Proteins of the transforming growth factor beta family
(TGF?) are involved in the regulation of proliferation,
differentiation, migration and adhesion of most cell types.
TGF?1, which represents the best-studied member of this
family, inhibits the proliferation and induces differentiation
of epithelial cells (Attisano and Wrana, 2002). On the
molecular level, binding of TGF? to type II TGF? receptors
leads to recruitment and transphosphorylation of type I TGF?
receptors and activation of receptor-activated Smad2 and
Smad3, which then hetero-oligomerise with the common
partner Smad4. This complex regulates the transcription of
several target genes [reviewed in (Moustakas and Heldin,
2002)]. Several non-Smad signalling mechanisms have been
described in mediating cellular effects of TGF?. These
include the mitogene-activated protein kinases ERK, JNK
and p38, the phosphatidylinositol 3-kinase (PI3-kinase)
(Bakin et al., 2000) and Ras- and Rho-GTPases (Derynck and
Zhang, 2003). Studies have shown that somatic mutations in
components of the TGF? signalling pathway are associated
with loss of proliferation control, malignant progression,
invasion and metastasis formation both in vitro and in vivo
(Akhurst and Derynck, 2001). In this context, TGF?1was
described to induce morphological, biochemical and
transcriptional changes towards a mesenchymal phenotype
designated as epithelial to mesenchymal transition (EMT)
(Fensterer et al., 2004; Ellenrieder et al., 2001b; Oft et al.,
1998; Boyer et al., 1996). One important feature of EMT is
the dissociation of the E-cadherin adhesion complex.
Cadherin molecules represent a family of calcium-
dependent transmembrane glycoproteins, which build adherens
junctions and contribute to cell-cell adhesion (Takeichi, 1991;
Kemler, 1993; Gumbiner, 1996). The cadherin isoforms
interact with catenins, which mediate cadherin linkage to the
actin cytoskeleton (Kemler, 1993). Either ?- or ?-catenin
(plakoglobin) binds directly to cadherin and ?-catenin, which
links this complex directly or indirectly to the actin-based
cytoskeleton (Hinck et al., 1994; Herrenknecht et al., 1991).
The assembly and maintenance of adherens junctions is under
tight control. Tyrosine phosphorylation of ?-catenin was
shown to be responsible for dissociation of E-cadherin/?-
catenin from ?-catenin and the actin cytoskeleton. The cellular
kinase Src (Behrens et al., 1993; Hamaguchi et al., 1993;
Takeda et al., 1995), as well as FER-kinase (Piedra et al., 2003)
or receptor kinases such as epidermal growth factor receptor
(EGFR) and hepatocyte growth factor receptor (cMET) are
Transforming growth factor beta (TGF? ?) has profound
growth-suppressive effects on normal epithelial cells, but
supports metastasis formation in many tumour types. In
most epithelial tumour cells TGF? ?1treatment results in
epithelial dedifferentiation with reduced cell aggregation
and enhanced cellular migration. Here we show that the
epithelial dedifferentiation, accompanied by dissociation of
the E-cadherin adhesion complex, induced by TGF? ?1
depended on phosphatidylinositol 3-kinase (PI3-kinase)
and the phosphatase PTEN as analysed in PANC-1 and
Smad4-deficient BxPC-3 pancreatic carcinoma cells.
TGF? ?1treatment enhanced tyrosine phosphorylation of ? ?-
and ? ?-catenin, which resulted in dissociation of the E-
cadherin/catenin complex from the actin cytoskeleton and
reduced cell-cell adhesion. The PI3-kinase and PTEN were
found associated with the E-cadherin/catenin complex via
? ?-catenin. TGF? ?1treatment reduced the amount of PTEN
bound to ? ?-catenin and markedly increased the tyrosine
phosphorylation of ? ?-catenin. By contrast, forced
expression of PTEN clearly reduced the TGF? ?1-induced
phosphorylation of ? ?-catenin. The TGF? ?1-induced ? ?-
catenin phosphorylation was also dependent on PI3-kinase
and Ras activity. The described effects of TGF? ?1 were
independent of Smad4, which is homozygous deleted in
BxPC-3 cells. Collectively, these data show that the TGF? ?1-
induced destabilisation of E-cadherin-mediated cell-cell
adhesion involves phosphorylation of ? ?-catenin, which is
regulated by E-cadherin adhesion complex-associated PI3-
kinase and PTEN.
Key words: Transforming growth factor beta, E-cadherin, Cell-cell
adhesion, ?-catenin tyrosine phosphorylation, Epithelial cells,
Pancreatic cancer, Invasion, Cell migration
TGF? ?-induced downregulation of E-cadherin-based
cell-cell adhesion depends on PI3-kinase and PTEN
Roger Vogelmann1,*, Marc-Daniel Nguyen-tat1, Klaudia Giehl2, Guido Adler1, Doris Wedlich3and
1Department of Internal Medicine I, 2Department of Pharmacology and Toxicology, University of Ulm, Robert-Koch-Strasse 8, 89070 Ulm,
3Institute of Zoology II, University of Karlsruhe, 76131 Karlsruhe, Germany
*Present address: Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA 94305, USA
‡Author for correspondence (e-mail: firstname.lastname@example.org)
Accepted 19 July 2005
Journal of Cell Science 118, 4901-4912 Published by The Company of Biologists 2005
Journal of Cell Science
(Hoschuetzky et al., 1994; Birchmeier et al., 1997).
In vivo, protein phosphorylation is a highly reversible and
dynamic process, in which the level of phosphorylation reflects
the sum of protein kinase and protein phosphatase activity.
Several lines of evidence suggest that phosphatases represent
important regulators of E-cadherin-mediated
adhesion. A direct association of different protein tyrosine
phosphatases (PTPs), such as PTP?, PTP?, LAR or SHP-2
with proteins of the E-cadherin/catenin complex has been
described (Brady-Kalnay et al., 1998; Müller et al., 1999).
Inhibition of PTPs results in destabilisation of cellular
adhesion and enhanced migration (Müller et al., 1999; Volberg
et al., 1992) and can be detected in many carcinomas (Streuli,
In this study, we examine the effect of TGF?1on cell-cell
adhesion and cell migration in pancreatic carcinoma cells.
Pancreatic adenocarcinomas belong to the most fatal cancers
because of their extensive invasion into surrounding tissues and
metastases formation (Kern et al., 2001; Poston et al., 1991).
We show that PI3-kinase and the phosphatase PTEN are
associated with the E-cadherin adhesion complex and play an
important role in TGF?1-induced phosphorylation of ?- and ?-
catenin, which results in a decrease of cell-cell adhesion with
a concomitant increase in cell migration.
to contribute to catenin phosphorylation
Materials and Methods
Monoclonal antibodies against E-cadherin (C20820), ?- (C21620)
and ?-catenin (C19220) were obtained from BD Bioscience.
Polyclonal antibodies against ?- (C-2081) and ?-catenin (C-2206) as
well as anti-?-actin (A-4700) antibody were purchased from Sigma-
Aldrich. An antiserum against PTEN (210-774-R100) was purchased
from Alexis Biochemicals. A HRP-coupled phosphotyrosine-specific
antibody was purchased from BD Bioscience (PY20:HRP). Anti-
TGF? receptor type II antibody (AF-241-NA), which inhibits TGF?1
binding, was purchased from R&D Systems. Antibody against
phospho-PTEN (Ser380) and anti-p110? were obtained from Cell
Signaling Technology. Anti-EGFP antibody was purchased from
Rockland. Polyclonal antiserum against the p85 subunit of PI3-kinase
was kindly provided by M. Thelen (Institute for Research in
Biomedicine, Bellinzona, Switzerland).
Human PTEN cDNA was amplified by PCR using cDNA established
from mRNA of PANC-1 cells. PCR was performed using specific
primers for PTEN according to the published sequence (Li et al.,
1997) (sense primer: 3?-ATGACAGCCATCATCAAAGA-5? and
antisense primer: 3?-TTGGATCCTCAGACTTTTGTAATTTG-5?)
with Pfu DNA polymerase (Promega). The PCR product was inserted
as a BamHI/EcoRI-fragment into the pEGFP-C2 vector (BD
Clontech). A C-terminally truncated cDNA of PTEN was produced
by excision of the NheI/XbaI-fragment from the pEGFP/PTEN-
construct. The sequence of individual plasmids was confirmed by
DNA sequencing (GATC-Biotech).
Cell culture, transfection and siRNA
PANC-1 cells were obtained from ATCC (CRL-1469) and BxPC-3
cells were available from ECACC (No. 93120816). Cells were
maintained in Dulbecco’s modified Eagles medium (DMEM)
supplemented with 10% fetal calf serum (Gibco Invitrogen)
containing 1% L-glutamine and 1% non-essential amino acids (PAA
Laboratories). For transfection, cells were treated with 15 ?g DNA
per 100 mm dish and DMRIE-C reagent according to the
manufacturer’s protocol (Gibco Invitrogen). Stably transfected cell
clones were selected by addition of 1.5 mg/ml G418 to growth
medium (PAA Laboratories). PANC-1 cells stably transfected with an
EGFP/H-Ras N17 expression construct were described before
(Fensterer et al., 2004). Inhibitory siRNA for silencing PTEN
expression was purchased from Cell Signaling Technology (#6250).
In 6-well plates PANC-1 cells were transfected with 250 nM siRNA
specific for PTEN or unrelated control sequence and DMRIE-C
transfection reagent. After 36 hours the medium was changed against
DMEM and another 6 hours later TGF?1was added for further 6
hours. Forty eight hours after transfection the cells were lysed in RIPA
buffer supplemented with protein and phosphatase inhibitors.
Human recombinant TGF?1 was used at a concentration of
10 ng/ml (TEBU). Pharmacological inhibitors were used in the
following concentrations: PD98059 (25 ?M), LY294002 (25 ?M),
PP1 (100 ?M) and FTI 277 (2 ?M). FTI 277 was purchased from
Calbiochem, all other inhibitors were from Alexis Biochemical.
SDS gel electrophoresis (SDS-PAGE) was performed according to
standard procedures as described previously (Menke et al., 2001). For
total protein analysis, cells were lysed in RIPA-buffer (50 mM Tris-
HCl pH 7.2, 150 mM NaCl, 1% Triton X-100, 0.5% sodium
deoxycholate, 0.1% SDS). NOP-buffer (10 mM Tris-HCl pH 7.4, 150
mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 1% Nonidet P40, 0.2% Triton
X-100) was used for co-immunoprecipitation experiments.
Immediately before use, the buffers were supplemented with
proteinase inhibitors: 5 ?M aprotinin, 1 mM Pefabloc, 5 ?M soya
trypsine inhibitor (STI; all from Roche Diagnostics). In case of protein
phosphorylation analysis, phosphatase inhibitors were added: 10 mM
sodium pyrophosphate, 25 mM ?-glycerophosphate, 2 mM sodium
orthovanadate (Sigma-Aldrich). Thirty micrograms of lysate were
analysed by SDS-PAGE and transferred onto nitrocellulose
membranes (Schleicher and Schuell). Immunoreactive proteins were
detected with a secondary horseradish peroxidase-coupled antibody
(Pierce) and visualised using enhanced chemiluminescence (ECL,
Pierce). For co-immunocoprecipitation experiments, 0.3-3.0 mg of
NOP-lysates were used for the ?MACS protein isolation system from
Miltenyi Biotec in accordance to manufactures instruction. The
immunoprecipitates were analysed by western blotting as described
To obtain Triton X-100-soluble and -insoluble fractions, cells were
incubated with Triton-lysis-buffer (1% Triton X-100, 0.3 M sucrose,
25 mM HEPES pH 7.4, 100 mM NaCl, 4.7 mM KCl, 1.2 mM
KH2PO4, 1.2 mM MgCl2, 5 ?M aprotinin, 1 mM Pefabloc, 5 ?M STI)
for 15 minutes on a rocking platform. After centrifugation, the
supernatant (Triton-soluble fraction) was collected. The Triton-
insoluble fraction was resuspended in SDS-lysis-buffer (20 mM Tris-
HCl pH 7.5, 2.5 mM EDTA, 1% SDS, 0.5 ?g/ml DNase I, 1 mM
Pefabloc, 5 ?M STI, 5 ?M aprotinin). Both fractions were
reconstituted to equal volumes and 15 ?l of each fraction were
analysed by western blotting.
Membrane preparations were carried out as described previously
(Lutz and Miller, 1993) with few modifications. Cells were scraped
into hypotonic buffer (10 mM Tris-HCl pH 7.4, 1 mM MgCl2, 5 ?M
aprotinin, 1 mM Pefabloc, 0.1 mM STI, 10 ?M pepstatin, 10 ?M
leupeptin) and mechanically broken using a Dounce homogenisator
(Braun). Nuclei were removed by centrifugation at 10,000 g for 30
seconds. Sodium chloride and saccharose were added to a final
concentration of 0.2 M and 0.3 M, respectively. Cytosolic and
membrane fractions were separated by ultracentrifugation for 1 hour
at 4°C and 150,000 g in a swinging bucket rotor. Membranes were
recovered from the interphase by aspiration, diluted with an equal
Journal of Cell Science 118 (20)
Journal of Cell Science
TGF? induces E-cadherin complex disassembly
volume of NOP buffer, and collected by centrifugation at 100,000 g
for 30 minutes at 4°C. The pellets were resuspended in NOP buffer.
For immunoprecipitation experiments 0.5 mg of membrane fraction
Protein phosphatase assays
PTEN dephosphorylation of ?-catenin was examined using anin vitro
phosphatase assay as described (Gu et al., 1999). Briefly, EGFP, WT
PTEN or PTEN?C were isolated from 1 mg of pEGFP, pEGFP/PTEN
or pEGFP/PTEN?C transfected PANC-1 cells lysed in RIPA buffer
with enhanced amounts of detergents to ensure dissociation of
associated proteins (50 mM Tris-HCl pH 7.2, 450 mM NaCl, 1.0%
Triton X-100, 1.0% sodium deoxycholate, 0.5% SDS). The proteins
were purified by immunoprecipitation with an anti-EGFP antibody as
described above and renatured by washing with renaturation buffer
(PBS pH 7.0 containing 2 mM MgCl2, 0.5 mM PMSF, 0.005% Tween
20, 10 mM DTT, 1 mM pefabloc, 5 ?M STI, 5 ?M aprotinin). Purity
was confirmed by SDS-PAGE and Coomassie blue staining.
Phosphorylated ?-catenin was obtained from BxPC-3 cell that had
been stimulated with EGF (10 ng/ml for 15 minutes) and 2 mM
sodium orthovanadate by immunoprecipitation from 0.5 mg of RIPA
lysate. PTEN phosphatase activity against ?-catenin was examined by
incubation of immunoprecipitated tyrosine phosphorylated ?-catenin
with immunoprecipitated EGFP, WT PTEN or PTEN?C in 30 ?l of
50 mM Tris buffer pH 7.0 containing 50 mM NaCl and 10 mM DTT
at 30°C for 30 minutes. Controls were incubated with
immunoprecipitates obtained without PTEN antibody. The reaction
was terminated by adding reducing SDS sample buffer and heating at
100°C for 5 minutes. The samples were fractionated by SDS-PAGE
Cells used for immunostaining were fixed and permeabilised in cold
acetone-methanol (1:1) for 15 minutes. Primary antibodies were
incubated for 1 hour at 37°C and visualised by incubation with a
secondary Cy-3- (Biomol) or Alexa-488-conjugated antibody
(Molecular Probes). Staining was examined by fluorescence
microscopy (DM RBE, Leica-micosystems) and images were
recorded using a CCD camera and analySIS 3.1-software (Soft-
Imaging System) or confocal laser microscopy (TCS-4, Leica-
Migration and aggregation studies
Migration assays were performed using cell culture inserts with
uncoated porous membranes or membranes coated with collagen type
I (8.0 ?m pore size, BD Bioscience) as described in (Giehl et al.,
2000b). To exclude TGF?1effects caused by cell proliferation, cell
growth was inhibited by mitomycin C-treatment (10 ?g/ml for 1
hour). After 48 hours of incubation with 20 ng/ml TGF?1or solvent,
the number of cells that had migrated through the pores towards
TGF?1was quantified by counting five independent visual fields in
the microscope (Zeiss) using a 20? objective. Three independent
assays were performed in triplicates.
To determine the Ca2+-dependent cell-cell adhesion, cell
aggregation was determined as described by Ozawa and Kemler
(Ozawa and Kemler, 1990). Cells were carefully detached with 0.01%
trypsin in HEPES-buffered saline (37 mM NaCl, 5.4 mM KCl, 0.34
mM NaH2PO4, 5.6 mM Glucose, 10 mM HEPES) containing 2 mM
CaCl2, centrifuged, washed twice with HEPES buffer with 2 mM
CaCl2and resuspended in the same buffer. Cells were singularised
using a pasteur pipette until no aggregates were monitored and were
allowed to aggregate by incubation with constant rotation of 70 rpm
for 30 minutes. Afterwards, the number of aggregates was determined
in an invert phase-contrast microscope (objective 10?; Zeiss). The
extent of cell aggregation was calculated by the formula
A=(No–Ne)/No, with Norepresenting the total particle number at the
start and Nethe total particle number after incubation for 30 minutes.
To examine the calcium dependency, EDTA and EGTA were added
to a final concentration of 5 mM each. E-cadherin dependence was
proved by addition of neutralising antibody against E-cadherin
DECMA-1 (Sigma) to a final concentration of 4 ?g/ml assay medium.
To remove preservatives of the antisera the solutions were replaced
by TBS using centricon centrifugation columns (Filtron,
Northborough, MA, USA). Three independent assays were performed
The mean values and s.e.m. were calculated from at least three
experiments, each performed in triplicate or quadruplicate. For
statistical analysis, the Wilcoxon non-parametric test was used and
P<0.02 was considered significant.
TGF?1treatment (10 ng/ml) of the pancreatic carcinoma cell
lines PANC-1 and BxPC-3 caused a dramatic change in cell
morphology from a more epithelial-like to a spindle-shaped
cell morphology typical for mesenchymal cells (Fig. 1A).
PANC-1 and BxPC-3 cells scattered with a loss of cell-cell
contacts typically observed in mesenchymal cells. Because E-
cadherin-mediated cellular adhesion contributes substantially
to epithelial cell morphology, we examined the impact of
TGF?1treatment on the cellular distribution of E-cadherin as
demonstrated in Fig. 1B. Control PANC-1 and BxPC-3 cells
incubated with the solvent only showed a typical E-cadherin
staining at membranes localised at the site of cell-cell contacts.
By contrast, TGF?1treatment resulted in a loss of E-cadherin
in membranes of contacting cells (Fig. 1B). In BxPC-3 cells
the E-cadherin immunofluorescence signal was redistributed
over the whole cell surface and into the cytoplasm. The
observed changes are independent of Smad4, which is
homozygous deleted in BxPC-3 cells. In PANC-1 cells, E-
cadherin was internalised on TGF?1treatment into punctuate
structures. Total protein lysates of TGF?1-treated PANC-1 and
BxPC-3 cells were analysed regarding protein levels of E-
cadherin, ?- and ?-catenin. As shown in Fig. 1C, E-cadherin
and ?-catenin total protein concentrations decreased after
TGF?1-stimulation in PANC-1 cells, whereas BxPC-3 cells
exhibited only a slight decrease in E-cadherin, ?- and ?-catenin
TGF?1treatment of PANC-1 and BxPC-3 cells reduces
cell aggregation and enhances cell migration
To analyse whether reduction of E-cadherin induced by TGF?1
alters cell-cell adhesion in pancreatic carcinoma cells, we
performed aggregation assays in the presence and absence of
TGF?1. As illustrated in Fig. 2A, both analysed cell lines
showed cellular aggregation under control conditions. In
PANC-1 cells, application of TGF?1reduced the diameter of
aggregates to 51% (±11%) compared with controls. The
additional incubation of TGF?1-treated cells with a
neutralising antibody against E-cadherin does not further
reduce cell aggregation (47.8±7.7%), indicating the
dependence of cell aggregation on E-cadherin. The diameter of
cell aggregates in TGF?1-treated cells is only slightly more
Journal of Cell Science
then the size of cell clusters after the removal of Ca2+
ions using EDTA/EGTA (30±9% compared with
untreated control cells), demonstrating that the TGF?1
effects most but not all of E-cadherin depend cell
aggregation. Similar ratios were observed for BxPC-3
(TGF?1: 48±12.2%, TGF?1 + neutralising antibody:
42.2±7.2%, EDTA/EGTA: 36.3±10.4%) (Fig. 2A).
In addition to the observed decrease in cell
aggregation, we asked whether TGF?1influences the
migratory behaviour of pancreatic carcinoma cells.
Cell migration was analysed using transwell
migration assays with collagen type I-coated or
uncoated porous membranes. As shown in Fig. 2B,
PANC-1 and BxPC-3 cells showed a threefold
increase in cell migration towards the TGF?1-
containing compartment compared with solvent-
containing wells. Interestingly, invasion of BxPC-3
cells was blocked by a collagen type I matrix. In a paper by
Ellenrieder et al. it has been shown that TGF? is able to
induce expression of metalloproteinase-2 (MMP-2) and the
urokinase plasminogen activator (uPA) system in a Smad4-
dependent way (Ellenrieder et al., 2001a). Thus, one might
speculate that the failure of Smad4-dependent gene
expression in BxPC-3 cells contributes to the reduced
invasion through collagen type I in BxPC-3 cells harbouring
a homozygous deletion in the Smad4 gene. PANC-1 cells,
which have Smad4 protein, were able to invade the collagen
type I matrix towards TGF?1 (2.5-fold) suggesting that
TGF?1-induced migration of pancreatic carcinoma cells is
independent of Smad4 signalling.
TGF?1induces dissociation of the E-cadherin/catenin
Next, we asked for the effect TGF?1 has on E-cadherin-
mediated cellular adhesion on a molecular level. As an
indicator, how much E-cadherin/catenin is associated with the
actin cytoskeleton, we analysed the E-cadherin/catenin
distribution in Triton X-100 soluble and insoluble fractions in
TGF?1-treated and untreated PANC-1 and BxPC-3 cells. As
shown in Fig. 3A, the amounts of E-cadherin, ?- and ?-catenin
were reduced in Triton-insoluble fractions already 30 minutes
after addition of TGF?1and were further decreasing in the
next 48 hours, indicating that the E-cadherin adhesion
complex was less associated with the actin cytoskeleton. In
the Triton-soluble fractions, the amount of E-cadherin
remained constant in PANC-1 cells and was only slightly
reduced in BxPC-3 after TGF?1 treatment (Fig. 3A). The
amounts of ?- and ?-catenin were also reduced in the Triton-
Journal of Cell Science 118 (20)
Fig. 1. PANC-1 and BxPC-3 cells were serum-starved for
24 hours and treated with solvent (–TGF?) or 10 ng/ml
TGF?1(+TGF?1) for 2 days. (A) The left panel shows
phase contrast pictures. (B) The right panel shows
immunofluorescence staining of E-cadherin. Bars, 20 ?m.
(C) Western blot analyses of E-cadherin, ?- and ?-catenin
in total lysates of PANC-1 and BxPC-3 cells treated with 10
ng/ml TGF?1for the indicated periods of time. Equal
loading was demonstrated by staining of ?-actin. Molecular
mass standards are given in Mr?103. Representative blots
out of four independent experiments are shown.
Fig. 2. (A) Cell aggregation assays were performed by incubation of
pancreatic carcinoma cells PANC-1 and BxPC-3 under constant
agitation in HEPES buffer plus CaCl2supplemented with solvent,
TGF?1(10 ng/ml), EDTA/EGTA (5 mM each) or TGF?1(10 ng/ml)
+ E-cadherin neutralising antibody (nAB=DECMA1, 4 ?g/ml). The
aggregation index was determined by A=(No–Ne)/No, Norepresents
the total particle number before and Nethe particle number after 30
minutes of incubation with constant rotation at 70 rpm. Mean values
±s.e.m. are shown of three independent experiments. (B) Cell
migration of PANC-1 and BxPC-3 was analysed using uncoated or
collagen type I-coated transwell cell culture inserts with 8 ?m pores.
After inhibition of cell proliferation by treatment with 10 ?g/ml
mitomycin C, 20 ng/ml TGF?1or solvent were added to the lower
compartment. After 48 hours of incubation the number of cells,
which had migrated through the pores, was estimated by counting 5
independent visual fields. Three independent assays were performed
in triplicate. Mean values ±s.e.m. are shown.
Journal of Cell Science
TGF? induces E-cadherin complex disassembly
soluble fraction of PANC-1 and BxPC-3 cells 30 minutes after
addition of TGF?1(Fig. 3A).
To further support the observation that TGF?1 treatment
causes a dissociation of the E-cadherin/catenin complex, we
immunoprecipitated E-cadherin in TGF?1-treated and control
cells. TGF?1treatment of PANC-1 cells for 6 hours resulted
in a dissociation of ?- and ?-catenin from the E-cadherin
complex (Fig. 3B).
PI3-kinase mediates TGF?1-induced disassembly of the
To determine the signalling pathways involved in TGF?1-
induced dissociation of the E-cadherin complex from the actin
cytoskeleton, we used pharmacological inhibitors of different
signalling molecules, which might be involved in this process.
In PANC-1 cells, only the phosphoinositide 3-kinase (PI3-
kinase) inhibitor LY294002 (25 ?M) caused significant
reduction of TGF?1-induced dissociation of ?- and ?-catenin
from E-cadherin (Fig. 3B). By contrast, inhibition of the
cellular kinase Src (100 ?M PP1) as well as inhibition of
MEK-1 (25 ?M PD98059) did not show any effect on TGF?1-
induced disassembly of the E-cadherin/catenin complex.
Treatment of cells with inhibitors alone had no effect on the
amount of co-immunoprecipitated ?- and ?-catenin (Fig. 3B).
To analyse whether PI3-kinase activity is necessary for
TGF?1-induced cell migration, we performed transwell
migration assays in the presence of the PI3-kinase inhibitor
LY294002 using uncoated inserts. As shown in Fig. 4A, 25 ?M
LY294002 completely inhibited TGF?1-induced migration of
PANC-1 and BxPC-3 cells. Inhibition of MEK-1-activity (25
?M PD98059) reduced TGF?1-induced cell migration but to a
smaller extent than the LY294002 compound. In contrast to the
PI3-kinase inhibitor LY294002, the MEK-1 inhibitor PD98059
also reduced the spontaneous migration rate of control cells.
Because TGF?1-induced cell migration was highly
dependent on PI3-kinase activity, we asked if PI3-kinase was
associated with the E-cadherin/catenin complex in pancreatic
carcinoma cells and if TGF?1may modulate this association.
By immunofluorescence studies, the p85? subunit of PI3-
kinase is localised to the cytoplasm of unstimulated PANC-1
cells (Fig. 4D). TGF?1treatment induced an enrichment of
PI3-kinase staining in areas of cell-cell contacts at the
membrane where the E-cadherin/catenin complex is localised.
Co-immunoprecipitation studies using ?-catenin antibodies for
the precipitation demonstrated that the p85? subunit of PI3-
kinase was associated with the E-cadherin/catenin complex in
TGF?1-treated and untreated PANC-1 and BxPC-3 cells (Fig.
4B). As expected from our E-cadherin co-immunoprecipitation
data, ?-catenin dissociated from ?-catenin 6-48 hours after
addition of TGF?1(Fig. 4B). In reverse, ?-catenin protein can
be detected when immunoprecipitating with p85? antibodies
further strengthening our observation that p85? is in a complex
with the E-cadherin/catenin complex (data not shown). In this
experiment, we used more stringent conditions by increasing
the concentration of detergents in the lysis buffer, which causes
a greater dissociation of protein complexes. Under these
conditions, neither E-cadherin nor ?-catenin can be co-
immunoprecipitated with p85?. These data suggest that ?-
catenin is important for the association of p85? with the E-
cadherin/catenin complex. Furthermore, we were able to show
Fig. 3. Triton-soluble and -insoluble protein fractions were analysed
regarding their amounts of E-cadherin, ?- and ?-catenin in PANC-1
and BxPC-3 cells after treatment with TGF?1(10 ng/ml) or solvent
for the time points indicated (A). Equal amounts of each fraction
were separated by SDS-PAGE and blotted onto nitrocellulose. E-
cadherin, ?- and ?-catenin were detected by immunostaining. The ?-
actin concentration served as control to prove equal loading.
Representative blots out of four independent experiments are shown.
(B) E-cadherin was precipitated from lysate of TGF?1-stimulated or
unstimulated PANC-1 cells and coprecipitated ?- and ?-catenin was
analysed by western blotting. After serum starvation for 24 hours,
cells were incubated for 90 minutes either with 100 ?M PP1 to
inhibit Src-kinase, 25 ?M PD98059 to inhibit MEK-1 or with 25 ?M
LY294002 to inhibit PI3-kinase and stimulated with 10 ng/ml TGF?1
or solvent for additional 6 hours. E-cadherin was precipitated from 1
mg of NOP lysate. The amount of co-immunoprecipitated ?- and ?-
catenin was examined by immunoblotting. The blots were restained
for E-cadherin to document equal amounts of precipitated protein.
Three independent experiments were performed.
Journal of Cell Science
that the enzymatic subunit of PI3-kinase p110? associated with
?-catenin as well (Fig. 4C).
PI3-kinase mediates TGF?1-induced ?- and ?-catenin
Tyrosine phosphorylation of catenin proteins is important for
regulation of E-cadherin complex assembly and disassembly
(Behrens et al., 1993). We analysed tyrosine phosphorylation
of ?- and ?-catenin after addition of TGF?1to PANC-1 and
BxPC-3 cells. To detect changes of tyrosine phosphorylation
levels of functional, membrane-associated catenins, ?- and ?-
catenins were immunoprecipitated from purified membrane
fractions of TGF?1-treated and solvent-treated control cells.
phosphotyrosine specific antibody. As shown in Fig. 5A,
tyrosine phosphorylation of ?-catenin increased continuously
from 30 minutes up to 6 hours and remained constant for at
least 48 hours in both PANC-1 and BxPC-3 cells. The ?-
catenin phosphorylation increased after 6 h of TGF?1
application in both cell lines and reached a maximum after 12
hours in PANC-1 cells and 48 hours in BxPC-3 cells,
was detected using a
respectively. E-cadherin phosphorylation was hardly detectable
in both cell lines after addition of TGF?1(data not shown). The
kinetics of catenin phosphorylation shown here were in
accordance with the observed disassembly of the E-
cadherin/catenin complex shown in Fig. 3.
Next we examined the role of PI3-kinase activity in TGF?1-
induced catenin phosphorylation. Immunoprecipitated ?- and
?-catenins were analysed for their level of tyrosine
phosphorylation in the presence or absence of the PI3-kinase
inhibitor LY294002 in TGF?1-treated and control cells. The
results presented in Fig. 5B show a marked inhibition of ?-
catenin phosphorylation in cells treated simultaneously with
TGF?1and LY294002, in contrast to cells treated with TGF?1
only or TGF?1in combination with the Src inhibitor PP1.
TGF?1-induced phosphorylation of ?-catenin was also
dependent on PI3-kinase activity as demonstrated in Fig. 5C.
TGF?-induced catenin phosphorylation requires Ras
The TGF?1-induced phosphorylation of ?- and ?-catenin in
BxPC-3 cells was independent of Smad4, which is
Journal of Cell Science 118 (20)
Fig. 4. (A) Transwell migration assays
were performed as described before.
The inhibitors LY294002 (25 ?M) or
PD98059 (25 ?M) were added to the
upper compartment. TGF?1(20 ng/ml)
or solvent was added to the lower
chamber. The number of migrated cells
was estimated after 48 hours of
incubation. Mean values ±s.d. of one
representative assay are shown out of
three independent experiments.
(B) The amount of PI3-kinase
associated with the E-cadherin
complex was analysed by co-
immunoprecipitation. Beta catenin was
precipitated from lysates of PANC-1 or
BxPC-3 cells treated for 30 minutes, 6
hours or 48 hours with TGF?1. The
amount of co-precipitated p85? was
analysed by western blotting. (C) The
amount of p110? co-precipitated with
?-catenin was analysed in PANC-1 or
BxPC-3 lysates. Beta-catenin was
precipitated from 2 mg of PANC-1 or 1
mg of BxPC-3 NOP lysate treated with
10 ng/ml TGF?1or solvent. One
representative blot out of three is
shown. (D) Immunolocalisation of
p85? was performed in PANC-1 cells
treated with 10 ng/ml TGF?1or
solvent (–TGF?1) for 3 hours. P85?
localisation was analysed with
confocal laser microscopy. Bar, 20 ?m.
Journal of Cell Science
TGF? induces E-cadherin complex disassembly
homozygous deleted in this cell line. We and others have
shown before that TGF?1stimulation of pancreatic carcinoma
cells resulted in the activation of the GTPase Ras (Giehl et al.,
2000a; Ellenrieder et al., 2001a; Fensterer et al., 2004; Mulder,
2000). It is well established that Ras-GTPases are important
regulators of PI3-kinase activity (Bar-Sagi and Hall, 2000;
Giehl, 2005). To test whether the observed effect of PI3-kinase
on TGF?1induced catenin phosphorylation depends on Ras
activity, we incubated PANC-1
farnesyltransferase inhibitor FTI 277 (2 ?M) prior to TGF?1
treatment to inhibit Ras
phosphorylation of ?-catenin was clearly reduced by FTI 277
(Fig. 5D). Furthermore, PANC-1 cells stably expressing
dominant-negative EGFP/H-Ras N17 did not show enhanced
tyrosine phosphorylation of ?-catenin after TGF?1stimulation
(Fig. 5D). These data suggest a role of Ras in TGF?1-induced
activation of PI3-kinase and in the regulation of the E-
cadherin/catenin adhesion complex assembly. The importance
of Ras in TGF?1-induced activation of PI3-kinase was
confirmed by the localisation of PI3-kinase in TGF?1-
stimulated EGFP/H-Ras N17 expressing PANC-1 cells. Fig. 5E
shows that treatment of PANC-1 cells stably expressing
EGFP/H-Ras N17 with TGF?1did not result in translocation
of p85? to sites of cell-cell contacts as demonstrated in control
cells (left column, compare also Fig. 4D).
cells with the
The phosphatase PTEN is associated with ?-catenin
and dephosphorylates ?-catenin
The phosphatase PTEN is a known regulator of PI3-kinase
signalling and has been described to be involved in stabilising
adherens junctions (Kotelevets et al., 2001). To analyse if
PTEN is involved in TGF?1-regulated E-cadherin/catenin
complex disassembly, we immunoprecipitated E-cadherin as
well as ?- and ?-catenin from RIPA cell lysates and examined
the amount of PTEN precipitated with E-cadherin, ?- and ?-
catenin. Under the stringent conditions of RIPA cell lysates,
indirectly associated proteins dissociate from the E-
cadherin/catenin complex, but protein interactions of directly
associated proteins are preserved. PTEN was primarily
Fig. 5. (A) Phosphorylation of ?- and ?-catenin after
TGF?1treatment (10 ng/ml) for 30 minutes, 6 hours
and 48 hours for BxPC-3 and additionally after 12
hours for PANC-1 cells was determined by
immunoprecipitation of the individual proteins from
isolated membrane fractions. Phosphorylated tyrosine
was detected using a phosphotyrosine-specific
antibody. Blots were restained with antibodies used for
immunoprecipitation to document equal amounts of
precipitated proteins. Representative blots out of
three independent experiments are shown.
(B) Phosphorylation of ?-catenin or (C) ?-catenin was
examined after treatment of PANC-1 cells with
TGF?1, the PI3-kinase inhibitor LY294002 or the Src
inhibitor PP1 (only for ?-catenin phosphorylation).
Beta-catenin or ?-catenin was precipitated from 1 mg
of PANC-1 RIPA-lysates treated with TGF?1, TGF?1
plus PP1 or TGF?1plus LY294002. Blots were
restained for ?- or ?-catenin to document equal
amounts of precipitated protein. (D) TGF?1-induced
?-catenin phosphorylation was analysed in the
presence of the farnesyltransferase inhibitor FTI 277
(2 ?M) to inhibit Ras activity. In addition PANC-1
cells, which stably expressed EGFP/H-Ras N17, were
analysed for ?-catenin phosphorylation in response to
10 ng/ml TGF?1or solvent. Beta-catenin
immunoprecipitated from 1 mg of PANC-1 RIPA
lysate was analysed regarding its phosphorylation by
western blotting with a phospho-specific antibody.
Equal amounts of ?-catenin were documented by
restaining the blots with ?-catenin antibody.
Representative blots are shown (n=3). (E) In addition
to the pictures shown in Fig. 4D, immunolocalisation
of p85? was performed in PANC-1 cells stably
expressing EGFP/H-Ras N17 treated with TGF?1or
solvent (–TGF?1) for 3 hours and analysed with
confocal laser microscopy. Bar, 20 ?m.
Journal of Cell Science
detected in ?-catenin precipitates and to a smaller extent in ?-
catenin precipitates, but no PTEN was found in association
with E-cadherin (Fig. 6A). Treatment of PANC-1 and BxPC-3
cells with TGF?1for 6 hours markedly reduced the amount of
PTEN bound to ?-catenin (Fig. 6B), whereas the small amount
of PTEN associated with ?-catenin was independent of TGF?1
treatment (data not shown). The total amount of PTEN protein
in lysates of PANC-1 and BxPC-3 cells did not change upon
TGF?1treatment (Fig. 6B).
To clarify the role of PTEN in TGF?1-induced E-
cadherin/catenin complex disassembly, we transiently
transfected PANC-1 cells with expression plasmids for wild
type PTEN (PTEN WT) or with a C-terminal deletion mutant
of PTEN (PTEN?C), which is assumed to be inactive in its
phosphatase function (Tolkacheva
Raftopoulou et al., 2004). As documented in Fig. 6C, the
TGF?1-induced decrease of ?- and ?-catenin proteins co-
immunoprecipitating with E-cadherin was inhibited in cells
expressing wild type PTEN, whereas the phosphatase inactive
form, PTEN?C, had minimal effect on TGF?1-induced
changes on the E-cadherin/catenin protein complex.
and Chan, 2000;
TGF?1-induced dissociation of PTEN from the E-
cadherin/catenin complex correlates with enhanced
In addition to the role of PTEN in E-cadherin/catenin
complex assembly, we examined
phosphorylation of ?-catenin was altered in PTEN-
transfected cells. The TGF?1-induced phosphorylation of ?-
catenin was diminished after transient transfection of wild
type PTEN in PANC-1 cells, whereas only minor changes in
?-catenin-phosphorylation were detectable after expression
of the phosphatase inactive mutant PTEN?C (Fig. 7A). To
determine if PTEN can directly dephosphorylate ?-catenin,
we established an in vitro phosphatase assay. Incubation of
immunoprecipitated EGFP-PTEN and the mutant form
EGFP-PTEN?C with tyrosine phosphorylated ?-catenin
revealed that PTEN, but not PTEN?C, could dephosphorylate
?-catenin at tyrosine residues in vitro (Fig. 7B). To confirm
the role of PTEN in catenin dephosphorylation, we
transfected PANC-1 cells with siRNA against PTEN. The
protein level of PTEN was nearly abolished in siRNA treated
cells as shown in two independent experiments, which
phosphorylation (Fig. 7C). In addition,
PANC-1 cells transfected with PTEN siRNA
demonstrated in Fig. 7C, TGF? did not
increase ?-catenin phosphorylation, which
was already enhanced by knock down of
The regulation of the phosphatase PTEN is
mainly unknown, some studies suggest that
the activity of PTEN is modulated by
regulating total protein levels of PTEN by
unphosphorylated protein (Birle et al., 2002).
Using an antibody specific for PTEN
phosphorylated at Ser380, we showed that
TGF?1treatment of PANC-1 cells resulted in
a significant reduction of phosphorylated
PTEN co-immunoprecipitating with ?-
phosphorylation was not significantly altered
by TGF?1(data not shown), probable due to
the huge amount of PTEN in PANC-1 cells.
The inhibition of TGF?1-induced Ras
activation by the incubation of the cells with
the farnesyltransferase inhibitor FTI277 did
not suppress the TGF?1 effect on the
phosphorylation of ?-catenin associated
PTEN (Fig. 7D). Also the PI3-kinase
inhibitor LY294002 was not able to inhibit
the TGF?1-induced modification of PTEN
phosphorylation in PANC-1 cells (data not
shown). Together with our observation that
total PTEN protein levels do not change
upon TGF?1treatment (see Fig. 6B), these
data indicate that TGF?1indirectly induces
PTEN dissociation from the E-cadherin
complex which correlates with reduced
PTEN phosphorylation and PTEN protein
with TGF?. As
degradation of the
7D). The total PTEN
Journal of Cell Science 118 (20)
Fig. 6. (A) Immunoprecipitation of E-cadherin, ?- or ?-catenin was performed from
500 ?g of PANC-1 RIPA lysate and PTEN was detected by immunoblotting. Equal
amounts of immunoprecipitated proteins were documented by restaining the blots
with the appropriated antibody. (B) Beta-catenin was precipitated from 2 mg of
BxPC-3 or PANC-1 lysates treated with 10 ng/ml TGF?1or solvent. Co-precipitated
PTEN was detected by western blotting. The blots were restained with anti-?-catenin
antibody to demonstrate equal amounts of protein. (C) E-cadherin was precipitated
from 0.5 mg of total lysate from PANC-1 cells transiently transfected with PTEN,
PTEN?C or vector alone (mock). Co-precipitated ?- and ?-catenin was determined
by western blotting. Restaining of the blot for E-cadherin confirmed equal amounts
of precipitated proteins. In all experiments representative blots out of three
independent studies are shown.
Journal of Cell Science
TGF? induces E-cadherin complex disassembly
Only little is known how TGF?1 affects E-cadherin/catenin
mediated cell-cell adhesion in tumour cells. Here we
demonstrate that PI3-kinase and the phosphatase PTEN
mediates the TGF?1induced decrease of E-cadherin mediated
cell-cell adhesion by phosphorylation of ?-catenin in pancreatic
carcinoma cells and that this effect is independent of the Smad
pathway. PI3-kinase was found in association with the E-
cadherin adhesion complex via ?-catenin. Whereas TGF?1
treatment did not alter the association of PI3-kinase with the
complex, PI3-kinase activity induced by TGF?1and probably
mediated by the GTPase Ras was necessary for tyrosine
phosphorylation of ?-catenin and to a minor extent of ?-catenin.
We could show for the first time that the phosphatase PTEN is
also associated with ?-catenin in the E-cadherin adhesion
complex and directly dephosphorylates ?-catenin. PTEN
disassociates from ?-catenin upon TGF?1 treatment, which
correlates with an increase in ?-catenin phosphorylation. As a
consequence of these modifications, the E-cadherin/catenin
complex disassembles and detaches from the actin cytoskeleton.
E-cadherin plays an important role in tumourigenesis and
metastasis formation (Birchmeier et al., 1996). Expressing
wild-type E-cadherin in tumourigenic pancreatic ?-cells in a
transgenic mouse model, progression from adenoma to
carcinoma could be inhibited (Perl et al., 1998). We have
shown before that the expression of E-cadherin in E-cadherin-
negative pancreatic carcinoma cells restored cellular
aggregation and reduced the metastatic potential of these E-
cadherin-transfected cells compared with parental cells (Seidel
et al., 2004). In our study, TGF?1 treatment of pancreatic
tumour cells did not only disassemble the E-cadherin/catenin
complex, but concomitantly decreased cell-cell adhesion and
increased cell migration.
Recently Tian and Phillips (Tian and Phillips, 2002)
demonstrated that TGF?1 treatment resulted in a direct
association of TGF? receptor type II with E-cadherin and ?-
catenin, enhanced ?-catenin tyrosine phosphorylation and
reduced E-cadherin/catenin complex assembly in a non-tumour
renal cell line (Tian and Phillips, 2002). In addition, the authors
showed an increase of ?-catenin associated with Smad3 and
Smad4 in cells treated with TGF?1. However, the proteins,
which phosphorylate ?-catenin at tyrosine residues, remained
unknown. In our study, we demonstrate for the first time that
the TGF?1-induced tyrosine phosphorylation takes place in
Smad4-deficient cells as well (BxPC-3), which shows that the
TGF?1effects described here are independent of Smad4.
Although important cellular effects of TGF?1are transmitted
by the Smad pathway, some Smad-independent effects have
been described, such as activation of RhoA, Ras and ERK, p38
MAPK or PI3-kinase (Imamichi et al., 2005; Mulder, 2000;
Bhowmick et al., 2001; Bakin et al., 2002). Whereas the
phosphorylation of ?-catenin is Smad4-independent in
pancreatic cancer cells, our data show that invasion of a
collagen matrix depends on Smad4. The expression of collagen
degrading proteases requires Smad4-induced gene transcription
(Ellenrieder et al., 2001a), suggesting the cooperation of two
separate TGF?-regulated signalling pathways for cellular
invasion through extracellular matrix. However, we cannot
exclude that other signalling mechanisms that are potentially
defect in BxPC-3 cells may also contribute to the observed
difference in cell migration and invasion.
The impact of ?-catenin phosphorylation on regulation of
the E-cadherin complex assembly is well known and was
demonstrated in different studies (Behrens et al., 1993;
Hamaguchi et al., 1993; Hoschuetzky et al., 1994). In
agreement with Tian and Phillips (Tian and Phillips, 2002), the
data shown here demonstrate
phosphorylation after TGF? treatment. In the present study, we
provide evidence that PI3-kinase and the phosphatase PTEN
Fig. 7. (A) Beta-catenin was immunoprecipitated from 1 mg of
PANC-1 lysate of cells transfected with pEGFP, pEGFP-PTEN or
pEGFP-PTEN?C, treated with 10 ng/ml TGF?1or solvent for 6
hours and analysed for its tyrosine phosphorylation. The blot was
restained for ?-catenin to demonstrate equal amounts of protein.
(B) For an in vitro phosphatase assay, tyrosine phosphorylated ?-
catenin was incubated for 30 minutes at 30°C with EGFP-PTEN-
constructs, which were immunoprecipitated with anti-EGFP
antibody. The amount of phosphorylated ?-catenin was analysed by
western blotting. Restaining of the blots with anti-?-catenin antibody
revealed equal amounts of phosphatase substrate and restaining with
anti-PTEN documents the presence of both EGFP-tagged PTEN
proteins. Representative assays out of four independent experiments
are shown. (C) Beta-catenin was immunoprecipitated from 1 mg of
lysate from PANC-1 cells transfected with siRNA for PTEN and
treated with TGF?1(10 ng/ml) or solvent (two independent
experiments each) or an unrelated control siRNA and analysed for its
tyrosine phosphorylation. Restaining for ?-catenin is shown to
document equal loading. PTEN protein expression was analysed by
western blots stained for PTEN. Representative assays are shown
(n=3). (D) Phosphorylation of Ser380 of PTEN, which was co-
immunoprecipitated with ?-catenin, was analysed after TGF?1
treatment. Beta-catenin was immunoprecipitated from 2 mg of
PANC-1 lysate treated with TGF?1for 30 minutes or 6 hours. In
addition PANC-1 cells were examined, which were pretreated with
the farnesyltransferase inhibitor FTI 277 (2 ?M) for 2 hours prior to
addition of TGF?1for 6 hours. Co-precipitated PTEN was analysed
regarding the phosphorylation at Ser380 with a phospho-specific
antibody. Restaining for ?-catenin documented equal amounts of
precipitated protein. A representative blot out of three independent
experiments is shown.
Journal of Cell Science
are important for tyrosine phosphorylation of ?- and ?-catenin
after TGF?1stimulation. We show that in pancreatic carcinoma
cells PI3-kinase is associated with the E-cadherin/catenin
complex probably via ?-catenin. This is in line with findings
reported by Woodfield et al. showing that p85? associates with
?-catenin in human keratinocytes (Woodfield et al., 2001) as
well as findings by Pece et al., who demonstrated in MDCK
cells that p85? can be co-immunoprecipitated with E-cadherin
(Pece et al., 1999). Our data show that in epithelial pancreatic
carcinoma cells TGF?1does not regulate the binding of PI3-
kinase protein to ?-catenin, but rather regulates its activity
necessary for ?- and ?-catenin tyrosine phosphorylation.
Recently, Yi and colleagues demonstrated that the PI3-kinase
subunit p85? is associated with the TGF? receptor type 2
(T?RII) in Cos7 cells. TGF?1 treatment induced the
association of TGF? receptor type 1 (T?RI) with this
T?RII/p85 complex followed by an increase of PI3-kinase
activity and Akt phosphorylation (Yi et al., 2005). In agreement
with the data from Woodfield et al., we were able to detect
T?RII but not T?RI associated with the E-cadherin/catenin
complex upon treatment with TGF? (data not shown).
Together, these data strongly support our hypothesis that
TGF?1mediates the disassembly of the E-cadherin adhesion
complex via PI3-kinase in a protein complex consisting of
T?RII, PI3-kinase and E-cadherin/catenin. A role for PI3-
kinase/Akt in TGF?1-induced EMT in mammary epithelial
cells has been shown also by Bakin et al. (Bakin et al., 2000).
The authors demonstrated that TGF?1treatment activated PI3-
kinase and Akt resulting in a delocalisation of E-cadherin from
adherens junctions and ZO-1 from tight junctions as well as
altered Smad4-regulated gene expression. In this study the
GTPase RhoA was suggested to mediate TGF?1activation of
Akt-kinase in mouse mammary cells.
In agreement with data published before (Giehl et al., 2000a;
Mulder, 2000), the data presented in this study further suggest
that Ras activity is important for TGF?1-induced tyrosine
phosphorylation of ?- and ?-catenin. Interestingly, Yi et al. (Yi
et al., 2005) showed in their study that the activation of PI3-
kinase via T?RII is indirect. Our data suggest that Ras could
be involved in the signalling cascade from TGF?1receptors
and activation of PI3-kinase upon TGF?1 treatment. In
agreement with data from the literature demonstrating that Ras
can activate PI3-kinase (Bar-Sagi and Hall, 2000; Giehl, 2005),
we show that the inhibition of Ras activation by pretreatment
of cells with a farnesyltransferase inhibitor as well as
expression of a dominant-negative Ras mutant abolished the
TGF?1-induced tyrosine phosphorylation of ?- and ?-catenin.
We propose that TGF?1activates PI3-kinase mediated by Ras,
which leads directly or indirectly to tyrosine phosphorylation
of ?-catenin and dissociation of the E-cadherin/catenin
In addition to PI3-kinase, we identified the phosphatase
PTEN as being involved in TGF?1-induced phosphorylation of
?-catenin. Different phosphatases have been described to
interact with the E-cadherin complex or individual components
of the complex. Among these are receptor phosphatases such
as PTP?, PTP?, or PTP LAR, which have been shown to
interact directly with the E-cadherin/catenin complex and
mediate dephosphorylation of ?-catenin as a prerequisite for
strong cell-cell adhesion (Aicher et al., 1997; Brady Kalnay et
al., 1995; Fuchs et al., 1996). Other studies showed that
cytoplasmic tyrosine phosphatases interact directly or
indirectly with the E-cadherin complex, such as SHP-1 and
SHP-2, PTP1B, PTP and Pez (Balsamo et al., 1998; Taddei et
al., 2002; Kotelevets et al., 2001; Müller et al., 1999). In this
study, we show for the first time that the phosphatase PTEN is
associated with the E-cadherin complex by binding to ?-
catenin. PTEN dephosphorylates ?-catenin at tyrosine residues
in pancreatic tumour cells, which was verified in vitro by
demonstrating that ectopically expressed PTEN is able to
decrease ?-catenin phosphorylation.
Indirect evidence that TGF? affects PTEN in pancreatic
cancer derives from a study of Ebert et al. (Ebert et al., 2002),
which demonstrated that PTEN expression was dramatically
reduced in human pancreatic cancer as well as in the pancreas
of TGF?1-transgenic mice (Ebert et al., 2002). In our study,
TGF?1 treatment results in dissociation of PTEN from ?-
catenin and the concomitant increase in ?-catenin
phosphorylation. The molecular mechanism by which PTEN
activity is regulated is yet unknown. Reduced phosphorylation
of PTEN at the serine residue 380, as shown here upon TGF?1-
treatment, has been suggested to reduce the protein stability of
PTEN leading to reduced phosphatase activity (Vazquez et al.,
2000). Although the protein phosphatase activity of PTEN is
not the main function for which PTEN is known for, which is
the conversion of the phospholipid PIP3 to PIP2 and
termination of PI3-kinase induced PIP3 production, it was
shown that PTEN mediates dephosphorylation of the proteins
focal adhesion kinase (FAK) and Shc and concomitantly
influences directed cell motility (Tamura et al., 1998; Gu et al.,
1999). In the present study tyrosine phosphorylation of ?-
catenin is directly reduced by PTEN, supporting the hypothesis
that PTEN activity is regulating cell-cell adhesion.
Interestingly, the lipid but not the protein phosphatase activity
of PTEN was necessary to revert Src-induced transformation
of MDCK cells (Kotelevets et al., 2001). In this study,
overexpression of Src in MDCK cells caused a decrease in cell-
cell adhesion, which could be reversed by forced expression of
wild type PTEN. However, PTEN expression had no significant
effect on ?-catenin phosphorylation, which is in contrast with
our data. Potentially, this could be due to the ectopic
overexpression of Src kinase in the MDCK cells used in their
In summary, our data demonstrate that treatment of
pancreatic tumour cells with TGF?1 resulted in reduced
cellular adhesion due to a dissociation of E-cadherin/catenin
complexes from the actin cytoskeleton. The reduction of E-
cadherin/catenin complexes was induced by tyrosine
phosphorylation of ?-catenin and ?-catenin followed by the
dissociation of ?- and ?-catenin from E-cadherin. We have
identified PI3-kinase as well as the phosphatase PTEN as
important mediators of
phosphorylation and cell
dephosphorylation of ?-catenin by PTEN was reduced after
TGF?1stimulation. These results suggest that PI3-kinase and
PTEN are crucial in controlling the invasive phenotype of
pancreatic tumour cells.
We thank S. Braeg, A. Birk and C. Laengle for excellent technical
assistance and Y. Imamichi for helpful discussion. We are grateful to
M. Thelen for providing antibody. The work was supported by the
Deutsche Forschungsgemeinschaft (SFB 518 and SFB 497).
Journal of Cell Science 118 (20)
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