functional regulation of slug / snail2 is dependent on gsk-3β-mediated phosphorylation
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Functional regulation of Slug ⁄ Snail2 is dependent onGSK-3b-mediated phosphorylationJin Young Kim1,*, Young Mee Kim1,*, Chang Hee Yang1, Somi K. Cho2, Jung Weon Lee3 andMoonjae Cho1,4
1 Department of Biochemistry, School of Medicine, Jeju National University, South Korea
2 Faculty of Biotechnology, College of Applied Life Sciences, Jeju National University, South Korea
3 Department of Pharmacy, College of Pharmacy, Seoul National University, South Korea
4 Institute of Medical Science, Jeju National University, South Korea
Keywords
epithelial–mesenchymal transition; GlcNAc;
GSK-3b; protein localization; protein
phosphorylation; Snail2
Correspondence
M. Cho, Department of Biochemistry,
School of Medicine, Cheju National
University, Jeju 690-756, South Korea
Fax: +82 64 725 2593
Tel: +82 64 754 3837
E-mail: moonjcho@jejunu.ac.kr
*These authors contributed equally to this
work
(Received 20 February 2012, revised 15
May 2012, accepted 11 June 2012)
doi:10.1111/j.1742-4658.2012.08674.x
Snail family proteins regulate transcription of molecules for cell–cell adhe-
sion during epithelial–mesenchymal transition (EMT). Based on putative
glycogen synthase kinase 3b (GSK-3b) phosphorylation sites within the
Slug ⁄Snail2, we explored the significance of GSK-3b-mediated phosphory-
lation in Slug ⁄Snail2 expression during EMT. Mutation of the putative
GSK-3b phosphorylation sites (S92 ⁄ 96A or S100 ⁄ 104A) enhanced the
Slug ⁄Snail2-mediated EMT properties of E-cadherin repression and vimen-
tin induction, compared with wild-type Slug ⁄Snail2. S92 ⁄96A mutation
inhibited degradation of Slug ⁄Snail2 and S100 ⁄ 104A mutation extended
nuclear stabilization. Inhibition of GSK-3b activity caused similar effects,
as did the phosphorylation mutations. Thus, our study suggests that GSK-
3b-mediated phosphorylation of Slug ⁄Snail2 controls its turnover and
localization during EMT.
Introduction
The Snail family proteins, such as Snail, Slug ⁄Snail2,EF1(ZEB-1), SIP1(ZEB-2) and Twist, contain four
tandem C2-H2 zinc finger motifs at the COOH-termi-
nus and a highly conserved SNAG repression domain
(one to nine amino acids) that is important for
co-repressor interaction at the NH2-terminus [1]. The
zinc finger binds to a DNA target sequence called the
E-box motif (CANNTG), which is usually found in
tandem in Snail target genes, including E-cadherin [2].
Recent studies have shown that the E-cadherin pro-
moters serve as direct targets for Snail1, and the sup-
pression of E-cadherin causes epithelial–mesenchymal
transition (EMT) and is a major contributor to the
acquisition of an invasive, highly migratory phenotype
during human tumor progression [3]. Snail1 is upregu-
lated in a number of human tumors, including breast,
colon and gastric cancer, and overexpression of Snail1
correlates with tumor grade, nodal metastasis and
tumor recurrence [4,5]. Similarly, another Snail family
member, Slug ⁄Snail2, is also upregulated in breast can-
cer and in malignant mesothelioma [6], is a mediator
of EMT and metastasis [7] and is shown to be induced
by fibroblast growth factor and hepatocyte growth
factor [8].
Abbreviations
b-TrCP, b-transducin repeat containing protein; CHX, cycloheximide; EMT, epithelial–mesenchymal transition; GAPDH, glyceraldehyde-3-
phosphate dehydrogenase; GFP, green fluorescent protein; GSK-3b, glycogen synthase kinase 3b; TGF, transforming growth factor;
WT, wild-type.
FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 1
Glycogen synthase kinase 3b (GSK-3b) is a ubiqui-
tously expressed serine ⁄ threonine kinase that is active
in resting epithelial cells [9]. GSK-3b can regulate
Snail1 function via phosphorylation at two consensus
motifs: phosphorylation at the first motif regulates its
ubiquitination by b-transducin repeat containing pro-
tein (b-TrCP), whereas phosphorylation at the second
motif controls its subcellular localization [10]. A non-
phosphorylated variant of Snail1 resides more stably
in the nucleus to exclusively induce EMT [5]. GSK-3bcan inhibit Snail1 expression via inhibiting its tran-
scription [11], as well as by regulating Snail1 degrada-
tion and nuclear translocation [5]. Snail1 is also
known to be stabilized by O-GlcNAc modification on
Ser112, which competes with O-phosphorylation in
hyperglycemic conditions [12]. Therefore, GSK-3bmaintains epithelial phenotypes via inhibiting the
expression and stabilization of Snail1 (mediator of
EMT) and thereby also helps in maintaining a high
E-cadherin expression [13]. However, the effects of
GSK-3b-mediated phosphorylation on Slug ⁄Snail2function remain undefined mainly because the subdo-
mains of Slug ⁄Snail2 with putative GSK-3b phos-
phorylation sites are different from those of Snail1.
Here, we report that GSK-3b-mediated phosphoryla-
tion appears to regulate the degradation and subcellu-
lar localization of Slug ⁄Snail2.
Results
Overexpression of Slug ⁄ Snail2 in MCF7 cells
caused EMT
The expressions of Snail1 and Slug ⁄Snail2 were ana-
lyzed in various cancer cell lines by RT-PCR. Of the
eight cancer cell lines, Huh7 hepatic and MDA-MB-
231 breast cancer cells showed the highest expression
of Slug ⁄Snail2 mRNA, compared with mRNA levels
in other carcinoma cell lines. Furthermore, Snail1
mRNA expression was high in SNU449 and Huh7
hepatic cancer cells and minimal in other cell lines.
The expression of both Snail1 and Slug ⁄Snail2 was
negligible in the MCF7 cell line (Fig. 1A); hence we
selected this cell line for further experiments to mini-
mize the compensatory effects of endogenous Snail1
while studying the expression of Slug ⁄Snail2. Overex-
pression of Slug ⁄Snail2 in MCF7 cells resulted in a
decrease in the mRNA and protein levels of E-cadher-
in, but an increase in the levels of the mesenchymal
marker vimentin, indicating that Slug ⁄Snail2 caused
EMT of the MCF7 cells (Fig. 1B,C).
Because O-GlcNAc modification at Ser112 in Snail1
stabilizes the protein and thus increases its repressor
function to attenuate E-cadherin mRNA expression
[12], we sought to determine whether Slug ⁄Snail2 was
A B
D
C
Fig. 1. Slug ⁄ Snail2 induces changes in epithelial and mesenchymal marker molecules. (A) Expressions of Snail1 or Slug ⁄ Snail2 were ana-
lyzed in various carcinoma cell lines. mRNAs were prepared to determine the expression of Snail1 or Slug ⁄ Snail2 mRNAs by RT-PCR. GAPDH
was used as a loading control. The bar graph indicates gene expression, as quantified by densitometry. Values shown represent the
mean ± standard deviation of three independent experiments. (B), (C) Expressions of Slug ⁄ Snail2 (Slug), E-cadherin (E-cad), a-smooth
muscle actin (a-SMA) and vimentin (Vim) in Slug ⁄ Snail2-MCF7 and vector control MCF7 cells were analyzed by RT-PCR (B) and western
blotting (C). HEK293 cells were transfected to express GFP-tagged Slug ⁄ Snail2 or Snail1 and immunoprecipitated using anti-GFP or anti-
Snail1 IgG that was probed with the anti-O-GlcNAc CTD110.6 IgM monoclonal antibody (D). Data shown represent three independent
experiments.
GSK-3b-mediated phosphorylation of Slug ⁄ Snail2 J. Y. Kim et al.
2 FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS
also modified with O-GlcNAc. To test this hypothesis,
HEK293 cells were transfected to express green fluo-
rescent protein (GFP) tagged Slug ⁄Snail2 and immu-
noprecipitated using anti-GFP IgG that was probed
with the O-GlcNAc-specific monoclonal CTD110.6.
We were unable to detect O-GlcNAc on Slug ⁄Snail2even with or without O-GlcNAc transferase overex-
pression (Fig. 1D). It is thus likely that O-GlcNAc
modification may not be involved in the functions of
Slug ⁄Snail2.
Mutations in the phosphorylation sites of
Slug ⁄ Snail2 enhance EMT
There are two GSK-3b phosphorylation motifs in
Snail1 that include the b-TrCP destruction motif
(DSGxxS) and the SxxxSxxxS motif (Fig. 2A). When
the Slug ⁄Snail2 sequence was compared with the Snail1
sequence to predict putative GSK-3b phosphorylation
motifs, we found a region with GSK-3b consensus
serine sequences (called the Slug domain) that was
A
B C
D
Fig. 2. Mutations in phosphorylation sites of Slug ⁄ Snail2 cause mesenchymal features. (A) Sequence comparison of Snail1 and Slug ⁄ Snail2
showing the positions of predicted GSK-3b phosphorylation sites. Schematic representations of Snail1 and Slug ⁄ Snail2 are shown at the top
and bottom. The amino acid sequences for Slug ⁄ Snail2 mutants used in this study are shown between the schematic diagrams. Consensus
GSK-3b phosphorylation sequences are indicated as SxxxSxxxS. (B) Various mutants of Slug ⁄ Snail2 were transiently expressed in MCF7
cells before analyzing the expression of Slug ⁄ Snail2 by RT-PCR and western blotting. (C) Expressions of E-cadherin (E-cad) and vimentin
(Vim) in MCF7 cells, after transient transfection with WT or mutant Slug ⁄ Snail2, were determined by RT-PCR and western blotting. GAPDH
were used an internal control to normalize data, and results are presented at the bottom as fold changes relative to mock-transfected MCF7
cells. Gene expressions were quantified by densitometry and are represented as values of mean ± standard deviation of three independent
experiments. (D) The E-cadherin promoter inserted into a reporter vector (pGL3 enhancer vector) was coexpressed with WT or with different
mutants of Slug ⁄ Snail2 or mock control plasmids in MCF7 cells. All constructs were cotransfected with 100 ng of pLR-null vector. Luciferase
activities were normalized to renilla (pLR-null) activity and are expressed as a percentage of the corrected luciferase activity of the mock con-
trol vector (means ± standard deviation of three independent experiments). *P < 0.05 and **P < 0.001 compared with the corresponding
WT Snail2 was considered significant.
J. Y. Kim et al. GSK-3b-mediated phosphorylation of Slug ⁄ Snail2
FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 3
distinct from the two GSK-3b phosphorylation motifs
in Snail1 (Fig. 2A). To understand the role of GSK-3bphosphorylation sites in the function of Slug ⁄Snail2,we mutated the serine residues to alanines (S87A,
S92 ⁄96A or S100 ⁄104A) (Fig. 2A). We then compared
the mRNA and protein expression in the wild-type
(WT) and various mutants of Slug ⁄Snail2 (Fig. 2B).
There were no meaningful differences at the transcrip-
tional level (mRNA level) between the WT and mutant
Slug ⁄Snail2; however, the protein amounts of Snail2
mutants were higher than that of WT. This indicates
that GSK-3b phosphorylation may affect the stability
of the Slug ⁄Snail2 protein. Furthermore, the decrease
in E-cadherin and the increase in vimentin at both the
mRNA and protein levels were more marked in the
phosphorylation mutants than in the mock- or WT
Slug ⁄Snail2-expressing cells (Fig. 2C). These observa-
tions indicate that GSK-3b-mediated phosphorylation
of Slug ⁄Snail2 may facilitate the degradation of Slug ⁄Snail2, thus leading to inhibition or termination of the
EMT process. We next analyzed the effects of the phos-
phorylation site mutations on the transcriptional activ-
ity of the E-cadherin promoter. To determine how the
Slug ⁄Snail2 WT or mutants affect the transcriptional
activities of the E-cadherin gene, using a human
E-cadherin promoter-luciferase construct (Ecad-Luc,
)365 to +48), pcDNA3-mock, -Slug ⁄Snail2 WT or
mutants were transiently cotransfected into MCF7 cells
prior to luciferase assay. The WT and Slug ⁄Snail2mutants showed more efficient repression of the Ecad-
Luc activity than that shown by the pcDNA3 control
vector (Fig. 2D). Compared with WT or S87A Slug ⁄Snail2, the S92 ⁄ 96A or S100 ⁄ 104A mutants of Slug ⁄Snail2 caused a prominent decrease in the Ecad-Luc
activity (Fig. 2D). Thus, the results of the phosphoryla-
tion of Slug ⁄Snail2 (presumably by GSK-3b) can
contribute to the stabilization of Slug ⁄Snail2, which
thereby causes suppression of E-cadherin during EMT.
Phosphorylation of Slug ⁄ Snail2 regulates its
subcellular localization and stabilization
We next examined how phosphorylation of Slug ⁄Snail2 affects its intracellular localization by immuno-
fluorescence analysis of cells transfected with WT or
mutant Slug ⁄Snail2 tagged with GFP. In addition to
GFP-WT Slug ⁄Snail2, the S92 ⁄ 96A mutants of Slug ⁄Snail2 localized in both the cytoplasm and the nucleus,
whereas the S100 ⁄ 104A mutant of Slug ⁄Snail2 mainly
localized in the nucleus (Fig. 3A). Using a cell extract
fractionation approach, we observed that WT Slug ⁄Snail2 localized both in the nucleus and in the cytosol
at a nucleus : cytosol ratio of 2 : 1 (Fig. 3B). However,
the S92 ⁄ 96A mutant of Slug ⁄Snail2 was more stable
and predominantly located in the nucleus rather than
the cytosol compared with WT Slug ⁄Snail2 (Fig. 3B).
The S100 ⁄ 104A mutant of Slug ⁄Snail2 was transfected
and cells were treated with cycloheximide (CHX). The
cytosol and nuclei fractions were separated and then
analyzed with western blot and the band intensity was
quantified (Fig. 3C).
The immunofluorescence and cellular fractionation
studies indicated that although the S92 ⁄ 96A or
S100 ⁄ 104A mutants of Slug ⁄Snail2 were more stable
and predominantly located in the nucleus, it was the
S100 ⁄ 104A mutant that caused more obvious nuclear
accumulation (Fig. 3A–C).
We then examined the half-lives of WT and mutant
Slug ⁄Snail2 following the treatment of MCF7 cells
with CHX, a protein synthesis inhibitor. The half-life
of the S92 ⁄ 96A mutant was longer than that of the
WT or mutant S100 ⁄ 104A (Fig. 3D), suggesting that
phosphorylation at Ser92 ⁄ 96 may be important for the
half-life of Slug ⁄Snail2. Therefore, it may be likely that
Ser92 ⁄ 96 phosphorylation negatively affects the sta-
bility of Slug ⁄Snail2, whereas Ser100 ⁄ 104 phosphoryla-
tion causes its cytosolic localization in addition to
stabilization, and that both phosphorylations
(Ser92 ⁄96 and Ser100 ⁄ 104) can lead to a negative
impact on E-cadherin suppression.
GSK-3b regulates Slug ⁄ Snail2 localization and
degradation
Using a pharmacological inhibitor against GSK-3b, wenext examined whether the function of Slug ⁄Snail2was affected by GSK-3b activity. Cells were transiently
transfected with WT Slug ⁄Snail2 and then treated with
50 lm LiCl, which is a GSK-3b inhibitor. The mRNA
level of Slug ⁄Snail2 was slightly elevated in cells trea-
ted with LiCl compared with that in untreated cells
(Fig. 4A), which is consistent with the enhanced
Slug ⁄Snail2 expression level (i.e. stabilization) caused
by mutations in the putative GSK-3b phosphorylation
sites at Ser92 ⁄ 96 (Figs 2B and 3B). Furthermore, LiCl
treatment had a similar effect on the protein levels of
Slug ⁄Snail2 as on its mRNA levels (Fig. 4B). As
shown in Fig. 3A, in the absence of LiCl treatment,
the WT and S92 ⁄ 96A mutants localized in both the
cytosol and the nucleus, whereas the majority of Slug
proteins in S100 ⁄ 104A mutant were located in the
nucleus. In the presence of LiCl treatment, however,
more Slug ⁄Snail2 was located inside the nucleus
(Fig. 4C, lower panel). After LiCl treatment, cytosol
and nuclear fractions were analyzed by western blot-
ting. More Slug ⁄Snail2 was found in LiCl-treated cells
GSK-3b-mediated phosphorylation of Slug ⁄ Snail2 J. Y. Kim et al.
4 FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS
A
B
D
C
Fig. 3. Phosphorylation of Slug ⁄ Snail2 affects its subcellular localization and stability. (A) The GFP-Slug ⁄ Snail2 proteins were transiently
expressed in MCF7 cells for 24 h. The subcellular localization of Slug ⁄ Snail2 proteins (green) and nucleus (blue, Hoechst 33342) were exam-
ined under a fluorescent microscope. (B) Following transient transfection of WT or of mutant Slug ⁄ Snail2 plasmids for 24 h, MCF7 cells
were lysed and fractionated as described in Materials and methods. Then, Slug ⁄ Snail2 protein in each fraction was analyzed by western blot-
ting. GAPDH and lamin B1 were used as controls for fractionation and for equal loading of proteins in the cytoplasmic and nuclear fraction,
respectively. (C) MCF7 cells that were treated with 30 lM CHX for different time intervals following transient transfection of WT or of
S100 ⁄ 104A Slug ⁄ Snail2 for 24 h and cytosol and nuclear fraction were analyzed by western blotting and measured band intensity by densi-
tometry. (D) MCF7 cells that were treated with 30 lM CHX for different time intervals following transient transfection of WT or of mutant
Slug ⁄ Snail2 for 24 h were analyzed by western blotting. Relative band intensity was plotted (lower panel).
J. Y. Kim et al. GSK-3b-mediated phosphorylation of Slug ⁄ Snail2
FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 5
A
D
F
G
E
B C
Fig. 4. GSK-3b phosphorylation appears to regulate Slug ⁄ Snail2 localization and degradation. (A), (B) LiCl (50 lM) was added to MCF7 cells
for 6 h following transient transfection with WT or with Slug ⁄ Snail2 mutants for 24 h. Expressions of Slug ⁄ Snail2 (Slug), E-cadherin (E-cad)
and vimentin (Vim) were analyzed by RT-PCR (A) or western blotting (B). (C) GFP-Slug ⁄ Snail2 protein was transiently expressed in MCF7
cells for 24 h and then treated with LiCl (50 lM) for 6 h. Representative images of fluorescent microscopy show GFP-Slug ⁄ Snail2 proteins
(green). (D) GFP-Slug ⁄ Snail2 protein was transiently expressed in MCF7 cells for 24 h and then treated with LiCl (50 lM) for 6 h. The cells
were lysed and fractionated as described in Materials and methods. Then, the Slug ⁄ Snail2 protein in each fraction was analyzed by western
blotting. (E) MCF7 cells were treated with 30 lM CHX for different time intervals following transient transfection with WT Slug ⁄ Snail2 for
24 h with or without 50 lM LiCl treatment before western blot analysis. Relative band intensity was plotted (lower panel). (F) LiCl was added
to MCF7 cells for 6 h following transient transfection with WT Slug ⁄ Snail2 for 24 h. Luciferase activities were normalized to renilla (pLR-null)
activity and expressed as a percentage of the corrected luciferase activity of WT Slug ⁄ Snail2 (mean ± standard deviation of three indepen-
dent experiments). *P < 0.5 compared with WT Slug ⁄ Snail2 was considered significant. (G) HEK293 cells were transiently transfected with
pcDNA mock, pcDNA-Slug WT or S92 ⁄ 96 ⁄ 100 ⁄ 104A mutant for 30 h prior to harvesting of whole cell extracts using lysis buffer as above.
The extract was immunoprecipitated with anti-Slug IgG and incubated with protein A ⁄ G-sepharose. The collected immunoprecipitates were
processed for western blots using anti-Slug or anti-phosphoserine IgG. Data shown represent three independent experiments.
GSK-3b-mediated phosphorylation of Slug ⁄ Snail2 J. Y. Kim et al.
6 FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS
in both cytosol and nuclear fractions (Fig. 4D). Further-
more, LiCl treatment of WT Slug ⁄Snail2-transfectedcells resulted in a longer half-life of the Slug ⁄Snail2protein (Fig. 4E) and in enhanced suppression of the
transcriptional activity of the E-cadherin promoter,
compared with that of untreated cells (Fig. 4F). To
confirm whether the serines in the mutated sequence
were phosphorylated, WT and mutant Slug ⁄Snail2(S92 ⁄ 96 ⁄ 100 ⁄ 104A) were immunoprecipitated by
anti-Slug and detected by anti-phosphoserine. The
mutant Slug ⁄Snail2 showed enhanced expression level
(Fig. 4G, upper panel) and less phosphorylation
(Fig. 4G, lower panel). These results confirm that
GSK-3b-mediated phosphorylation of Slug ⁄Snail2 con-
trols the intracellular localization of Slug ⁄Snail2toward the cytosol, leading to inefficient E-cadherin
suppression (i.e. inhibition of EMT).
TGF-b exerts its effects on Slug ⁄ Snail2 through
GSK-3b inhibition
Given that the transforming growth factor b (TGF-b)signaling pathway is known to cause EMT, we investi-
gated the effect of TGF-b treatment on Slug ⁄Snail2expression and function. Compared with basal expres-
sion levels, Slug ⁄Snail2 mRNA and protein expression
levels were enhanced by TGF-b treatment of MCF7
cells (Fig. 5A). Accordingly, E-cadherin promoter
activity decreased upon TGF-b treatment alone (81%),
compared with that of untreated cells (100%), and
concomitant LiCl treatment in the presence of TGF-b
treatment caused enhanced suppression of the E-cadh-
erin promoter activity (68.3%, Fig. 5B).
To determine the relationship between GSK-3b,Slug ⁄Snail2 and vimentin in endogenous proteins, we
examined the MCF7-derived cancer stem cell line
(MCF7 ⁄CSC), which has partial mesenchymal cell
properties [14]. MCF7 ⁄CSC cells showed higher expres-
sion levels of Slug ⁄Snail2 and vimentin (Fig. 5C).
When GSK-3b was overexpressed in MCF7 ⁄CSC cells,
the level of Slug ⁄Snail2 protein and vimentin decreased
dramatically (Fig. 5C). These observations in
MCF7 ⁄CSC cells suggest that GSK-3b-mediated phos-
phorylation may suppress Slug ⁄Snail2 expression, lead-
ing to inhibition of the EMT process.
Discussion
This study provides evidence that GSK-3b-mediated
phosphorylation of Slug ⁄Snail2 results in localization
and degradation in the cytosol, leading to efficient
E-cadherin suppression and inhibition of EMT.
Slug ⁄Snail2 has a short region that contains the puta-
tive GSK-3b phosphorylation residues, indicating the
involvement of GSK-3b in the regulation of Slug ⁄Snail2 activity. In the present study, Slug ⁄Snail2appeared to be regulated by GSK-3b-mediated phos-
phorylation. Our conclusion is based on the following
findings: (a) the alanine mutants of the putative
GSK-3b phosphorylation residues caused alterations in
the Slug ⁄Snail expression level, stability, intracellular
localization and its function in E-cadherin suppression,
A B C
Fig. 5. TGF-b appears to exert its effect on Slug ⁄ Snail2 through GSK-3b inhibition. (A) TGF-b (10 ngÆmL)1) was added to MCF7 cells for 6 h
following transient transfection with WT Slug ⁄ Snail2 for 24 h. Protein expression of Slug ⁄ Snail2 was analyzed by western blotting. (B) Cells
were treated with 10 ng TGF-b for 6 h. In some cases, cells were pretreated with 10 ng TGF-b for 1 h before a 6-h LiCl treatment. Lucifer-
ase activities were normalized to renilla (pLR-null) activity and expressed as a percentage of the corrected luciferase activity of WT Slug ⁄Snail2 (mean ± standard deviation of three independent experiments). *P < 0.05 and **P < 0.005 compared with WT Slug ⁄ Snail2 were con-
sidered significant. (C) MCF and MCF ⁄ CSC cells were transiently transfected with pCDNA-GSK-3b for 24 h with or without LiCl treatment,
and expressions of Slug ⁄ Snail2 (Slug) and vimentin were analyzed by western blotting. Data shown represent three independent experi-
ments.
J. Y. Kim et al. GSK-3b-mediated phosphorylation of Slug ⁄ Snail2
FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 7
and (b) treatment with the GSK-3b inhibitor LiCl
mimicked the mutations of the putative GSK-3b phos-
phorylation residues in terms of E-cadherin suppres-
sion and restricted Slug ⁄Snail2 mainly to the nucleus,
leading to enhanced E-cadherin suppression. Thus,
GSK-3b-mediated phosphorylation of Slug ⁄Snail2 may
play inhibitory roles in the EMT process.
High expression of Snail1 is often found in breast
cancer and in malignant mesothelioma [6] and meta-
static cancer cells [15]. E-cadherin promoters are
directly targeted by Snail1, and hence the repression of
E-cadherin expression is a major contributor to the
acquisition of an invasive, highly migratory phenotype
via the triggering of EMT [16,17]. Downregulation of
E-cadherin in most carcinomas is a transient and
highly dynamic event, where Twist, Snail1 and Slug ⁄Snail2 have binding affinities toward the E-box motif
(CANNTG) that is usually found in the target genes
of the Snail family proteins [18]. Post-translational
modifications of Snail1 are a well-known mechanism
to regulate its functions. O-GlcNAc modification of
Ser112 is known to stabilize Snail1 by inhibiting its
phosphorylation [12]. The competition between O-Glc-
NAc modification and O-phosphorylation of Ser ⁄Thrresidues is important in regulating the function of
many signaling proteins and in degrading certain pro-
teins such as b-catenin [19]. Examples of the competi-
tion between O-GlcNAc and O-phosphorylation for
regulation of their signaling activities include Ser473 of
Akt1 and Ser85 of paxillin [20,21]. Snail1 proteins have
a highly conserved b-catenin-like consensus motif and
destruction box. GSK-3b phosphorylates motif 2 of
Snail1 and thereby induces its nuclear export, whereas
subsequent phosphorylation by GSK-3b at motif 1
results in the association of Snail1 with b-TrCP and
thus leads to the degradation of Snail1 [5,10,22]. It is
thus likely that phosphorylation by GSK-3b is a deter-
mining step in Snail1 metabolism [23]. The phosphory-
lation of Ser96, Ser104 and Ser107 in the destruction
box of Snail1 proteins is a prerequisite for the ubiqu-
itin-ligase b-TrCP; phosphorylation of Ser104 and
Ser107 can prime GSK-3b phosphorylation of Ser96
and Ser100 [23] and the primed phosphorylation is
carried out by casein kinase 1 [24].
In this study, we have demonstrated the inhibitory
effects of Slug ⁄Snail2 phosphorylation by GSK-3b on
EMT. However, our data showed that no O-GlcNAc
modification is found in Slug ⁄Snail2, indicating that
phosphorylation of Slug ⁄Snail2 may be a regulatory
mechanism for its expression, stability and intracellular
localization. We demonstrated that Slug ⁄Snail2 translo-
cated to the cytosol upon Ser100 ⁄ 104 phosphorylation
and presumably underwent proteasomal degradation,
but further phosphorylations at Ser92 ⁄ 96 resulted in its
accumulation in the cytosol. Slug ⁄Snail2 displays only
one of the distinct GSK-3b consensus motifs, whereas
Snail1 exhibits two such motifs (Fig. 2A). The first
GSK-3b phosphorylation sequence in Slug ⁄Snail2 is
located on the serine-rich domain site at Ser87 and
Ser92 ⁄ 96, which plays a regulatory role in the stability of
Slug ⁄Snail2, and the second site is located at Ser100 ⁄ 104which, depending on GSK-3b activity, regulates the
export of nuclear Slug ⁄Snail2 into the cytosol. Ser87 is
not a part of the classic GSK-3b recognition sequence
(SxxxS) and the effect of this mutation was not dramatic,
although it still affected the E-cadherin promoter activ-
ity (Fig. 4A) and expression of EMT-related molecules
(Fig. 2C). It may thus be likely that Ser87 phosphoryla-
tion alone is not enough for Slug ⁄Snail2 function.TGF-b1 is a well-known multifunctional cytokine
that plays important roles in EMT to enhance the met-
astatic potential [25] and causes an early and sustained
ERK5 activation that is required for the functional
inactivation of GSK-3b and for the stabilization of the
TGF-b1 target Snail1 [26]. In the present study, we
demonstrated that TGF-b1 induced the expression of
the Slug ⁄Snail2 protein, suggesting that TGF-b1 also
regulates the degradation of the Slug ⁄Snail2 protein,
presumably through GSK-3b inactivation.
Taken together, our study suggests that GSK-3b-mediated phosphorylation can regulate the function of
Slug ⁄Snail2 in controlling EMT. Our study not only
reveals a molecular mechanism underlying Snail2-
induced EMT, but also has valuable implications in
the development of effective treatment strategies for
metastatic cancer progression.
Materials and methods
Plasmids and antibodies
The following primary antibodies were used for standard
western blots: anti-Slug ⁄Snail2, anti-glyceraldehyde-3-phos-phate dehydrogenase (GAPDH), anti-vimentin (Cell Signal-
ing Technology, Beverly, MA, USA), anti-E-cadherin (BD
Biosciences, San Diego, CA, USA), anti-phosphoserine and
anti-lamin B1 (Abcam, Cambridge, UK). pEGFP-C3 was
purchased from Clontech (Palo Alto, CA, USA), pGL3 from
Promega (Madison, WI, USA), and pCDNA-GSK-3b was
provided by E. H. Cho (Seoul City University, South Korea).
Cell culture and transfection
Human breast adenocarcinoma (MCF7) cells or human
embryonic kidney 293 (HEK293) cells (ATCC, Manassas,
VA, USA) were cultured in RPMI with 10% fetal bovine
GSK-3b-mediated phosphorylation of Slug ⁄ Snail2 J. Y. Kim et al.
8 FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS
serum (Gibco, Grand Island, NY, USA). MCF7 cells
transfected with pcDNA3-Slug ⁄ Snail2 WT or S87A,
S92 ⁄ 96A or S100 ⁄ 104A mutant were selected by G418
(AG Scientific, San Diego, CA, USA) after transfection
using Lipofectamine� 2000 (Invitrogen, Carlsbad, CA,
USA) according to the manufacturer’s protocol. MCF7
and HEK293 cells were transiently transfected with
pEGFP-C3-Slug ⁄Snail2 WT, mutant plasmids or pCDNA-
GSK-3b using Lipofectamine� 2000 for 24 h before
analysis [27]. In some cases, cells were treated with 50 lm
LiCl (Sigma Chemical Co., St Louis, MO, USA) for 6 h
after transient transfection for 24 h as described above.
Cell images were acquired using a phase-contrast micro-
scope (BX41, Olympus, Tokyo, Japan).
Western blots and antibodies
Cells transfected with the indicated plasmids or treated with
LiCl were washed twice with ice-cold NaCl ⁄Pi and har-
vested in RIPA buffer (50 mm Tris ⁄HCl pH 8.1, 150 mm
NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS
and protease inhibitors). To measure the half-life of Slug ⁄Snail2, MCF7 cells were treated with 30 lm CHX for
different time intervals following transient transfection with
WT Slug ⁄ Snail2 for 24 h with or without 50 lm LiCl treat-
ment before western blot analysis. The amount of total
protein was measured using the bicinchoninic acid assay
(Pierce, Rockford, IL, USA). Samples containing 30 lg of
total protein were electrophoresed on 12% SDS-polyacryla-
mide gels and transferred onto a poly(vinylidene difluoride)
membrane. The membrane was incubated with specific anti-
bodies. The primary antibodies used included anti-Slug ⁄Snail2 (1 : 1000 dilution in NaCl/Tris containing 5%
skimmed milk anti-rabbit; Cell Signaling Technology), anti-
GAPDH (1 : 5000 dilution in NaCl ⁄Tris-T, anti-rabbit;
Cell Signaling Technology), anti-vimentin (1 : 1000 dilution
in NaCl ⁄Tris-T, anti-rabbit; Cell Signaling Technology),
anti-E-cadherin (1 : 1000 dilution in NaCl ⁄Tris-T, anti-rab-bit; BD Biosciences) and anti-lamin B1 (1 lgÆmL)1 dilution
in NaCl ⁄Tris-T, anti-rabbit; Abcam). Primary antibody
incubation was followed by washing in 0.1% Tween-20
NaCl ⁄Tris solution (TTBS) and then incubating with a sec-
ondary horseradish peroxidase-labeled anti-rabbit and
mouse IgG (1 : 5000; Vector Laboratories, Burlingame,
CA, USA) at room temperature. Signals were detected
using an enhanced chemiluminescent substrate (West-Zol,
iNtRON Biotechnology Inc., Seoul, South Korea).
For phosphoserine detection, HEK293 cells at 70% con-
fluence was transiently transfected with pcDNA mock,
pcDNA-Slug WT or S92 ⁄ 96 ⁄ 100 ⁄ 104A mutant for 30 h,
prior to harvesting of whole cell extracts using lysis buffer
as above. The extracts (1.0 mg for each condition) were
immunoprecipitated with anti-Slug IgG (2.0 mg for each
condition) overnight at 4 �C and incubated with protein
A ⁄G-sepharose (30 mL for each condition of 50% slurry,
Upstate Biotech, Lake Placid, NY, USA) for 2 h at 4 �C.The immunoprecipitates were washed with ice-cold lysis
buffer twice and NaCl ⁄Pi twice. The collected immunopre-
cipitates were boiled with 2· SDS ⁄PAGE sample buffer for
5 min, and then the samples were processed for western
blots using anti-Slug or anti-phosphoserine IgG.
RT-PCR analysis
Total RNA was isolated from cells using the TRIzol
reagent (Invitrogen) according to the manufacturer’s
instructions. Reverse transcription was carried out using
the reverse transcription system (Promega). PCR primers
for amplification were as follows: of E-cadherin, forward
5¢-TCCCATCAGCTGCCCAGAAA-3¢, reverse 5¢-TGACT
CCTGTGTTCCTGTTA-3¢; of vimentin, forward 5¢-AAT
GGCTCGTCACCTTCGTGAAT-3¢, reverse 5¢-CAGATT
AGTTTCCCTCAGGTTCAG-3¢; of GAPDH, forward
5¢-GAAGGTGAAGGTCGGAGTC-3¢, reverse 5¢-GAAGAT
GGTGATGGGATTTC-3¢; of Slug ⁄Snail2, forward 5¢-CCCGTTAACATGCCGCGCTCTTTC-3¢, reverse 5¢-TTTCTCGAGTCAGCGGGGACATCC-3¢. PCR was performed
using Taq polymerase (iNtRON Biotechnology Inc.).
PCR was initiated by incubating the samples at 95 �Cfor 5 min, followed by 30 cycles of 1 min denaturation
at 95 �C, 1 min annealing at 57 �C and 1 min elongation at
72 �C. Samples were analyzed by electrophoresis on 1% aga-
rose gels containing 0.002% nucleic acid staining solution
(RedSafe�; Biotechnology Inc., Seoul, South Korea).
Plasmid construction and site-directed
mutagenesis
A recombinant plasmid was made by inserting the human
SNAI2 gene into the pEGFP-C3 plasmid vector (Promega)
using BamHI and the XhoI restriction sites. The following
primers (Integrated DNA Technologies, Bioneer, Seoul,
South Korea) were used: Snail2-BamH1 forward
5¢-TTTGGATCCATGCCGCGCTCCTTC-3¢, Snail2-Xho1
reverse 5¢-GGGCTCGAGTCAGTGTGCTACACA-3¢ and
Snail2-Xho1 forward 5¢-AAACTCGAGATGCCGCGCT
CCTTC-3¢, Snail2-BamH1 reverse 5¢-TTTGGATCCCGG
TGTGCTACACAGCA-3¢. Mutagenesis PCR within the
Slug/Snail2 gene was performed using the following primer
sequences: S87A, forward 5¢-TCTTTGGGGCGAGTG
GCTCCCCCTCCTCCATCT-3¢, reverse 5¢-AGATGGAG
GAGGGGGAGCCACTCGCCCCAAAGA-3¢; S92 ⁄ 96A,
forward 5¢-CCTCCTCCAGCTGACACCTCCGCAAAG-
GACCAC-3¢, reverse 5¢-GTGGTCCTTTGATTCGGTGT-
CAGCTGGAGGAGGGGG-3¢; S100 ⁄ 104A, forward 5¢-TCCTCCAAGGACCACGCTGGCTCAGAAAGCCCC-3¢,reverse 5¢-GGGGCTTTCTGAGCCAGCGTGGTCCTTG-
GAGGA-3¢. Mutagenesis resulted in the formation of a
unique Dpn1 restriction site within the recombinant plas-
mid.
J. Y. Kim et al. GSK-3b-mediated phosphorylation of Slug ⁄ Snail2
FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 9
Generation of luciferase-reporter constructs and
transfection
To clone the E-cadherin promoter region in pGL3, E-cadher-
in genomic DNA was amplified with the primer sets
5¢)365GCGGTACCCTTGGGTGAAAGAGTGAGCC)3453¢(forward) and 5¢+48GCAGATCTTGAACTGACTTCCGC
AAGC+303¢ (reverse), and each of the primers had a Kpn1
and BglII site. The PCR products were cut with Kpn1 and
BglII and then cloned into the pGL3 enhancer vector
(Promega).
MCF7 cells were transfected with pcDNA3 or pcDNA3-
Slug ⁄Snail2 plasmid (4 lg), reporter construct (2 lg) in the
pGL3 enhancer vector, and 100 ng renilla luciferase null vec-
tor (as an internal control for transfection efficiency) using
10 lL Lipofectamine 2000 (Invitrogen), according to the
manufacturer’s protocol. One day after the transfection, cells
were harvested to be used in the luciferase assay for measur-
ing the activities of firefly and renilla luciferases using a lumi-
nometer (Luminmax-c; Biomedical Science Co. Ltd, Seoul,
South Korea) and the Dual Luciferase Reporter Assay Sys-
tem (Promega). The firefly luciferase activity was normalized
by using the renilla luciferase activity. The pGL3 enhancer
(promoterless) vector was used in all experiments as a nega-
tive control and as a baseline for data comparisons. The
pGL3 control vector (containing the SV40 promoter and the
SV40 enhancer) was used in all experiments as a positive con-
trol.
Subcellular fractionation
To obtain cytoplasmic and nuclear fractions, cells were
washed with ice-cold NaCl ⁄Pi and collected by scraping.
Cells were then pelleted by centrifugation at 300 g for
5 min at 4 �C. The cytosolic and nuclear fractionations
were performed using a nuclear extraction kit (Cayman
Chemical Co., Ann Arbor, MI, USA). The purity of the
fractions was confirmed by western blotting with an anti-
GAPDH IgG for the cytoplasmic fraction and with an
anti-lamin B1 IgG for the nuclear fraction.
Statistical analysis
All experiments were independently performed at least three
times. Data are presented as values of means ± standard
deviation. Data were analyzed using Student’s t test.
P < 0.05 was considered statistically significant.
Acknowledgements
This research was supported by the Basic Science
Research Program through the National Research
Foundation of South Korea (NRF) funded by the
Ministry of Education, Science and Technology (2011-
0003911 to Dr. MoonJae Cho).
References
1 Shih JY & Yang PC (2011) The EMT regulator slug
and lung carcinogenesis. Carcinogenesis 32, 1299–1304.
2 Comijn J, Berx G, Vermassen P, Verschueren K, van
Grunsven L, Bruyneel E, Mareel M, Huylebroeck D &
Van Roy F (2001) The two-handed E box binding zinc
finger protein SIP1 downregulates E-cadherin and
induces invasion. Mol Cell 7, 1267–1278.
3 Creighton CJ, Chang JC & Rosen JM (2010) Epithe-
lial–mesenchymal transition (EMT) in tumor-initiating
cells and its clinical implications in breast cancer.
J Mammary Gland Biol Neoplasia 15, 253–260.
4 Blanco MJ, Moreno-Bueno G, Sarrio D, Locascio A,
Cano A, Palacios J & Nieto MA (2002) Correlation of
Snail expression with histological grade and lymph node
status in breast carcinomas. Oncogene 21, 3241–3246.
5 Zhou BP, Deng J, Xia W, Xu J, Li YM, Gunduz M &
Hung MC (2004) Dual regulation of Snail by GSK-3b-mediated phosphorylation in control of epithelial–
mesenchymal transition. Nat Cell Biol 6, 931–940.
6 Catalano A, Rodilossi S, Rippo MR, Caprari P &
Procopio A (2004) Induction of stem cell factor ⁄ c-Kit ⁄slug signal transduction in multidrug-resistant malig-
nant mesothelioma cells. J Biol Chem 279, 46706–46714.
7 Casas E, Kim J, Bendesky A, Ohno-Machado L, Wolfe
CJ & Yang J (2011) Snail2 is an essential mediator of
Twist1-induced epithelial–mesenchymal transition and
metastasis. Cancer Res 71, 245–254.
8 Kang XQ, Zang WJ, Bao LJ, Li DL, Song TS, Xu XL
& Yu XJ (2005) Fibroblast growth factor-4 and hepato-
cyte growth factor induce differentiation of human
umbilical cord blood-derived mesenchymal stem cells
into hepatocytes. World J Gastroenterol 11, 7461–7465.
9 Papkoff J & Aikawa M (1998) WNT-1 and HGF
regulate GSK3 [beta] activity and [beta]-catenin
signaling in mammary epithelial cells. Biochem Biophys
Res Commun 247, 851–858.
10 Zhou BP & Hung MC (2005) Perspectives Wnt,
Hedgehog and Snail. Cell Cycle 4, 772–776.
11 Bachelder RE, Yoon SO, Franci C, de Herreros AG &
Mercurio AM (2005) Glycogen synthase kinase-3 is an
endogenous inhibitor of Snail transcription. J Cell Biol
168, 29–33.
12 Park SY, Kim HS, Kim NH, Ji S, Cha SY, Kang JG,
Ota I, Shimada K, Konishi N & Nam HW (2010)
Snail1 is stabilized by O-GlcNAc modification in
hyperglycaemic condition. EMBO J 29, 3787–3796.
13 Doble BW & Woodgett JR (2007) Role of glycogen
synthase kinase-3 in cell fate and epithelial–mesenchy-
mal transitions. Cells Tissues Organs 185, 73–84.
14 Van Phuc P, Khuong TTT, Le Van Dong TDK &
Tung T (2010) Isolation and characterization of breast
cancer stem cells from malignant tumours in
Vietnamese women. J Cell Anim Biol 4, 163–169.
GSK-3b-mediated phosphorylation of Slug ⁄ Snail2 J. Y. Kim et al.
10 FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS
15 Elloul S, Silins I, Trope CG, Benshushan A, Davidson
B & Reich R (2006) Expression of E-cadherin transcrip-
tional regulators in ovarian carcinoma. Virchows Arch
449, 520–528.
16 Cano A, Perez-Moreno MA, Rodrigo I, Locascio A,
Blanco MJ, del Barrio MG, Portillo F & Nieto MA
(2000) The transcription factor snail controls epithelial–
mesenchymal transitions by repressing E-cadherin
expression. Nat Cell Biol 2, 76–83.
17 Langer EM, Feng Y, Zhaoyuan H, Rauscher FJ III,
Kroll KL & Longmore GD (2008) Ajuba LIM
proteins are snail ⁄ slug corepressors required for
neural crest development in Xenopus. Dev Cell 14,
424–436.
18 Lopez D, Niu G, Huber P & Carter WB (2009) Tumor-
induced upregulation of Twist, Snail, and Slug represses
the activity of the human VE-cadherin promoter. Arch
Biochem Biophys 482, 77–82.
19 Kamemura K & Hart GW (2003) Dynamic interplay
between O-glycosylation and O-phosphorylation of nu-
cleocytoplasmic proteins: a new paradigm for metabolic
control of signal transduction and transcription. Prog
Nucleic Acid Res Mol Biol 73, 107–136.
20 Kang ES, Han D, Park J, Kwak TK, Oh MA, Lee S,
Choi S, Park ZY, Kim Y & Lee JW (2008) O-GlcNAc
modulation at Akt1 Ser473 correlates with apoptosis of
murine pancreatic [beta] cells. Exp Cell Res 314,
2238–2248.
21 Kwak TK, Kim H, Jung O, Lee SA, Kang M, Kim HJ,
Park JM, Kim SH & Lee JW (2010) Glucosamine treat-
ment-mediated O-GlcNAc modification of paxillin
depends on adhesion state of rat insulinoma INS-1 cells.
J Biol Chem 285, 36021–36031.
22 Wu Y, Deng J, Rychahou PG, Qiu S, Evers BM &
Zhou BP (2009) Stabilization of snail by NF-[kappa]B
is required for inflammation-induced cell migration and
invasion. Cancer Cell 15, 416–428.
23 Yook JI, Li XY, Ota I, Fearon ER & Weiss SJ (2005)
Wnt-dependent regulation of the E-cadherin repressor
snail. J Biol Chem 280, 11740–11748.
24 Xu Y, Lee S, Kim H, Kim N, Piao S, Park S, Jung Y,
Yook J, Park B & Ha N (2010) Role of CK1 in
GSK3b-mediated phosphorylation and degradation of
Snail. Oncogene 29, 3124–3133.
25 Korpal M & Kang Y (2010) Targeting the transforming
growth factor-[beta] signalling pathway in metastatic
cancer. Eur J Cancer 46, 1232–1240.
26 Marchetti A, Colletti M, Cozzolino AM, Steindler C,
Lunadei M, Mancone C & Tripodi M (2008) ERK5 ⁄MAPK is activated by TGF [beta] in hepatocytes and
required for the GSK-3 [beta]-mediated Snail protein
stabilization. Cell Signal 20, 2113–2118.
27 Yang CH, Song B-C & Cho M (2012) A natural
mutation of Hepatitis B virus X gene affects cell cycle
progression and apoptosis in Huh7 cells. J Korean Soc
Appl Biol Chem 55, 229–236.
J. Y. Kim et al. GSK-3b-mediated phosphorylation of Slug ⁄ Snail2
FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 11
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