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
´

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: [email protected]
(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

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

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-

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

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.

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
–+
–

+
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
+
+
+
++

–

(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.

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

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.

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

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

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

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

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)

)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

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

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,

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

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

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

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,

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