Báo cáo khoa học: Proteolysis of the tumour suppressor hDlg in response to osmotic stress is mediated by caspases and independent of phosphorylation pot

Proteolysis of the tumour suppressor hDlg in response to
osmotic stress is mediated by caspases and independent
of phosphorylation
Francisco A. In
˜
esta-Vaquera
1
, Francisco Centeno
2
, Paloma del Reino
1
, Guadalupe Sabio
3
, Mark
Peggie
3
and Ana Cuenda
1,3
1 Departamento de Inmunologı
´
a y Oncologı
´
a, Centro Nacional de Biotecnologı
´
a-CSIC, Madrid, Spain
2 Departamento Bioquı
´
mica y Biologı
´
a Molecular, Universidad de Extremadura, Ca
´

ceres, Spain
3 MRC Protein Phosphorylation Unit, University of Dundee, UK
Mammalian cells respond to changes in the osmolarity
of the medium by activating multiple signalling path-
ways, with p38 mitogen-activated protein kinases
(MAPKs) critical for both early response and long-term
cellular adaptation to prolonged hyperosmotic exposure
[1]. Although all four p38 MAPKs (p38a, p38b, p38c
and p38d) are activated in response to hyperosmotic
stress, activation of the isoform p38c is particularly
rapid and strong compared with other p38s [2,3].
Recently, we described a novel regulatory pathway for
the adaptation of cells to a hyperosmolar environment
that acts parallel to the classical p38a pathway, which
involves the protein kinase p38c and its substrate hDlg,
and modulates the composition of the cytoskeletal
protein by phosphorylating one of its components [3].
hDlg (also called Dlg1 and dlgh1) is the human
orthologue of the Drosophila tumour suppressor Dlg,
and belongs to the membrane-associated guanylate
kinase family of scaffold proteins, whose members
have a similar structural organization composed of a
basic core of a variable number of PDZ domains, a
SH3 domain and a catalytically inactive guanylate
kinase-like region [4]. Functions of hDlg are related to
the establishment and maintenance of cell polarity and
the adhesion integrity of intestinal epithelial cells [5,6].
Moreover, gene-targeted mice lacking full-length hDlg
show defects in the morphogenesis of the kidney and
urogenital tracts [7,8].

Evidence suggests that alterations in hDlg function
may contribute to the development of cancer. The
Keywords
apoptosis; caspase; human disc-large;
osmotic shock; p38-mitogen activated
protein kinase
Correspondence
A. Cuenda, Departamento de Inmunologı
´
ay
Oncologı
´
a, Centro Nacional de
Biotecnologı
´
a-CSIC, Campus de
Cantoblanco-UAM, 28049-Madrid, Spain
Fax: +34 91 372 0493
Tel: +34 91 585 5451
E-mail:
(Received 6 August 2008, revised 29
October 2008, accepted 7 November 2008)
doi:10.1111/j.1742-4658.2008.06783.x
Human disc-large (hDlg) is a scaffold protein critical for the maintenance
of cell polarity and adhesion. hDlg is a component of the p38c MAP
kinase pathway, which is important for the adaptation of mammalian cells
to changes in environmental osmolarity. Here we report a strong decrease
in the levels of hDlg protein in the human epithelial cell line HeLa when
exposed to osmotic shock. This is independent of the phosphorylation state
of hDlg, is prevented by preincubating the cell with the caspase inhibitor

z-VAD and is part of the apoptotic process triggered by cellular stress.
Although, both caspase 3 and caspase 6 are strongly activated by osmotic
shock, the time course of caspase 6 activation parallels hDlg degradation,
suggesting that this caspase may be responsible for the proteolysis. Mutat-
ing hDlg Asp747 to Ala abolishes caspase-induced cleavage, but does not
affect the early stage of apoptosis or cell attachment. Our findings show
that osmotic stress triggers hDlg degradation through a mechanism differ-
ent from the one mediated by proteasomes, and we identify hDlg as a
caspase substrate during the apoptotic process, although its proteolysis
may not be implicated in the progression of early apoptosis.
Abbreviations
GST, glutathione S-transferase; hDlg, human disc large; HPV, human papillomavirus; MAPK, mitogen-activated protein kinase; PSI,
proteasome inhibitor I.
FEBS Journal 276 (2009) 387–400 ª 2008 The Authors Journal compilation ª 2008 FEBS 387
expression of hDlg in epithelial-derived cancers (such
as cervical, gastric and colon cancers) is extremely low
or even absent [9], in addition, hDlg binds to oncopro-
teins expressed by viruses such as, human papillomavi-
rus (HPV), human T-cell leukaemia virus type 1 and
human adenovirus type 9 [10]. The binding of HPV E6
protein to hDlg causes a decrease in hDlg protein
levels by inducing its proteasome-mediated degradation
[11]. In epithelial cell lines, this degradation is highly
dependent on the state of hDlg phosphorylation and
the degree of isolation of the cell. Hyperphosphoryla-
tion of hDlg makes it more susceptible to degradation
induced by the HPV E6 oncoprotein [12], whereas in
isolated cells, infected or not with HPV, the degrada-
tion of hDlg is constitutive in the cytoplasm [13].
In addition to the suggestion that hDlg phosphory-

lation may modulate its protein levels in cells, in recent
years, phosphorylation has emerged as a mechanism
for regulating hDlg’s function as a scaffold protein
[5,14,15]. Accordingly, we have shown that hDlg is
hyperphosphorylated in response to cellular stress such
as osmotic shock or UV radiation. This phosphoryla-
tion is mediated by p38c MAPK and triggers its
dissociation from the cytoskeletal protein GKAP,
therefore releasing it from the cytoskeleton into the
cytoplasm [3].
Our aim in this study was to gain a better under-
standing of the role of hDlg phosphorylation by
p38c when cells are exposed to hyperosmotic stress.
Here we analyse whether the phosphorylation of hDlg,
triggered by osmotic shock, could also control levels of
hDlg protein in the human epithelial cell line HeLa. We
report a strong decrease in hDlg protein, although this
event is independent of its phosphorylation state. More-
over, this hDlg proteolysis was dependent on caspase
activation during the apoptosis process in the cells.
Results
Osmotic shock causes a decrease of hDlg protein
As mentioned previously, hDlg degradation seems to
be regulated by phosphorylation and cell density
[12,13,16]. Moreover, we have reported that when cells
are exposed to osmotic shock, endogenous hDlg is
hyperphosphorylated by the protein kinase p38c [3].
Therefore, we initiated experiments to determine
whether hDlg degradation is affected by phosphory-
lation mediated by p38c in a cell density-dependent

manner. We first checked the regulation of hDlg
phosphorylation by hyperosmotic stress at different
cell densities using a phosphospecific antibody, which
recognizes phosphorylated serine 158 (S158), a hDlg
residue that becomes phosphorylated by p38c follow-
ing sorbitol treatment [3,17]. As expected, we observed
an increase in hDlg phosphorylation upon sorbitol
stimulation under all experimental conditions
(Fig. 1A). Moreover, a significant increase in basal
hDlg phosphorylation ( 26%) was observed in con-
fluent cells, but this may be due to an increase in the
total amount of hDlg protein ( 30%) when cells were
100% confluent, consistent with the stabilization of
this protein upon cell–cell contact (Fig. 1A) [16].
Once we had confirmed that hDlg was phosphory-
lated, we examined the effect of osmotic stress on hDlg
protein levels and whether this was dependent on cell
density. To avoid cell death caused by long exposure
to hyperosmotic shock, cells were treated for 60 min
with sorbitol and then released into fresh medium for
9 or 14 h. Under these conditions, we detected a large
decrease in hDlg protein levels, of 75% at 9 h and
85% at 14 h after the treatment with sorbitol ceased.
Moreover, hDlg loss was similar in cells 50 or 100%
confluent, indicating that cell density does not affect
the decrease in hDlg protein caused by osmotic shock
(Fig. 1B). We found that the levels of hDlg in HeLa
cells treated with sorbitol decrease progressively with
time after stress, falling to 30% at 6 h after release
from sorbitol treatment (Fig. 1C). For comparison,

exposure of cells to UV radiation, which also triggers
hDlg phosphorylation mediated by p38c as well as its
degradation [3,18], causes a decrease in hDlg protein
levels similar to that observed after osmotic sock treat-
ment (Fig. 1D). No strong stable accumulation of
hDlg cleavage fragments could be detected at these
time points (not shown), indicating that either the deg-
radation of this molecule is very rapid and at multiple
sites or the antibodies used do not recognize the
epitope of the cleavage fragments.
Osmotic shock-induced degradation of hDlg is
mediated by caspase
We measured the activation of different proteases after
hyperosmotic shock or UV treatment in HeLa cells to
determine which might be involved in hDlg degrada-
tion. As shown in Fig. 2A, only caspase 3 and cas-
pase 6 activities were significantly induced. Other
protease activities, including caspase 8, calpain or
cathepsin B, were not affected. Activation of the effec-
tor caspase 3 and caspase 6 suggests that hDlg could
be proteolysed by one of them. To check this, HeLa
cells were treated with or without the general caspase
inhibitor z-VAD prior to and during exposure to
stress. As shown in Fig. 2B, z-VAD blocked hDlg
degradation and the activation of caspase 3 and
Caspase-dependent degradation of hDlg F. A. In˜ esta-Vaquera et al.
388 FEBS Journal 276 (2009) 387–400 ª 2008 The Authors Journal compilation ª 2008 FEBS
caspase 6 (Fig. 2C). Other protease inhibitors, such as
the calpain inhibitor MDL 28170 or the cathepsin
inhibitor 3-Met adenine, did not affect hDlg degrada-

tion (data not shown). Because it has been shown that
the reduction in hDlg levels may be proteasome depen-
dent, we also treated the cells with the proteasome
inhibitor proteasome inhibitor I (PSI), and found that
this did not prevent hDlg degradation (Fig. 2B),
although it did prevent degradation of the kinase
SGK1, the steady-state level of which is very low
because of its degradation by proteasomes (Fig. 2D)
[19]. These results indicate that the degradation of
hDlg upon cellular stress is mediated by caspases and
not by the proteasome.
In addition, when we examined the time course of
activation of caspases by cellular stress, we found that
activation of caspase 6 is greater and more sustained
than that of caspase 3 (Fig. 2E). Moreover, caspase 6
activation reached its maximum at 3 or 6 h after UV
or sorbitol treatment, respectively, and then decreased.
Whereas caspase 3 was transiently activated, being
maximal 30–60 min after exposure to either stimulus.
These results show that the loss of hDlg over the time
course of sorbitol or UV treatment (Fig. 1C,D) corre-
lated with the kinetics of caspase activation, particu-
larly of caspase 6, supporting that hDlg cleavage is
caspase dependent.
Phosphorylation by p38c does not modulate hDlg
proteolysis
To determine whether hDlg degradation induced by
osmotic stress was dependent on phosphorylation by
p38c, we first expressed wild-type glutathione S-trans-
ferase (GST)–hDlg, which behaves similarly to endoge-

nous hDlg (Fig. S1), or different GST–hDlg mutants,
in which each of the in vivo p38c-phosphorylation
sites were mutated individually to Ala to prevent
phosphorylation (S158A, T209A, S431A and S442A)
[3]. We found that the amount of GST–hDlg wild-type
and of the different GST–hDlg mutants decreased
equally following osmotic shock treatment (Fig. 3A).
A
Sorbitol
++
hDlg (S158)
hDlg (total)
Cell density 50% 100%
B
hDlg (total)
GAPDH
Cell density
50% 100%
Time (h)
Sorbitol
+
09
14
––
––
+
09
14
hDlg
GAPDH

0
20
40
60
80
100
0246
hDlg protein level (%)
GAPDH
hDlg
0361
Time after sorbitol treatment (h)
03 6 14
Time after UV treatment (h)
9
D
hDlg protein level (%)
03691215
0
20
40
60
80
100
C
Time after sorbitol release (h) Time after UV treatment (h)
Fig. 1. Osmotic shock causes a decrease in
hDlg protein levels in a manner not depen-
dent on cell density. (A) HeLa cells were
exposed for 15 min to 0.5

M sorbitol.
Endogenous hDlg was immunoprecipitated
from 0.4 mg of cell lysate and pellets were
immunoblotted using an antibody that
recognizes phosphorylated hDlg [hDlg
(S158)] or total hDlg (anti-hDlg). (B) Cells
were exposed to hyperosmotic stress
(0.5
M sorbitol) for 60 min, and then
released into sorbitol-free medium for 0, 9
or 14 h. Levels of hDlg were analysed by
immunoblot using hDlg Ig. (C) (Upper) HeLa
cells treated as in (B). Endogenous levels of
hDlg were analysed, by immunoblot with
hDlg Ig, 1, 3 and 6 h after sorbitol had
been washed out. (Lower) hDlg levels were
quantified as described in Materials and
methods. Values are means (± SE) of three
independent experiments. (D) HeLa cells
were exposed to UV irradiation (200 JÆm
)2
),
followed by 3, 6, 9 or 14 h incubation.
Endogenous levels of hDlg were analysed
as in (C). Quantification values are means
(± SE) of two independent experiments.
Immunoblots are shown as one representa-
tive experiment. Endogenous GAPDH level
was used as a loading control. Lines in this
figure are duplicates.

F. A. In˜ esta-Vaquera et al. Caspase-dependent degradation of hDlg
FEBS Journal 276 (2009) 387–400 ª 2008 The Authors Journal compilation ª 2008 FEBS 389
These results suggest that the phosphorylation of indi-
vidual sites does not modulate hDlg degradation and
prompted us to examine whether it was controlled by
phosphorylation at more than one site. We then per-
formed the same experiment using a hDlg mutant
[GST–hDlg(A
6
)] in which all sites phosphorylated by
Control Sorbitol UV-C
Caspase 3 1.25 +
0.068 13.22 + 0.35 6.11 0.53
Caspase 6 1.71 +
0.26 7.74 + 1.11 4.73
+
+
+
+
1.17
Caspase 8 1.43 +
0.03 1.67 + 0.13 1.82 0.23
Calpain 0.33 +
0.03 0.55 + 0.01 0.8 0.05
CathepsinB 0.61 +
0.08 0.47 + 0.05
+
0.36 0.02
0
3

6
9
12
0369
12
0
5
10
15
20
25
Fold caspase activation
0369
12
Time after treatment
(
h
)
Sorbitol
UV-C
Caspase 6
Sorbitol
UV-C
Caspase 3
E
Sorbitol
zVAD
PSI
–+
–

+
+
–
+
+
–
–
–
–
–
–
–
–
hDlg
GAPDH
–+
–
–
–
–
–
–
–
–
–
–
UV-C
+
+
+

+
B
0
25
50
75
100
hDlg protein
level (%)
0
5
10
15
Caspase activity
(Fluor
–1
·min
–1
·mg protein
–1
)
Sorbitol
zVAD
UV-C
+
+
+
++

–

––
––
––
–
Caspase 3
+
+
+
++

Caspase 6
50
37
SGK1
PSI
–
+
D
A
C
Caspase-dependent degradation of hDlg F. A. In˜ esta-Vaquera et al.
390 FEBS Journal 276 (2009) 387–400 ª 2008 The Authors Journal compilation ª 2008 FEBS
p38c in vitro and in vivo were mutated to Ala and
therefore could not be phosphorylated under stress.
The mutated phosphorylation sites were the four sites
regulated by stresses (S158, T209, S431 and S442), one
site constitutively phosphorylated in cells (S122) and
one site phosphorylated in vitro by p38c (S447) [3].
Surprisingly, both hDlg wild-type and mutant disap-
peared to a similar extent in cells treated with sorbitol

(Fig. 3B), indicating that phosphorylation of hDlg by
p38c does not regulate its degradation induced by
osmotic stress in HeLa cells.
To confirm these findings and verify whether hDlg
degradation was dependent on its state of phosphoryla-
tion, we treated cells with different MAPK pathway
inhibitors to block hDlg phosphorylation [17]. We then
examined hDlg loss in cells treated with inhibitors and
exposed to sorbitol. Both p38MAPK inhibitors,
BIRB0796, which at high concentrations inhibits all p38
and JNK isoforms [17], and SB203580, which inhibits
the isoforms p38a ⁄ b, failed to abolish hDlg degradation
(Fig. 3C). BIRB0796 at high concentrations (1 and
10 lm), but not SB203580, efficiently blocked hDlg
phosphorylation [17] (data not shown). Because osmotic
stress also may cause the activation of other MAPKs
such as ERK1 ⁄ 2, ERK5 or JNK, we investigated
whether these kinases were involved in hDlg degrada-
tion. Treatment of cells with PD184352 at low concen-
tration abolishes the activation of ERK1 ⁄ 2 and at high
concentrations abolishes the activation of ERK5; treat-
ment with SP600125 blocks JNK activity along with
other many protein kinases such as SGK1, PRAK,
AMPK, CHK, CDK2 or S6K1 [20]. None of these
inhibitors blocked the decrease in hDlg protein levels
(Fig. 3C) triggered by osmotic shock (or UV, data not
shown), although they inhibited the different MAPKs
activations (Fig. S2). These results suggest that other
MAPK family members activated by cellular stress do
not control the disappearance of hDlg.

Fig. 2. hDlg degradation is mediated by caspases. (A) HeLa cells were treated with 0.5 M sorbitol for 60 min or with UV irradiation
(200 JÆm
)2
), followed by 1 h incubation in stimulus-free media, proteases activity was determined by fluorescence emitted from specific
peptide cleavage, as described in Materials and methods. Values are means (± SE) of three independent experiments. (B) Cells were prein-
cubated in the absence or in the presence of 30 l
M caspase inhibitor z-VAD or 60 lM proteasome inhibitor PSI, 60 min before treatment
with sorbitol or UV irradiation, as in (A), followed by 14 h incubation in stimulus-free media (in the continued absence or presence of prote-
ase inhibitor). Endogenous levels of hDlg were analysed and quantified as in Fig. 1C. (C) HeLa cells were preincubated with (grey bars) or
without (black bars) the pan-caspase inhibitor z-VAD for 1 h before treatment with sorbitol or UV irradiation, as in (A). Values are means
(± SE) of three independent experiments. (D) HeLa cells were incubated with or without 60 l
M PSI and endogenous levels of SGK1 were
analysed by western blotting using SGK1 Ig. (E) HeLa cells (circles) or HEK293 cells (triangles) were exposed, as in (A), to sorbitol (black
circles or triangles) or to UV irradiation (empty circles or triangles), then released from the stimuli for the times indicated before assaying
caspase activities. Values are means (± SE) of two independent experiments.
C
Sorbitol
BIRB0796 (µM)
SB203580 (µM)
PD184352 (µM)
SP600125 (µM)
+++
+
–
++ +
+
–
++
–
–

–
–
–
–
10
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–

–
–
0.1 1 10
–
10
–
–
2
–
––
–
10
–
–
–
–
–
–
hDlg
GAPDH
Fig. 3. The decrease in hDlg protein level is not regulated by p38c phosphorylation. (A) HeLa cells were transfected with either GST–hDlg
wild-type or different GST–hDlg mutants in which in vivo p38c phosphorylation sites had been mutated to Ala, and then exposed 0.5
M sorbi-
tol for 60 min. Overexpressed GST–hDlg were analysed 6 h after sorbitol had been washed out, as in Fig. 1. (B) Cells were transfected with
GST–hDlg wild-type or GST–hDlg(A
6
) mutant. Cells were treated as in (A) and GST–hDlg was analysed 1, 3 and 6 h after sorbitol release.
Lines indicate duplicates. (C) Cells were preincubated for 2 h with or without different kinase inhibitors, at the concentrations indicated. Cells
were exposed to 0.5
M sorbitol for 60 min, and then incubated for 9 h in sorbitol-free medium (in the continued absence or presence of

inhibitor). Endogenous levels of hDlg were analysed as in Fig. 1C. GAPDH or p38a levels were used as loading controls.
F. A. In˜ esta-Vaquera et al. Caspase-dependent degradation of hDlg
FEBS Journal 276 (2009) 387–400 ª 2008 The Authors Journal compilation ª 2008 FEBS 391
To determine whether hDlg phosphorylation regu-
lates hDlg degradation and to exclude the possibility
that other phosphorylation sites, different from those
for p38c, may modulate hDlg degradation, we treated
cells with the general kinase inhibitor staurosporine
[21]. To test whether staurosporine could inhibit hDlg
phosphorylation, cells were preincubated with or with-
out this compound for 1 h before exposure to sorbitol.
Phosphorylation of endogenous hDlg was blocked
completely by 1 lm staurosporine (Fig. 4A). However,
preincubation of cells with staurosporine failed to
block the decrease in hDlg protein level, and caused a
significant increase in hDlg degradation (Fig. 4B).
Given the above results, we decided to check whether
incubation of cells with staurosporine alone caused
hDlg loss. As shown in Fig. 4C, staurosporine also
caused hDlg degradation in a concentration- and time-
dependent manner.
hDlg is degraded in apoptotic cells
These findings suggest that hDlg degradation might be
related to the apoptosis of the cell, because stauro-
sporine is a potent inducer of caspase-dependent cell
apoptosis [21] (data not shown). Therefore, we estab-
lished that, after cellular stress, cells undergo apopto-
sis. HeLa, or HEK293 cells for comparison, were
exposed to either sorbitol or UV, and apoptosis was
determined 3 or 14 h after exposure to the stimulus.

As shown in Fig. 5A,  70% of HeLa cells underwent
apoptosis 14 h post stimulus, whereas only  15% of
HEK293 cells started to die 14 h after sorbitol or UV
treatment. Basal levels of apoptosis in control cells,
not exposed to stress, were also significantly higher in
HeLa cells than in HEK293 cells (Fig. 5A). Accord-
ingly, when we examined the levels of hDlg protein in
HEK293 cells that had been exposed to sorbitol or
UV, we could not detect any protein degradation even
14 h after the stimulus (Fig. 5B). However, neither
0
25
50
75
100
hDl gprotein level (%)
Staurosporine (µM)
2
0110
1
10
GAPDH
hDlg
0
Staurosporine (µM)
7
2
727Time (h)
C
B

0
25
50
75
100
hDlg protein level (%)
Staurosporine (µM)
Sorbitol
00 1 10
–
++ +
GAPDH
hDlg
Staurosporine (µM)
Sorbitol
0
01
–+ +
hDlg (S158)
hDlg (total)
A
hDlg (T209)
hDlg (S431)
hDlg (S442)
Fig. 4. The broad-spectrum kinase inhibitor staurosporine enhances
hDlg degradation. (A) Cells were incubated for 1 h with or without
the indicated concentration of staurosporine and then exposed for
15 min to 0.5
M sorbitol. hDlg was immunoprecipitated and analy-
sed using antibodies that recognize phosphorylated hDlg at four

different residues, hDlg (S158), hDlg (T209), hDlg (S431) or hDlg
(S442), or an antibody that recognizes total hDlg (anti-hDlg). (B)
HeLa cells were preincubated for 1 h with or without the pan-
kinase inhibitor staurosporine at the concentrations indicated, and
treated with 0.5
M sorbitol for 60 min, and then incubated for 3 h in
sorbitol-free medium (in the continued absence or presence of the
inhibitor). Endogenous levels of hDlg were analysed by immuno-
blotting (lower) and the percentage of protein level quantified
(upper) as described in Materials and methods. Values are means
(± SE) of three independent experiments. (C) HeLa cells were trea-
ted without or with 1 or 10 l
M staurosporine for 2 h (upper and
lower) or 7 h (lower). Endogenous levels of hDlg were analysed
as described previously and the percentage of hDlg protein was
quantified (lower). Values are means (± SE) of two independent
experiments.
Caspase-dependent degradation of hDlg F. A. In˜ esta-Vaquera et al.
392 FEBS Journal 276 (2009) 387–400 ª 2008 The Authors Journal compilation ª 2008 FEBS
caspase 3 nor caspase 6 were activated in HEK293
cells under any of the experimental conditions
described (Fig. 2E), which is consistent with the lack
of hDlg degradation observed in this cell line. The low
percentage of HEK293 cells undergoing apoptosis
(compared with HeLa cells), and the the lack of hDlg
loss in HEK293 cells, support the idea that hDlg deg-
radation in HeLa cells is part of the apoptotic event
triggered by cellular stress.
Changes in hDlg localization upon hyperosmotic
shock exposure

Although hDlg is normally localized in adherens junc-
tions at sites of cell–cell contact, its cellular distribu-
tion is different in confluent and subconfluent cells,
hence our next question was whether this localization
would change during apoptosis induced by sorbitol. As
expected, when HeLa cells were 50% confluent, hDlg
was localized diffusely throughout the cytosol and at
the membrane. hDlg localization changed when cells
reached confluency; in this condition, hDlg was present
mainly at the membrane, whereas the amount found in
the cytoplasm decreased markedly (Fig. 6A). These
results were confirmed by subcellular fractionation
analysis; we found that the amount of hDlg in the
cytoplasm is greater in subconfluent cells, but in
confluent cells hDlg is mainly in the membrane frac-
tion (Fig. 6B). After exposing the cells to sorbitol
(Fig. 6A,B), the total amount of hDlg decreased in
both 50% and 100% confluent cells, and this decrease
was equal in all cell compartments (Fig. 6B). These
results show that hDlg localizes to the plasma mem-
brane when cells reach confluency and establish cell–
cell contact, and that its degradation occurs in all cell
compartments in which hDlg is present.
In addition, in CaCo-2 cells, derived from human
colonic adenocarcinoma and in which hDlg is
degraded in response to osmotic stress (Fig. S3),
hDlg was found mostly in areas of cell–cell contact
and a substantial and gradual loss from the mem-
brane was observed after osmotic shock treatment
(Fig. 6C). The more compact localization of hDlg is

partially lost and hDlg is localized more diffusely
throughout the cytoplasm in the vicinity of the mem-
brane (Fig. 6Ca–c). However, pretreatment of the cell
with the p38MAPK inhibitor BIRB0796 causes a sig-
nificant delay in the loss of Dlg from the membrane
(Fig. 6Cd–f), whereas pretreatment with the caspase
inhibitor z-VAD, in both the absence and presence
of BIRB0796, preserved the membrane localization
of hDlg under all experimental conditions (Fig. 6Cg–
l). Because cell–cell contacts are largely preserved at
these initial time points, it is suggested that hDlg
Sorbitol
0
20
40
60
80
03
14
UV-C
03
14
Time after stimuli treatment (h)
Apoptosis(%)
A
B
Time after stimuli treatment (h)
C0 3 6 14
Sorbitol
UV-C

hDlg
GAPDH
hDlg
GAPDH
Fig. 5. Cellular stresses induce apoptosis in
HeLa cells. (A) HeLa (black bars) or HEK293
cells (grey bars) were exposed to 0.5
M
sorbitol for 60 min or to UV irradiation
(200 JÆm
)2
) followed by 3 or 14 h incubation
in stimulus-free media before quantitative
analysis of apoptosis by propidium iodide ⁄
annexin V. Values are means (± SE) of three
independent experiments. (B) HEK293 cells
were exposed to 0.5
M sorbitol for 60 min
or to UV irradiation (200 JÆm
)2
), followed by
0, 3, 6 or 14 h incubation. Endogenous
levels of hDlg were analysed as described
in Fig. 1.
F. A. In˜ esta-Vaquera et al. Caspase-dependent degradation of hDlg
FEBS Journal 276 (2009) 387–400 ª 2008 The Authors Journal compilation ª 2008 FEBS 393
A
Control
Sorbitol
Cell density

50% 100%
Control
Sorbitol
hDlg hDlg + DAPI
hDlg hDlg + DAPI
hDlg hDlg + DAPI hDlg hDlg + DAPI
hDlg
InsR
Calpain
Cell density
50% 100%
Cytop CytopCytopCytopMemb MembMembMemb
Control
Sorbitol
Control
Sorbitol
B
No inhibitor
zVAD
Treatment
None
1 h Sorbitol
1 h Sorbitol
+
3 h release
BIRB0796 + zVAD
hDlg
hDlg
hDlg
hDlg

C
a
b
c
d
e
f
g
h
i
j
k
l
BIRB0796
Caspase-dependent degradation of hDlg F. A. In˜ esta-Vaquera et al.
394 FEBS Journal 276 (2009) 387–400 ª 2008 The Authors Journal compilation ª 2008 FEBS
cleavage represents a step in apoptosis that may
precede cell–cell detachment.
Identification of hDlg caspase-cleavage sites
These findings suggest that hDlg is a caspase target.
To confirm this and identify the caspase(s) cleavage
site(s) on hDlg, experiments were performed using
mutations that affect putative caspase 3 and ⁄ or cas-
pase 6 sites. Within their substrates, caspases recog-
nize a core tetrapeptide motif (P
4
P
3
P
2

P
1
) that
contains an essential aspartic acid residue required
for the cleavage reaction at position P
1
[22]. One
well-defined substrate-recognition motif for caspases,
in particular caspase 3, is DXXD, whereas for cas-
pase 6 it is (I ⁄ V ⁄ L)EXD [22]. Analysis of the hDlg
protein sequence indicates that it contains several
putative caspase-cleavage sites, DVRD
255
and
DGRD
750
, which are typical caspase 3 recognition
sequences, YEVD
747
, which may be another potential
caspase site, and QSVD
397
, which has been previ-
ously reported as an unusual caspase 3 cleavage site
[18]. We generated different hDlg mutants in which
the aspartic acid residue predicted as being required
for caspase cleavage was mutated to Ala (Fig. 7A).
These constructs were transfected to HeLa cells and
the effects of sorbitol (and UV) treatment on GST–
hDlg wild-type or mutant GST–hDlg degradation

were compared. As shown in Fig. 7B, 3 h after stim-
ulation ceased, only the mutation D747A blocked
the degradation of GST–hDlg, although wild-type
hDlg and the other hDlg mutant forms were
degraded to the same extent after treatment. Quanti-
fication of hDlg protein confirmed that D747A was
the only mutant not cleaved after cellular stress
(Fig. 7C). These results identify the sequence YEVD
as a possible site of hDlg cleavage in early apoptotic
cells.
Effect of hDlg cleavage on cell–cell detachment
and early stage of apoptosis
To evaluate the role of hDlg during these processes,
HeLa cells were transfected with hDlg wild-type or
mutant hDlgD747A, which is not degraded, or mutant
hDlgD397A, as a control. We tried to generate cell
lines stably overexpressing wild-type or hDlg mutants.
However, none of the attempts was successful. There-
fore, the transfection procedure was optimized to
Control Sorb. UV-C
Tubulin
Tubulin
Tubulin
Tubulin
Tubulin
hDlg(WT)
hDlg(D255A)
hDlg(D397A)
hDlg(D255A/D750A)
hDlg(D747A)

B
C
0
20
40
60
80
100
hDl
g
-WT -D255A -D397A -D747A -D255A/D750A
hDlg protein level (%)
A
PDZ1 PDZ3 SH3 GUKPDZ2
hDlg
DVRD
255
QSVD
397
YEVD
747
DGRD
750
Fig. 7. Identification of the hDlg-caspase-cleavage site. (A) Sche-
matic representation of hDlg and the putative caspase-cleavage
sites. (B) Cells were transfected with GST–hDlg wild-type or differ-
ent GST–hDlg mutants, in which different caspase-cleavage sites
have been mutated to Ala, and then exposed to 0.5
M sorbitol for
60 min or UV irradiation (200 JÆm

)2
). GST–hDlg were analysed 3 h
after UV treatment or after sorbitol had been washed out, as in
Fig. 1. The endogenous tubulin level was use as the loading con-
trol. Immunoblots are shown as one representative experiment. (C)
Percentage GST–hDlg protein level was quantified as before: hDlg
protein from control cells (black bars), cells treated with sorbitol
(dark grey bars) or treated with UV (light grey bars). Values are
means (± SE) of three to four independent experiments.
Fig. 6. Localization of hDlg in apoptotic cells. (A) HeLa cells were grown at 50 or 100% confluency, exposed or not to 0.5 M sorbitol for
60 min, stained with hDlg Ig 3 h after sorbitol release, and subjected to immunofluorescence microscopy. Nuclei are stained with DAPI.
Similar results were obtained in three independent experiments. (B) HeLa cells were exposed to 0.5
M sorbitol for 60 min, and then released
in sorbitol-free medium for 0 or 9 h. Cells were subjected to cellular fractionation as indicated in Materials and methods and 10–30 lgof
protein from cytoplasm and membrane fractions were immunoblotted using the antibodies indicated. (C) CaCo-2 cells were preincubated
with or without the kinase inhibitor BIRB0796 (1 l
M) and in the presence or absence of z-VAD (30 lM). Cells were exposed to hyperosmotic
stress for 1 h followed by 0 or 3 h incubation in sorbitol-free medium.
F. A. In˜ esta-Vaquera et al. Caspase-dependent degradation of hDlg
FEBS Journal 276 (2009) 387–400 ª 2008 The Authors Journal compilation ª 2008 FEBS 395
result in  50% hDlg-transfected HeLa cells, which is
a level tolerated by the cells. Using these cells, we
examined the effect of hDlg mutation on the progres-
sion of both cell detachment and apoptosis induced by
sorbitol. As shown in Fig. 8, none of these processes
was significantly affected by the mutation D747A of
hDlg, indicating that its proteolysis is not sufficient for
the regulation of early apoptotic events.
Discussion
Our aim was to gain a better understanding of the role

of hDlg phosphorylation in the adaptation of cells to
stress, by determining whether stress could regulate
hDlg degradation in cells exposed to changes in envi-
ronmental osmolarity. We analysed the extent of hDlg
phosphorylation and hDlg protein levels in subconflu-
ent and confluent cells exposed to hyperosmolarity.
Our results show that hDlg is phosphorylated in
response to osmotic stress, although the degree of
hDlg phosphorylation upon sorbitol treatment is
 40% higher in subconfluent cells than in confluent
cells. This is probably because of the difference in
hDlg localization observed. In confluent cells, hDlg is
present mainly in the membrane, at sites of cell–cell
contact. However, in subconfluent cells, hDlg localiza-
tion is identical to its physiological kinase p38c, and
both are localized diffusely throughout the cytosol,
nucleus and membrane [3]. Under these conditions,
hDlg may be more accessible to the kinase and this
may facilitate its phosphorylation after sorbitol treat-
ment. In addition, we have also shown that hDlg is
largely degraded in cells exposed to hypertonicity and
that this is not dependent on cell density.
As mentioned previously, our objective was to inves-
tigate in more depth the role of phosphorylation in
hDlg degradation, but the results obtained from exper-
iments using hDlg mutated at different p38c phosphor-
ylation sites or using several kinase inhibitors,
including the general kinase inhibitor staurosporine,
demonstrated that under these conditions hDlg degra-
dation was not regulated by its phosphorylation state.

In addition, we showed that hDlg degradation is
blocked by z-VAD, a general caspase inhibitor, and
this indicates that hyperosmotic shock-induced loss of
hDlg is mediated by caspases during apoptosis. The
caspase-dependent cleavage of many key structural
and regulatory proteins contributes to the typical
morphological changes, including the dismantling of
cell–cell contact, seen during apoptosis. As mentioned
previously, hDlg is a scaffold protein, which has been
implicated in the maintenance of cell polarity and cell
adhesion [4]. We report that hDlg is proteolysed by
caspases during the apoptosis of HeLa (Fig. S3) trig-
gered by hyperosmolarity and also in CaCo-2 cells and
mouse embryonic fibroblasts. However, the mechanism
by which there is a different degree of apoptosis, and
therefore of hDlg degradation, in HeLa cells than in
HEK293 cells is unknown. We speculate that in HeLa
cells prolonged exposure to hypertonicity induces
apoptosis because of a lack of or the dysfunction of
the component(s) needed for the adaptive response of
these cells to stress. For example, it has been described
that, in some cell types, the lack of the restoration of
cell volume after cell shrinkage is associated with the
concomitant appearance of apoptosis [23]. However,
the difference in the degree of apoptosis observed
between HeLa and HEK293 cells may be due to a dif-
ference in the sensitivity of these cells to the strength
0
5
10

15
SorbitolControl
Caspase 3 activity
(Fluor
–1
·min
–1
·mg protein
–1
)
hDlg–D747A
hDlg–D397A
hDlg–WT
None
B
3
4
5
6
7
06
Time after sorbitol release (h)
hDlg–D747A
hDlg–D397A
hDlg–WT
None
Number of attached cell (x10
–5
)
A

Fig. 8. Quantification of cells attached and apoptotic cells after sor-
bitol treatment. (A) Cell attachment and (B) caspase 3 activation
were measured as indicated in Materials and methods, at the times
indicated – (A) 0 and 6 h or (B) 0 and 2 h – in nontransfected and
hDlg wild-type or hDlg–D397A or hDlg–D747A transfected HeLa
cells. All other experimental details are described in the text. Values
are means (± SE) of three independent experiments performed in
triplicate.
Caspase-dependent degradation of hDlg F. A. In˜ esta-Vaquera et al.
396 FEBS Journal 276 (2009) 387–400 ª 2008 The Authors Journal compilation ª 2008 FEBS
of the stimulus, because we found that prolonged
exposure of HEK293 cells to hypertonicity is deadly,
whereas returning the cells to normotonicity after a
brief time (1 h) prevents apoptosis (Feijoo & Cuenda,
unpublished results).
Here we show that caspase 3 and caspase 6 are
strongly activated (12- and 20-fold, respectively) in
HeLa cells exposed to hyperosmotic shock. Caspase 3
appears to act globally, as required for multiple pro-
teolytic events, suggesting that it is the primary exe-
cutioner caspase. By contrast, caspase 6 plays
relatively minor or highly specialized roles during the
execution phase of apoptosis [24]. We observed a
parallel activation of these proteases, the activation
of caspase 3 being transient and the activation of cas-
pase 6 being stronger and sustained over time. In
addition, the length of activation of caspase 6 is simi-
lar to the time course of hDlg degradation, indicating
that this caspase may be responsible for hDlg prote-
olysis under our experimental conditions. However,

we cannot rule out a role for caspase 3 in hDlg
cleavage because some caspase 6 substrates are also
cleaved by other caspases, typically caspase 3,at
other cleavage sites [22]. Further work is required to
establish the identity of the caspase responsible for
hDlg under stressful conditions.
Caspases are among the most specific proteases, with
an unusual and absolute requirement for cleavage after
aspartic acid [22]. Recognition of at least four amino
acids N-terminal to the cleavage site is necessary for
efficient catalysis: caspase 3 recognizes DXXD and
caspase 6 (I ⁄ V ⁄ L)EXD [22,25]. Our findings suggest
that the sequence YEVD
747
is required for caspase
cleavage of hDlg in HeLa cells, as mutation of D747
to Ala prevented hDlg proteolysis by stress, whereas
mutation of other potential caspase-cleavage sites did
not block degradation. It has been suggested that
ternary structural elements may also influence caspase
substrate recognition [26]. This may explain the
presence of the Tyr (Y) in position P4 (at the YEVD
cleavage motif), a residue found in substrates specific
for caspase 1, but never in caspase 3 or caspase 6
substrates [22,25].
Cleavage of hDlg at YEVD
747
would generate a
peptide of  100 kDa, however, we were unable to
detect any hDlg fragment of that size. This may be

because of a lack of recognition by the antibodies used
in this study, but also because of the further proteoly-
sis of the fragment by other caspase(s), because we
have found that at later time points (such as 9 h) the
YEVA
747
mutant also becomes proteolysed (results not
shown) indicating that this site is an early cleavage site
and there are more caspase-cleavage sites in the hDlg
molecule. In addition, the sequence YEVD
747
is
located at the C-terminus of hDlg, more specifically at
the guanylate kinase domain, the region by which
hDlg binds to proteins such as GKAP, which target it
to the cytoskeleton [3]; cleavage at this site would
release hDlg from this cellular compartment. These
findings indicate that when apoptosis is initiated in
HeLa cells exposed to hypertonicity, activation of
caspases causes the proteolysis of hDlg at YEVD
747
and this releases it from the cortical cytoskeleton at
the membrane into the cytoplasm where hDlg may be
further proteolysed.
In immunolocalization experiments we observed
that, after osmotic shock treatment, the more compact
localization of hDlg at the cell–cell contact region is
lost and it is more diffusely localized throughout the
cytoplasm at the vicinity of the membrane. We cannot
exclude the possibility that hDlg phosphorylation may

contribute to its change in cellular localization because
in cells pretreated with the p38MAPKs inhibitor, hDlg
is not phosphorylated [17] and a significant delay in its
release from the vicinity of the membrane to the cyto-
skeleton is observed. These results are in agreement
with previous findings in which phosphorylation of
hDlg, catalysed by p38c, triggers its dissociation from
the scaffold protein GKAP and its release from the
cytoskeleton [3]. However, given that it has been
shown previously that hDlg is associated with the sub-
membranous cytoskeleton at cell–cell contact via an
E-cadherin-induced assembly of the cortical cytoskele-
ton [27], we suggest that the change in hDlg localiza-
tion may be caused by the possible loss of E-cadherin
or other protein(s) in the hDlg complex under stressful
conditions, and the cortical cytoskeleton is therefore
disrupted and hDlg released into the cytoplasm.
The lack of effect of an hDlg mutant, which cannot
be proteolysed at initial apoptosis, on cell detachment
or early apoptosis suggests that hDlg or its cleavage
fragments do not have an active role in apoptotic pro-
gression. However, at later times, this mutant is also
degraded and, given the importance of the hDlg
protein level in several processes, we cannot discount
the possibility that disruption of the scaffolding ability
of hDlg by caspase cleavage may be one of the major
steps in apoptosis, which is the dismantling of the cell–
cell contacts, therefore causing cell–cell detachment
under osmotic stress.
In conclusion, our studies demonstrate that hDlg is

proteolysed in vivo by caspases via a different mecha-
nism to that described previously, which was
proteasome- and phosphorylation-dependent [12,13];
and we show that hDlg needs to be cleaved during the
apoptotic process.
F. A. In˜ esta-Vaquera et al. Caspase-dependent degradation of hDlg
FEBS Journal 276 (2009) 387–400 ª 2008 The Authors Journal compilation ª 2008 FEBS 397
Materials and methods
Antibodies, inhibitors and DNA constructs
All hDlg Ig were generated as described previously [3].
b-tubulin Ig was purchased from Zymed (Cambridge, UK)
and GAPDH Ig from Fitzgerald (Concord, MA, USA).
Other antibodies were as described previously [3,17,28].
SB203580, SP600125, staurosporine and PSI were
obtained from Calbiochem (Nottingham, UK) and z-VAD
from BD Bioscience (San Jose, CA, USA). PD184352 [20]
and BIRB0796 [17] were custom synthesized by N. Shpiro
and R. Ma
´
rquez.
All DNA constructs for expression of hDlg wild-type or
mutated at the p38c phosphorylation residues were gener-
ated as described previously [3]. pCR2.1 hDlg was mutated
at the possible caspase cleavage sies (Asp255, Asp397,
Asp750 and Asp747) using the QuickChange site directed
mutagenesis method using KOD Hot Start DNA Polymer-
ase (Novagen, Darmstadt, Germany). The resulting hDlg
mutations were digested with NotI (NEB, Ipswich, MA,
USA) and ligated into the same site in pEBG-2T.
Cell stimulation, transfection and cell lysis

Experiments in this study were performed using a strain of
easily transfectable HeLa, HEK293 or CaCo-2, cultured as
described previously [2,3]. Cells were incubated in Dul-
becco’s modified Eagle’s medium for 12 h in the absence of
serum before stimulation with 0.5 m sorbitol or UV radia-
tion (200 JÆm
)2
) as indicated, then lysed in lysis buffer as
described previously [2,3]. Lysates were centrifuged at
13 000 g for 10 min at 4 °C, the supernatants were
removed, quick frozen in liquid nitrogen and stored at
)80 °C until use. When required, cells were preincubated
for 2 h with either kinase or protease inhibitors as indicated
in the figures, prior to stimulation with the above-men-
tioned agonists. For transfection experiments, each 6 cm
dish of HeLa cells was transfected with 2–12 lg poly(ethy-
lenimine) (Polysciences, Eppelheim, Germany) and 1–6 lg
of plasmid DNA, as described previously [29]. Cells were
either lysed or stimulated for 24–36 h after transfection.
Immunoblotting, immunoprecipitation and immunofluores-
cence staining were performed as described previously [2,3].
Subcellular fractionation was performed using the
ProteoExtractÔ, subcellular Proteome Extration Kit from
Calbiochem (Nottingham, UK). Immunoblotting with the fol-
lowing antibodies against the indicated marker proteins were
carried out as control: anti-calpain for the cytoplasmic frac-
tion and anti-(insulin receptor) for the membrane fraction.
Proteases activities
Caspases, calpain and cathepsin B activities were deter-
mined using peptide substrates that emit fluorescence once

cleaved by the specific protease. Cytosolic extracts were
obtained from control, sorbitol- or UV-treated cells and the
protease assay was performed at 37 °C in a reaction buffer
containing 20 mm Hepes pH 7.5, 10% (v ⁄ v) glycerol, 2 mm
ditiothreitol, 20 lm fluorogenic peptide substrate (with the
exception of cathepsin B activity measurement where
200 lm of peptide substrate was used).
The substrates used for caspase activities were: N-acetyl-
Tyr-Val-Ala-Asp-7-amino-4-metylcoumarin for caspase 1,
N-acetyl-Asp-Glu-Val-Asp-7-amino-4-metylcoumarin for
caspase 3, N-acetyl-Val-Glu-Ile-Asp-7-amino-4-trifluoro-
metylcoumarin for caspase 6 and N-acetyl-Ile-Glu-Thr-
Asp-7-amino-4-trifluoromethylcoumarin for caspase 8.
7-Amino-4-metylcoumarin formation was monitored using
an k
exc
380 nm and k
em
440 nm and 7-amino-4-trifluorome-
tylcoumarin was monitored using a k
exc
440 nm and k
em
505 nm.
The substrate used for calpain activity was N-succinyl-
Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (k
exc
380 nm
and k
em

440 nm) and the measurements were carried out in
the presence of 100 lm Ca
2+
in the reaction buffer.
The substrate used for assaying cathepsin B activity was
Z-Arg-Arg-7-amino-4-methylcoumarin (k
exc
380 nm and
k
em
440 nm) and the measurements were carried out in the
presence of 0.6 mm Ca
2+
and Mg
2+
in the reaction buffer.
Propidium iodide/annexin V assay
HeLa or HEK293 cells were exposed to UV radiation
(200 JÆm
)2
) or to hyperosmotic shock (0.5 m sorbitol) for
1 h and then released into fresh media for 3 or 14 h before
counting apoptotic cells using an annexin V ⁄ FITC detec-
tion kit (Oncogene, Inc. Boston, MA, USA) according to
manufacturer’s instructions.
Cell adhesion assay
Cells were plated subconfluently (3.5 · 10
5
cells in a 6-cm
dish) and transfected as described above with the plasmid

DNA. Twenty-four hours after transfection, cells were incu-
bated in serum-free medium for a further 12 h, before stim-
ulation with 0.5 m sorbitol (60 min) and then incubated in
sorbitol-free medium (6 h). Detached cells were washed
with NaCl ⁄ P
i
and cells remaining attached to the dish
were treated with trypsin and counted using CasyTon
(Casy-Technology, Innovatis, Bielefeld, Germany).
Protein level quantification was performed by densito-
metry using ChemiDoc XRS system and the program
quantity one from BioRad (Hercules, CA, USA).
Acknowledgements
We thank J. Mateos for technical support, N. Shpiro
and R. Marquez for the synthesis of PD184352 and
Caspase-dependent degradation of hDlg F. A. In˜ esta-Vaquera et al.
398 FEBS Journal 276 (2009) 387–400 ª 2008 The Authors Journal compilation ª 2008 FEBS
BIRB0796, to The Sequencing Service (School of
Life Sciences, University of Dundee, Scotland) for
DNA sequencing, and to the protein production and
antibody purification teams (Division of Signal
Transduction Therapy, University of Dundee), coor-
dinated by Dr H. McLauchlan and J. Hastie, for
generation and purification of antibodies. FAI-V was
supported by fellowship from the Spanish Govern-
ment (Beca FPU, Ministerio de Educacion y Cien-
cia). The work in the author’s laboratory is
supported by the Medical Research Council UK,
pharmaceutical companies that support the Division
of Signal Transduction Therapy (Astra-Zeneka,

Boehringer-Ingelheim, GlaxoSmithKline, Merck &
Co. Inc, Merck KgaA and Pfizer), Ministerio de
Educacion y Ciencia (MEC) Spain (SAF2004-1933)
and (BFU2007-67577), and Junta de Extremadura
(IIPR04A092).
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Supporting information
The following supplementary material is available:
Fig. S1. Exogenous GST–hDlg degradation is medi-
ated by caspases.
Fig. S2. Blockade of different MAPK pathway activa-
tion by different kinase inhibitors.
Fig. S3. Osmotic shock causes a decrease in hDlg levels
in CaCo-2 cells and mouse embryonic fibriblasts.
This supplementary material can be found in the
online version of this article.
Please note: Wiley-Blackwell is not responsible for
the content or functionality of any supplementary
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than missing material) should be directed to the corre-
sponding author for the article.

Caspase-dependent degradation of hDlg F. A. In˜ esta-Vaquera et al.
400 FEBS Journal 276 (2009) 387–400 ª 2008 The Authors Journal compilation ª 2008 FEBS