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The Journal of Neuroscience, June 1, 1999, 19(11):4229-4237
Presenilin 1 Facilitates the Constitutive Turnover of
 -Catenin: Differential Activity of Alzheimer's Disease-Linked PS1
Mutants in the -Catenin-Signaling Pathway
David E.
Kang1,
Salvador
Soriano1,
Matthew
P.
Frosch2, 3,
Tucker
Collins3,
Satoshi
Naruse4,
Sangram S.
Sisodia4,
Gil
Leibowitz5,
Fred
Levine5, and
Edward H.
Koo1
Departments of 1 Neurosciences and
5 Pediatrics, University of California, San Diego, La
Jolla, California 92093, 2 Center for Neurological
Diseases, Brigham and Women's Hospital, Boston, Massachusetts 02115, 3 Department of Pathology, Harvard Medical School, and
Department of Pathology, Brigham and Women's Hospital, Boston,
Massachusetts 02115, and 4 Department of Pharmacological
and Physiological Science, University of Chicago, Chicago, Illinois
60637
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ABSTRACT |
Although an association between the product of the familial
Alzheimer's disease (FAD) gene, presenilin 1 (PS1), and  -catenin has been reported recently, the cellular consequences of this interaction are unknown. Here, we show that both the full length and
the C-terminal fragment of wild-type or FAD mutant PS1 interact with -catenin from transfected cells and brains of transgenic mice,
whereas E-cadherin and adenomatous polyposis coli (APC) are not
detected in this complex. Inducible overexpression of PS1 led to
increased association of -catenin with glycogen synthase kinase-3
(GSK-3), a negative regulator of -catenin, and accelerated the
turnover of endogenous -catenin. In support of this finding, the
-catenin half-life was dramatically longer in fibroblasts deficient
in PS1, and this phenotype was completely rescued by replacement of PS1, demonstrating that PS1 normally stimulates the
degradation of -catenin. In contrast, overexpression of FAD-linked PS1 mutants (M146L and  X9) failed to enhance the association between
GSK-3 and -catenin and interfered with the constitutive turnover
of -catenin. In vivo confirmation was demonstrated in the brains of transgenic mice in which the expression of the M146L mutant PS1 was correlated with increased steady-state levels of endogenous -catenin. Thus, our results indicate that PS1 normally promotes the turnover of -catenin, whereas PS1 mutants partially interfere with this process, possibly by failing to recruit GSK-3 into the PS1--catenin complex. These findings raise the intriguing possibility that PS1--catenin interactions and subsequent
activities may be consequential for the pathogenesis of AD.
Key words:
presenilin; -catenin; glycogen synthase kinase-3; immunoprecipitation; turnover; half-life; Alzheimer's disease
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INTRODUCTION |
Inherited mutations in the gene
coding for presenilin 1 (PS1) cause the most aggressive form of
Alzheimer's disease (AD) and account for a large proportion of
familial early-onset AD (FAD) (Sherrington et al., 1995 ). However,
neither the physiological nor the aberrant activities associated with
FAD-linked presenilin gene products are clearly understood. PS1, a six
to eight multipass transmembrane protein (Doan et al., 1996; De
Strooper et al., 1997; Lehmann et al., 1997) enriched in
nuclear, endoplasmic reticulum (ER), and Golgi membranes (Kovacs et
al., 1996; Li et al., 1997), undergoes constitutive endoproteolytic
processing into relatively stable N- and C-terminal fragments in the ER
(Thinakaran et al., 1996; J. Zhang et al., 1998). PS1
deficiency in mice results in an embryonic lethal phenotype associated
with severe malformations of the axial skeleton and cerebral hemorrhage
(Shen et al., 1997; Wong et al., 1997). In cell culture, we have
likewise demonstrated that inhibition of PS1 expression in Ntera2
neuronal precursor cells results in the loss of neuronal
differentiation and the concomitant increase in cell death (Hong et
al., 1999). In Caenorhabditis elegans, PS1
facilitates signaling mediated by the Lin-12/Notch family of receptors
(Levitan and Greenwald, 1995). However, the molecular processes by
which PS1 exerts these functions are unknown.
Recent studies have documented the interaction of PS1 with -catenin,
suggesting that PS1 may be involved in modulating the Wnt--catenin
signaling pathway (Zhou et al., 1997; Yu et al., 1998). The binding of
Wnt ligands to cell-surface receptors initiates a cascade of
intracellular signals resulting in the inactivation of glycogen
synthase kinase-3 (GSK-3) and the translocation of -catenin
into the nucleus (Orsulic and Peifer, 1996). Subsequently, the
association of -catenin with the T-cell factor/lymphoid enhancer factor-1 family of transcription factors mediates the expression of
downstream genes (Behrens et al., 1996). In the absence of Wnt
stimulation, -catenin is rapidly targeted for degradation via the
ubiquitin-proteosome pathway, a step that requires the phosphorylation
of -catenin by GSK-3 and the binding to adenomatous polyposis
coli (APC) (Aberle et al., 1997; Morin et al., 1997). It was
shown recently that axin negatively regulates -catenin by bridging
-catenin and GSK-3 together in the same complex and thereby
facilitating the phosphorylation of -catenin by GSK-3 (Hart et
al., 1998; Ikeda et al., 1998).
Recent studies have documented apparently conflicting results regarding
the effects of PS1 proteins on the "stability" of -catenin.
However, of the two recent reports, one examined the turnover of
epitope-tagged -catenin and the generation of putative -catenin
proteolytic fragments (Z. Zhang et al., 1998), whereas the other
analyzed steady-state levels of endogenous, cytosolic -catenin
(Murayama et al., 1998). Because -catenin exists in multiple
cellular pools (Ozawa and Kemler, 1992; Papkoff, 1997), it is
conceivable that measuring a partial, static, or transfected pool of
-catenin can yield different outcomes. In this study, we examined
the turnover rate and steady-state levels of endogenous, full-length
-catenin in cultured cells and transgenic mice. Our results indicate
that PS1 constitutively stimulates the turnover of endogenous
-catenin, whereas FAD-linked PS1 mutations interfere with this activity.
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MATERIALS AND METHODS |
Antibodies. PS1 polyclonal antibodies including J27
(against residues 27-42), 4627 (against residues 457-467),  PS1Loop
(against residues 319-442), and the PSN2 monoclonal antibody (against
residues 31-56) were used in this study (Thinakaran et al., 1996; J. Zhang et al., 1998). Additional monoclonal antibodies include
-catenin, E-cadherin, and GSK-3 (Transduction Laboratories,
Lexington, KY) and APC (Ab-1 and Ab-5; Calbiochem, La Jolla, CA). The
-catenin polyclonal and actin monoclonal antibodies were purchased
from Sigma (St. Louis, MO).
Generation of cell lines. EcR293 cells (Invitrogen, San
Diego, CA) inducibly expressing PS1 variants [PS1 wild type (WT), M146L, and X9] have been described previously (J. Zhang et
al., 1998). At least three independent clones of each construct were generated, and representative results of two clones are shown. Induction was typically performed with 0.1-5 µM
muristerone. PS1  / and /+ fibroblast cultures were
prepared from the skin of embryonic day 15.5 fetal mice (Wong et al.,
1997). Cells were enzymatically dissociated by the addition of 0.25%
trypsin and 20 µg/ml DNase I in HBSS at 37°C for 30 min. After
dissociation by repeated trituration, cells were spun at 200 × g and resuspended in DMEM containing 10% FCS, and the
suspension was plated into 25 cm2 flasks to yield an
initial plating density of 4-5 × 104
cells/cm2. Cells (80-90% confluent) were passaged
by trypsinization and cultured as above. Immortalization of
PS1 / fibroblasts was accomplished by expression of SV40
T antigen. In brief, the retroviral vector plasmid expressing SV40 T
antigen was constructed by replacing the chloramphenical
acetyltransferase gene in the vector LoCRNL0 with the SV40 T antigen
cDNA, and the virus was produced as a VSV-G pseudotype as
described previously (Wang et al., 1996). Primary PS1 /
fibroblasts were infected by adding the virus in the presence of 4 mg/ml polybrene. Transformed PS1-deficient fibroblasts were then
transfected with human PS1 WT or X9 mutant subcloned into pIREShygro
(Clontech, Palo Alto, CA) or vector control, and stable transfectants
were selected by hygromycin resistance.
Immunoprecipitations, Western blotting, and metabolic
labeling. Cultured cells were lysed in 1% NP-40 lysis
buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.02%
sodium azide, 1% NP-40, 100 µg/ml [amino ethyl benzenesulfonyl
fluoride (AEBSF), and 10 µg/ml leupeptin] on ice for 20 min.
In experiments involving GSK-3, 1% NP-40 lysis buffer was
supplemented with 1 mM sodium vanadate and 20 mM sodium fluoride for inhibition of phosphatase activity.
Lysates were precleared with normal rabbit serum and protein A
sepharose (for rabbit polyclonal antibodies) or with anti-mouse IgG
agarose (for mouse monoclonal antibodies) and then incubated overnight
at 4°C with primary antibody in the presence of protein A sepharose
or anti-mouse IgG agarose. Immune complexes were washed twice for 15 min each at room temperature in 1 ml of 1% NP-40 lysis buffer and
heated to 70°C for 20 min in 2× Laemmli buffer. After separation in
SDS-polyacrylamide gels (SDS-PAGE), proteins were transferred onto
nitrocellulose membranes. Coimmunoprecipitated proteins were detected
by the incubation of the cognate primary antibody followed by
HRP-conjugated secondary antibody and enhanced chemiluminescence.
For -catenin turnover experiments, overnight cultures of fibroblasts
or EcR293 cells with or without muristerone induction were incubated in
methionine-free medium for 20 min, followed by metabolic labeling with
100 µCi/ml [35S]methionine for 15 min and
chasing for 0, 0.5, 1, or 2 hr. At each time point, cells were lysed in
1% NP-40 lysis buffer and immunoprecipitated with a monoclonal
antibody directed against the C terminal of -catenin.
Immunoprecipitates were washed twice in radioimmunoprecipitation assay
buffer (50 mM Tris, 150 mM NaCl, 0.02% sodium
azide, 1% NP-40, 0.5% deoxycholate, and 0.1% SDS), resuspended in
2× Laemmli buffer, and separated in 8% SDS-PAGE.
Dried gels were either exposed to film or quantitated by
phosphorimaging (Bio-Rad, Hercules, CA). All cell culture experiments were performed at least three times. The results are presented either
from representative experiments or as an average of all experiments as indicated.
Generation of transgenic mice and homogenization of mouse
brains. The transgene constructs were assembled using a portion of
the human platelet-derived growth factor (PDGF) B-chain promoter (Sasahara et al., 1991) to drive expression of PS1 cDNAs (WT or M146L
mutation). A 3.4 kb fragment containing the promoter, cDNA, and other
required sequences was then excised and used for pronuclear injection
performed in the Transgenic Mouse Facility (Department of Pathology,
Brigham and Women's Hospital, Boston, MA). Transgenic lines were
expanded via breeding with inbred FVB/N mice. Genomic DNA was isolated
from tail snips of 3- to 4-week-old animals using a standard proteinase
K digestion procedure. RNA was isolated from brain tissue using TRIzol
reagent (Life Technologies, Gaithersburg, MD). For verification of
transgenic animals, PCR reactions were run with three primers: a
forward PCR primer (FP1320, 5'-GGCCAGAAGAGGAAAGGCT-3') that anneals
equally well to the endogenous mouse and human PDGF B-chain promoter
portion of the transgene and two reverse primers specific to either the
PS1 cDNA (RP1841, 5'-GTACAGTATTGCTCAGGTGGTTGT-3') or the mouse
genomic PDGF B-chain gene (RP-B2, 5'-AGTCTGCTATCTACCCACTCGCT-3'). The
transgene and endogenous mouse gene products of this reaction are 521 and 355 bp, respectively. For reverse transcription (RT)-PCR to
examine mRNA expression, first-strand cDNA synthesis was performed with
oligo-dT12-18 and reverse transcriptase. The primers for
the subsequent amplification consist of a common forward primer recognizing both human and mouse PS1 (RT-FP,
5'-GAGCTGCTGTCCAGGAACTTTC-3') and two reverse primers specific to
either the 3'-untranslated region of the transgene (RT-tRP,
5'-TCACTGCATTCTAGTTGTGGTTTGT-3') or the mouse PS1 3'-untranslated
region (RT-mRP, 5'-GAAACATCCATGTTCTAACTGCAGA-3). The transgene
(400 bp) and endogenous mouse (360) mRNA gene products are resolved on
a 1.9% agarose gel. Two controls were included to ensure that
transgene-specific PCR products accurately reflect transcription: (1)
DNase I treatment before reverse transcriptase to degrade any possible
contaminating genomic DNA and (2) inclusion of RT samples to
demonstrate that any signal required the generation of cDNA from mRNA.
Half brains (excluding the cerebellum) from transgenic mice expressing
the human wild-type or FAD M146L mutant PS1 and from nontransgenic
littermates were homogenized by >60 strokes in a micro-Dounce
homogenizer in buffer (0.5% Triton X-100, 50 mM
NaCl, 10 mM HEPES, pH 6.8, 3 mM
MgCl2, 300 mM sucrose, 100 µg/ml
AEBSF, and 10 µg/ml leupeptin). The Triton X-100 insoluble material
was precipitated twice by centrifugation and discarded. The supernatant of the second spin was then used for all subsequent procedures. Protein concentrations were measured by the micro-BCA method (Pierce, Rockford, IL).
Affinity precipitation of -catenin from brain. To
generate recombinant glutathione S-transferase (GST)-PS1
loop fusion protein, the putative loop region of PS1 (amino acid
residues 263-407) was subcloned into pGEX4T-1 plasmid (Pharmacia,
Piscataway, NJ), transformed into Escherichia coli
strain HB101, and induced with isopropyl--D-thiogalactopyranoside. GST or the
GST-PS1 loop fusion proteins were purified from bacterial lysates
using glutathione agarose beads in accordance with the manufacturer's
instructions (Pharmacia). For affinity precipitations with recombinant
GST fusion proteins, clarified mouse brain lysates were first
precleared with GST-agarose beads, and 1 µg of GST-PS1 loop fusion
protein was added to the lysates (200 µg) in the presence of
glutathione agarose beads. After 1 hr of incubation at 4°C,
precipitates were washed three times with 1% NP-40 buffer and
subjected to SDS-PAGE and immunoblotting for -catenin.
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RESULTS |
Association of the PS1 full length and C-terminal
fragment with -catenin but not with cadherin or APC
To examine the role of PS1 in the -catenin pathway, we
generated stably transfected EcR293 cells capable of inducible
overexpression of wild type or FAD mutant PS1 variants (M146L and
X9). Figure 1 shows the inducibility
profile of EcR293 cells stably transfected with wild-type PS1 or the
FAD-linked X9 mutant. Induction of wild-type PS1 with increasing
amounts of muristerone resulted in a dose-dependent increase in both
the N-terminal fragment (NTF) and full-length (FL) PS1, whereas the
X9 mutant led to a diminution of endogenous NTF concomitant to the
appearance of full-length X9 protein (Fig. 1). Consistent with
previous reports (Zhou et al., 1997; Yu et al., 1998), our
immunoprecipitation studies revealed -catenin in immune complexes of
wild-type PS1 or FAD-linked PS1 mutants (M146L and X9), whereas
preimmune serum failed to detect any PS1--catenin complexes under
identical conditions (see Figs. 1, 2b, 3). The amount
of -catenin complexed to wild-type PS1 or X9 mutant was directly
correlated with the level of PS1 protein induction by muristerone in
EcR293 cells (Fig. 1). Interestingly, in cells expressing wild-type
PS1, the amount of PS1--catenin complex correlated better with the
amount of PS1 fragments than with the appearance of the full-length
protein. Extraction of proteins by 1% NP-40 revealed that -catenin
associated with both the full length and the C-terminal fragment
(CTF) of PS1 but not with the NTF, demonstrating that the
-catenin-binding domain is contained within the CTF (Fig.
2a,c).
Nevertheless, extraction of proteins in 0.5%
3[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPS) revealed both the CTF and NTF in -catenin immune complexes, consistent with previous studies demonstrating the interaction between
the CTF and NTF in nondenaturing conditions (data not shown) (Capell et
al., 1998). However, E-cadherin, the major transmembranous -catenin-binding partner, was absent from the PS1--catenin
complex (Fig. 2b), a finding that is consistent with the
subcellular localization of PS1 to perinuclear structures and of
cadherins to the cytoskeleton and plasma membrane. Moreover, we were
unable to detect PS1 in APC immune complexes (Fig. 2c).
Conversely, APC was not detected in PS1 immune complexes under
experimental conditions in which APC was easily detected in -catenin
immune complexes (Fig. 2d). Thus, these data indicate that
PS1--catenin complexes do not contain APC or E-cadherin.
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Figure 1.
Induction profile of PS1 proteins in stably
transfected EcR293 cells and coimmunoprecipitation of -catenin with
PS1 variants. Stably transfected EcR293 cells were treated with
increasing amounts of muristerone (Mur; 0-5
µM for 20 hr) to induce expression of wild-type PS1 or
the X9 mutant, and cells were lysed in 1% NP-40 buffer.
Top, An immunoblot of PS1 visualized by a monoclonal
antibody against the N terminal of PS1 (PSN2;
PS1-N). The full length (~45 kd) and N-terminal
fragment (~29 kd) of wild-type PS1 and the truncated X9 protein
(~42 kd) are shown. Bottom, The same cell lysates
immunoprecipitated with polyclonal antibody against the C terminal of
PS1 (4627), followed by immunoblotting for -catenin
(-cat) using a monoclonal antibody directed against
the C terminal of -catenin (PS1-C).
IP, Immunoprecipitation.
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Figure 2.
The full length and the C-terminal fragment of
PS1 interact with -catenin but not with E-cadherin or APC.
a, Stably transfected EcR293 cells were treated with or
without muristerone (2 µM for 20 hr), and cell lysates
were immunoprecipitated with a polyclonal antibody directed against
-catenin and analyzed for the presence of the FL, NTF, and CTF of
PS1. The PS1 FL and NTF were detected by the PSN2 monoclonal antibody
specific for the N terminal of PS1 (PS1-N). The
CTF was detected by the PS1Loop antibody. b,
Left, Cell lysates were immunoprecipitated with a
polyclonal antibody against the N terminal of PS1 (J27;
PS1-N) or J27 preimmune serum
(Pre) for detection of -catenin.
Right, An antibody directed against the C-terminal end
of PS1 (4627; PS1-C) fails to coimmunoprecipitate
E-cadherin (E-cad). C, Cell lysates were
immunoprecipitated with antibody against -catenin or APC and probed
with PS1 loop antibody for the presence of PS1 CTF. D,
Cell lysates were immunoprecipitated with an antibody against the N
terminal of PS1 (J27; PS1-N) or -catenin, and
immune complexes were analyzed for the presence of APC. Molecular
weights from prestained protein standards are shown in kilodaltons on
the right.
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Recruitment of GSK-3 by wild-type PS1 but not by X9
and M146L mutants
In most cell types, -catenin-mediated signaling is tightly
regulated by rapid and constitutive degradation of -catenin via the
ubiquitin-proteosome pathway, a process that requires the phosphorylation of -catenin by GSK-3 (Aberle et al., 1997). Thus,
we assessed whether GSK-3 might be in complex with PS1 and
-catenin. In rapidly dividing cells, GSK-3 coprecipitated with
the full length and the NTF of wild-type PS1 (Fig.
3), in contrast to the interaction of
-catenin with the CTF of PS1 (Fig. 2a,c).
Notably, overexpression of PS1 led to a robust increase in the amount
of the -catenin-GSK-3 complex (Fig. 3), suggesting that PS1
facilitates the -catenin-GSK-3 interaction, perhaps by serving
as a scaffold on which both -catenin and GSK-3 are recruited into
the same complex. In contrast, sequestration of GSK-3 was markedly
reduced in cells overexpressing the two different FAD-linked PS1
mutants (M146L and X9), although the binding to -catenin was
unaffected (Fig. 3). Moreover, overexpression of the M146L or X9
mutant did not appreciably increase the amount of -catenin in
GSK-3 immune complexes, suggesting that these PS1 mutants do not
enhance complex formation between -catenin and GSK-3 (Fig.
3).
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Figure 3.
Recruitment of GSK-3 into the
PS1--catenin complex of wild-type PS1 but not by X9 and
M146L mutants. Stably transfected EcR293 cells were treated with or
without muristerone (2 µM for 20 hr) to induce the
expression of PS1 variants and were lysed in 1% NP-40 buffer. Equal
amounts of protein from uninduced and induced parallel cultures were
subjected to immunoprecipitation with an antibody against PS1 (4627;
PS1-C) or GSK-3. Immune complexes were detected by
immunoblotting with antibodies specific for -catenin, GSK-3, or
PS1 (PSN2) as indicated on the left. Note that
PS1-GSK-3 complexes are detected only in wild-type
PS1-overexpressing cells. Concomitantly, the amount of
-catenin-GSK-3 complex is prominently increased by induction of
wild-type PS1 but not by M146L and X9 mutants. The two bottom
panels show coimmunoprecipitation of -catenin with wild-type
PS1 and mutants (M146L and X9) and the expression level of PS1
variants after induction by muristerone as detected by immunoblotting
(PSN2; PS1-N). Molecular weights are shown in
kilodaltons on the right in this and all subsequent
figures.
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X9 and M146L mutants interfere with constitutive
turnover of endogenous -catenin
Because GSK-3-mediated phosphorylation of -catenin leads to
-catenin degradation, we examined whether the differential interaction of wild-type and FAD mutant PS1 with GSK-3 alters the
turnover of -catenin. No detectable differences were observed in the
steady-state levels of -catenin in cell lines expressing wild-type
and mutant PS1 after 24 hr of induction (data not shown). However,
pulse-chase experiments revealed that overexpression of wild-type PS1
promoted the degradation of -catenin, whereas both FAD mutants
(M146L and X9) significantly delayed the constitutive turnover of
-catenin. Figure 4a shows a
representative experiment in which induction of wild-type PS1 decreased
the half-life of -catenin by ~15 min, whereas induction of the
X9 mutant increased the half-life of -catenin by >1 hr (from
~30 to >90 min) compared with that in uninduced control cells.
Because the constitutive half-life of -catenin appeared to be 20-30
min, we then measured the amount of -catenin remaining after 30 min
of degradation in cells inducibly expressing wild-type or mutant PS1.
In multiple experiments (n = 3), levels of -catenin
were reduced by ~40% after induction of wild-type PS1, whereas
-catenin levels were increased by ~50 and ~75% after induction
of M146L and X9 mutants, respectively, compared with levels in their
uninduced controls (Fig. 4b; ANOVA comparison of wild-type,
M146L, and X9, F = 10.593; p = 0.0108; post hoc Tukey, mutants compared with wild type,
*p < 0.05). These results indicate that overexpression
of M146L and X9 mutants partially interferes with the constitutive
turnover of endogenous -catenin.
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Figure 4.
Wild-type PS1 facilitates and FAD-linked PS1
mutants perturb the constitutive turnover of endogenous -catenin.
A, Parallel cultures of muristerone-induced and
uninduced cells were metabolically labeled with
[35S]methionine for 20 min and chased for 0, 0.5, 1, and 2 hr. Top, Lysates were immunoprecipitated with a
-catenin monoclonal antibody and analyzed by autoradiography.
Bottom, The decay rate of -catenin from the actual
autoradiograms was plotted for control and muristerone-induced cells
expressing wild-type PS1 or the X9 mutant. B, Cells
treated with or without muristerone for 20 hr were metabolically
labeled with [35S]methionine for 20 min, chased
for 0 or 30 min, and immunoprecipitated for -catenin. The stability
of endogenous -catenin is expressed as the proportion of -catenin
remaining after 30 min of degradation (chase period) in
muristerone-induced cells normalized to that in uninduced parallel
cultures. The values and SE bars represent the averages of three
independent experiments from two different stable clones, each
expressing wild-type, M146L, or X9. Statistical analysis is
performed by ANOVA (F = 10.593;
p = 0.0108; post hoc Tukey, mutants
compared with wild type, *p < 0.05).
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Association of PS1 with -catenin in the brains of
transgenic mice
To determine whether the PS1--catenin complexes are unique to
cultured cells, we turned to brain tissue obtained
from transgenic mice overexpressing human PS1 (WT and M146L mutation).
The presence and expression of transgene was established by genotyping
and RT-PCR of mRNA from brain homogenate (Fig.
5a). In addition, N- and
C-terminal PS1 fragments derived from the transgene were detected by
Western blotting of brain tissue (Fig. 5b,c).
Specifically, the NTF was readily visualized using PSN2 monoclonal
antibody that only recognizes human and not mouse PS1 (Fig.
5b). The transgene-derived CTF was recognized by the slower
mobility as compared with that of endogenous PS1 on an SDS-PAGE gel
(Fig. 5c), as reported previously (Thinakaran et al., 1996).
In coimmunoprecipitation experiments from brain homogenates of
heterozygous transgenic mice and control nontransgenic littermates,
antibodies specific for the N and C terminals of PS1 were able to
coprecipitate -catenin (Fig.
6a). This observation
indicated that the association of PS1 with -catenin occurs in
vivo. Both the wild-type and M146L mutant PS1 coprecipitated endogenous -catenin to a comparable degree in the brains of
transgenic animals. Moreover, a recombinant GST-PS1 loop fusion
protein affinity precipitated -catenin from mouse brains, whereas
GST alone failed to precipitate -catenin under identical conditions
(Fig. 6b), indicating that -catenin specifically
interacts with PS1 within the hydrophilic loop region.
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Figure 5.
Expression of the human PS1 M146L mutation in
transgenic mice. A, Confirmation of the transgene in
offsprings is made by PCR of genomic DNA obtained from tailsnips.
Top, Transgene-positive animals give two bands at 355 and 521 bp (lane 3), whereas animals without the
transgene give only the smaller band (lane 2). A
negative control is included in each set of PCR reactions (lane
1). Bottom, mRNA expression from the human PS1
transgene in brain tissue is shown by RT-PCR. Heterozygous transgenic
animals (Tg/) show two bands after RT-PCR. Only one band is seen in
nontransgenic littermates (/). The RT lanes are
negative controls for the reverse-transcriptase reaction to ensure
complete digestion of the DNA template. B,
The full length and the N-terminal fragment of human PS1
(hPS1) in brains of transgenic mice expressing the M146L
mutation (lines A, B) are visualized by
immunoblotting for the human-specific antibody against the N terminal
of PS1 (PSN2). C, The CTF from both human and endogenous
PS1 can be seen using an PS1Loop antibody. As reported, the
transgenic human PS1 CTF (upper arrowhead in
doublet) migrates higher than the endogenous
(lower arrowhead) species. Three different lines of
transgenic animals expressing M146L are illustrated (lines
A, B, D) to show differences in
expression level.
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Figure 6.
Association of PS1 with -catenin in
vivo and correlation of soluble -catenin levels with PS1
M146L FAD-linked mutant protein expression in the brains of transgenic
mice. A, Triton X-100 soluble brain extracts from M146L
transgenic and nontransgenic littermates were immunoprecipitated
with an antibody against the N terminal
(J27; PS1-N) or C terminal (4627;
PS1-C) of PS1 and immunoblotted for -catenin.
B, Triton X-100 soluble mouse brain extract (200 µg)
was affinity precipitated with recombinant GST or GST-PS1Loop fusion
protein and immunoblotted for -catenin. C, Triton
X-100 soluble brain lysates (30 µg) were subjected to SDS-PAGE,
immunoblotted using -catenin monoclonal antibody, and quantitated by
phosphorimaging. The amount of -catenin in each lane
was normalized to the amount of actin present in the same
lane. The amount of soluble -catenin, expressed as the fold
increase in -catenin in transgenic mice with respect to that in
their nontransgenic littermates, was plotted against the amount of
human M146L PS1 mutant CTF. Linear regression analysis shows a
significant positive correlation between the amount of human M146L
mutant PS1 CTF and the amount of -catenin (r = 0.889; p = 0.0179).
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Having established that PS1 can associate with -catenin in
vivo, we next asked whether the stabilization of -catenin by mutant PS1 seen in tissue cultured cells can also be detected in
vivo. Analysis of three independent transgenic mouse lines expressing the PS1 M146L mutation indicated a trend toward higher amounts of Triton X-100 soluble -catenin in the brain of transgenic animals as compared with that in nontransgenic littermates (data not
shown). However, regression analysis showed that the elevation in
steady-state levels of -catenin was dependent on the level of PS1
M146L mutant expression (Fig. 6c). In other words, levels of
mutant PS1 protein were positively and significantly correlated with
levels of -catenin in brain and provide in vivo support of our investigations in vitro. In two different lines of
transgenic mice expressing wild-type human PS1, we observed an ~25%
reduction in steady-state -catenin levels compared with that in
control littermates; however, limitation of sample size precluded
meaningful statistical analysis (data not shown). These in
vivo results together with -catenin turnover studies therefore
suggest that wild-type PS1 normally promotes the degradation of
-catenin that we hypothesize is caused by the recruitment of
GSK-3 into the complex, whereas FAD-linked PS1 mutants perturb the
constitutive turnover of -catenin by failing to sequester
GSK-3.
PS1 is necessary for rapid and constitutive degradation of
endogenous -catenin
Our finding that overexpression of wild-type PS1 promoted the
turnover of endogenous -catenin whereas mutant PS1 increased the
stability and steady-state levels of endogenous -catenin prompted us
to examine whether the lack of PS1 might differentially alter this
activity. Thus, we cultured fibroblasts from homozygous (/) and
hemizygous (/+) PS1-deficient mouse embryos (Wong et al.,
1997). In support of the concept that PS1 facilitates the degradation
of -catenin, PS1 / cells showed dramatically reduced turnover of -catenin compared with that of PS1 /+
cells. Although the half-life of -catenin was <30 min in
PS1 /+ cells, it was longer than 2 hr in
PS1-deficient cells (Fig.
7a). This reduced turnover of
-catenin was correlated with more than threefold higher levels at
steady state in PS1-deficient cells as compared with that in
hemizygous cells (data not shown). To test directly whether the
prolonged half-life in PS1 / cells is indeed caused by
the lack of PS1, we stably transfected PS1 / cells with
wild-type PS1 or the PS1 X9 FAD mutation. Pulse-chase analysis
showed that wild-type PS1 completely rescued the defective -catenin
turnover phenotype of PS1 / cells, restoring the
half-life from >2 hr to <30 min (Fig. 7b). On the other
hand, expression of the PS1 X9 mutant only partially reversed this
phenotype as compared with wild-type PS1 (Fig. 7b). This
finding, together with corresponding effects of PS1 overexpression,
provides compelling evidence that PS1 normally promotes the rapid
degradation of cytosolic -catenin.
View larger version (46K):
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|
Figure 7.
Defective turnover of -catenin in cells
genetically deficient in mouse PS1 and rescue by human PS1.
A, Immortalized fibroblasts derived from PS1 /+ and
PS1 / embryonic day 15.5 embryos were pulse labeled with
[35S]methionine for 15 min and chased for 0, 0.5, 1, and 2 hr. Lysates were immunoprecipitated with a -catenin
monoclonal antibody and analyzed by autoradiography. B,
PS1 / fibroblasts stably transfected with vector control, wild-type
PS1, or PS1 X9 mutant were pulse labeled with
[35S]methionine for 15 min and chased for 0, 30, and 90 min. Lysates were immunoprecipitated with a -catenin
monoclonal antibody and analyzed by autoradiography.
Arrows indicate immunoprecipitated -catenin.
|
|
| |
DISCUSSION |
Although a molecular link between PS1 and -catenin was recently
reported, the functional consequences of these interactions are
unknown. Our studies not only confirmed the initial reports of the
PS1--catenin interaction but also provided a number of novel
insights into the molecular and cellular consequences of this
interaction. First, we showed that both wild-type and mutant PS1
proteins sequester a pool of -catenin excluded from E-cadherin and
APC. Second, we showed that wild-type PS1 promotes while X9 and
M146L mutants interfere with the constitutive turnover of endogenous
-catenin. In support of these findings, the amount of
GSK-3--catenin complex was preferentially enhanced by
overexpression of wild-type PS1 but not PS1 mutants. Moreover, the
finding that wild-type PS1 facilitates -catenin turnover was
confirmed in cells genetically deficient in PS1, in which
the half-life of -catenin was dramatically prolonged but corrected
to a normal rate after replacement of PS1. Lastly, these in
vitro results were supported by observations in transgenic mice
brain tissue in which PS1--catenin association can be detected and
in which expression of mutant PS1 augments the steady-state levels of
endogenous -catenin.
In our coprecipitation experiments, -catenin was detected in immune
complexes of endogenous as well as exogenously expressed PS1 in
cultured cells and brain tissues. In addition, we showed that this
interaction occurs in the C-terminal half of PS1, because the full
length and the CTF but not the NTF of PS1 associate with -catenin.
This finding is consistent with the recent observation that residues
322-450 of PS1 interact with -catenin (Murayama et al., 1998).
Under mild detergent conditions (e.g., CHAPS, digitonin), the NTF and
CTF of PS1 (Capell et al., 1998; Thinakaran et al., 1998), together
with -catenin (Yu et al., 1998), stay as a complex. Under the NP-40
lysis used in our experiments, however, only the CTF of PS1 was
selectively detected in -catenin immune complexes. In addition, our
studies indicated that the two major -catenin-binding proteins,
E-cadherin and APC, were absent from this complex. This observation is
consistent with a previous study demonstrating that APC and E-cadherin
form independent complexes with -catenin in vitro
(Rubinfeld et al., 1995) and the recent finding that PS1 interacts with
a domain in -catenin that binds E-cadherin (Hulsken et al., 1994;
Murayama et al., 1998).
A recent study showed that both GSK-3 and tau associate with PS1 via
residues 250-298, a domain within the NTF of PS1, in transient
cotransfection experiments (Takashima et al., 1998). In our study using
stably transfected cells, we confirmed the association of GSK-3 with
PS1 in the N-terminal region of PS1 and extended this finding to show
functional changes in the turnover rate of -catenin. An unexpected
finding from our coprecipitation studies was that wild-type PS1 but not
FAD-linked PS1 mutants increased the amount of the
GSK-3--catenin complex. This result was confirmed by the
observation that the association between GSK-3 and two different PS1
mutants (M146L and X9) was virtually undetectable by our
coimmunoprecipitation assays. The association of GSK-3 was via the
full length and the NTF of wild-type PS1, in contrast to the
interaction of -catenin with the CTF. Thus, the region in PS1
responsible for the binding to GSK-3 is distinct from that of
-catenin. Our results are somewhat different from those in a recent
report demonstrating that binding to GSK-3 was increased in two PS1
mutations (C263R and P264L) (Takashima et al., 1998). However, because
both C263R and P264L mutations lie within the putative
GSK-3-binding site, it is possible that the two different sets of
mutations used in these two studies may have differential activity with
regard to GSK-3 interaction. Nonetheless, these observations taken
together indicate that PS1 might function as a membrane-associated
scaffold on which both -catenin and GSK-3 are assembled. This
activity is similar to that seen in axin, a cytosolic molecule that
assembles -catenin, GSK-3, and APC into a multiprotein complex
and negatively regulates -catenin stability (Hart et al., 1998;
Ikeda et al., 1998).
Although APC was not detected in the PS1--catenin complex in our
study, overexpression of wild-type PS1 nevertheless enhanced the
degradation of endogenous -catenin. This observation was confirmed
by the striking differences in turnover rates of endogenous -catenin
in rodent PS1 / cells with and without transfection by
wild-type human PS1. Furthermore, in EcR293 cells, overexpression of
two different PS1 mutants (M146L and X9) delayed the turnover of
-catenin. This finding from cultured cells was confirmed in vivo in transgenic mice expressing the M146L mutant PS1, in which the amount of Triton X-100 soluble -catenin is directly correlated with the mutant transgene expression. Taken together, these data conclusively demonstrate that PS1 is required for rapid and
constitutive turnover of the cytosolic, and presumably signaling, pool
of -catenin. Furthermore, we hypothesize that PS1 mutants (M146L and
X9) contain only partial activity, and in cells previously
expressing wild-type PS1, the mutant proteins exert a
dominant-negative activity on -catenin turnover. We postulate that
PS1 mutants interfere with the normal activity of endogenous PS1 to
mediate the rapid turnover of -catenin perhaps by delaying the
phosphorylation of -catenin by GSK-3. This latter activity is
believed to be required for efficient degradation of -catenin via
the ubiquitin-proteosome pathway (Aberle et al., 1997). It remains to
be seen whether all FAD-linked PS1 mutations exert a similar activity
on the turnover of endogenous -catenin, an issue highlighted by the
GSK-3 results reported recently (Takashima et al., 1998).
Finally, it is important to note that our results are in apparent
disagreement with a recent report demonstrating increased -catenin
stability secondary to wild-type PS1 expression, an effect that was
partially lost with PS1 mutations (Z. Zhang et al., 1998).
However, different experimental approaches between the studies may
account for the divergent results. For example, Yankner and colleagues
(Z. Zhang et al., 1998) analyzed the turnover rate of myc-tagged -catenin that was transiently cotransfected with
PS1 plasmids, whereas our study examined the turnover of endogenous
-catenin after inducible expression of PS1 proteins in stably
transfected cell lines. Moreover, our study determined the turnover of
full-length -catenin in PS1-deficient cells by pulse-chase paradigm,
whereas Z. Zhang et al. (1998) focused on apparent -catenin
proteolytic products from steady-state pools by Western blotting.
Although such -catenin-related proteolytic fragments require
further characterization, the appearance of these
species indicates processes that are clearly distinct from conventional
degradation and turnover of -catenin per se. On the other hand, a
recent study by Takashima and colleagues (Murayama et al., 1998) showed
that transient transfection of PS1 in COS cells results in reduced
levels of endogenous cytosolic -catenin that were further reduced by
transient transfection of PS1 mutants, findings that are in partial
agreement with our studies in stably transfected inducible cells. Thus,
apparent discrepancies between different studies highlight the
importance of defining the pools of -catenin under examination and
interpreting the findings accordingly. In this study, as an initial
step in studying the role of PS1 in the -catenin pathway, we have
analyzed the turnover rate and steady-state levels of endogenous,
full-length -catenin.
Our studies have shown that the PS1--catenin interactions lead to
corresponding changes in the processing of proteins known to function
within the -catenin-signaling pathway. Specifically, alterations in
turnover of endogenous -catenin resulting from expression of two
different PS1 mutations were seen in stably transfected cells, in
PS1 knock-out cells, and in transgenic animals. These
findings, therefore, provide compelling evidence that PS1 normally
modulates signaling within the -catenin pathway. Moreover, the
functional changes in -catenin turnover induced by PS1 mutations suggest the intriguing possibility that this pathway may be involved in
the pathobiology of AD. From studies of sel-12 in C. elegans and PS1-deficient animals, it has been proposed that PS1
might function as a mediator of the Notch/Lin-12-signaling pathway
(Levitan and Greenwald, 1995), although evidence of a direct molecular link has not been presented. On the other hand, it has been reported that Wingless/Wnt and Notch pathways may interact at the level of
disheveled (Axelrod et al., 1996), a negative regulator of GSK-3
activity. Taken together, these observations lead us to hypothesize
that the loss of PS1--catenin interaction might underlie the
embryonic lethal phenotype of PS1 null mutants.
| |
FOOTNOTES |
Received Feb. 1, 1999; revised March 12, 1999; accepted March 17, 1999.
This work was supported in part by the National Institutes of Health
Grant NS28121 and by the Paul Beeson Physician Faculty Scholar Award in
Aging from the American Federation for Aging Research. We thank Drs.
Barbara Ranscht and Jim Posakany for helpful discussions and critique,
Drs. Sreeganga Chandra and Gorazd Stokin for experimental advice, Dr.
Dennis Selkoe for providing the 4627 antibody, and Dr. Hiroshi Mori for
providing the PSN2 antibody.
This work was presented at the 6th International Conference on
Alzheimer's Disease and Related Disorders, July 18-23, 1998, Amersterdam, The Netherlands.
Parts of this paper have been published previously
[Neurobiol Aging (1998) 19:S187].
Correspondence should be addressed to Dr. Edward Koo, Department of
Neurosciences 0691, University of California, San Diego, La Jolla, CA
92093-0691.
| |
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TOP
ABSTRACT
INTRODUCTION
