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INTRODUCTION |
The familial form of Alzheimer's disease
(FAD)1 has been found to be
largely attributed to mutations in the presenilin proteins, presenilin
1 (PS1) and presenilin 2 (PS2). The role of PS proteins in Alzheimer's
disease is of interest because of the strong causal relationship to the
disease (reviewed in Ref. 1). The mechanism(s) through which FAD PS
leads to Alzheimer's pathogenesis is not clearly defined, although
recent advances have been made regarding PS structure and function. PS
are polytopic membrane proteins to which a variety of functions have
been linked including protein processing and transport, as well as for
intracellular signaling in cell fate determination, survival,
apoptosis, and response to stress (reviewed in Refs. 2-4).
Association between PS1 and a number of proteins, including
-catenin
(5-7), has been reported. Both PS1 and
-catenin have been
identified as components of a high molecular weight membrane complex
present in the endoplasmic reticulum and Golgi apparatus (8). The large
hydrophilic loop located between transmembrane domains 6 and 7 of the 8 transmembrane PS1 contains the domain(s) responsible for
-catenin
binding (7, 9, 10). Furthermore, glycogen synthase kinase-3
(GSK-3
), the serine/threonine kinase that regulates
-catenin
levels, has also been shown to associate with PS1 (11). The regulation
of cytoplasmic
-catenin levels is pivotal to the Wnt signal
transduction pathway. Wnt signaling regulates cell fate determination
during development and cell proliferation in adult tissues (reviewed in
Ref. 12). In the absence of Wnt signal, no signal transduction occurs
due to the rapid degradation of
-catenin.
-Catenin is targeted
for ubiquitination and proteosome degradation following phosphorylation
by GSK-3
. The activity of the
-catenin complex is regulated by
other proteins present, such as Axin and adenomatous polyposis coli
(APC), which facilitate the phosphorylation of
-catenin by GSK-3
.
The ability of Axin and APC to facilitate
-catenin phosphorylation
is mediated by phosphorylation of both proteins by GSK-3
. Upon
stimulation of the Wnt receptor, GSK-3
is inhibited, resulting in an
increase in the cytosolic level of
-catenin rendering the protein
available for transport to the nucleus where it activates Wnt target genes.
PS1 also participates in the regulation of cytoplasmic
-catenin
levels, although the nature of this modulation is unclear. Many
insights regarding the role of PS1 in
-catenin regulation have come
from comparisons of normal PS1 with those bearing FAD mutations.
However, conflicting results have prevented firm conclusions. FAD PS1
appears to increase
-catenin degradation (13, 14) although contrary
results have also been reported (15, 16). This loss of
-catenin
signal in a neuronal background has been linked to increased
susceptibility to apoptosis (13). FAD PS1 was also shown to
compromise the ability of
-catenin to translocate to the nucleus, a
state that could potentiate apoptosis (16). We have identified three
GSK-3
consensus phosphorylation sites in the PS1 hydrophilic loop
domain. Similar to Axin or APC, phosphorylation of PS1 by GSK-3
may
serve to regulate
-catenin phosphorylation, thereby regulating
-catenin/Tcf gene expression. To test this hypothesis, we have
characterized the role of each GSK-3
phosphorylation consensus motif
in PS1/
-catenin association and identified one to be critical.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Human embryonic kidney (HEK) 293T cells were
grown in DMEM-21 containing 10% fetal bovine serum, 1%
L-glutamine, and antibiotics (50 units/ml penicillin and 50 µg/ml streptomycin). Cells were maintained in a humidified 37 °C
incubator with 5% CO2. Transfection was carried out by
subconfluent seeding of 293HEK cells into six-well tissue culture
plates and transfecting DNA using the calcium phosphate precipitation method.
Expression Vector Construction--
A cDNA encoding PS1 was
amplified by standard polymerase chain reaction techniques and
subcloned into a C-terminal Myc-His6-tagged mammalian
expression vector, pcDNA3.1/Myc-His(
) (Invitrogen). Site-directed
mutagenesis of the serine and threonine residues found in PS1, which
conform to the aforementioned (S/T)XXXS consensus phosphorylation site were generated by using a two-primer pair method
protocol outlined by the QuikChangeTM site-directed
mutagenesis kit (Stratagene). The mutagenic primer pairs were as
follows: 5'-TATAATGCAGAAAGCAGAGAAAGGGAG-3' and
3'-ATATTACGTCTTTCGTCTCTTTCCCTC-5' for site T320R;
5'-GAAAGGGAGGCACAAGACACTGTTG-3' and
3'-CTTTCCCTCCGTGTTCTGTGACAAC-5' for site S324A;
5'-TATAATGCAGAAAGCAGAGAAAGGGAGGCACAAGACACTGTTG and
3'-ATATTACGTCTTTCGTCTCTTTCCCTCCGTGTTCTGTGACAAC
for both sites T320R,S324A;
5'-CTAGGGCCTCATCGCGCTACACCTGAG-3' and
3'-GATCCCGGAGTAGCGCGATGTGGACTC-5' for site S353A;
5'-CTACACCTGAGGCACGAGCTGCTGTCC-3' and
3'-GATGTGGACTCCGTGCTCGACGACAGG-5' for site S357A;
5'-CTAGGGCCTCATCGCGCTACACCTGAGGCACGAGCTGCTGTCC-3' and
3'-GATCCCGGAGTAGCGCGATGTGGACTCCGTGCTCGACGACAGG-5'
for both site S353A,S357A; 5'-GGTTGGTAAAGCCGCAGCAACAGCC-3'
and 3'-CCAACCATTTCGGCGTCGTTGTCGG-5' for site S397A;
5'-GCAACAGCCAGAGGAGACTGGAAC-3' and
3'-CGTTGTCGGTCTCCTCTGACCTTG-5' for site S401R;
5'-CCAACCATTTCGGCGTCGTTGTCGGTCTCCTCTGACCTTG-3' and
3'-GGTTGGTAAAGCCGCAGCAACAGCCAGAGGAGACTGGAAC-5'
for both site S397A,S401R. The fidelity of polymerase chain
reaction replication and introduction of each corresponding PS1
mutation (underlined) was confirmed by DNA sequence analysis.
Luciferase Reporter Assay--
For reporter gene assays, 293HEK
cells were transfected with 0.1 µg of Tcf-luciferase reporter gene
plasmid, TOPFLASH, (Upstate Biotechnology, Inc.) and the indicated
amount of each expression construct. The total concentration of
transfected DNA was kept constant by supplementation with empty vector.
Cells were harvested 24 h post-transfection, and the reporter
activity was determined with the Luciferase Assay System (Promega).
In Vitro and in Vivo Phosphorylation--
Assessment of the PS1
consensus phosphorylation motif was carried out in vitro
according to the method described by Dong et al. (17) with
only minor changes. Briefly, synthetic peptide (30-50
µM) containing either the Ser353,
Ser357 motif (NH2-GPHRSTPESRAAV-COOH) or an
altered motif to substitute the serine residues for alanine
(NH2-GPHRATPEARAAV-COOH) (S353A,S357A), was
incubated with 5 units of kinase, 10 µM ATP, and 2 µCi
of [
-32P]ATP at 111 TBq/mmol (Amersham Pharmacia
Biotech) for 60 min at 37 °C. Incorporation of radiolabeled
phosphate was determined by adsorption of the peptide to P81 membranes
and scintillation counting. Synthetic PS1 peptides used for in
vitro kinase studies were obtained from American Peptide Co.
GSK-3
(rabbit), produced by recombinant methods, was purchased from
Sigma or New England Biolabs. Other kinases were acquired as follows:
casein kinase II from Sigma, protein kinase C from Promega, p38
from
Upstate Biotechnology, and p38
produced at Scios Inc. Control
reactions using cognate substrates for each kinase were performed in an identical fashion. In vivo phosphorylation was carried out
using 293HEK cells transiently transfected with wild type or GSK-3
mutant PS1 DNA. Twenty-seven hours post-transfection, cell
monolayers were washed, incubated for 1 h in phosphate-free
medium supplemented with 10% dialyzed fetal bovine serum,
washed, and radiolabeled for 4 h with 0.5 mCi/ml
[32P]orthophosphate (Amersham Pharmacia Biotech). Cell
lysates were prepared in 10 mM HEPES, 10 mM
KCl, 500 mM NaCl, 1% Nonidet P-40, 50 mM NaF,
0.2 mM sodium orthovanadate, 10 mM imidazole,
and protease inhibitor mixture (Roche Molecular Biochemicals) prepared
without EDTA. Lysates were incubated overnight at 4 °C with nickel
beads (Qiagen), washed first in complete lysis buffer and then in lysis buffer without 500 mM NaCl. Protein bound to the beads was
eluted with lysis buffer lacking 500 mM NaCl and
supplemented with 100 mM imidazole. The elute was adjusted
to 2 mM EDTA, and PS1 was immunoprecipitated with a
tetra-His monoclonal antibody followed by electrophoresis of the
precipitate on a 12% NuPage Tris/glycine gel (Novex) as described
below. The gel was dried and exposed for 48 h using a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Sample
dephosphorylation was carried out using calf intestinal alkaline
phosphatase (New England Biolabs) by adding 300 units of enzyme during
the final hour of the immunoprecipitation step.
Cell Death Assays--
Human 293HEK cells (3 × 105) were transiently transfected with 0.01 mg of a green
fluorescent protein reporter plasmid (pGFP) plus the indicated
concentration of test plasmid in six-well tissue culture dishes.
Twenty-four hours post-transfection, the cells were induced to undergo
apoptosis. Cells were then fixed in 3.7% formaldehyde and were
visualized by microscopy. Approximately 300 green fluorescent
protein-positive cells were assessed from each transfection
(n = 3) from three randomly selected fields, and the
mean of these was used to calculate percentage of apoptosis. Viable or
apoptotic cells were distinguished based on morphological alterations
typical of adherent cells undergoing apoptosis including becoming
rounded, condensed, membrane-blebbing, and detached from the culture dish.
Coimmunoprecipitation and Western Blot Analysis--
HEK293
cells were transiently transfected with the indicated constructs. Cells
were harvested 24-48 h post-transfection. For coimmunoprecipitation,
cells in 100-mm plates were washed twice with ice-cold
phosphate-buffered saline and lysed in 1 ml of lysis buffer (50 mM HEPES, 150 mM NaCl, 2 mM EDTA,
0.1% Nonidet P-40, and protease inhibitor mixture
(CompleteTM, Roche Molecular Biochemicals). Cells were
lysed on ice for 15 min and then spun at 14,000 rpm for 20 min, and the
supernatants were collected. Lysates were precleared with 50 µl of
protein G-agarose beads (Roche Molecular Biochemicals) and
immunoprecipitated with 5 µg of tetra-His monoclonal antibody
(Qiagen). Immunoprecipitates were washed five times in lysis buffer and
resolved on either 4-12% or 10% NuPage Bis-Tris gels (Novex). For
Western blots, lysates were prepared in an identical manner. The PS1
monoclonal antibody directed to the C-terminal "loop" domain
(3.6.1) was obtained by immunizing mice with a synthetic peptide
spanning PS1 residues 309-331. The rat anti-human PS1 N-terminal
antibody was purchased from Chemicon. Comparison of PS1 CTFs from
His-tagged and untagged constructs was made by transfecting 293HEK
cells with each construct for 24 h as described above, after which
lysates were analyzed by Western blot using antibodies directed to PS1 NTF or CTF. The same samples were immunoprecipitated with anti-His antibody, and then the precipitates were probed by Western blot with
the PS1 C-terminal antibody 3.6.1. The BCA method (Pierce) was used to
normalize each sample such that equivalent amount of protein was
applied to the gel. After gel separation, proteins were transferred to
membranes and blotted with antibody.
-Catenin and GSK-3
monoclonal antibodies were purchased from Transduction Laboratories.
The monoclonal antibody specific for activated GSK-3
Y279/Y216 was
purchased from BIOSOURCE International.
Cytosolic and Nuclear Fractionation--
HEK293 cell cultures in
100-mm plates were washed twice in ice-cold PBS. Cells were washed once
with ice-cold hypotonic buffer (10 mM Hepes, pH 7.4, 10 mM KCl, 2 mM MgCl), resuspended in 500 µl of
hypotonic buffer supplemented with 1 mM dithiothreitol, 5 µM cytochalasin B, and protease inhibitors
(CompleteTM; Roche Molecular Biochemicals), and allowed to
swell on ice for 15 min. Cells were Dounce homogenized, and the nuclei
(and membranes) were removed by centrifugation at 2,000 rpm. The
supernatant was collected and further spun at 100,000 × g for 45 min to provide the cytosolic fraction. The pelleted
fraction was washed twice in hypotonic buffer, vortexed, lysed by
vigorous vortexing in cell lysis buffer, and spun at 14,000 rpm, and
the supernatant was collected to provide the nuclear fraction. Protein
concentrations in the cytosolic and nuclear fractions were determined
using the BCA reaction.
A
Measurements--
Cultures of 293HEK cells stably
expressing the APP Swedish (APPswe) mutation were transiently
transfected with wild type PS1, FAD-PS1, or PS1 site-directed mutants
with 2 µg of DNA per well of a six-well plate. Each cDNA was
transfected in triplicate, and after 8 h, the medium was replaced
by 1 ml of DMEM-21 containing 0.5% bovine serum albumin, 1%
L-glutamine, and antibiotics (50 units/ml penicillin and 50 µg/ml streptomycin). Supernatants were collected 24 h
post-transfection. The supernatants were clarified of cell debris and
assayed for A
1-40 and A
1-42 by sandwich ELISA. Monoclonal
antibodies developed by Scios used in the ELISA assays included:
1101.1, raised to residues 12-22 of the A
sequence; a C-terminal
A
40-specific antibody, 1702.1; and a C-terminal A
42-specific antibody.
Pulse-Chase Analysis--
HEK293 cells were transiently
transfected with either mock cDNA, wild type PS1, FAD-PS1, or PS1
site-directed mutants with 2 µg of DNA per well of a six-well plate
as previously described. Twenty-four-hour post-transfection cells were
washed twice with ice-cold PBS, and growth medium was replaced with
methionine- and cysteine-free medium supplemented with 0.5%
bovine serum albumin, 1% L-glutamine, and antibiotics (50 units/ml penicillin and 50 µg/ml streptomycin) and incubated at
37 °C for 60 min. At this time, 100 mCi/ml
[35S]methionine/cysteine was added and incubated for a
further 20 min. Medium was then removed, and cells were washed and
replaced with complete growth medium and chased for 0, 1.5, and
3 h. Cells were rinsed two times with ice cold phosphate-buffered
saline and lysed in radioimmune precipitation buffer (50 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM EDTA, 1%
sodium deoxycholate, 1% Triton X-100, 0.1% SDS) supplemented with
protease inhibitors on ice for 15 min. The cell lysates were spun at
14,000 rpm and immunoprecipitated with anti-
-catenin monoclonal
antibody, as described above. Immunoprecipitates were resolved on a
4-12% NuPage Bis-Tris gel (Novex) and analyzed by a PhosphorImager.
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RESULTS |
PS1 Hydrophilic Loop Domain Contains GSK-3
Consensus
Phosphorylation Sites--
PS1 and PS2 encode structurally similar
proteins that share a high degree of homology. Each contain six or
eight transmembrane domains with both N- and C-terminal hydrophilic
regions and a large hydrophilic loop domain. From sequence alignment
studies, it has been previously shown that the hydrophilic loop domain has the greatest sequence divergence, suggesting that the loop domain
may mediate PS1 and PS2 functional differences.
Alignment of the hydrophilic loop domain of PS1 from several species
revealed that the loop domain contained a highly conserved sequence
containing three potential GSK-3
consensus phosphorylation sites,
two of which are conserved in all examined species (Fig. 1A). The sequence
(S/T)XXXS is known to be a consensus sequence for a GSK-3
phosphorylation site. In the hydrophilic loop region of PS1, residues
263-407, there are three possible phosphorylation sites:
TERES324,
STPES357 and
SATAS401. Of the three sites, only
STPES357 and
SATAS401 are conserved across all
examined species. Fig. 1B shows an alignment of PS1 and PS2
hydrophilic loop domains and demonstrates that neither of the GSK-3
consensus phosphorylation sites are present in PS2.

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Fig. 1.
Sequence analysis of PS1 hydrophilic loop
domain. A, the hydrophilic loop domain of PS1 and its
amino acid sequence homology across species. Alignments were done with
Megalign (DNASTAR) software. Residues that fit the consensus sequence
for GSK-3 phosphorylation are indicated in boldface type
and are boxed. B, alignment of PS1 and PS2
hydrophilic loop domains. The residues that fit the consensus sequence
for GSK-3 phosphorylation, present in PS1 but absent from PS2, are
indicated in boldface type and are
boxed. C, alignment of amino acid sequences
recognized as GSK-3 phosphorylation consensus motifs.
Shading indicates amino acids that are identical in all
substrates. D, schematic representation of full-length PS1.
The arrows indicate the location of GSK-3 consensus
phosphorylation residues that were mutated.
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In addition to
-catenin, both APC and Axin are also GSK-3
substrates, and their ability to bind
-catenin in vitro
is enhanced by phosphorylation. The sites necessary for GSK-3
phosphorylation have been identified, and mutating these sites leads to
a disruption in the association between
-catenin and APC or Axin.
Alignment of the amino acid sequence of Axin, APC,
-catenin, and PS1
reveals a conserved consensus motif (Fig. 1C). Since this
motif is conserved in PS1 and not PS2, it seems likely that these
identified GSK-3
consensus phosphorylation sites mediate a specific
PS1 function(s). Specifically, we sought to address whether the
GSK-3
motifs present on PS1 are used to mediate PS1 binding to
-catenin. The general location of each GSK-3
consensus
phosphorylation motif on the PS1 hydrophilic loop domain is
schematically shown in Fig. 1D.
Characterization of PS1 Mutants--
Each of the three consensus
GSK-3
phosphorylation motifs present on PS1 was mutated. Within each
GSK-3
motif, the critical serine and threonine codons of all three
(S/T)XXXS consensus sites were either individually or both
mutated. Table I summarizes the 14 different PS1 mutants we generated, the specific residues mutated, and
the nomenclature used to reference each mutant. Prior to analyzing the
effect of mutating the PS1 GSK-3
motifs relative to
-catenin
binding and function, we first assessed their influence on two other
PS1 functions: endoproteolysis and A
production.
PS1 is synthesized as a 46-kDa polypeptide that undergoes
endoproteolytic processing to generate an ~26-kDa N-terminal fragment and ~20-kDa C-terminal fragment (CTF), which are the predominant in vivo detected PS1 species (18-20). Epitope mapping and
radiosequence analysis have shown that PS1 endoproteolysis occurs
within the hydrophilic loop domain (21). Therefore, we determined
whether the consensus site mutations altered PS1 endoproteolysis,
reflecting a requirement for GSK-3
phosphorylation. To confirm
expression and to evaluate effects on the endoproteolytic processing of
PS1, subconfluent 293HEK cell cultures were transiently transfected with wild type or mutant PS1, each tagged with a His epitope to distinguish exogenous from endogenous PS1. Western blot analysis of
cell lysates with anti-His monoclonal antibody demonstrated the
presence of PS1 holoprotein migrating at ~46 kDa, as well as
increased amounts of the ~20-kDa CTF, compared with mock transfected cell lysates (Fig. 2A,
upper panel). These results demonstrate that the
mutation of any or all of the GSK-3
phosphorylation consensus sites
on the PS1 loop domain does not affect the regulated endoproteolytic
processing of PS1. Western blot analysis of 293HEK cells transiently
expressing tagged or untagged PS1 using PS1 N- and C-terminal specific
antibodies and immunoprecipitation with an anti-His-antibody followed
by Western blot with PS1 C-terminal specific antibody documented the
authenticity of the tagged PS1-CTF (Fig. 2A), reflecting
endoproteolysis and replacement of endogenous PS1-CTF, although
endoproteolysis was somewhat inefficient compared with untagged
exogenous PS1 (Fig. 2A, lower
panels).

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Fig. 2.
Endoproteolysis and A
generation by PS1 GSK-3 phosphorylation
motif mutants. A, effects of GSK-3 mutations on PS1
endoproteolysis. Subconfluent cultures of 293HEK cells were transiently
transfected with wild type or the indicated mutant PS1 cDNAs.
Twenty-four hours post-transfection, cell extracts were prepared, and
expression and endoproteolysis were assessed by protein immunoblot
analysis with anti-His monoclonal antibody (upper
panel). FL, full-length PS1; NTF and
CTF, PS1 N- and C-terminal fragments, respectively. The
asterisk denotes an irreproducible band recognized variably
by anti-His antibody (refer also to Fig. 2A,
lower right panel, and Fig.
4D). Lysates from transiently expressed wild type PS1
constructs, tagged and lacking the His tag, were compared by Western
blot using PS1 N- and C-terminal specific antibodies (lower
left and center panels) and by
immunoprecipitation with an anti-His antibody followed by Western blot
with the PS1 C-terminal antibody (lower right
panel). B, quantification of PS1 mutant effects
on A generation. Subconfluent cultures of 293HEK cells stably
expressing APPswe were transiently transfected with the indicated
presenilin wild type, FAD, or mutant cDNAs. Twenty-four hours
post-transfection, the conditioned medium was collected, and
quantitative analysis of secreted A 1-40 and A 1-42 was performed
using two-site ELISAs. Results are the mean ± S.D. of a
representative experiment performed in duplicate.
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We next determined whether the GSK-3
phosphorylation sites on PS1
altered the production of A
. PS1 has been shown to influence
-secretase function. This has been best demonstrated in cells harboring PS1 targeted gene disruption where the levels of A
are
greatly reduced (22). Furthermore, mutations in PS1 associated with FAD
have been shown to elevate A
42 levels and many have been mapped to
the loop domain (1). Thus, we were curious if the mutations introduced
in PS1 at the GSK-3
motifs would also result in increased levels of
A
42. To measure A
production, 293HEK cell cultures stably
expressing
APP carrying the Swedish mutation (APPswe) were
transiently transfected with wild type, FAD PS1 (E280G), or "GSK-3
" mutant PS1. Twenty-four hours post-transfection, ELISA
quantification revealed an increase in A
42 in conditioned medium collected from cells expressing PS1-FAD E280G mutant
(Fig. 2B). In contrast, A
42 production was not
significantly changed in cells transfected with either wild type or
with PS1 mutated at the conserved GSK-3
motifs (Fig. 2B).
This suggests that mutation of the GSK-3
consensus phosphorylation
sites does not influence the production of A
42.
Interaction of PS1 with
-Catenin Requires Ser353 or
Ser357--
It has previously been shown that the
association between
-catenin and APC or Axin can be disrupted by
mutating their GSK-3
consensus phosphorylation sites (12, 23, 24).
Since PS1 has been shown to interact with
-catenin (5-7) and we
have identified GSK-3
phosphorylation sites on PS1, we examined
whether mutating these sites to nonfunctional sequences for kinase
recognition would affect the association between PS1 and
-catenin.
Subconfluent 293HEK cell cultures were transfected with expression
constructs that directed the synthesis of His epitope-tagged wild type
or PS1 GSK-3
mutant. Consistent with previous reports (6-7),
immunoprecipitation of wild type PS1 quantitatively coprecipitated
endogenous
-catenin (Fig.
3A). Likewise, PS1 mutant
constructs bearing the mutations at residues T320R, S324A, S397A, or
S401A also efficiently coimmunoprecipitated endogenous
-catenin.
However, coimmunoprecipitation of endogenous
-catenin was
dramatically reduced following expression of PS1 mutants bearing
substitutions at residues Ser353 and Ser357
(Fig. 3A). All of the constructs showed similar levels of
PS1 expression (Fig. 3A, lower panel),
as well as equal amounts of total and active GSK-3
(data not shown).
Both Ser353 and Ser357 comprise a single
GSK-3
phosphorylation motif. To determine whether the association
could be attributed to a single residue, we tested two additional
constructs, PS1-S353A and PS1-S357A, each bearing a single point
mutation at the serine residues within this motif.
Coimmunoprecipitation analysis after transient expression of each
mutant demonstrated that the ability of PS1 to associate with
-catenin was virtually abolished when either Ser353 or
Ser357 were mutated (Fig. 3B). Together, these
results clearly demonstrate that the highly conserved consensus
sequence STPES357, containing Ser353 and
Ser357, is sufficient and necessary for the association
between PS1 and
-catenin.

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Fig. 3.
GSK-3 consensus
phosphorylation sites on PS1 and interaction with
-catenin. Subconfluent cultures of 293HEK
cells were transiently transfected with the indicated His
epitope-tagged wild type or mutant PS1 expression vectors. Following
24-36 h, extracts were prepared and normalized to ensure equal protein
levels in all samples; the lysates were immunoprecipitated
(IP) with a control monoclonal antibody (C) or an
anti-His monoclonal antibody (His). Following separation by
SDS-polyacrylamide gel electrophoresis and transfer to polyvinylidene
difluoride membranes, the membranes were immunoblotted with an
anti- -catenin monoclonal antibody to detect association with
endogenous -catenin. A, Western blot of
-catenin coimmunoprecipitated with PS1, wild type, and with
GSK-3 PS1 mutants (upper) and of exogenous PS1 expression
(lower). B, Western blot of -catenin
coimmunoprecipitated with wild type and PS1-(S353A) or PS1-(S357A).
C, Western blot of -catenin coimmunoprecipitated with
wild type and FAD PS1.
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Recent reports have suggested that PS1-FAD mutant effects may be
attributed to an altered association between PS1-FAD and
-catenin
(7, 13-15). We examined the association between PS1-FAD mutants and
endogenous
-catenin. The results shown in Fig. 3C clearly
demonstrate that equivalent quantities of endogenous
-catenin and
PS1 could be coimmunoprecipitated from 293HEK cells transiently transfected with PS1-WT or PS1-FAD (M146V and E280G) expression constructs. Thus, PS1-FAD mutations do not appear to influence binding
to
-catenin.
The Ser353, Ser357 PS1 Motif Can Be
Phosphorylated by GSK-3
--
To directly demonstrate that the
Ser353, Ser357 site is recognized by GSK-3
,
we carried out in vitro kinase studies. A synthetic peptide
with the Ser353, Ser357 motif centered within
the sequence was assessed as a substrate for purified recombinant
GSK-3
. An identical peptide in which Ser353 and
Ser357 were replaced with alanine residues served as a
control substrate. Phosphorylation was observed with only the
Ser353, Ser357-containing substrate following
incubation (Fig. 4A).
Insignificant phosphorylation was seen when the serine residues were
replaced with alanine residues. This result argues against
phosphorylation at the threonine 354 site, a residue shared by both
peptides. The selectivity of Ser353, Ser357
phosphorylation by GSK-3
was determined. We compared levels of
phosphate incorporation onto the Ser353, Ser357
peptide by GSK-3
against four other serine/threonine kinases: p38
, p38
, protein kinase C, and casein kinase II. Only GSK-3
was able to phosphorylate this site (Fig. 4B). Therefore, it
appears that phosphorylation of the Ser353,
Ser357 motif is selective for GSK-3
.

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Fig. 4.
In vitro and in vivo
phosphorylation of PS1 by GSK-3 .
A, in vitro kinase reactions were performed using
synthetic peptides corresponding to either the wild type
Ser353, Ser357 GSK-3 motif
(NH2- GPHRSTPESRAAV-COOH) or mutated
(NH2-GPHRATPEARAAV-COOH) and GSK3 . Reactions, run in
duplicate, were incubated for 0 or 60 min, after which incorporation of
32PO3 was monitored. B, in
vitro reactions to address selectivity were carried out with wild
type peptide and 10 units of kinase for 60 min. GSK-3 , p38 ,
p38 , protein kinase C, and casein kinase II were tested on the PS1
wild type substrate and on their cognate substrates (not shown).
C, immunoprecipitation of PS1 CTF phosphorylated in
vivo. Phosphorylation was assessed by
[32P]orthophosphate-labeling 293HEK cells transiently
expressing wild type (WT) PS1; PS1 carrying Ser353 and
Ser357 mutations; or PS1 carrying Thr320,
Ser324, Ser353, Ser357,
Ser397, and Ser401 GSK-3 site mutations. PS1
CTF in cell lysates was purified by nickel affinity chromatography and
immunoprecipitation. Wild type PS1 was dephosphorylated with alkaline
phosphatase (PS1 WT phosphatase). D, Western blot of wild
type and GSK-3 mutant PS1 CTF expressed in 293HEK cells detected by
anti-His antibody.
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We next evaluated the in vivo phosphorylated state of wild
type PS1, PS1 mutated at the Ser353, Ser357
GSK-3
site, and PS1 mutated at all three GSK-3
consensus sites. Wild type and mutant PS1 DNA were transiently expressed in 293HEK cells, during which time protein was radiolabeled with
[32P]orthophosphate. Each PS1 CTF was purified by virtue
of a His epitope added to the C terminus using nickel affinity
chromatography and immunoprecipitation with anti-His antibody. Wild
type PS1 expression generated a phosphorylated CTF that was sensitive
to alkaline phosphatase (Fig. 4C). The phosphorylated wild
type CTF migrated slightly slower upon gel electrophoresis as expected (Fig. 4, C and D). In contrast to the
phosphorylated CTF observed with wild type PS1 expression, only trace
amounts of phosphorylated CTF were produced by the Ser353,
Ser357 mutant, and virtually no phosphorylated CTF was seen
with PS1 mutated at all three GSK-3
motifs (Fig. 4C).
Wild type and both mutants as shown by Western blot analysis (Fig.
4D) expressed approximately equivalent amounts of CTF. The
PS1 mutant harboring mutations at all three GSK-3
motifs was
expressed at slightly higher levels but was not phosphorylated. These
data indicate that PS1 is phosphorylated at the GSK-3
sites in
vivo.
Disruption of PS1/
-Catenin Interaction Does Not Affect
-Catenin Stability or Tcf/Lef-mediated Signaling--
In response
to activation of the Wnt signaling pathway,
-catenin stability is
increased, enabling translocation to the nucleus, where it regulates
the transcription of Wnt-responsive genes. Several recent conflicting
reports have suggested that the effect of wild type and FAD-PS1 on
-catenin signaling arise from either disruption of processes
affecting cytoplasmic accumulation of
-catenin (13) or processes
involving nuclear translocation of
-catenin (16). To assess the
effect of disrupting PS1 and
-catenin association on
-catenin
signaling, we examined endogenous
-catenin levels by Western blot
analysis of cytosolic and nuclear fractions from 293HEK cells
transiently transfected with either wild type PS1, FAD-PS1, or the
GSK-3
mutant PS1-(S353A,S357A). No apparent differences were
observed between the basal levels of endogenous cytosolic or nuclear
-catenin in untransfected, wild type, or FAD-PS1-expressing cultures
(Fig. 5, A and B).
In 293HEK cells transfected with the PS1-(S353A,S357A) mutant, which disrupts the association between PS1 and
-catenin, both cytosolic and nuclear levels of
-catenin were similar to those of wild type
PS1-expressing cultures (Fig. 5, A and B). This
conclusion is most apparent when
-catenin levels from individual
experiments are normalized to GSK-3
(Fig. 5, histograms).
These results demonstrate that disruption of the association between
PS1 and
-catenin has no apparent adverse affect on cytosolic levels
of
-catenin or on nuclear localization of
-catenin.

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Fig. 5.
PS1/ -catenin
association in regulating -catenin stability
and Tcf/Lef-mediated signaling. Subconfluent cultures of 293HEK
cells were transiently transfected with the indicated wild type, FAD, or site-directed GSK-3 mutants of PS1. Twenty-four hours
post-transfection, cytosolic (A) and nuclear (B)
extracts were prepared, and the levels of endogenous -catenin were
determined by immunoblotting with an anti- -catenin monoclonal
antibody. Data presented in the histogram is representative of three
independent experiments. C, pulse-chase analysis of
-catenin degradation. Subconfluent cultures of 293HEK cells were
transiently transfected with the indicated wild type, FAD, or
site-directed GSK-3 mutants of PS1. Twenty-four hours
post-transfection, cells were pulse-chase-labeled with
[35S]methionine/cysteine and analyzed by
immunoprecipitation of endogenous -catenin with an anti- -catenin
monoclonal antibody. D, subconfluent cultures of 293HEK
cells were transiently transfected with 100 ng of the
pTOPFLASH-luciferase reporter gene plasmid along with 0.5 µg of Wnt-1
and the indicated wild type, FAD, or site-directed mutants of PS1. In
all experiments, cells were harvested 24 h post-transfection, and
levels of luciferase activity were determined according to the
manufacturer's instructions (Promega). Results are the mean ± S.D. of a representative experiment performed in duplicate normalized
for -galactosidase expression. E, -catenin induction
of the Wnt signaling cascade. -Catenin (0.75 µg) was coexpressed
with pTOPFLASH-luciferase and PS1 plasmids in 293HEK cells using an
identical procedure to that described above in D.
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As mentioned earlier, the regulation of
-catenin stability is a
crucial control point in Wnt-mediated signaling pathways. To determine
whether dissociation of the interaction between PS1 and
-catenin
affects the function of
-catenin by altering its stability, we
assessed
-catenin degradation by pulse-chase analysis. Subconfluent
293HEK cells were transiently transfected with an expression construct
that encoded His epitope-tagged versions of either wild type PS1, FAD
PS1, or GSK-3
mutant PS1-(S353A,S357A) or PS1-(T320R,S324A).
Endogenous
-catenin was degraded at a similar rate in cells
transfected with vector alone, wild type PS1, or FAD PS1, which
contains the E280G mutation associated with FAD (Fig. 5C).
Endogenous
-catenin in cells expressing PS1-(S353A,S357A) which is
unable to associate with
-catenin had an equivalent half-life. Thus,
disruption of the binding between PS1 and
-catenin does not affect
the stability of
-catenin.
To directly assess the role of PS1 and its association with
-catenin
in a
-catenin signaling cascade, we determined the effects of wild
type PS1, FAD PS1, and several PS1 GSK-3
mutants in a Tcf-luciferase
reporter assay. In the prevailing model, following the initiation of a
Wnt signaling cascade, GSK-3
is inhibited, leading to decreased
phosphorylation and accumulation of cytosolic
-catenin. This allows
-catenin to translocate to the nucleus and regulate gene expression
via interaction with Tcf/Lef transcription factors. To measure PS1
activity or involvement, we cotransfected 293HEK cells with an
expression vector encoding Wnt-1 together with wild type PS1, FAD PS1,
GSK-3
mutant PS1-(S353A, S357A), or PS1-(T320R,S324A). Upon
expression, Wnt-1 acts in an autocrine/paracrine fashion to activate
its receptor, frizzled, on 293HEK and to initiate the
-catenin
signaling cascade (25). Expression of Wnt-1 alone induced Tcf-mediated
luciferase activity in a dose-dependent manner (data not
shown) with maximum induction of luciferase activity being 6-8-fold
compared with control vector (Fig. 5D). Cotransfection of
Wnt-1 with wild type PS1, FAD PS1, or control PS1 GSK-3
mutant (T320A,S324A) did not significantly affect Tcf-mediated luciferase activity (Fig. 5D). Similarly, coexpression of Wnt-1 with
PS1-(S353A,S357A) or FAD PS1 (S353A,S357A), which fail to associate
with
-catenin, had little effect on Wnt-1-induced Tcf-luciferase
activity (Fig. 5D). Identical results were obtained when
cells were cotransfected with
-catenin and the same PS1 constructs
(Fig. 5E). Taken together, these results clearly demonstrate
that dissociation of PS1 and
-catenin binding has no significant
effect on
-catenin stability or on Wnt-1- or
-catenin-mediated
gene expression.
Disruption of PS1/
Catenin Binding Does Not Influence
PS1-FAD-increased Susceptibility to Apoptosis--
One of the
pathological characteristics of PS-associated FAD is an increased
susceptibility of neuronal cells to inducers of apoptosis (2-4). We
next determined whether disruption of PS1 and
-catenin binding
affected cell viability by altering cell vulnerability to apoptosis.
Subconfluent cultures of HEK293 cells were cotransfected with green
fluorescent protein and wild type PS1, FAD PS1, or PS1 GSK-3
mutants
(Fig. 6A). Following treatment
with etoposide, all cell cultures displayed a progressive increase in
the number of apoptotic cells (Fig. 6A). Cells treated with
etoposide exhibited characteristic morphologies of cells undergoing
apoptosis, becoming rounded and condensed and detaching from the
culture dish (Fig. 6B). Consistent with previous reports, cells expressing PS1-FAD mutants showed a significant enhancement in
their susceptibility to etoposide, with increased numbers of apoptotic
cells present at 12 h after exposure to etoposide (Fig. 6A). Expression of either wild type PS1-(S353A,S357A) or FAD
PS1-(S353A,S357A), which no longer associate with
-catenin, did not
affect cell susceptibility to apoptosis. Thus, dissociation of the
binding between
-catenin and either wild type PS1 or FAD PS1 does
not alter cell vulnerability to apoptosis.

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Fig. 6.
PS1/ -catenin
association and cell survival. A, subconfluent cultures
of 293HEK cells were transiently transfected with wild type, FAD or the
indicated site-directed mutants of PS1, together with a plasmid
encoding green fluorescent protein (pGFP) as an indicator of
transfection. Following 24 h, cells were either untreated or
treated with etoposide (25 µg/ml) for the indicated time, and the
percentage of apoptosis was assessed by morphological evaluation of
pGFP-positive cells by fluorescent microscopy. B, morphology
of 293HEK cells under standard culture conditions and cells treated
with etoposide. Typical apoptotic features displayed by
etoposide-treated cells include cell shrinkage, cytosolic and nuclear
condensation, and detachment from the culture vessel
(arrow).
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DISCUSSION |
PS1 has been shown to associate with
-catenin (5-7); however,
the functional significance of this interaction has been unclear. Likewise, the influence of FAD PS1 on
-catenin biology and how this
might result in a pathophysiological state has not been fully resolved.
PS1 and
-catenin have independently been shown to be associated with
cell survival signaling (2, 26, 27). Consequently, it has been
suggested that FAD PS1 causes dysregulation of
-catenin such that
survival signaling is compromised toward apoptosis. This idea has
appeal given the highly reproducible observation that FAD PS1 and FAD
PS2 increase cell susceptibility to apoptosis (2-4). Whether FAD PS1
establishes a proapoptotic state by reducing
-catenin signaling
through destabilizing this protein (13), by impeding its nuclear
translocation (16), or by some other mechanism is controversial.
In this study, we sought to further define the physical nature of PS1
and
-catenin interaction as well as the role of this association in
Wnt signaling. Three key observations can be derived from this work.
First,
-catenin binding requires either of two serine residues
located in the hydrophilic loop domain of PS1, Ser353 and
Ser357. Mutating either of these serine residues
essentially abolishes the in vivo phosphorylation of PS1 as
well as the ability of PS1 to bind to
-catenin. Furthermore,
Ser353 and Ser357 comprise a consensus
recognition site for phosphorylation by GSK-3
, a serine/threonine
kinase known to regulate multiple members of the Wnt signaling complex.
Second, ablating PS1/
-catenin association has no effect on PS1
function as it relates to endoproteolysis and A
production. Third,
-catenin stability and signaling are unaltered in the absence of PS1
binding. Moreover, there is no increased propensity toward apoptosis
when PS1 is unable to bind efficiently to
-catenin.
Since the observation that PS1, but not PS2, binds to
-catenin (9),
subsequent reports have grossly mapped the domain of
-catenin
interaction to the hydrophilic loop of PS1 (7, 10). Upon alignment of
the hydrophilic loop domain of PS1 for several species, we identified a
highly conserved sequence containing three GSK-3
consensus
phosphorylation sites ((S/T)XXXS) (28). Two of these
consensus motifs are fully conserved across species, and both are
absent in PS2. We anticipated that these GSK-3
site(s) on PS1 might
be involved in
-catenin association. Several members of the Wnt
signaling complex are regulated by GSK-3
, including Axin, APC, and
-catenin. Phosphorylation of Axin and APC by GSK-3
enhances the
efficiency of
-catenin binding to these proteins, thereby
facilitating the phosphorylation of
-catenin by GSK-3
(12, 28).
Therefore, we evaluated the potential role of these three PS1 GSK-3
consensus sites in
-catenin binding using site-directed mutagenesis
of the serine/threonine residues phosphorylated by GSK-3
within each
motif. Our results identified one GSK-3
motif as critical for
PS1/
-catenin association. Mutating either Ser353 or
Ser357 of this consensus site essentially abolished
PS1/
-catenin interaction in vivo. Our result is in
agreement with that of Murayama et al. (7), who mapped the
site to 322-450 of PS1 by deletion mutagenesis and
coimmunoprecipitation to analyze association of PS1 and
-catenin in vivo. Our data are somewhat incompatible with the
findings of Saura et al. (10), who localized the binding to
331-351 on PS1. In the study by Saura et al., however,
portions of the PS1 loop domain were expressed as fusion proteins in
E. coli and assessed for
-catenin binding in
vitro. Thus, PS1 was removed from its normal structural context.
Because mutating Ser353 and/or Ser357 greatly
reduces but does not completely abolish
-catenin binding, it is
possible that residual binding occurs through minor adjacent sites on
PS1, such as at residues 331-351. Nonetheless, our data support the
primary site of in vivo association with
-catenin at this
GSK-3
consensus motif in the PS1 loop domain.
Several independent reports support our conclusion that the
Ser353, Ser357 motif on PS1 is phosphorylated
by GSK-3
. The C-terminal loop domain fragment of PS1 in COS cells
has been shown to be phosphorylated at serine residues (29, 30). Also,
Takashima et al. (11) demonstrated an in vivo
association of PS1 and GSK-3
using coimmunoprecipitation. We were
able to demonstrate selective in vitro phosphorylation at
the Ser353, Ser357 residues of the consensus
motif by GSK-3
but not by four other common serine/threonine
kinases. Importantly, we showed that the Ser353,
Ser357 consensus site is phosphorylated in vivo.
Mutations at these residues prevent phosphorylation of PS1 CTF in
293HEK cells. Together, these observations demonstrate the PS1 loop is
a substrate for GSK-3
in vivo.
The Ser353, Ser357 PS1 point mutants that lack
the ability to efficiently bind to
-catenin provided us with a means
to address the functional significance of this interaction. Several
activities were measured relevant to each of these two proteins. With
regard to PS1 function, we demonstrated that both A
production and
endoproteolysis were normal when
-catenin binding was prevented.
This result agrees with that of Saura et al. (10), who
deleted the entire loop domain of PS1 and found no effect on
endoproteolysis or A
generation. More interesting are the results we
obtained examining the effects of PS1 dissociation on
-catenin
stability and cell survival signaling. No change in either free
cytosolic or nuclear
-catenin was observed in the absence of PS1
association. Similarly, no significant differences were seen in either
cellular compartment between exogenous wild type and FAD PS1. The
effects of PS1 FAD on
-catenin levels have been highly
controversial, and little consensus exists between numerous
investigations including this one (13-16). This inconsistency is most
likely attributed to the technical difficulties of measuring a small
pool of cytosolic
-catenin against a background of plasma
membrane-associated
-catenin that is roughly 10-fold larger (16).
Differences in the methods used to assay
-catenin might also be a
contributing factor to this controversy.
The most important issue this study addresses is the role of
PS1/
-catenin interaction in Wnt signaling. Using the
Ser353, Ser357 mutant, we were able to
demonstrate normal
-catenin signaling when the association between
PS1 and
-catenin is largely abolished. This result is supported by
an observation of Nishimura et al. (16), who found identical
levels of nuclear
-catenin in PS1+/+ and PS1
/
fibroblasts after
exposure to Li+ to induce the Wnt signaling cascade. In
addition, we found no difference in Wnt signaling between wild type PS1
and PS1 FAD. While Nishimura et al. (16) elegantly showed
defective nuclear translocation of
-catenin in association with FAD
PS1, this study did not include a functional measure for
-catenin.
Therefore, it seems possible that while there may exist deficiencies in
-catenin trafficking in the FAD PS1 background, sufficient levels of
-catenin apparently reach the nuclear compartment to allow for
normal induction of Tcf/Lef-mediated transcription. We were also able
to demonstrate using the Ser353, Ser357 point
mutants that disrupting the physical association between PS1 and
-catenin has no impact on cell viability. Previously, it has been
proposed that FAD PS1 destabilizes
-catenin, promoting a
proapoptotic state (13). While we observed increased susceptibility to
apoptosis with expression of E280G FAD PS1 compared with wild type PS1,
no further increase was seen with E280G PS1 bearing mutations at
Ser353 and Ser357. Similarly, wild type and
Ser353, Ser357 mutant PS1 displayed identical
responses to apoptotic insult. Hence, we conclude that PS1/
-catenin
interaction is not essential for maintenance of cell survival. In the
work of Zheng et al. (13), loss of
-catenin signaling was
shown to increase neuronal apoptosis; however, this effect was not
directly linked to
-catenin instability due to FAD PS1. We suggest
that the FAD PS1 effects observed on
-catenin transport, stability,
and cell survival signaling involve a mechanism separate from the
ability of PS1 to associate with
-catenin.
In summary, it appears that the interaction between PS1 and
-catenin
requires GSK-3
phosphorylation at serine residues within a consensus
motif located on the PS1 loop domain. When these residues are altered
to eliminate their potential for phosphorylation in vivo,
binding between PS1 and
-catenin is essentially abolished. No
negative consequences resulted from the disruption of the
PS1/
-catenin interaction with respect to A
production,
-catenin stability, Wnt signaling, or cell survival. This indicates
that association of PS1 with
-catenin is nonessential for these
functions and that alterations in the interaction between these two
proteins are unlikely to contribute to the pathogenesis of familial
Alzheimer's disease.