Department of Molecular Embryology, Max-Planck Institute of
Immunobiology, Stuebeweg 51, D-79108 Freiburg, Germany
* Present address: Stephan Bek, Aventis Pharma Deutschland, Functional Genomics,
Industriepark Hoechst, G879/029, D-65926 Frankfurt/Main, Germany
Author for correspondence (e-mail:
kemler{at}immunbio.mpg.de)
Accepted 9 September 2002
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Summary |
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Key words: Casein kinase II, ß-Catenin, -Catenin, Protein kinase, Caderin adhesion complex
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Introduction |
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ß-catenin is implicated in quite different cellular processes, which
requires a fine-tuned regulation of its function, so it is very likely that
ß-catenin is a substrate for other yet to be identified protein kinases.
Indeed, it was recently reported that CKII phosphorylates ß-catenin
(Song et al., 2000), and that
CKII and ß-catenin co-immunoprecipitate with Dvl proteins, the mammalian
homologues of Drosophila Dishevelled (Dsh). From these results it was
concluded that CKII participates in Wnt signaling and may act as a positive
regulator in this pathway although the underlying molecular mechanisms are at
present poorly understood.
CKII exists as a constitutively active tetramer that contains two catalytic
( or
') and two regulatory (ß) subunits
(Pinna and Meggio, 1997
;
Allende and Allende, 1995
).
Although more than 160 substrates have been identified to date, the regulation
of this ubiquitously expressed pleiotrophopic kinase remains unclear. A
nuclear shift of CKII-
' during G1-phase and in
proliferating cells (Seldin and Leder,
1995
; Keliher et al.,
1996
; Landesman-Bollag et al.,
1998
; McKendrick et al.,
1999
; Ahmed, 1994
)
points towards a role of CKII in mitotic control and proliferation. However,
due to the broad subcellular distribution, it is generally assumed, that CKII
is controlled by different interaction partners and in different subcellular
compartiments (for a review, see Faust and
Montenarh, 2000
).
In a search for protein kinases that use ß-catenin as substrate we confirmed that CKII also phosphorylates ß-catenin. We have now identified amino acid (aa) residues in ß-catenin phosphorylated by CKII and performed a mutational analysis to obtain first insights into the biological function of this post-translational modification. By comparing wild-type (wt) and Ser/Thr-mutated (Ser/Thr-mutant) ß-catenin in kinase assays in vitro and in vivo we provide evidence here that CKII regulates the best studied functions of ß-catenin (i.e. its central role in the E-cadherin adhesion complex and its tight control of cytoplasmic stability), which is a prerequisite for the canonical Wnt signaling pathway.
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Materials and Methods |
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Antibodies and reagents
Mouse monoclonal antibodies against -catenin, ß-catenin,
Protein kinase A (PkA), and GSK-3ß were obtained from Transduction
Laboratories (Lexington, KY), rabbit polyclonal antibodies against CKII were
from Santa Cruz Biotechnologies, and rat monoclonal antibodies against
hemagglutinin (HA) (clone 3F10) were from Roche. Anti-GST antibodies were from
Sigma, and rabbit polyclonal anti-Ki67 was from Novocastra Laboratories
(Newcastle). Anti-myc antibodies (Evan et
al., 1985
) were purified from supernatants of hybridoma clone 9E10
grown in DMEM containing 3% FCS. Fluorescein-conjugated secondary antibodies
were purchased from Dianova. 3000 mCi/ml [
-32P]ATP was from
Amersham Pharmacia Biotech.
Immunofluorescence
293 cells grown on collagen- or poly-L-lysine-coated coverslips were washed
in PBS, pH 7.4, and fixed in 3% paraformaldehyde/PBS, pH 7.4 at room
temperature (RT) for 20 minutes. Free aldehyde groups were blocked with 1 M
glycine/PBS, pH 8.5, for 5 minutes. Cells were permeabilized with 0.5% Triton
X-100 for 5 minutes. Incubation was performed with primary antibodies at 2
µg/ml for 1 hour at 37°C and with fluorescein-conjugated secondary
antibodies (Dianova) for 1 hour at 37°C in the dark. Cells were kept in a
mounting solution (50% glycerol; 50% PBS; 100 µg/ml
1,4-diazabicyclo-[2,2,2]octone) and digital images were taken with a
computer-controlled digital C4880 camera (Hamamatsu, Japan) on an Axioskop
microscope (Zeiss, Jena). Camera and microscope were controlled by the
computer program Openlab (Improvision, Coventry, UK).
Immunobiochemistry
Cells grown to 80% confluency were washed with PBS and lysed in 500 µl
CSK buffer [150 mM NaCl, 10 mM PIPES (pH 6.8), 3 mM MgCl2, 0.5%
Triton X-100, 10 mM NaF, 1 mM sodium vanadate, 10 µg/ml leupeptin, 10
µg/ml phenylmethylsulfonyl fluoride (PMSF)] on ice for 20 minutes. Cell
debris was removed by centrifugation at 16,000 g for 10
minutes. The amount of total protein was measured using the BCA-Kit (Pierce)
and equal amounts of total protein were used for each analysis. Immunoblot
analysis was performed as described (Aberle
et al., 1996b), but using PVDF membranes (Millipore).
Immunoprecipitation experiments were done as described
(Hoschützky et al., 1994
)
with the following modifications: lysis and precipitations were performed in
CSK buffer and the immunocomplexes were incubated overnight at 4°C.
Sequential detergent extraction was performed as described
(Ramsby and Makowski, 1999).
Briefly, after washing the cells three times in PBS, cytosolic and soluble
cytoskeletal proteins were released with 0.015% digitonin, prior to the
extraction of membrane and organelle-components with 0.5% Triton X-100.
Nuclear and cytoskeletal proteins were subsequently solubilized with 1% SDS,
prior to 10 fold dilution with normal CSK-buffer. All other steps were carried
out on ice.
For transient transfection experiments 1x106 293 cells cultured in 9-cm culture dishes for 5-7 hours were incubated overnight with a calcium-phosphate coprecipitate containing 5 µg wt or Ser/Thr-mutant ß-catenin DNA; 4 µg DNA for mouse Axin-1 fused to a Myc tag; 2 µg Myc-GSK-3ß DNA; 2 µg Myc-tagged CKII DNA, or 2 µg Myc-tagged ERK2. Cells were washed three times with PBS and cultured for 36 hours before analysis.
For pulse-chase experiments 2.5x105 293 cells stably expressing either wt or Ser/Thr-mutant ß-catenin were cultured in 3.5-cm culture dishes for 6 hours. Cells were then washed twice with PBS, starved for 1 hour in cysteine- and methionine-free medium, incubated with [35S]cysteine/[35S]methionine (150 Ci/ml) for 1 hour, washed and chased for 5 hours. Cells were lysed in 100 µl CSK buffer containing 0.5% SDS and boiled for 10 minutes. The lysates where then diluted 10-fold and equal amounts of incorporated radioactivity were subjected to immunoprecipitation experiments. Precipitates were resolved by SDS-PAGE and the intensities of the products were quantified using the phospho-imager.
Precipitation and elution of ß-catenin associated
kinase-activity
CMT cells grown to 80% confluency in 9-cm dishes were lysed in 500 µl
CSK buffer and immunoprecipitations were usually done overnight at 4°C
with anti-ß-catenin antibodies coupled to protein A-Sepharose
(Amersham-Pharmacia Biotech). Precipitates were washed three times in ice-cold
PBS containing 0.5% Triton X-100, 150 mM NaCl, 10 mM NaF, 2 mM sodium
molybdate, 1 mM sodium vanadate. Kinase activities present in the
immunocomplexes were eluted by applying ascending salt concentrations
(200-1000 mM NaCl) in distilled water containing phosphatase inhibitors (10 mM
NaF, 10 mM sodium molybdate, 1 mM sodium vanadate). After brief centrifugation
the supernatants were diluted to physiological salt concentrations (100 mM
NaCl) and the volumes were adjusted to 400 µl. In control assays cell
lysates were incubated with 1 µg mouse IgG of unrelated specificity.
Kinase assays
For in vitro kinase assays with eluted kinase activity, the indicated GST
fusion proteins immobilized on glutathione-Sepharose 4B (Amersham Pharmacia)
were incubated with 100 µl of the eluate at RT for 30 minutes. After three
washes with PBS containing 0.5% Triton X-100, 150 mM NaCl, 10 mM NaF, 2 mM
sodium molybdate and 1 mM sodium vanadate, kinase assays were performed in 50
µl kinase buffer [20 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 1 mM
CaCl2, 1 µM ATP] including phosphatase inhibitors and 7 µCi
[-32P]ATP at RT for 20 minutes. Reactions were stopped by
adding 20 µl of stop buffer [10 mM EDTA (pH 8.0), 10 mM sodium phosphate
(pH 8.0), 10 mM Na4P2O7] and washed three
times with cold PBS containing 0.5% Triton X-100, 1000 mM NaCl, 10 mM NaF, 2
mM sodium molybdate, 1 mM sodium vanadate. Radioactive gels were Coomassie
Blue-stained and radioactivity was quantified with a BAS 1000 Bioimaging
Analyzer (Fuji), while Coomassie Blue staining was quantified using the NIH
Imager program version 1.59. CKII was specifically inhibited with the
following peptides: (Arg)3 Ala Asp Asp Ser (Asp)5
(competitor); and (Arg)3 Ala Asp Asp Ala (Asp)5
(control).
Pre-phosphorylation experiments
To pre-phosphorylate recombinant ß-catenin with CKII, 2 µg of GST
ß-catenin linked to GSH beads was incubated in CKII kinase buffer (20 mM
Tris-HCl, 50 mM KCl, 10 mM MgCl2, pH 7.5) with 20 mM
non-radioactive ATP and 1 U of recombinant CKII (New England Biolabs) at
25°C for 20 minutes. Reactions were stopped by adding 50 µl stop-buffer
(see above). To remove all residual kinase-activity, the reactions were
extensively washed 5 times with 1 ml ice-cold PBS containing 1000 mM NaCl,
0.5% Triton X-100 and phosphatase-inhibitors. The NaCl concentration was
reduced by 2 additional washing steps with PBS containing 100 mM NaCl.
Pre-phosphorylation with GSK-3ß was done in the same way, but including 5
mM DTT in the assay-buffer, using 0.1 U of recombinant GSK-3ß (New
England Biolabs), and performing the reaction at 30°C.
Expression and purification of recombinant proteins
All GST fusion proteins were expressed in E. coli BL21 (DE3).
Affinity purification on GSH-Sepharose beads (Amersham Pharmacia) was carried
out as described (Aberle et al.,
1996b). Proteins were dialyzed against 20 mM HEPES (pH 8.0). For
ß-catenin fragments including only aa residues 302-535 or 535-683,
concentration with Centricon ultrafiltration cartridges was required.
After purification from E. coli BL21 (Amersham Pharmacia Biotech)
GST-Axin was loaded on a PD10 column to get rid of some major degradation
products. The eluted and pooled fractions were concentrated as described
above. ß-catenin-His6 was expressed in E. coli BL21
(DE3) and affinity purified on Talon-Beads (Clontech) as described
(Aberle et al., 1996b). All
recombinant proteins were aliquoted and stored at -20°C.
GST pull-down assays
If not indicated otherwise, 2 µg of GST-protein linked to GSH-beads was
incubated with recombinant His6-tagged protein pre-absorbed with
GSH-beads. Reactions were carried out in 500 µl association-buffer (20 mM
Tris-HCl, 10 mM MgCl2, 0.1% Triton X-100, 100 mM KCl) at RT for 30
minutes. Protein complexes were collected by brief centrifugation (1 minute,
800 g, 4°C), and washed three times with cold PBS
containing 0.5% Triton X-100, 160 mM NaCl, 10 mM NaF, 2 mM sodium molybdate, 1
mM sodium vanadate.
Constructs
GST expression constructs pGEX4T1MMBC, pGEX4T1BNTERM (coding for
ß-catenin in residues 1-119) and pGEX4T1BCTERM (aa 683-781) were
previously described (Aberle et al.,
1996b). To obtain pGEX4T1B1-302, pSKßtot was digested with
BamHI/BglII and the resulting 906 bp-fragment coding for the
first 302 aa residues of ß-catenin was cloned into BamHI-cleaved
pGEX4T1. PGEX4T1B302-535 was obtained by cloning the 1176-bp
BglII-fragment into BamHI-cleaved pGEX4T1. The resulting
plasmid coding for residues 302-781 was digested with
SpeI/XhoI and blunt-end ligated to obtain a construct coding
for ß-catenin missing aa residues 535-781.
For construction of pGEX4T1B535-683 a 741 bp-fragment coding for residues
535-781 was amplified from pGEX4T1MMBC by PCR, using the following primers:
ß535ff (5'-GCGGGATCCCGAC-TAGTTCAGCTG-3') and ßctermrev
(5'-TTACAGGTCAGTAT-CAAACCAGGC-3'). The fragment was cloned into
BamHI/EcoRV digested pGEX4T1BCTERM
(Aberle et al., 1996b
).
The plasmids coding for Myc-tagged mouse Axin 1 and Myc-tagged ERK2 where kindly provided by L. Zeng (Columbia University, New York, NY) and P. E. Shaw (The Beatson Institute for Cancer Research, Glasgow, UK).
For the generation of C-terminal HA-tagged ß-catenin, oligonucleotides encoding the influenza virus HA-tag were inserted into pCS2+ after EcoRI restriction of the vector. cDNA coding for ß-catenin aa residues 1-781 was then cloned in-frame in front of the HA-tag region.
Site-directed mutagenesis was performed as described
(Higuchi, 1990). For all three
mutants the same wt primers were used:
ß1ff:5'-ATGGCTACTCAAGCTGACC-3', and ß1875rev:
5'-GGCCTCT-GCAGCCTCCTTGTCC-3'.
For generation of the single mutants the following mutagenic primer pairs were used: M1ff: 5'-CTGGCAGCAGCAGGCT-TACTTGGATTCTGG-3' and M1rev: 5'-CCAGAATCCAAGT-CAGCCTGCTGCTGCCAG-3'. M2: M2ff: 5'-ATGTTCCCTGAG-GCTCTAGATGAGGGC-3', and M2rev: 5'-GCCCTCATCTAGAGC-CTCAGGGAACAT-3'. M3: M3ff: 5'-GGCATGCAGATCCCATC-CCAGCAGTTTGACGCT-3', and M3rev: 5'-AGCGTCAAACTG-GTCGGATGGGATCTGCATGCC-3'.
After NcoI digestion, the PCR products were cloned into
pCS2+ß-catenin-HA. To generate the Ser/Thr-mutant,
HA-ß-catenin-M3 was first subcloned after into HA-ß-catenin-M2 after
SphI/SpeI digestion of the vector. A
HincII/SpeI-fragment containing both mutations, M2 and M3,
was then cloned into HA-ß-catenin-M1. HA-tagged wt ß-catenin and
Ser/Thr-mutant constructs were cloned into the pBUD CE4 vector (Invitrogen)
under control of the EF-1 promoter. For stable transfection of 293
cells the calcium-phosphate method was used.
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Results |
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Phosphorylation of ß-catenin was clearly reduced in the presence of
CKII-specific competitor peptides (compare
Fig. 1A, lanes 3 and 4), but
not with control peptides containing mutated CKII motifs
(Fig. 1A, co). These results
provide evidence that the heparin-mediated inhibition of
ß-catenin-phosphorylation is CKII-specific, and therefore CKII activity
was measured in immunocomplexes collected with anti-ß-catenin from cell
lysates. The results indicated further that CKII and ß-catenin are
associated in vivo. Consistent with that, specific in vivo associations
between the two proteins were found in 293- and NIH 3T3 cells in
immunoprecipitations with anti-CKII- antibodies and subsequent
immunoblots with anti-ß-catenin (Fig.
1B, lanes 2,4). Conversely, when Myc-tagged CKII-
was
transiently expressed in 293 cells, CKII could also be co-precipitated with
anti-ß-catenin antibodies from cell lysates, whereas an unrelated kinase
could not (Fig. 1B, lanes 7,8).
This association might well be a direct interaction, since recombinant CKII is
able to interact directly with GST-ß-catenin in vitro
(Fig. 1C, lane 5). This
interaction becomes enhanced in the presence of ATP
(Fig. 1C, lane 6), similar to
previous observations for the association of CKII with other target proteins
(Muslin et al., 1996
;
Pawson, 1995
). Taken together,
these results provide good evidence that CKII interacts with and
phosphorylates ß-catenin.
To determine which part of ß-catenin is phosphorylated by CKII, various ß-catenin deletion constructs were expressed and the purified GST fusion-proteins were subjected to in vitro kinase assays (Fig. 2A). Heparin was included in parallel as inhibitor of CKII phosphorylation. As shown in Fig. 2B, strong phosphorylation was observed in the N-terminal (aa residues 1-119/1-302) and C-terminal (aa residues 683-781) regions of ß-catenin. Remarkably, in contrast to the C-terminal region, the N-terminal phosphorylation was efficiently inhibited by heparin, indicating that CKII preferentially phosphorylates the N-terminal part of ß-catenin. These results are consistent with the localization of three conserved CKII-consensus motifs in the N-terminal region of ß-catenin around the GSK-3ß recognition motifs (Fig. 2C).
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CKII regulates the cytoplasmic stability of ß-catenin
In the following the phosphorylation of ß-catenin by CKII was
investigated with respect to its biological functions, the best studied of
which are its central role in the E-cadherin-catenin adhesion complex and as a
Wnt signaling component. In these experiments mutant forms of ß-catenin
were included where one or more of the three CKII consensus motifs were
destroyed by single or combined aa substitutions (i.e. aa S29A;
T102
A; T112
Q) (Fig.
3A). GST fusion proteins with either single or combined mutations
were tested in kinase assays in vitro. No phosphate-incorporation was observed
when ß-catenin mutated in all three CKII consensus motifs
(Ser/Thr-mutant) was tested with recombinant CKII
(Fig. 5D, lane 7), and single
aa substitutions drastically reduced phosphorylation (not shown).
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The cytoplasmic turn-over of wt and mutant ß-catenin was compared in
pulse-chase experiments with 293 cells stably expressing HA-tagged wt or
Ser/Thr-mutant ß-catenin (Fig.
3). At each time-point, HA-tagged ß-catenin was
immunoprecipitated from cell lysates containing comparable amounts of
incorporated radioactivity and quantitated. The autoradiograph in
Fig. 3A shows that the
time-dependent degradation of mutant ß-catenin was clearly delayed as
compared to the wt form. Three independent pulse-chase experiments were
quantified using the phospho-imager (Fig.
3B). The half-life of HA-tagged wt ß-catenin is similar to
that previously determined for endogenous ß-catenin
(Aberle et al., 1997). In
comparison, Ser/Thr-mutant ß-catenin showed a significant stabilization
by more than two-fold (Fig.
3B). From these results it is concluded that functional
CKII-motifs are required for the control of the cytoplasmic amount of
ß-catenin. These results suggested further that CKII is part of the
multi-protein complex which controls the cytoplasmic amount of ß-catenin.
To test this possibility Myc-tagged Axin, or Myc-GSK3ß were transiently
expressed in 293 cells, endogenous CKII was immunoprecipitated and the
precipitates were probed with anti-Myc antibodies
(Fig. 4A). Both, Myc-Axin
(Fig. 4A, lane 2) and
Myc-GSK3ß (Fig. 4A, lane
4) were found associated with endogenous CKII suggesting that CKII is a
component of the ß-catenin degradation machinery. It is well established
that phosphorylation of ß-catenin by GSK3ß enhances binding of
ß-catenin to Axin and APC. A similar notion can be taken from the results
depicted in Fig. 4B, since
ß-catenin mutated in its CKII-motifs can only poorly associate with Axin.
In these experiments HA-tagged wt or Ser/Thr-mutant ß-catenin and
Myc-Axin were transiently expressed in 293 cells, comparable amounts of
immunoprecipitates were collected with anti-Myc antibodies
(Fig. 4B, lanes 4-6), and
precipitates were probed for ß-catenin
(Fig. 4B, lanes 1-3). The
introduction of one mutation in ß-catenin already decreased the affinity
to Axin (Fig. 4B, lane 2) and
binding was further decreased when all three CKII-motifs were mutated
(Fig. 4B, lane 3). These
results demonstrate that ß-catenin mutated in its CKII-motifs can poorly
associate with Axin and suggested that both GSK3ß and CKII act
synergistically in controlling the degradation of ß-catenin. To address
this question, wt or Ser/Thr-mutant GST-ß-catenin was phosphorylated with
either CKII, GSK-3ß, or both in vitro. The reaction was stopped with a
high-salt wash (500 mM NaCl, 0.5% Triton X-100), and bound kinases were eluted
and examined by immunoblot analysis with antibodies as indicated
(Fig. 5). CKII bound to wt
ß-catenin, but binding was clearly reduced with mutant ß-catenin
(compare Fig. 5A, lanes 3 and
7).
|
Importantly, enhanced binding of GSK-3ß to ß-catenin was observed
when ß-catenin was pre-phosphorylated by CKII
(Fig. 5B, compare lanes 4 and
5). Pre-phosphorylation of Ser/Thr-mutant ß-catenin with CKII did not
enhance binding of GSK-3ß to mutant ß-catenin
(Fig. 5B, lane 9) and binding
of GSK-3ß to wt and Ser/Thr-mutant ß-catenin was comparable
(Fig. 5B, lanes 4,8). In
Fig. 5D the
[-32P]ATP-incorporation in these experiments is depicted,
demonstrating enhanced phosphate incorporation in wt ß-catenin when both
kinases were used (lane 5) and the lack of incorporated phosphate in the CKII
mutant (lane 7). Comparable amounts of wt and Ser/Thr-mutant ß-catenin
were used in these experiments, as monitored with anti-GST antibodies
(Fig. 5C).
Several conclusions can be drawn from these results. Most notably, CKII and GSK-3ß bind and phosphorylate wt ß-catenin synergistically and pre-phosphorylation by CKII enhances binding of GSK-3ß. In the Ser/Thr-mutant CKII can still bind, although to a reduced amount, but no phosphate incorporation is observed here. Thus, the CKII consensus motifs in ß-catenin are substrates for the enzyme. Mutations in the CKII motifs do not affect binding and phosphorylation by GSK-3ß, but the enhanced binding in the wt when both kinases were used is not observed for the mutant form. Altogether, a sequential action of CKII and GSK-3ß in phosphorylation of ß-catenin is suggested, and the role of CKII in controlling the protein turnover is underlined.
CKII regulates the interaction of ß-catenin with
-catenin
Since Ser/Thr-mutant ß-catenin becomes stabilized, it was of interest
to examine how the subcellular distribution of mutant ß-catenin compares
to wt protein. For this, 293 cells stably expressing HA-tagged wt or
Ser/Thr-mutant ß-catenin were subjected to sequential detergent
extractions to separate the cytosolic, membrane-organelle and cytoskeletal
fractions of cells. Immunoblot analysis with anti-HA antibodies revealed the
expected subcellular distribution of transfected HA wt ß-catenin, with
the major content in the membrane-organelle fraction, similar to that of
endogenous wt ß-catenin in untransfected 293 cells
(Fig. 6A). In comparison, the
cytoplasmic pool of Ser/Thr-mutant ß-catenin was drastically increased
and little mutant protein was detected in the cytoskeletal fraction
(Fig. 6A). In these
preparations the HA antibody crossreact most likely with c-terminal
degradation products from HA ß-catenin, whereas a 65 kDa-band from the
membrane-organelle fractions is of unrelated specifity. The reduced amount of
Ser/Thr-mutant ß-catenin in the cytoskeletal fraction was of particular
interest and pointed to the possibility of an altered interaction of the
mutant form with -catenin.
|
Therefore, in vitro association experiments were carried out with
recombinant - and ß-catenin
(Fig. 6B,C). Wild-type
ß-catenin bound to increasing amounts of
-catenin. However, little
-catenin interacted with Ser/Thr-mutant ß-catenin
(Fig. 6B). Interestingly,
pre-phosphorylation of wt ß-catenin with CKII in vitro even enhanced
binding of
-catenin, but this was not the case for the mutant form of
ß-catenin (Fig. 6C). These
results provide strong evidence that phosphorylation of ß-catenin by CKII
regulates the interaction between
- and ß-catenin. They further
explain the reduced amount of Ser/Thr-mutant ß-catenin in the
cytoskeletal fraction and its increase in the cytosol as seen in
Fig. 6A. This difference in
sub-cellular localization could influence cell behaviour. To test this
possibility, wound-healing experiments were performed with 293 cells stably
expressing wt and Ser/Thr-mutant ß-catenin
(Fig. 7A). Remarkably, the
wound closure was much faster in 293 cells expressing Ser/Thr-mutant
ß-catenin compared to those expressing the wt form. The enhanced wound
closure is very unlikely due to differences in cell proliferation between the
two cell types, as monitored with the proliferation marker Ki-67
(Fig. 7B). Instead, these
results suggest that 293 cells expressing mutant ß-catenin exhibit higher
migratory potential.
|
This is also underlined by the subcellular distribution of HA-tagged wt or Ser/Thr-mutant ß-catenin in these stably transfected cells (Fig. 7C). In comparison to HA wt ß-catenin, which is strongly localized at cell-cell-contact sites, Ser/Thr-mutant ß-catenin shows a more intense and diffuse staining in the cytoplasm. Since the switch of ß-catenin from the membrane to the cytoplasm is required for cell-migration, this also reflects the higher migratory potential of the cells expressing Ser/Thr-mutant ß-catenin.
A similar distribution can also be seen for -catenin in both cell
lines. However, significant amounts of
-catenin in mutant cells are
still localized at the membrane due to the association with endogenous
ß-catenin. In line with such a view, an increased staining for the
mesenchymal marker vimentin in cells expressing Ser/Thr-mutant ß-catenin
could be observed (S.B., unpublished).
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Discussion |
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CKII regulates ß-catenin stability
It is well established that GSK-3ß regulates the cytoplasmic turnover
of ß-catenin in a multiprotein complex and that the stability and
composition of this complex is highly dynamic and depends on phosphorylation
(Rubinfeld et al., 1996;
Hart et al., 1998
;
Ikeda et al., 1998
;
Fagotto et al., 1999
;
Kawahara et al., 2000
;
Strovel et al., 2000
). Here we
propose that CKII participates together with GSK-3ß in controlling the
cytoplasmic stability of ß-catenin and is part of the multiprotein
complex. We show that CKII can associate with GSK-3ß and Axin and that
Ser/Thr-mutant ß-catenin binds less efficiently to Axin and GSK-3ß.
Moreover, Ser/Thr-mutant ß-catenin has a longer half-life
(Fig. 3) as has been observed
with ß-catenin which is not phosphorylated by GSK-3ß
(Aberle et al., 1997
). A
synergistic phosphorylation by CKII and GSK-3ß was observed when wt and
Ser/Thr-mutant ß-catenin were compared in in vitro kinase assays
(Fig. 5). Pre-phosphorylation
by CKII enhanced binding of GSK-3ß and increased phosphorylation of
ß-catenin. The CKII-dependent higher binding and phosphorylation
capabilities of GSK-3ß are not observed in mutant ß-catenin,
suggesting a sequential action of CKII and GSK-3ß in phosphorylating
ß-catenin. Taking this together with the biochemical data, it is
concluded that CKII phosphorylation of ß-catenin stabilizes its binding
to Axin and GSK-3ß and thus enhances the activity of GSK-3ß. Our
findings are in line with the demonstrated involvement of CKII in the
proteolytic degradation of other proteins, e.g. IkB
and lens connexin
45.6 (Bren et al., 2000
;
Yin et al., 2000
). Altogether
it is concluded that a combined action of CKII and GSK-3ß controls the
cytoplasmic turnover of ß-catenin.
Using the Wnt1-expressing mouse mammary epithelial cell line C57MG, a
Wnt1-dependent increase of CKII expression resulting in stronger
phosphorylation of ß-catenin. Association of both proteins with Dvl
proteins (the mammalian homologues of Drosophila Dishevelled) was
previously observed, and it was suggested that CKII acts as a positive
regulator of Wnt signaling (Song et al.,
2000). Our results do not support such a view, but one cannot
exclude that CKII activity on ß-catenin at the membrane and in the
cytoplasm vary in different cell types, which raises the question how this can
be achieved by a widely distributed and nearly always active kinase.
Possible explanations come from GSK-3ß, which is differentially
regulated by insulin and Wnt signaling. As a consequence of different
post-translational modifications (e.g. phosphorylation on serine-9) and
complex formation (e.g. the APC-Axin-Frat/GBP-Dvl-complex), wnt-signals are
able to accumulate ß-catenin in the cytoplasm, whereas insulin-signals do
not (Ding et al., 2000).
Although no extracellular signal are confirmed to regulate CKII-activity,
similar mechanisms can be discussed for CKII-mediated regulation of
ß-catenin. CKII-phosphorylation of ß-catenin also might be regulated
through the interaction with different protein-complexes in the respective
subcellular compartments. In line with this, CKII-activity can be regulated
through the interaction with p53 in the nucleus and with p47(phox)
(Guerra et al., 1997
;
Kim et al., 2001
).
CKII regulates the interaction between - and
ß-catenin
Ser/Thr-mutant and wt ß-catenin exhibit different sub-cellular
distributions when stably expressed in 293 cells. The relative amount of
Ser/Thr-mutant ß-catenin in the cytosolic fraction is higher, consistent
with the prolonged half-life of the mutant protein. Significantly, only little
mutant ß-catenin distributes to the cytoskeletal fraction, indicating
that mutant ß-catenin does not fulfil properly its role in the
cadherin-catenin complex. Such a view is supported by our immunofluorescence
data showing that Ser/Thr-mutant ß-catenin is preferentially distributed
in the cytoplasm whereas wt ß-catenin is mostly localized to the cell
membrane. This suggested that the affinity of Ser/Thr-mutant ß-catenin to
either E-cadherin and/or -catenin might be reduced. To test this
possibility protein interaction assays were performed with recombinant
proteins. Binding of E-cadherin to wt or Ser/Thr-mutant ß-catenin was
unchanged (as was the binding of another ß-catenin interaction partner,
LEF-1; S.B., unpublished). However, mutant ß-catenin bound less
efficiently to
-catenin and, even more importantly, pre-phosphorylation
of wt ß-catenin with CKII enhanced its binding to
-catenin
(Fig. 6B,C). These results
provide strong evidence that CKII regulates binding of ß-catenin to
-catenin and thus add a new molecular mechanism to modulate the
E-cadherin-catenin cell adhesion complex. In 293-cells the catenins are
propably sequestered to another cadherin, since these cells do not express
E-cadherin. This view is supported by immunoprecipitation-experiments in
35S-labeled 293-cells. After separation of the anti ß-catenin
immuno-complex, we could detect three bands running at the size of E-cadherin,
- and ß-catenin, as could be observed in cells containing
E-cadherin (S.B., unpublished). Again, the biochemical data are underlined by
the distribution of
-catenin, which shows significant similarity with
the staining of HA-tagged wt or Ser/Thr-mutant ß-catenin in
immunofluorescence analysis. However, significant amounts of
-catenin
in mutant cells is still localized at the membrane due to the association with
endogenous ß-catenin.
From the N-terminal aa-sequence of ß-catenin, a amphipathic
-helix can be predicted, which has been confirmed for the aa 134-161
(Graham et al., 2000
). However
for formation of the ß-catenin-
-catenin heterodimer a change in
the overall secondary structure of ß-catenin is necessary
(Huber and Weis, 2001
). The
introduction of negatively charged phosphate-residues in neighbouring
sequences from ß-catenin might help to provide the surface for this
hydrophobic interactions. Whereas Tyr 142 in ß-catenin is absolutely
needed for the interaction with the hydrophobic core of the binding region
(Aberle et al., 1996b
;
Huber et al., 1997
;
Pokutta and Weis, 2000
), the
residues targeted by CKII might regulate the strength of the interaction.
Related to the interaction with -catenin, a hydrophobic binding
pocket within the armadillo repeats 3 and 4 in ß-catenin, is critical for
Axin-binding (Graham et al.,
2000
; von Kries et al.,
2000
). N-terminal phosphorylation of ß-catenin by CKII might
provide the structure of the hydrophobic interaction surface. Binding domains
for E-cadherin, APC and LEF-1/Tcf in ß-catenin are not or are only
partially located in this region. Consequently, the binding to these proteins
was not influenced after CKII phosphorylation of ß-catenin in vitro
(S.B., unpublished).
Our biochemical results provide convincing evidence that phosphorylation of
ß-catenin by CKII modulates the biological function of ß-catenin.
The wound-healing experiments additionally substantiate this. Cells expressing
Ser/Thr-mutant ß-catenin exhibit higher migratory potential as compared
to cells expressing the wt protein. An increase in cell proliferation is
unlikely to account for the enhanced wound healing; instead, cells expressing
mutant ß-catenin appear to have adopted a more mesenchymal phenotype. The
enhanced migratory potential of cells expressing Ser/Thr-mutant ß-catenin
is likely due to the inefficient binding of mutant protein to -catenin,
which could affect the cytoskeletal architecture of the cells. Although other
explanations are possible, this view is supported by the subcellular
distribution of HA-tagged wt or Ser/Thr-mutant ß-catenin and
-catenin. Mutations in aa residues in ß-catenin which are targets
for CKII could thus be relevant in tumorigenesis. Notably, aa residue Ser29,
which we found to be phosphorylated by CKII, is mutated in some gastric-cancer
cell lines, and results in an accumulation of ß-catenin
(Polakis, 2000
;
Park et al., 1999
).
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Acknowledgments |
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