-Box can be targeted by
both Cdc20 and Cdh1. We have obtained preliminary results sug-
gesting that Cdh1 may be effective in decreasing the protein stabil-
ity of DNp63a (Fig. 4B). Treating cells with proteasome inhibitor
MG132 rescued the destabilization of DNp63a in Cdh1-overx-
pressing cells. Importantly, mutation of the putative D-box motif
stabilized DNp63a in Cdh1-overexpressing cells (Fig. 4C, left pa-
nel). Moreover, knocking down Cdh1, but not Cdc20, increased
DNp63a (Fig. 4C, right panel). Together, these results indicate that
DNp63a may be a target of Cdh1 for proteosome degradation. De-
tail analysis of interaction of DNp63a and APC/C, however, is war-
rant to further delineate the role of DNp63a and its interaction
with APC/C in regulating mitotic exit.
In conclusion, we found that DNp63a expression is cell cycle-
dependent and degraded at mitotic exit. Knocking down DNp63a
induced earlier mitotic exit. Failure to degrade DNp63a may result
in delay of mitotic exit and contribute to genomic instability in
We appreciate the kind gifts from Drs. James DiRenzo (Dart-
mouth-Hitchcock Medical Center, USA), Kurt Engeland (University
of Leipzig, Leipzig, Germany), Patrick Ling (Queensland University
of Technology, Australia), Serigo Moreno (University of Salamanca,
Spain), Ralph Wäsch (Albert-Ludwigs University Medical Center,
Freiburg, Germany) and Randy Y.C. Poon (The Hong Kong Univer-
sity of Science and Technology, Hong Kong). This work was sup-
ported by Research Grant Council, Hong Kong (HKU7770/07 HKU
7778/09 M), the AoE NPC (AoE/M-06/08) and CRCG grant of Uni-
versity of Hong Kong.
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