© 2006 Japanese Cancer Association
Cancer Sci|November 2006|vol. 97|no. 11|
Blackwell Publishing Asia
Human homolog of Drosophila tumor suppressor
Scribble negatively regulates cell-cycle progression
from G1 to S phase by localizing at the basolateral
membrane in epithelial cells
Kazunori Nagasaka,1 Shunsuke Nakagawa,1,3 Tetsu Yano,1 Shin Takizawa,1 Yoko Matsumoto,1 Tetsushi Tsuruga,1
Keiichi Nakagawa,2 Takeo Minaguchi,1 Katsutoshi Oda,1 Osamu Hiraike-Wada,1 Hajime Ooishi,1 Toshiharu Yasugi1
and Yuji Taketani1
1Department of Obstetrics and Gynecology, and 2Department of Radiology, Graduate School of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo
(Received April 12, 2006/Revised July 23, 2006/Accepted July 29, 2006/Online publication September 8, 2006)
Drosophila tumor suppressor Scribble has been identified as an
apical-basolateral polarity determinant in epithelia. A human
homolog of Drosophila Scribble, human Scribble (hScrib), has been
identified as a protein targeted by human papillomavirus E6 for the
ubiquitin-mediated degradation dependent on E6AP, a cellular
ubiquitin-protein ligase. Human Scribble is classified as a LAP
protein, having leucine-rich repeats (LRRs) and PDZ domains. We
investigated whether hScrib, which is thought to have a role in
polarity determination based on the data of its Drosophila homolog,
is involved in cell-cycle regulation and proliferation control of
epithelia. Transfection of hScrib inhibits cell-cycle progression
from G1 to S phase, and it up- and down-regulates expression of
adenomatous polyposis coli and cyclins A and D1, respectively.
Knockdown of hScrib expression by siRNA leads to cell-cycle
progression from G1 to S phase. We explored functional domain
mapping to reveal which domains of hScrib are critical for its cellular
proliferation control and localization at the basolateral membrane.
We found that LRRs and PDZ domain 1 are indispensable for hScrib
to inhibit cell growth by blocking cell-cycle progression and to keep
its proper localization. These data indicate that basolateral
membrane localization of hScrib is closely related to its proliferation
control. Our findings suggest the possibility that hScrib is involved
in signal transduction to negatively regulate cell proliferation by
localizing at the basolateral membrane of epithelial cells through
LRRs and PDZ domains. (Cancer Sci 2006; 97: 1217–1225)
architecture is based on proper localization of junctional proteins.(1)
Loss of cell polarity due to aberrant expression and localization
of junctional proteins causes overgrowth of epithelia.(2) Mutation
of a tumor suppressor gene whose encoded protein regulates
cell-cycle progression is also characteristic of cancer tissues.
Maintenance of tissue polarity and regulation of cell cycle are
tightly linked to keep normal growth control and differentiation
in epithelia.(3) Loss of tissue architecture and disruption of the
cell-cycle control system leads to uncontrolled proliferation of
epithelial cells and subsequent tumor formation.(4)
Drosophila tumor suppressor Scribble has recently been identified
as an apical-basal polarity determinant in epithelia.(5) Drosophila
Scribble localizes at the septate junction, which is functionally
identical to the vertebrate tight junction, and loss of scribble
mutation causes mislocalization of adherens junction to the
basolateral membrane.(5) Follicle cells homozygous for scribble
mutation are rounded, rather than columnar, and are often multilayered,
rather than sheet-like.(6) In Drosophila scribble mutant, disorganized
rganized tissue architecture and cell polarity are hallmarks
of epithelia. Construction of organized epithelial tissue
epithelial tissue overproliferates and causes massive tumors in
imaginal discs, brain, and follicles.(6) Recently, scribble has been
identified as a novel regulator of S-phase entry in Drosophila by a
genetic screen for dominant hypomorphic cyclin E mutation.(7)
The human homolog of Drosophila tumor suppressor Scribble,
hScrib, has recently been identified as a target of the high-risk
human papillomavirus (HPV) E6 oncoprotein for ubiquitin-
mediated degradation dependent on the cellular ubiquitin-protein
ligase E6AP.(8) The high-risk E6s, but not low-risk HPV E6s,
have a conserved PDZ domain-binding sequence (threonine-X-
leucine/valine) at their C-terminal region.(8,9) Through this motif,
high-risk E6s bind to the PDZ (named for its occurrence in the
PSD-95, discs large and ZO-1 proteins) domains of hScrib.(10)
The ternary complex formation of hScrib, E6 and E6AP leads to
ubiquitin transfer to hScrib dependent on the E3 activity of E6AP.(8)
hScrib tagged with multi-ubiquitin branches is recognized at
the 26S proteosome and is degraded. We recently reported
that the expression of hScrib is down-regulated according to
the progression of disease during cervical carcinogenesis.(11)
Decreased expression of hScrib without down-regulation of
hScrib mRNA transcription in the high-grade cervical intraepi-
thelial neoplasm, which is a precursor lesion of cervical can-
cer, supports the possibility that the ubiquitin-mediated
degradation of hScrib depending on HPV E6 and E6AP is
involved in the development of cervical cancer.(11)
Human and Drosophila Scribbles have identical protein structures,
consisting of 16 canonical leucine-rich repeats (LRRs) at their
N-terminal region and four PDZ domains. Recently, proteins
having 16 LRRs and one or four PDZ domains have been grouped
as LAP (leucine-rich and PDZ) proteins.(12) LAP proteins are
involved in the establishment of epithelial cell polarity, by
localizing at the basolateral wall of epithelia.(5,13,14) LAP proteins
have no membrane-localization signals by themselves and interaction
with other transmembrane proteins through their LRRs or PDZ
domains is necessary for them to localize at the basolateral wall
of epithelia.(13,15,16) The LRRs and PDZ domains are also thought
to be critical in the control of cell polarity and inhibition of
tumorous overproliferation of epithelial cells.
In this study, we explored a possible role of hScrib in the
regulation of cell-cycle progression and cell proliferation. Next
we analyzed whether LRRs and/or PDZ domains of hScrib have
a critical role in the proper functioning of cell growth control
through the regulation of cell-cycle progression.
3To whom correspondence should be addressed.
© 2006 Japanese Cancer Association
Materials and Methods
Construction of green fluorescent protein (GFP)-fusion hScrib expression
plasmids. The full-length cDNA of hScrib was subcloned into
the multicloning site of the pEGFP-C1 vector (BD Biosciences–
Clontech, Palo Alto, CA, USA). To generate the deletion mutants
of GFP-fusion hScrib, the following cDNA sequences were
amplified with polymerase chain reaction (PCR): LRR + PDZ
1–4 (amino acids 1–1191); LRR + PDZ 1–3 (amino acids 1–1096);
LRR + PDZ 1–2 (amino acids 1–953); LRR + PDZ 1a (amino
acids 1+818); LRR + PDZ 1b (amino acids 1+747); LRR (amino
acids 1–495); LRR∆LAPSDb (amino acids 1–421); LRR∆LAPSDa-
b (amino acids 1–390); and PDZ 1–4 (amino acids 731–1191).
The amplified PCR products were digested with restriction
enzymes and subcloned into the pEGFP-C1 vector. GFP-
PDZ 1–2 GRGI mutant, in which critical amino acid sequence
GLGI to the structure of PDZ domain pocket in PDZ domain 1
is substituted by GRGI, was generated by Quick change site
directed mutagenesis kit (Stratagene) using the following
Mammalian cell transfection and immunofluorescence analysis.
NIH3T3 cells or Madin–Darby canine kidney (MDCK) cells
were cultured on coverslips in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine serum
(FBS) at 37°C in a humidified incubator with 5% CO2. For
transfection, 5 × 102 cells/cm2 were seeded onto glass coverslips
the day before transfection in six-well plates and subconfluent
cells were transfected with 1.5 µg of various plasmids using
Polyfect reagent (Qiagen, Valencia, CA, USA). Transfected
subconfluent MDCK cells with various clones of GFP-fusion
hScrib expression plasmids containing various parts of hScrib
cDNA subcloned into pEGFP-C1 vector (BD Biosciences–
Clontech) were grown on coverslips in the culture medium.
Forty-eight hours later, cells were washed three times with
phosphate-buffered saline (PBS) and then fixed with 3.7%
formaldehyde in PBS for 10 min. Cells were then washed three
times with PBS, rinsed with distilled water, and made permeable
with acetone at −20°C for 10 min. Cells were washed with PBS
and incubated with the diluted the anti-hScrib C-terminus antibody
(Scrib C-20; Santa Cruz Biotechnology, Santa Cruz, CA, USA)
for 30 min at room temperature. Cells were then washed three
times with PBS and incubated with the rhodamine-conjugated
secondary antigoat antibody (Sigma, St Louis, MO, USA), and
then washed three times with PBS.
Cells were mounted on a slide glass and examined under a
confocal fluorescence microscopy (Carl Zeiss LSM 540 micro-
scope). Images were captured with a CCD camera. For each
GFP-fusion hScrib expression clone, at least three independent
experiments were performed.
Colony forming assay. NIH3T3, Hela, and MDCK cells (2 × 105
cells/cm2) were transfected with 4 µg of various GFP-fusion hScrib
expression plasmids using Polyfect reagent according to the
manufacturer’s instruction (Qiagen). After incubation for 30 h,
cells were trypsinized and diluted 1:30, and cultured in DMEM
supplemented with 10% FBS and G418 (1000 µg/mL) for 2
weeks. Cells were fixed with 100% methanol and stained with
0.4% Giemsa reagent (Gibco-BRL) in PBS. Several independent
colonies were isolated, and it was confirmed that they expressed
appropriate GFP-fusion hScrib proteins by immunoblotting assay.
For each GFP-hScrib construct, at least two independent experiments
Flow cytometry. NIH3T3 cells were grown at a density of
1 × 105 cells/mL in the culture medium. Cells were transfected
with GFP expression vector pEGFP-C1 vector (BD Biosciences–
Clontech) or pEGFP-C1-hScrib full-length plasmid. After 24 h,
cells were further cultured in DMEM containing 0.4% FBS for
12 h, and then the medium was changed to fresh DMEM containing
10% FBS. After culturing for 12 h, cells were trypsinized and
collected by centrifugation and then washed twice with PBS.
Cells were resuspended in PBS containing 1% formaldehyde for
1 h at 4°C. Cells were washed twice again in PBS and resuspended
in buffer containing ethanol and PBS at the ratio of 7:3 at −20°C
overnight. After washing twice with PBS, cells were incubated
in ribonuclease solution (0.25 mg/mL) (Qiagen) for 30 min at 37°C,
followed by staining with propidium iodide (50 µg/mL) (Dojindo,
Japan) on ice for 30 min in the dark. Gates were established for
purification of the GFP expressing cells. Approximately 100 000
stained cells with propidium iodide were analyzed for GFP
fluorescence and cell-cycle distribution on EPICS XL Flow
cytometry (Beckman Coulter), as described previously.
BrdU incorporation analysis in NIH3T3 cells transfected with pEGFP-
C1-hScrib expression plasmids. NIH3T3 cells were transfected with
various pEGFP-C1-hScrib expression plasmids. Twelve hours
later, cells were cultured in DMEM containing 0.4% FBS for
12 h. The medium was changed to fresh medium containing
10% FBS and 50 µM bromodeoxyuridine (BrdU; Roche). After
culturing for 16–18 h, cells were fixed with 3.7% formaldehyde
in PBS for 30 min, treated with methanol at −20°C for 10 min
and then treated with 1 M HCl for 10 min. Cells were washed
with PBS and incubated with anti-BrdU antibody (1:10 dilution,
BrdU detection kit, Roche) for 1 h at room temperature. Cells
were then washed six times with PBS and incubated with
rhodamine-conjugated antimouse antibody (Sigma) for 1 h and
then washed three times with PBS.
Cells were mounted on a slide glass and examined under the
confocal fluorescence microscope (Carl Zeiss LSM 540). For
each pEGFP-C1-hScrib expression construct, at least three
independent experiments were performed.
Western blotting. MDCK or NIH3T3 cells transfected with GFP-
fusion hScrib expression plasmids were grown in DMEM
medium supplemented with 10% FBS. The transfection efficiency
was determined to be over 80% for each cell line. After 24 h,
cells cultured in DMEM containing 0.4% FBS for 12 h were then
changed to fresh DMEM containing 10% FBS. After culturing
for 12 h, cell extracts were made in the NP-40 lysis buffer
containing 100 mM Tris (pH 8.0), 100 mM NaCl, 1% NP-40,
and protease inhibitor cocktail (Sigma). Protein concentration
was determined by standard Bradford assay. The cell extracts
were fractionated by SDS-PAGE and electrophoretically transferred
onto the polyvinylidene difluoride membranes (Millipore, Bedford,
The anti-GFP antibody (Zymed, South San Francisco, CA, USA)
was used at dilution of 1:1000 to detect the expression of GFP-
fusion hScrib. For adenomatous polyposis coli (APC), cyclin A,
cyclin D1, and α-tubulin proteins, proteins were visualized by
Western blotting using a 1 : 1000 dilution of an anti-APC (C-20,
Santa Cruz Biotechnology), and a 1:500 dilution of an anticyclin
A, anticyclin D1 (Santa Cruz Biotechnology), and antitubulin
(Calbiochem, San Diego, CA, USA). The level of protein
expression was analyzed by the STORM 860 according to
the manufacturer’s recommendation (Molecular Dynamics,
Sunnyvale, CA, USA).
siRNA transfection. All small interfering RNA (siRNA) were
obtained from Qiagen as purified. A target sequence of hScrib
siRNA was designed using HiPerformance-2 for silencing siRNA
duplexes from Qiagen. The sequence was submitted to a
BLAST search against the human genome sequence to ensure
that no gene of the human genome was targeted. An siRNA
against human Discs large (hDlg) was used as a positive control,
as described previously.(17)
The target sequence against hScrib was 5′-CAG GAT GAA
GTC ATT GGA ACA. Several cell-lines, Caco2, HaCaT and
293T cells were transfected by the use of 5 µL of X-treme
GENE siRNA transfection reagent (Roche) per mL and a final
siRNA concentration of 10 nM according to the manufacturer’s
Nagasaka et al.
Cancer Sci| November 2006| vol. 97| no. 11|
© 2006 Japanese Cancer Association
instructions. Alexa 568-labeled negative control siRNA (Qiagen)
was used as a measure of transfection efficiency. The transfection
efficiency was determined to be 80–90% for each cell line. The
cells were fixed at 72 h post-transfection for immunofluorescence
or were lyzed in NP-40 buffer for Western blot analysis.
Protein structure of hScrib and expression of GFP-fusion hScrib in
the epithelial cell line MDCK. Human Scribble is classified as a
LAP4 protein, containing 4 PDZ domains at its central region in
addition to 16 canonical LRRs at its N-terminal region. Between
16 LRRs and 4 PDZ domains, hScrib has two conserved regions
of LAP proteins that are called LAP specific domains (LAPSD).
LAPSDa consists of 38 amino acid residues related to LRR, while
LAPSDb consists of 24 residues not related to LRR (Fig. 1A).
We constructed a GFP-fusion hScrib expression plasmid and
examined its localization in the MDCK epithelial cell line. GFP-
fusion hScrib localized broadly at the basolateral membrane of
MDCK epithelial cells (Fig. 1B). Localization and expression of
GFP-fusion hScrib was identical to that of an adherens junction
marker, E-cadherin, but not to that of a tight junction marker,
ZO-1 (Fig. 1B). Localization and expression of GFP-fusion
hScrib was identical to that of endogenous hScrib revealed by
immunofluorescence analysis with the anti-hScrib polyclonal
antibody, which indicated that the endogenous hScrib localizes
at the adherens junction (Fig. 1B,C).
Expression and localization of GFP-fusion hScrib mutants in MDCK
cells. We constructed GFP-fusion hScrib deletion mutants lacking
C-terminal region, PDZ domains, LAPSDa, and LAPSDb (Fig. 2),
and examined their expression by Western blotting (Fig. 3). We
also subcloned hScrib cDNA sequence encoding PDZ domains
fusion of hScrib. (A) Schematic representation of
hScrib. Leucine-rich repeats (yellow ovals), LAP
(leucine-rich and PDZ domain) specific domain
a (LAPSD a) (blue oval), LAPSD b (red oval), and
PDZ domains (green boxes) are indicated. (B,C)
Immunofluorescence images: a. expression of GFP-
fusion hScrib in MDCK cells; b. endogenous
expression of hScrib analyzed by the anti-hScrib
polyclonal antibody; c. merged image of a and b
indicates that GFP-fused hScrib colocalizes with
the endogenous hScrib. (Original magnification × 200;
bar = 20 µm).
Protein structure and expression of GFP-
encoding these hScrib domains were subcloned
into the pEGFP C-1 vector.
Generation of GFP-fused hScrib domain
deletion constructs. The
© 2006 Japanese Cancer Association
1–4 into pEGFP expression vector and tested its expression by
Western blotting (Figs 2,3). These pEGFP-hScrib expression
plasmids and GFP expression vector were transfected into MDCK
cells and confirmed to express the proteins with expected size
Minimal domains of hScrib necessary for its basolateral membrane
localization in MDCK cells. We transfected GFP-fused hScrib LRR +
PDZ 1–4 lacking the C-terminal region into MDCK cells. GFP-
fused LRR + PDZ 1–4 localized at the basolateral membrane of
MDCK cells (Fig. 4A). Colocalization of GFP-tagged signal of
hScrib LRR + PDZ 1–4 and the immunofluorescence signal
of endogenous hScrib analyzed with the anti-hScrib polyclonal
antibody indicated that the C-terminal region of hScrib is
dispensable for the proper localization of hScrib (Fig. 4A). We
further analyzed whether PDZ domains of hScrib are critical for
its proper basolateral membrane localization. GFP-fused hScrib
LRR + PDZ 1–4 lacking PDZ domain 4 and the C-terminal region
localized at the basolateral membrane, showing an identical
localization pattern to the endogenous hScrib (data not shown).
Likewise, GFP-fused hScrib LRR + PDZ 1–2 lacking PDZ domains
3 and 4 and the C-terminal region also showed colocalization
with the endogenous hScrib (data not shown). Furthermore,
GFP-fused LRR + PDZ 1 lacking PDZ domains 2–4 and the C-
terminal region showed identical localization to the endogenous
hScrib (Fig. 4B). In contrast, loss of PDZ domain 1 had a great
impact on the localization of hScrib. GFP-fused LRR lacking
PDZ domains 1–4 and the C-terminal region showed a totally
different localization pattern from that of GFP-fused LRR +
PDZ 1–4, LRR + PDZ 1–3, LRR + PDZ 1–2, and LRR + PDZ 1.
GFP-fused LRR of hScrib was faintly expressed at the
basolateral membrane. It localizes mainly in the cytoplasm, but
not in the nucleus (Fig. 4C). GFP-fused PDZ domains 1–4 of
hScrib showed no membrane-associated localization and were
expressed in the cytoplasm and nucleus (Fig. 4D).
hScrib inhibits proliferation of cells through its LRRs and PDZ
domains. We analyzed whether hScrib negatively regulates
proliferation of mammalian cells. We transfected pEGFP-full
length hScrib, which encodes GFP-fused hScrib, and pEGFP
vector into mouse NIH3T3 cells. Two weeks after the transfection,
G418-resistant colonies were stained with Giemsa reagent, and
the number of stained colonies was counted. NIH3T3 cells
transfected with pEGFP-full-length hScrib had a quarter of the
drug-resistant colony count of the cells transfected with pEGFP
vector alone (control) (Fig. 5A,B). The relative number of
deletion constructs. The hScrib cDNA encoding
the hScrib domains subcloned into the pEGFP
C-1 vector were transfected and expressed in
MDCK cells. All the pEGFP hScrib domain constructs
expressed the protein with expected size.
Arrowheads indicate the bands corresponding to
the expressed GFP-fused hScrib domain constructs.
Characterization of hScrib domain and
essential for its proper basolateral membrane localiz-
ation (original magnification × 200; bar = 20 µm).
(A) GFP-fused hScrib LRR + PDZ 1–4. (B) GFP-fused
hScrib LRR + PDZ domain 1. (C) GFP-fused hScrib
LRR. (D) GFP-fused hScrib PDZ domains 1–4.
All the GFP-fused hScrib domain constructs
were transfected and analyzed in MDCK cells.
Under the immunofluorescence image of X-Y section,
the image of Z-section is shown. a. Immuno-
fluorescence analysis of the expression of GFP-fused
hScrib domain construct. b. Immunofluorescence
analysis of the expression of endogenous hScrib. c.
Merged image of a. and b.
Analysis of critical domain of hScrib
Nagasaka et al.
Cancer Sci| November 2006| vol. 97| no. 11|
© 2006 Japanese Cancer Association
G418-resistant colonies of cells transfected with pEGFP-full-
length hScrib was calculated as the percentage of the number of
drug-resistant colonies of cells transfected with pEGFP vector
(Fig. 5B). The cells transfected with pEGFP-hScrib LRR + PDZ 1–4,
LRR + PDZ 1–3, LRR + PDZ 1–2, and LRR + PDZ 1 formed
approximately a quarter of the drug-resistant colony count of the
cells transfected with pEGFP vector alone (Fig. 5B). In contrast,
the cells transfected with pEGFP hScrib-LRR, LRR∆LAPSDb,
LRR∆LAPSDa-b, or PDZ 1–4 showed no obvious differences in
relative number of the drug-resistant colonies (Fig. 5B).
Next, we analyzed whether hScrib has a role in the inhibition
of proliferation of epithelial cells in Hela cells and MDCK cells.
The transfection of hScrib inhibited the formation of the drug-
resistant colonies to 25% of the control in these cell lines
hScrib negatively regulates cell-cycle progression from G0/G1 to S phase
depending on its LRRs and PDZ domains. We investigated whether
hScrib inhibits proliferation of epithelial cells through cell-cycle
regulation. NIH3T3 cells were transfected with pEGFP-full-
length hScrib or pEGFP vector. We compared cell-cycle profiles
of the cells transfected with pEGFP-full-length hScrib or pEGFP
vector by flow cytometry (Fig. 7). The percentage of cells in S
phase in NIH3T3 cells transfected with pEGFP-full-length
hScrib was significantly lower than that of cells transfected with
pEGFP vector alone (25.6 ± 1.9% vs 39.6 ± 0.3%; P < 0.05).
The percentage of cells in G0/G1 phase in NIH3T3 cells
transfected with pEGFP-full-length hScrib was high (62.1 ± 4.7%),
compared with that in the cells transfected with pEGFP vector
alone (48.3 ± 1.1%; P < 0.05).
Next, we analyzed which domains of hScrib are critical for its
cell-cycle regulatory function. We transfected pEGFP hScrib
expression mutants into serum-starved NIH3T3 cells. Cell-cycle
progression of quiescent NIH3T3 cells transfected with pEGFP
hScrib expression mutants was induced by addition of serum to
the medium. Progression of the cell cycle was analyzed by the
incorporation of BrdU into DNA in the cells transfected with
pEGFP hScrib expression mutants or pEGFP vector. Approxi-
mately one half of the cells transfected with pEGFP vector
alone (control) incorporated BrdU by the induction of cell-cycle
progression in the presence of serum (Figs 8,9A). In contrast,
the cells transfected with pEGFP full-length hScrib showed lower
rate of BrdU incorporation (P < 0.05 vs control) (Figs 8,9B). The
cells transfected with pEGFP hScrib LRR + PDZ 1–4,
LRR + PDZ 1–3, LRR + PDZ 1–2 (Figs 8,9C), or LRR + PDZ 1
showed lower rates of BrdU incorporation than the cells
transfected with pEGFP vector (P < 0.05).
To determine the importance of PDZ domain 1 in the control
of cell-cycle progression, we constructed a GFP-PDZ 1–2 GRGI
mutant, in which the amino acid sequence GLGI critical to the
expression of hScrib. (A) Representative G418 resistant colony form-
ation in NIH3T3 cells transfected with pEGFP vector alone (control) or
pEGFP-hScrib expression plasmid. The drug-resistant colonies were
stained with Giemsa reagent. (B) Colony formation assay using the
pEGFP-hScrib cDNA encoding hScrib domain constructs in NIH3T3 cells.
Results are shown as the mean percentage of the control ± SEM.
*P < 0.05 vs control.
Inhibition of proliferation of mouse NIH3T3 cells by over-
overexpression of hScrib. Colony formation assays were done by
transfection of pEGFP vector (control) or pEGFP-hScrib in Hela cells
and MDCK cells. Results are shown as the mean percentage of the
control ± SEM. *P < 0.05 vs control.
Inhibition of the proliferation of Hela and MDCK cells by
from G1 to S phase. The cell-cycle profiles of NIH3T3 cells transfected
with (A) pEGFP vector or (B) pEGFP-hScrib were analyzed by flow
cytometry. The result is representative of three independent
experiments. The percentage of cells in S phase was lower in the cells
transfected with pEGFP-hScrib than the cells transfected with pEGFP
Human Scribble negatively regulates cell-cycle progression
© 2006 Japanese Cancer Association
structure of PDZ domain pocket in PDZ domain 1 is substituted
by GRGI. Approximately half of the cells transfected with
pEGFP in this GFP-PDZ 1–2 GRGI mutant incorporated BrdU
by the induction of cell-cycle progression in the presence of
serum (Fig. 9D). The cells transfected with pEGFP-hScrib LRR
(Figs 8,9E), LRR∆LAPSDb, LRR∆LAPSDa-b, or PDZ 1–4
(Figs 8,9F) showed higher rates of BrdU incorporation than the
cells transfected with pEGFP full-length hScrib (P < 0.05).
These cells showed relatively lower BrdU incorporation compared
with the cells transfected with pEGFP vector, although not
reaching statistical significance.
Our previous study revealed that hScrib interacts with tumor
suppressor APC through PDZ domain 1 and 4 of hScrib and the
C-terminal PDZ domain-binding motif of APC.(18) We investi-
gated whether cell-cycle arrest from G1 to S phase induced by
the transfection of hScrib up-regulates APC levels in the trans-
fected cells. We also analyzed whether hScrib transfection reduces
cyclin D1. Cells transfected with GFP-hScrib(FL) showed up-
regulation of APC (Fig. 10). In those cells, levels of cyclin A
and cyclin D1 were reduced (Fig. 10). In cells transfected with
GFP-hScrib LRR + PDZ 1(1–818aa) and GFP-hScrib LRR + PDZ 2,
APC was up-regulated and cyclins A and D1 were reduced, as
for cells transfected with GFP-hScrib(FL) (Fig. 10). In contract,
cells transfected with GFP-hScrib PDZ 1–4, GFP-hScrib
LRR + PDZ 1(1–747aa), and GFP-hScrib LRR showed no up
regulation of APC and nor reduction of cyclins A and D1.
BrdU incorporation assay revealed that LRR + PDZ 1 is the
minimal domain for the negative cell-cycle regulatory function
of hScrib. These results were confirmed by flow cytometry.
The percentage of cells in S phase in NIH3T3 cells transfected
with GFP-hScrib(FL), LRR + PDZ 1–2, and LRR + PDZ 1 was
significantly lower than the cells transfected with GFP
alone (Fig. 11). In contrast, the percentage of S phase in cells
transfected with GFP-hScrib LRR and LRR + PDZ 1–2 GRGI
mutant was the same as cells transfected with GFP alone
Knockdown of hScrib leads to progression of cell cycle from G1 to
S phase. We investigated the involvement of hScrib in cell-cycle
regulation by a knockdown technique with siRNA against
hScrib. The siRNA against hScrib successfully down- regulated
the expression of hScrib in 293T, Caco-2, and HaCaT cells
(Fig. 12A). BrdU incorporation assay showed that BrdU was
incorporated in Caco-2 cells transfected with hScrib siRNA, but
it was not incorporated into non-transfected cells showing
normal expression by immunofluorescence analysis with anti-
hScrib antibody (Fig. 12A). Cell profiles of Caco-2 cells
transfected with hScrib siRNA or control siRNA were analyzed
by the flow cytometry. Knockdown of hScrib by siRNA led to
cell-cycle progression of G1 to S phase (Fig. 12B).
Loss of scribble mutation leads to disruption of apical-
basolateral polarity and tumorous overgrowth of imaginal discs,
follicles, and brain in Drosophila epithelia.(6,19) The Drosophila
Scribble is thought to exert its inhibitory effect on the prolifer-
ation of epithelia through negative regulation of the cell cycle.
Recently, scribble has been identified to be a novel negative
regulator of S phase entry by a genetic screen for dominant
modifier of a cyclin E hypomorphic mutation, DmcycEjp.(7) Cell-
cycle progression from G1 to S phase by extracellular signals is
critical for epithelial cells to proliferate properly according to
their environments. Progression of cell cycle is regulated by the
activity of cyclin-dependent kinases (CDK) and their cyclin
partners.(20) Phosphorylation of tumor suppressor Rb by cyclin
D/Cdk4 and cyclin E/Cdk2 inactivates pRb and results in
dissociation of Rb from its binding partner E2F. E2F then acts
as a transcription factor of S phase genes, including cyclin E, its
own.(21) hScrib has been reported to have a role in the rescue of
apical-basolateral polarity defect and overgrowth of Drosophila
epithelia with scribble mutation.(22) Expression of hScrib in
Drosophila scribble mutant effectively suppressed the giant
larvae phenotype and neoplastic overgrowth of Drosophila scribble
neural tissues. These results suggest that hScrib can substitute
the functions of Drosophila Scribble and that it has a role in the
inhibition of proliferation of epithelial cells through regulation
of cell-cycle progression from G1 to S phase.
In this study, we found that hScrib suppresses the prolifera-
tion of mammalian epithelial cells by regulating cell-cycle entry
from G1 to S phase. Knockdown of hScrib expression leads to
cell-cycle progression from G1 to S phase (Figs 11,12). These
data clearly indicate that the human homolog of Drosophila
tumor suppressor Scribble is involved in cell-cycle regulation
through intracellular signal transductions as is the Drosophila
How is the junctional protein hScrib connected to the cell-
cycle regulation or signal transduction pathway? Our analysis of
critical domains of hScrib for inhibition of cell proliferation,
cell-cycle regulation and intracellular localization gave us some
clues to solve this problem. hScrib requires at least LRRs and
PDZ domain 1 to exert its inhibitory effect on cellular proliferation
and cell-cycle regulation. Furthermore, it needs LRRs and at
least one PDZ domain to localize at the original location, the
basolateral membrane of epithelia, more specifically at the adherens
PDZ domains are approximately 90 residue repeats found in
scaffolding proteins implicated in ion-channel and hormone
receptor clustering. The class 1 PDZ domains recognize the
conserved binding motif, T/S-X-L/V motif, of the C-terminal
region of their binding partner.(23) Our previous work revealed
that the PDZ domain 1 of hScrib is essential for interaction with
the C-terminal region of APC.(18) Disruption of the PDZ domain
pocket motif in PDZ domain 1 of GFP-hScrib LRR + PDZ 1–2
(GRGI mutant) caused the loss of the inhibitory role of hScrib
in cell-cycle regulation, which strongly indicates that the interaction
of hScrib with APC is critical for its inhibition of cell growth
and negative regulation of cell-cycle progression. Western blotting
of lysates of cells transfected with GFP-hScrib mutants clearly
showed that its inhibition of cell-cycle entry from G1 to S phase
(i.e. reduction of cyclins A and D1), depends on the up-regulation
of APC (Fig. 6).(24)
Leucine-rich repeats are generally 20–29 residues long and
contain a conserved 11-residue consensus sequence L-X-X-L-
X-L-X-X-N/C-X-L. Crystal structure analysis revealed that
LRRs correspond to structural units, each consisting of a β
strand and an α helix.(25) The structural units are allied so that
all β strands and α helices are parallel to a common axis forming
a horseshoe-shaped structure with parallel β sheets lining the
pEGFP-hScrib expression plasmid clones. Results are shown as the
mean percentage of the control ± SEM. *P < 0.05 vs control, LRR,
LRR∆LAPSDb, LRR∆LAPSDa-b, and PDZ 1–4.
BrdU incorporation analysis in NIH3T3 cells transfected with
Nagasaka et al.
Cancer Sci|November 2006|vol. 97| no. 11|
© 2006 Japanese Cancer Association
inner concave part and α helices flanking the outer curved
structure.(25) The architecture of the LRRs provides the versatile
framework for the interaction with the binding partner.(25,26)
hScrib could interact with two different binding partners
independently through its LRRs and PDZ domain 1, resulting
in localization at the adherens junction.
Analysis of domain mapping of Drosophila Scribble revealed
that both LRRs and PDZ domains are required for Scribble to
localize efficiently at the septate junction of epithelial cells.(27)
Loss of LRRs and PDZ domains of Drosophila Scribble leads to
mislocalization of Scribble in nucleus and cytoplasm, respectively,
of epithelia.(27) Furthermore, only the Scribble proteins containing
both LRRs and PDZ domains have the ability to target Mira, the
cell fate scaffolding protein, to the basolateral cortex of mitotic
neuroblasts and to establish mitotic spindle and cell size asymmetry
in neuroblasts.(27) The LRRs are essential to the organization of
the polarized epithelial monolayer, while loss of PDZ domains
leads to disruption of septate junctions and overproliferation in
the absence of epithelial disorganization.(28) These data suggest
a two-step model for the localization of Drosophila Scribble, in
which LRRs bring Scribble to the plasma membrane and PDZ
domains enhance membrane-associated Scribble to the septate
junction.(28) Our data showed that the PDZ domain of hScrib
alone, which is its binding domain with APC, is not enough to
exert its proper localization at the adherens junction and inhibitory
activity of cell growth and cell-cycle progression.
Taken together, the Drosophila findings and our data suggest
that hScrib targets the adherens junctions of epithelia by a
cells transfected with pEGFP-hScrib expression
clones. Cell-cycle regulatory function of hScrib
was analyzed by the rate of BrdU incorporation
in serum-starved NIH3T3 cells transfected with
pEGFP-hScrib expression clones. a. Immunofluorescence
image of GFP-fused hScrib domain construct. b.
Immunofluorescence image of BrdU-incorporated
cells. c. Merged image of a. and b. NIH3T3 cells
were transfected with (A) pEGFP vector, (B)
pEGFP-hScrib expression plasmid, (C) pEGFP-hScrib
LRR + PDZ 1–2 expression plasmid, (D) pEGFP-
hScrib LRR + PDZ 1–2 GRGI, which was a mutant in
which the conserved Gly-Leu-Gly-Ile pocket motif
in PDZ domain 1 was replaced with Gly-Arg-Gly-Ile
expression plasmid, (E) pEGFP-hScrib LRR expression
plasmid, and (F) pEGFP-hScrib PDZ 1–4 expression
BrdU incorporation analysis in NIH3T3
© 2006 Japanese Cancer Association
two-step mechanism. In the first step, LRRs bring hScrib to the
plasma membrane. In the second step, PDZ domains restrict the
localization of hScrib at the adherens junction, resulting in its
involvement in the transduction of extracellular signals towards
the cytoplasm and nucleus of epithelial cells. hDlg is a human
homolog of Drosophila neoplastic tumor suppressor, in which
mutation causes disruption of tissue architecture and overgrowth
of epithelia. Human Dlg, which is targeted by high-risk HPV E6
for the ubiquitin-mediated degradation like hScrib, interacts
with tumor suppressor APC through its PDZ domain and is
involved in cell-cycle regulation cooperatively with APC.(10,29)
Our previous study showed that hScrib interact with APC
through its PDZ domains and thereby join the Wnt signaling
pathway.(18) It is also reported that hScrib interacts with βPIX
exchange factor and that the interaction between them is
Cell lysates generated from NIH3T3 cells transfected with GFP or each
GFP-fusion hScrib expression clones were examined by Western
blotting with an anti-APC antibody, anticyclin A antibody, anticyclin D1
antibody. An antitubulin antibody was used to equilibrate loading for
each lane. Cell lysates from GFP-fusion hScrib expression clones that
have its LRRs and at least one complete PDZ domain expressed 5–7-fold
less cyclin A and D1 than lysates from GFP-transfected cells. In
contrast, LRR + PDZ 1b1+747 in which the conserved hydrophobic pocket
was partially deleted had no effect on the regulation of cyclins A and
Effect of hScrib on the activity of the APC-cyclin D pathway.
S transition of the cell cycle. Caco-2 cells
transfected with hScrib siRNA were double
stained for anti-hScrib antibody (green) and
BrdU (red). The knockdown effects of hScrib
siRNA were evaluated by Western blotting. (B)
hScrib siRNA transfection increases the S phase
population as determined by flow cytometry.
Caco-2 cells transfected with negative control
siRNA or hScrib siRNA were labeled with pro-
pidium iodide and analyzed by flow cytometry.
Percentages of cells in the three phases from
control siRNA transfection were set to zero, and
the bars represent the changes for hScrib siRNA
(A) Effect of hScrib on the G1 to
mutant hScrib were analyzed by flow cytometry to examine the effects
of hScrib on cell-cycle progression. Gates were established such that
only cells with high GFP fluoresce. Note that LRR + PDZ 1–2 GRGI, a
mutant in which the conserved Gly-Leu-Gly-Ile pocket motif in PDZ
domain 1 was replaced with Gly-Arg-Gly-Ile, has no effect on cell-cycle
regulation of G1/S transition. (B) The approximate percentages of cells
in each of the major phases of the cell cycle were calculated using the
Multicycle software. The average difference in the percentage of cells
(Y-axis) in each phase of the cell cycle (X-axis) among GFP-hScrib
mutants from three different experiments is shown. Percentages of
cells transfected with GFP as a control were set to zero in the three
phases (G1, S, and G2/M), and changes of cell-cycle distribution were
shown for cells transfected with GFP-hScrib full-length and mutants.
(A) NIH3T3 cells transfected with GFP only, GFP-hScrib(FL),
Nagasaka et al. Download full-text
Cancer Sci|November 2006|vol. 97| no. 11|
© 2006 Japanese Cancer Association
involved in neural transmission.(15) Drosophila scribble has been
shown to exhibit genetic interaction with scad (αPS3-integrin-
cell adhesion), phyllopod (signaling), dEB1 (microbubule-binding
protein-cytoskeletal), and moira (chromatin remodeling).(7)
Human scribble could interact with the human homolog of
these cell adhesion, signaling, cytoskeletal, and chromatin
remodeling genes, and thereby could be involved in the
common pathway to negatively regulate the entry of cell cycle
from G1 to S phase.
Our data on the functional domain mapping of hScrib indicates
that hScrib is a tumor suppressor in mammalian epithelia, having
a role in the inhibition of cell proliferation through cell-cycle
regulation. Localization at the adherens junction is required
for hScrib to exert its proper inhibitory role in the proliferation
of epithelia, depending on its LRRs and at least one PDZ
domain. Further work on the identification of its binding
partner through LRRs or PDZ domains will reveal the
mechanisms of how hScrib controls cell growth and cell-cycle
This work was supported by a grant from the Mitsui Life Social Welfare
Foundation (to S. N.) and by a Grant-in-Aid for Scientific Research (no.
16591645, to S. N.) from the Ministry of Education, Science and Culture,
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