Substitution of a Glycogen Synthase Kinase-3beta Phosphorylation Site in Presenilin 1 Separates Presenilin Function from beta -Catenin Signaling*

Ford Kirschenbaum, Shu-Chi Hsu, Barbara CordellDagger, and Justin V. McCarthy

From Scios Inc., Sunnyvale, California 94086

Received for publication, May 31, 2000, and in revised form, November 27, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The majority of cases with early onset familial Alzheimer's disease have been attributed to mutations in the presenilin 1 (PS1) gene. PS1 protein is a component of a high molecular weight membrane-bound complex that also contains beta -catenin. The physiological relevance of the association between PS1 and beta -catenin remains controversial. In this study, we report the identification and functional characterization of a highly conserved glycogen synthase kinase-3beta consensus phosphorylation site within the hydrophilic loop domain of PS1. Site-directed mutagenesis, together with in vitro and in vivo phosphorylation assays, indicates that PS1 residues Ser353 and Ser357 are glycogen synthase kinase-3beta targets. Substitution of one or both of these residues greatly reduces the ability of PS1 to associate with beta -catenin. By disrupting this interaction, we demonstrate that the association between PS1 and beta -catenin has no effect on Abeta peptide production, beta -catenin stability, or cellular susceptibility to apoptosis. Significantly, in the absence of PS1/beta -catenin association, we found no alteration in beta -catenin signaling using induction of this pathway by exogenous expression of Wnt-1 or beta -catenin and a Tcf/Lef transcriptional assay. These results argue against a pathologically relevant role for the association between PS1 and beta -catenin in familial Alzheimer's disease.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -catenin (5-7), has been reported. Both PS1 and beta -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 beta -catenin binding (7, 9, 10). Furthermore, glycogen synthase kinase-3beta (GSK-3beta ), the serine/threonine kinase that regulates beta -catenin levels, has also been shown to associate with PS1 (11). The regulation of cytoplasmic beta -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 beta -catenin. beta -Catenin is targeted for ubiquitination and proteosome degradation following phosphorylation by GSK-3beta . The activity of the beta -catenin complex is regulated by other proteins present, such as Axin and adenomatous polyposis coli (APC), which facilitate the phosphorylation of beta -catenin by GSK-3beta . The ability of Axin and APC to facilitate beta -catenin phosphorylation is mediated by phosphorylation of both proteins by GSK-3beta . Upon stimulation of the Wnt receptor, GSK-3beta is inhibited, resulting in an increase in the cytosolic level of beta -catenin rendering the protein available for transport to the nucleus where it activates Wnt target genes.

PS1 also participates in the regulation of cytoplasmic beta -catenin levels, although the nature of this modulation is unclear. Many insights regarding the role of PS1 in beta -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 beta -catenin degradation (13, 14) although contrary results have also been reported (15, 16). This loss of beta -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 beta -catenin to translocate to the nucleus, a state that could potentiate apoptosis (16). We have identified three GSK-3beta consensus phosphorylation sites in the PS1 hydrophilic loop domain. Similar to Axin or APC, phosphorylation of PS1 by GSK-3beta may serve to regulate beta -catenin phosphorylation, thereby regulating beta -catenin/Tcf gene expression. To test this hypothesis, we have characterized the role of each GSK-3beta phosphorylation consensus motif in PS1/beta -catenin association and identified one to be critical.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 [gamma -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-3beta (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, p38beta from Upstate Biotechnology, and p38alpha 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-3beta 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. beta -Catenin and GSK-3beta monoclonal antibodies were purchased from Transduction Laboratories. The monoclonal antibody specific for activated GSK-3beta 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.

Abeta 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 Abeta 1-40 and Abeta 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 Abeta sequence; a C-terminal Abeta 40-specific antibody, 1702.1; and a C-terminal Abeta 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-beta -catenin monoclonal antibody, as described above. Immunoprecipitates were resolved on a 4-12% NuPage Bis-Tris gel (Novex) and analyzed by a PhosphorImager.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PS1 Hydrophilic Loop Domain Contains GSK-3beta 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-3beta 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-3beta 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-3beta 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-3beta 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-3beta 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-3beta 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-3beta consensus phosphorylation residues that were mutated.

In addition to beta -catenin, both APC and Axin are also GSK-3beta substrates, and their ability to bind beta -catenin in vitro is enhanced by phosphorylation. The sites necessary for GSK-3beta phosphorylation have been identified, and mutating these sites leads to a disruption in the association between beta -catenin and APC or Axin. Alignment of the amino acid sequence of Axin, APC, beta -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-3beta consensus phosphorylation sites mediate a specific PS1 function(s). Specifically, we sought to address whether the GSK-3beta motifs present on PS1 are used to mediate PS1 binding to beta -catenin. The general location of each GSK-3beta 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-3beta phosphorylation motifs present on PS1 was mutated. Within each GSK-3beta 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-3beta motifs relative to beta -catenin binding and function, we first assessed their influence on two other PS1 functions: endoproteolysis and Abeta production.


                              
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Table I
Summary of GSK-3beta phosphorylation site-specific mutant constructs

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-3beta 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-3beta 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 Abeta generation by PS1 GSK-3beta phosphorylation motif mutants. A, effects of GSK-3beta 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 Abeta 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 Abeta 1-40 and Abeta 1-42 was performed using two-site ELISAs. Results are the mean ± S.D. of a representative experiment performed in duplicate.

We next determined whether the GSK-3beta phosphorylation sites on PS1 altered the production of Abeta . PS1 has been shown to influence gamma -secretase function. This has been best demonstrated in cells harboring PS1 targeted gene disruption where the levels of Abeta are greatly reduced (22). Furthermore, mutations in PS1 associated with FAD have been shown to elevate Abeta 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-3beta motifs would also result in increased levels of Abeta 42. To measure Abeta production, 293HEK cell cultures stably expressing beta APP carrying the Swedish mutation (APPswe) were transiently transfected with wild type, FAD PS1 (E280G), or "GSK-3beta " mutant PS1. Twenty-four hours post-transfection, ELISA quantification revealed an increase in Abeta 42 in conditioned medium collected from cells expressing PS1-FAD E280G mutant (Fig. 2B). In contrast, Abeta 42 production was not significantly changed in cells transfected with either wild type or with PS1 mutated at the conserved GSK-3beta motifs (Fig. 2B). This suggests that mutation of the GSK-3beta consensus phosphorylation sites does not influence the production of Abeta 42.

Interaction of PS1 with beta -Catenin Requires Ser353 or Ser357-- It has previously been shown that the association between beta -catenin and APC or Axin can be disrupted by mutating their GSK-3beta consensus phosphorylation sites (12, 23, 24). Since PS1 has been shown to interact with beta -catenin (5-7) and we have identified GSK-3beta phosphorylation sites on PS1, we examined whether mutating these sites to nonfunctional sequences for kinase recognition would affect the association between PS1 and beta -catenin. Subconfluent 293HEK cell cultures were transfected with expression constructs that directed the synthesis of His epitope-tagged wild type or PS1 GSK-3beta mutant. Consistent with previous reports (6-7), immunoprecipitation of wild type PS1 quantitatively coprecipitated endogenous beta -catenin (Fig. 3A). Likewise, PS1 mutant constructs bearing the mutations at residues T320R, S324A, S397A, or S401A also efficiently coimmunoprecipitated endogenous beta -catenin. However, coimmunoprecipitation of endogenous beta -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-3beta (data not shown). Both Ser353 and Ser357 comprise a single GSK-3beta 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 beta -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 beta -catenin.



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Fig. 3.   GSK-3beta consensus phosphorylation sites on PS1 and interaction with beta -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-beta -catenin monoclonal antibody to detect association with endogenous beta -catenin. A, Western blot of beta -catenin coimmunoprecipitated with PS1, wild type, and with GSK-3beta PS1 mutants (upper) and of exogenous PS1 expression (lower). B, Western blot of beta -catenin coimmunoprecipitated with wild type and PS1-(S353A) or PS1-(S357A). C, Western blot of beta -catenin coimmunoprecipitated with wild type and FAD PS1.

Recent reports have suggested that PS1-FAD mutant effects may be attributed to an altered association between PS1-FAD and beta -catenin (7, 13-15). We examined the association between PS1-FAD mutants and endogenous beta -catenin. The results shown in Fig. 3C clearly demonstrate that equivalent quantities of endogenous beta -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 beta -catenin.

The Ser353, Ser357 PS1 Motif Can Be Phosphorylated by GSK-3beta -- To directly demonstrate that the Ser353, Ser357 site is recognized by GSK-3beta , 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-3beta . 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-3beta was determined. We compared levels of phosphate incorporation onto the Ser353, Ser357 peptide by GSK-3beta against four other serine/threonine kinases: p38alpha , p38beta , protein kinase C, and casein kinase II. Only GSK-3beta was able to phosphorylate this site (Fig. 4B). Therefore, it appears that phosphorylation of the Ser353, Ser357 motif is selective for GSK-3beta .



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Fig. 4.   In vitro and in vivo phosphorylation of PS1 by GSK-3beta . A, in vitro kinase reactions were performed using synthetic peptides corresponding to either the wild type Ser353, Ser357 GSK-3beta motif (NH2- GPHRSTPESRAAV-COOH) or mutated (NH2-GPHRATPEARAAV-COOH) and GSK3beta . 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-3beta , p38alpha , p38beta , 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-3beta 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-3beta mutant PS1 CTF expressed in 293HEK cells detected by anti-His antibody.

We next evaluated the in vivo phosphorylated state of wild type PS1, PS1 mutated at the Ser353, Ser357 GSK-3beta site, and PS1 mutated at all three GSK-3beta 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-3beta 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-3beta motifs was expressed at slightly higher levels but was not phosphorylated. These data indicate that PS1 is phosphorylated at the GSK-3beta sites in vivo.

Disruption of PS1/beta -Catenin Interaction Does Not Affect beta -Catenin Stability or Tcf/Lef-mediated Signaling-- In response to activation of the Wnt signaling pathway, beta -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 beta -catenin signaling arise from either disruption of processes affecting cytoplasmic accumulation of beta -catenin (13) or processes involving nuclear translocation of beta -catenin (16). To assess the effect of disrupting PS1 and beta -catenin association on beta -catenin signaling, we examined endogenous beta -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-3beta mutant PS1-(S353A,S357A). No apparent differences were observed between the basal levels of endogenous cytosolic or nuclear beta -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 beta -catenin, both cytosolic and nuclear levels of beta -catenin were similar to those of wild type PS1-expressing cultures (Fig. 5, A and B). This conclusion is most apparent when beta -catenin levels from individual experiments are normalized to GSK-3beta (Fig. 5, histograms). These results demonstrate that disruption of the association between PS1 and beta -catenin has no apparent adverse affect on cytosolic levels of beta -catenin or on nuclear localization of beta -catenin.



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Fig. 5.   PS1/beta -catenin association in regulating beta -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-3beta mutants of PS1. Twenty-four hours post-transfection, cytosolic (A) and nuclear (B) extracts were prepared, and the levels of endogenous beta -catenin were determined by immunoblotting with an anti-beta -catenin monoclonal antibody. Data presented in the histogram is representative of three independent experiments. C, pulse-chase analysis of beta -catenin degradation. Subconfluent cultures of 293HEK cells were transiently transfected with the indicated wild type, FAD, or site-directed GSK-3beta mutants of PS1. Twenty-four hours post-transfection, cells were pulse-chase-labeled with [35S]methionine/cysteine and analyzed by immunoprecipitation of endogenous beta -catenin with an anti-beta -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 beta -galactosidase expression. E, beta -catenin induction of the Wnt signaling cascade. beta -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.

As mentioned earlier, the regulation of beta -catenin stability is a crucial control point in Wnt-mediated signaling pathways. To determine whether dissociation of the interaction between PS1 and beta -catenin affects the function of beta -catenin by altering its stability, we assessed beta -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-3beta mutant PS1-(S353A,S357A) or PS1-(T320R,S324A). Endogenous beta -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 beta -catenin in cells expressing PS1-(S353A,S357A) which is unable to associate with beta -catenin had an equivalent half-life. Thus, disruption of the binding between PS1 and beta -catenin does not affect the stability of beta -catenin.

To directly assess the role of PS1 and its association with beta -catenin in a beta -catenin signaling cascade, we determined the effects of wild type PS1, FAD PS1, and several PS1 GSK-3beta mutants in a Tcf-luciferase reporter assay. In the prevailing model, following the initiation of a Wnt signaling cascade, GSK-3beta is inhibited, leading to decreased phosphorylation and accumulation of cytosolic beta -catenin. This allows beta -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-3beta 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 beta -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-3beta 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 beta -catenin, had little effect on Wnt-1-induced Tcf-luciferase activity (Fig. 5D). Identical results were obtained when cells were cotransfected with beta -catenin and the same PS1 constructs (Fig. 5E). Taken together, these results clearly demonstrate that dissociation of PS1 and beta -catenin binding has no significant effect on beta -catenin stability or on Wnt-1- or beta -catenin-mediated gene expression.

Disruption of PS1/beta -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 beta -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-3beta 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 beta -catenin, did not affect cell susceptibility to apoptosis. Thus, dissociation of the binding between beta -catenin and either wild type PS1 or FAD PS1 does not alter cell vulnerability to apoptosis.



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Fig. 6.   PS1/beta -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).



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PS1 has been shown to associate with beta -catenin (5-7); however, the functional significance of this interaction has been unclear. Likewise, the influence of FAD PS1 on beta -catenin biology and how this might result in a pathophysiological state has not been fully resolved. PS1 and beta -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 beta -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 beta -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 beta -catenin interaction as well as the role of this association in Wnt signaling. Three key observations can be derived from this work. First, beta -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 beta -catenin. Furthermore, Ser353 and Ser357 comprise a consensus recognition site for phosphorylation by GSK-3beta , a serine/threonine kinase known to regulate multiple members of the Wnt signaling complex. Second, ablating PS1/beta -catenin association has no effect on PS1 function as it relates to endoproteolysis and Abeta production. Third, beta -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 beta -catenin.

Since the observation that PS1, but not PS2, binds to beta -catenin (9), subsequent reports have grossly mapped the domain of beta -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-3beta 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-3beta site(s) on PS1 might be involved in beta -catenin association. Several members of the Wnt signaling complex are regulated by GSK-3beta , including Axin, APC, and beta -catenin. Phosphorylation of Axin and APC by GSK-3beta enhances the efficiency of beta -catenin binding to these proteins, thereby facilitating the phosphorylation of beta -catenin by GSK-3beta (12, 28). Therefore, we evaluated the potential role of these three PS1 GSK-3beta consensus sites in beta -catenin binding using site-directed mutagenesis of the serine/threonine residues phosphorylated by GSK-3beta within each motif. Our results identified one GSK-3beta motif as critical for PS1/beta -catenin association. Mutating either Ser353 or Ser357 of this consensus site essentially abolished PS1/beta -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 beta -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 beta -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 beta -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 beta -catenin at this GSK-3beta consensus motif in the PS1 loop domain.

Several independent reports support our conclusion that the Ser353, Ser357 motif on PS1 is phosphorylated by GSK-3beta . 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-3beta using coimmunoprecipitation. We were able to demonstrate selective in vitro phosphorylation at the Ser353, Ser357 residues of the consensus motif by GSK-3beta 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-3beta in vivo.

The Ser353, Ser357 PS1 point mutants that lack the ability to efficiently bind to beta -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 Abeta production and endoproteolysis were normal when beta -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 Abeta generation. More interesting are the results we obtained examining the effects of PS1 dissociation on beta -catenin stability and cell survival signaling. No change in either free cytosolic or nuclear beta -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 beta -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 beta -catenin against a background of plasma membrane-associated beta -catenin that is roughly 10-fold larger (16). Differences in the methods used to assay beta -catenin might also be a contributing factor to this controversy.

The most important issue this study addresses is the role of PS1/beta -catenin interaction in Wnt signaling. Using the Ser353, Ser357 mutant, we were able to demonstrate normal beta -catenin signaling when the association between PS1 and beta -catenin is largely abolished. This result is supported by an observation of Nishimura et al. (16), who found identical levels of nuclear beta -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 beta -catenin in association with FAD PS1, this study did not include a functional measure for beta -catenin. Therefore, it seems possible that while there may exist deficiencies in beta -catenin trafficking in the FAD PS1 background, sufficient levels of beta -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 beta -catenin has no impact on cell viability. Previously, it has been proposed that FAD PS1 destabilizes beta -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/beta -catenin interaction is not essential for maintenance of cell survival. In the work of Zheng et al. (13), loss of beta -catenin signaling was shown to increase neuronal apoptosis; however, this effect was not directly linked to beta -catenin instability due to FAD PS1. We suggest that the FAD PS1 effects observed on beta -catenin transport, stability, and cell survival signaling involve a mechanism separate from the ability of PS1 to associate with beta -catenin.

In summary, it appears that the interaction between PS1 and beta -catenin requires GSK-3beta 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 beta -catenin is essentially abolished. No negative consequences resulted from the disruption of the PS1/beta -catenin interaction with respect to Abeta production, beta -catenin stability, Wnt signaling, or cell survival. This indicates that association of PS1 with beta -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.


    ACKNOWLEDGEMENTS

We thank Dr. Roel Nusse of Stanford University Medical Center for the Wnt-1 cDNA, Dr. Ken Kosik of Brigham and Women's Hospital and Harvard Medical School for the murine beta -catenin expression plasmid, and Dr. Wenlin Zeng of Scios Inc. for providing the p38alpha kinase. We also thank Stephanie Rogers for DNA sequencing.


    FOOTNOTES

* This work was supported through a collaboration with Lilly.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Scios Inc., 820 W. Maude Ave., Sunnyvale, CA 94086. Tel.: 408-616-8230; Fax: 408-616-8317; E-mail: cordell@sciosinc.com.

Published, JBC Papers in Press, December 4, 2000, DOI 10.1074/jbc.M004697200


    ABBREVIATIONS

The abbreviations used are: FAD, familial Alzheimer's disease; GSK-3beta , glycogen synthase kinase-3beta ; PS1 and PS2, presenilin-1 and -2, respectively; CTF, C-terminal fragment; APP, beta -amyloid precursor protein; Abeta , beta -amyloid protein; APC, adenomatous polyposis coli; HEK, human embryonic kidney; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; ELISA, enzyme-linked immunosorbent assay; WT, wild type.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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