JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M508657200 on December 19, 2005

J. Biol. Chem., Vol. 281, Issue 8, 4787-4794, February 24, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/8/4787    most recent
M508657200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mi, K.
Right arrow Articles by Johnson, G. V. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mi, K.
Right arrow Articles by Johnson, G. V. W.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

The Low Density Lipoprotein Receptor-related Protein 6 Interacts with Glycogen Synthase Kinase 3 and Attenuates Activity*

Kaihong Mi, Philip J. Dolan, and Gail V. W. Johnson1

From the Department of Psychiatry, University of Alabama at Birmingham, Birmingham, Alabama 35294-0017

Received for publication, August 5, 2005 , and in revised form, November 28, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Glycogen synthase kinase 3 (GSK3) is a widely expressed Ser/Thr protein kinase that phosphorylates numerous substrates. This large number of substrates requires precise and specific regulation of GSK3 activity, which is achieved by a combination of phosphorylation, localization, and interactions with GSK3-binding proteins. Members of the Wnt canonical pathway have been shown to influence GSK3 activity. Through a yeast two-hybrid screen, we identified the Wnt canonical pathway co-receptor protein low density lipoprotein receptor-related protein 6 (LRP6) as a GSK3-binding protein. The interaction between the C terminus of LRP6 and GSK3 was also confirmed by in vitro GST pull-down assays and in situ coimmunoprecipitation assays. In vitro assays using immunoprecipitated proteins demonstrated that the C terminus of LRP6 significantly attenuated the activity of GSK3beta. In situ, LRP6 significantly decreased GSK3beta-mediated phosphorylation of tau at both primed and unprimed sites. Finally, it was also demonstrated that GSK3beta phosphorylates the PPP(S/T)P motifs in the C terminus of LRP6. This is the first identification of a direct interaction between LRP6 and GSK3, which results in an attenuation of GSK3 activity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Glycogen synthase kinase 3 (GSK3)2 is a widely expressed protein kinase with high expression in the brain, and specifically within neurons (for a review, see Ref. 1). GSK3 is a unique Ser/Thr protein kinase that phosphorylates both primed (target Ser/Thr is 4 amino acids N-terminal to a prephosphorylated Ser/Thr) and unprimed (target Ser/Thr is flanked by a Pro) substrates (for a review, see Ref. 2). A screen of a rat brain cDNA library revealed that GSK3 is encoded by two independent genes, GSK3{alpha} and GSK3beta, with molecular masses of 51 and 47 kDa, respectively (3). The two genes display 85% overall sequence identity, which is even higher in the catalytic domain (93%). In the brain, although GSK3{alpha} mRNA level is higher than GSK3beta, GSK3beta protein level is higher (4). The poor relationship between transcription and translation in some tissues indicates that these two isoforms are subject to differential regulation, but little is known about the isoform-specific functions.

More than 40 proteins have been reported to be phosphorylated by GSK3, including over a dozen transcription factors (reviewed in Ref. 2). This large number of substrates illustrates the great potential of GSK3 to affect many cellular functions and suggests that the activity of GSK3 must be carefully regulated by individual mechanisms for each substrate. Although the mechanisms regulating GSK3 are not fully understood, precise control appears to be achieved by a combination of phosphorylation, localization, and interactions with GSK3-binding proteins (reviewed in Ref. 2). Protein complexes that contain GSK3 are of major importance in regulating its actions. The best characterized of these complexes is involved in the Wnt canonical pathway, where GSK3-binding proteins control access to the GSK3 substrate, beta-catenin, and generate a high degree of specificity in regulating the actions of GSK3. In the absence of Wnt signal, adenomatous polyposis coli, beta-catenin, GSK3, casein kinase I (5), and other proteins all bind to the scaffold protein axin to form a complex (6). In this complex, beta-catenin is phosphorylated by casein kinase I and subsequently by GSK3, which allows the beta-transducing repeat-containing protein, an F-box protein in the E3 ubiquitin ligase complex, to bind and tag beta-catenin for proteasome-mediated degradation (7, 8). When specific Wnts, such as Wnt 3a and Wnt 8, bind to the appropriate Frizzled (Fz) receptors, Dishevelled is activated; this, in concert with the GSK3-binding protein, frequently rearranged in advanced T-cell lymphoma (FRAT), facilitates disruption of the axin-based complex. This decreases the phosphorylation of beta-catenin and results in beta-catenin accumulation and activation (for a review, see Ref. 9). This is a classical example of how GSK3-binding proteins can regulate the action of GSK3 toward individual substrates.

In addition to the Fz receptors, Wnt canonical signaling requires single span transmembrane proteins that belong to a subfamily of low density lipoprotein (LDL) receptor-related proteins (LRPs): vertebrate LRP5 and -6 and their Drosophila ortholog Arrow (10–12). Unlike Fz, which is required for multiple Wnt pathways (13, 14), Arrow and LRP5/6 appear to be specifically required for Wnt/beta-catenin signaling (12, 15). Although the mechanism for LRP5/6 involvement in Wnt signaling has not been fully elucidated, recent studies suggest that the C terminus of LRP5/6 binds to axin and localizes axin and other bound proteins of the destruction complex to the plasma membrane. This is followed by axin degradation and dispersal of proteins of the destruction complex (16, 17). Furthermore, a study demonstrated that a single PPP(S/T)P motif, which is reiterated five times in the LRP5/6/Arrow intracellular domain, can fully activate the Wnt pathway (16). Wnt signaling stimulates and requires phosphorylation of the PPP(S/T)P motif (16), although it has not been determined which kinases and/or phosphatases are involved in this modification. Although this is the prevailing model for LRP5/6/Arrow function, it does not explain the observation that the intracellular domain alone of LRP5/6 can activate and potentiate the Wnt 3a-induced LEF-1 signal, although it is not membrane-anchored (18).

Members of the Wnt canonical pathway have also been shown to influence GSK3 activity toward substrates outside of the pathway, with the microtubule-associated protein tau being a notable example (reviewed in Ref. 2). Tau is phosphorylated by GSK3 at both unprimed and primed motifs. It has been demonstrated that primed phosphorylation of tau at Thr231 by GSK3 plays an essential role in decreasing the ability of tau to both bind and stabilize microtubules (19). In contrast, GSK3-mediated phosphorylation of unprimed sites on tau (e.g. Ser396/404) may be more pathological and contribute to the formation of filamentous inclusions in Alzheimer disease and other neurodegenerative disorders (20). Previously, our group demonstrated that axin potently inhibits tau phosphorylation by GSK3beta, probably by sequestering GSK3beta away from tau (21). Dishevelled has also been shown to decrease tau phosphorylation by GSK3beta (22), despite the fact that Dishevelled and GSK3beta do not interact (8). Interestingly, studies showed that FRAT-1 inhibits GSK3-mediated tau phosphorylation (23). Further, antisense-induced knock-down of Dickkopf-1, a negative modulator of the Wnt pathway that functions through binding to the co-receptor LRP6 (24, 25), attenuates the increased tau phosphorylation in beta-amyloid peptide-treated neurons (26). However, whether LRP6 directly influences GSK3 activity has not been elucidated.

Here we identify LRP6 as a GSK3-binding protein through a yeast two-hybrid assay. The interaction between LRP6 and GSK3 was confirmed by an in vitro GST pull-down assay and coimmunoprecipitation assays. The interaction of LRP6 with GSK3 significantly attenuated the activity of GSK3, and LRP6 was found to be a GSK3 substrate. Overall, this study suggests that LRP6 plays a unique and important role in and out of Wnt canonical signaling through regulating GSK3 activity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—Chinese hamster ovary (CHO) cells were maintained in Ham's F-12/Dulbecco's modified Eagle's medium (Cellgro) supplemented with 5% fetal bovine serum (Hyclone), 100 units/ml penicillin (Invitrogen), 100 µg/ml streptomycin (Invitrogen), and 2 mM L-glutamine (Invitrogen). Cells were grown in a humidified atmosphere containing 5% CO2 at 37 °C.

Constructs and Recombinant Protein Preparation—The tau construct that does not contain exons 2 and 3 (T4) was described previously (27). Human GSK3beta-HA was a gift from Dr. J. R. Woodgett. The plasmid encoding glutathione S-transferase (GST) fusion beta-catenin in a bacterial expression vector was a gift from Dr. W. I. Weis (27). To make the GSK3{alpha}-HA construct, human brain mRNA (Clontech) was used as the template for RT-PCR of GSK3{alpha} (SuperscriptTM one-step reverse transcription-PCR (Invitrogen)). The following primers were used in the reaction: forward, 5'-GCG AAT TCC GTT ATG AGC GGC GGC GGG CCT TCG-3'; reverse, 5'-GCG AAT TCC AGC ACA CTG GCG GCC GTA-3'. The PCR product was digested with EcoRI and subcloned into the same vector as GSK3beta-HA. The cytosolic fragment of the C terminus of human LRP6 (LRP6-C3) was described previously (18). Using LRP6-C3 in pCMV5A (18) as a template, PCR was performed to subclone the LRP6-C3 fragment into corresponding vectors. The following primers were used for each PCR: forward (5'-CGC GGA TCC AGC ATG GGA CCA GCT TCT GTG CCT CTT GGT TAT GTG-3') and reverse (5'-GCG CGT CGA CTT CTA GGA GGA GTC TGT ACA GGG AGA GGG TGG CGG TGG GT-3') for subcloning LRP6-C3 into pGEX-6P-2 (Amersham Biosciences) (GST-LRP6-C3); forward (5'-CCG GAA TTC GGG ACC AGC TTC TGT GCC TCT TGG TTA TGT-3') and reverse (5'-GGG ATC CTC AGG AGG AGT CTG TAC AGG GAG AG-3') for subcloning LRP6-C3 into pEGFP-C1 (BD Biosciences) (GFP-LRP6-C3). The PCR products were digested and ligated into the corresponding cloning sites in vectors. For subcloning LRP6-C3 into pGBD-C1 vector (Clontech), LRP6-C3 was cut out from LRP6-C3 in pCMV5A by BamHI and SalI and ligated into the corresponding cloning sites in the vector. We also mutated all five PPP(S/T)P motifs in GST-LRP6-C3 to PPPAP to make GST-mut-LRP6-C3. The mutatgenesis reactions were carried out using either the GeneEditorTM in vitro site-directed mutagenesis system from Promega or the QuikChangeTM site-directed mutagenesis kit from Stratagene. Plasmid integrity was verified using DNA sequencing analysis. GST-LRP6-C3, GST-mut-LRP6-C3, and GST-beta-catenin were purified according to the manufacturer's protocol, with the exception that cleared bacterial lysates were incubated with preblocked Fast Flow Glutathione-Sepharose beads (Amersham Biosciences) for 1 h with rotation at 4 °C. For controls, GST was prepared in the same manner. For functional studies, GST-LRP6-C3 was treated on column with 60 units of Prescission protease (Amersham Biosciences) in cleavage buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, pH 7.0) overnight at 4 °C to remove the GST, and the LRP6-C3 was eluted. Where indicated, the fusion protein was left intact and eluted with glutathione buffer (20 mM Tris, 10 mM reduced glutathione, pH 8). After elution, GST-beta-catenin, GST-LRP6-C3, GST-mut-LRP6-C3, and LRP6-C3 were dialyzed into phosphate-buffered saline. To determine the concentration of the purified protein, the samples were diluted in 2x SDS stop buffer (0.25 M Tris-HCl, pH 7.5, 2% SDS, 25 mM dithiothreitol, 5 mM EDTA, 5 mM EGTA, 10% glycerol, and 0.01% bromphenol blue) and electrophoresed on an 8% SDS-polyacrylamide gel, in addition to aliquots containing known amounts of bovine serum albumin (BSA) (Fisher). The gel was Coomassie-stained, destained, and dried. The gel was scanned and quantitated using UNSCANIT software (Silk Scientific, Inc.), and the amount of recombinant protein in the sample was determined as a function of the BSA standards.

Yeast Two-hybrid Screening—The MATCHMAKER yeast two-hybrid system and an adult human brain cDNA library were purchased from Clontech. The bait for library screening was the intracellular domain of human LRP6 (residues 1416–1613), which was fused to the GAL4 DNA binding domain by subcloning into the pGBD-C1 vector. The adult human brain cDNA library (Clontech) in the pGAD-C2 vector, which has a transcription activation domain, was used in the screen. Prior to the screening, the LRP6-C3 construct in the pGBD-C1 vector was first transformed into the Y190 yeast hosts (Clontech), and subsequently the yeast Y190 cells carrying LRP6-C3 were transformed with the human brain cDNA library using the lithium acetate/single-stranded carrier DNA/polyethylene glycol protocol (28). A small fraction of the transformation reaction was plated on synthetic dropout medium –Leu, –Trp, to estimate the total number of transformants. The remainder was plated on synthetic dropout medium –Leu, –Trp, –Ade to identify ADE2 reporter gene-positive colonies, which were subsequently transferred to synthetic dropout medium –Leu, –Trp, –His plates (Teknova) for further selection.

Transient Transfections and Collection—CHO cells were transiently transfected using FuGENE 6 transfection reagent (Roche Applied Science) according to the manufacturer's protocol. Forty-eight hours after transfection, cells were harvested in lysis buffer (0.5% Nonidet P-40, 150 mM NaCl, 10 mM Tris-Cl (pH 7.4), 1 mM EGTA, and 1 mM EDTA, 0.1 µM okadaic acid, 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ml each of aprotinin, leupeptin, and pepstatin). After sonication on ice for 10 s, cellular debris was removed by centrifugation. Protein concentrations of supernatants were then determined by the bicinchoninic acid assay (Pierce).

Immunoblotting, Pull-down Assays, and Immunoprecipitation— Cells were collected as described above. After determination of protein concentrations, samples were diluted in 2x SDS buffer and electrophoresed on 8% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Nitrocellulose membranes were blocked for 1 h with 5% nonfat dry milk in TBST (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.05% Tween 20) and then probed with antibody overnight at 4 °C. Blots were then rinsed with TBST, incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Jackson Immunoresearch) for 1 h at room temperature, rinsed with TBST, and developed with enhanced chemiluminescence (Amersham Biosciences). To evaluate the phosphorylation state of tau, the following antibodies were used: AT180 (Pierce/Endogen), which recognizes tau phosphorylated on Thr231 (29, 30) (numbering based on the longest human tau isoform (31)) and PHF-1 (a gift from Dr. P. Davies), which recognizes tau phosphorylated on Ser396/404(32, 33). Total tau levels were determined using a combination of the phospho-independent monoclonal tau antibodies Tau 5 (a gift from Dr. L. Binder) (34) and 5A6 (35). Levels of transfected GSK3 and GFP-LRP6-C3 were detected using monoclonal HA (Transduction Laboratories) and monoclonal GFP (Roche Applied Science) antibodies, respectively.

The in vitro pull-down assay was carried out as described previously (36). Equal amounts of GST- or GST-LRP6-C3-conjugated glutathione beads were incubated for 2 h with lysates from CHO cells transiently transfected with GSK3 and washed eight times with phosphate-buffered saline containing 350 mM NaCl (high salt) and 0.2% Triton X-100 prior to immunodetection by Western blotting. Alternatively, GST- or GST-LRP6-C3-conjugated beads were incubated with 200 ng of recombinant GSK3beta (Upstate%20Biotechnology">Upstate Biotechnology, Inc., Lake Placid, NY) and washed in the same manner.

For immunoprecipitation, 100 µg of the total lysate was diluted to a final concentration of 0.5 mg/ml and subjected to immunoprecipitation at 4 °C with preconjugated monoclonal anti-GFP antibody (Roche Applied Science) M-280 sheep anti-mouse magnetic IgG beads (Dynal Biotech) for 2 h on a rotational shaker. Precipitates were washed at least five times and boiled in 2x SDS stop buffer. The supernatants were collected, resolved on 12% SDS-polyacrylamide gels, and blotted as described above. Five micrograms of total protein of each condition were probed to check expression levels.

GSK3 Activity Assays—Cell lysates were incubated for 3 h at 4 °C with magnetic IgG beads, which were precoupled with 1.5 µg of the monoclonal anti-GSK3beta antibody (BD Biosciences) (to immunoprecipitate endogenous GSK3beta) or with 1 µg of the monoclonal anti-GFP antibody (to immunoprecipitate GFP-LRP6-C3 and the associated GSK3beta). After washing IgG beads containing the immunomobilized GSK3beta four times with phosphate-buffered saline containing 350 mM NaCl (high salt) and 0.5% Nonidet P-40, the beads were washed two additional times with GSK3 kinase buffer (20 mM Tris-Cl, pH 7.5, 5 mM MgCl2, and 1 mM dithiothreitol). GSK3beta activity assays were performed as described using phosphoglycogen synthase peptide 2 or recombinant tau as substrates (37) with the exception that GSK3beta activity was normalized to GSKbeta levels in the sample, and the data were expressed as percentages of control (GFP alone). The same immunoprecipitation was performed to detect phospho-Ser9 GSK3beta (Cell Signaling) in each condition.

Recombinant in Vitro GSK3beta Activity Assays—GST-beta-catenin, purified as described above, was bound to washed glutathione beads for 4 h and washed twice with GSK3 kinase buffer. Sixty units of recombinant GSK3beta (New England Biolabs) was preincubated with or without LRP6-C3 (cleaved from GST as described above) in the presence of 1 mg/ml BSA in GSK3 kinase buffer for 30 min at 4 °C. Preliminary experiments were conducted to determine optimal conditions for functional assay readouts, and a 40-fold molar excess of LRP6-C3 over GSK3beta was used for all experiments. Following preincubation, reaction components were added to final concentrations of 125 µM ATP, 0.182 µCi/µl [{gamma}-32P]ATP, and 1 mg/ml BSA in GSK3 kinase buffer. Washed GST-beta-catenin bound to beads was then incubated in the reaction mix for 30 min at 30 °C. After removing the reaction supernatant and washing the beads twice with GSK3 kinase buffer, the beads were boiled with 30 µlof 2x SDS buffer, and the supernatants were resolved on an 8% polyacrylamide gel. The gels were dried and analyzed by phosphorimaging (Storm 840 (Amersham Biosciences) and ImageQuant software). GST-LRP6-C3 and GST-mut-LRP6-C3 in vitro phosphorylation by recombinant GSK3beta was performed in a similar manner except that the purified GST-LRP6-C3 or GST-mut-LRP6-C3 was incubated with recombinant GSK3beta alone without binding to the glutathione beads and without the addition of the BSA. For comparison, recombinant tau was incubated with recombinant GSK3beta under identical conditions.


Figure 1
View larger version (42K):
[in this window]
[in a new window]
  		FIGURE 1.
LRP6 interacts with GSK3 in yeast. Double transformed yeast were grown on culture medium glucose plates that lacked leucine and tryptophan (–LEU –TRP) to check the transformation efficiency or that lacked leucine, tryptophan, adenine, and histidine (–LEU –TRP –ADE –HIS) to check the interaction. Indicated double transformed yeast grew well in the –Leu –Trp plates with sequential 1:10 dilutions. Only LRP6-C3-BD (LRP6-C3 fused to GAL4 DNA-binding domain) and GSK3-AD (GSK3 fused to transcription activation domain) co-transformed yeast grew in –Leu –Trp –Ade –His plates, which demonstrates that LRP6-C3 and GSK3 interact.

 
Data Analysis—Data were analyzed using analysis of variance between individual groups. Values were considered significantly different when p was <0.05. Results were expressed as mean ± S.E.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The C Terminus of LRP6 Directly Interacts with GSK3—Previous studies have shown that the Wnt canonical pathway co-receptors LRP5 and -6 can bind to axin and recruit it to the membrane, resulting in the degradation of axin and propagation of the Wnt signal (16, 17). Although this is one mechanism by which LRP5/6 facilitates canonical Wnt signaling, our previous study showed that membrane targeting was not essential for the C terminus of LRP5/6 to constitutively activate the Wnt canonical pathway and synergistically increase LEF-1 activation in response to Wnt 3a (18). These findings suggested that LRP5/6 can facilitate the propagation of the Wnt signal through mechanisms other than recruitment of axin to the membrane. To determine which other proteins may be involved in the LRP5/6 signaling cascades, a yeast two-hybrid screen was carried out by using the intracellular domain of human LRP6 (LRP6-C3) as the bait, which was described previously (18). An adult human brain cDNA library was used in the screen. We screened ~2.5 million clones and identified four clones as GSK3 by nucleotide sequencing analyses. GSK3 interacted strongly with LRP6-C3 in yeast (Fig. 1). We also identified nine other unique clones, some of which are in the Wnt canonical pathway. However, none of them were axin. To confirm the interaction between LRP6-C3 and GSK3 in vitro, we made a GST-fused LRP6-C3 construct. Purified GST and a GST-LRP6-C3 fusion protein were conjugated to glutathione beads. CHO cells were transfected with GSK3{alpha} or GSK3beta, and lysates from the transfected cells were incubated with GST- or GST-LRP6-C3-conjugated beads. Both GSK3{alpha} and GSK3beta were specifically pulled down by LRP6-C3 in vitro (Fig. 2). To rule out an indirect interaction between LRP6-C3 and GSK3, a pull-down assay was performed using recombinant GSK3beta and recombinant GST-LRP6-C3 (Fig. 2C), which confirmed a direct protein-protein interaction. Further co-immunoprecipitation assays also revealed that LRP6-C3 interacts directly with both GSK3{alpha} and GSK3beta (Fig. 3).


Figure 2
View larger version (34K):
[in this window]
[in a new window]
  		FIGURE 2.
The C terminus of LRP6 interacts with both GSK3{alpha} and GSKbeta in vitro. CHO cells were collected 48 h after transfection with the indicated HA-tagged GSK3 constructs. 500 µg of the GSK3{alpha} cell lysates (A) or 200 µg of the GSK3beta cell lysate (B) was incubated with GST or GST-LRP6-C3 proteins conjugated to glutathione beads for 2 h. Both GSK3{alpha} and GSK3beta were specifically pulled down by LRP6-C3. Aliquots of the lysates (3 µg for GSK3{alpha} (A) and 4 µg for GSK3beta (B)) were run in the first lane of each blot to show input. Blots were probed with an anti-HA antibody. C, GST- or GST-LRP6-C3-conjugated beads were incubated with 200 ng of recombinant GSK3beta (rGSK3beta), washed, and blotted with a GSK3beta antibody. D, Coomassie-stained gel showing the amount of GST and GST-LRP6-C3 protein in the assay.

 
LRP6-C3 Decreases GSK3beta Phosphorylation of a Primed Peptide and Recombinant Tau in Vitro—Since LRP6-C3 interacts with GSK3beta, the effect of this interaction on the activity of GSK3beta was subsequently evaluated. In this assay, endogenous GSK3beta was used, because CHO cells express high levels of GSK3beta and this approach reduced the number of variables in the assay (Fig. 4A). To immunoprecipitate similar amounts of GSK3beta, 30 µg of the lysate was used when GSK3beta was immunoprecipitated directly, and 150 µg of the lysate was used when GSK3beta was co-immunoprecipitated by using the anti-GFP antibody to bring down LRP6-C3 (Fig. 4B). GSK3beta was immunoprecipitated from GFP- or GFP-LRP6-C3-transfected cell lysates with a monoclonal GSK3beta antibody and used to 32P-phosphorylate substrates in vitro. Fractionation and cytochemical analyses revealed that GFP-LRP6-C3 was present in both the cytosol and nucleus (data not shown). When GSK3beta was immunoprecipitated from CHO cell lysates and used in the assay, there was no significant difference in GSK3beta activity between the lysates from GFP- and GFP-LRP6-C3-transfected cells (Fig. 4C). This is probably due to the fact that only a small fraction of the GSK3beta immunoprecipitated was bound by LRP6-C3. Indeed, when the GSK3beta immunoprecipitates were probed for LRP6-C3, none could be detected at a normal exposure (Fig. 4B), and it was only visible when the blots were significantly overexposed (Fig. 4B). Therefore, to determine the effects of LRP6-C3 on GSK3beta activity, GFP-LRP6-C3 was immunoprecipitated with the GFP antibody, and GSK3beta activity was measured in the precipitates. When the values were normalized to the levels of GSK3beta in the immunoprecipitates, LRP6-C3 significantly inhibited GSK3beta activity toward the primed peptide substrate (Fig. 4C). Assays were also carried out using recombinant tau as a substrate. A representative phosphorylated recombinant tau autoradiograph is shown in Fig. 4D, and the corresponding quantitated data from three separate experiments are shown in Fig. 4E, demonstrating that LRP6-C3 also attenuates GSK3beta phosphorylation of an unprimed substrate. These data clearly demonstrate that the C terminus of LRP6 inhibits GSK3beta activity. Interestingly, only a small amount of the GSK3beta that co-precipitated with LRP6-C3 was phosphorylated on Ser9, compared with the phospho-Ser9 immunoreactivity that was present in the fractions in which GSK3beta was immunoprecipitated directly (Fig. 4B), indicating that LRP6 preferentially binds to the dephosphorylated, more active form of GSK3beta. It should be noted that the levels of phospho-Ser9 GSK3beta (Ser9) were equivalent in the lysates from GFP- and GFP-LRP6-C3-transfected cells (Fig. 4A).


Figure 3
View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 3.
The C terminus of LRP6 interacts with both GSK3{alpha} and GSK3beta in situ. A, cell lysates transfected with the indicated constructs were immunoprecipitated (IP) with the GFP antibody to precipitate the GFP-LRP6-C3 protein. The HA antibody was used to detect the exogenously expressed GSK3 proteins in the immunoprecipitate. Both GSK3{alpha} and GSK3beta interacted with LRP6-C3. B, the expression levels of GFP-LRP6-C3, GSK3{alpha}, and GSK3beta are shown by using GFP and HA antibodies, respectively.

 
Recombinant LRP6-C3 Is an in Vitro Substrate of GSK3beta—In a previous study (16), it was revealed that the C terminus of LRP6 was phosphorylated by a hitherto unidentified kinase at the PPP(S/T)P motif, which is reiterated five times. To determine whether GSK3beta can phosphorylate the C terminus of LRP6, GST-LRP6-C3 was incubated in the presence of recombinant GSK3beta and [32P]ATP (Fig. 5). These data demonstrate that LRP6-C3 is efficiently phosphorylated by GSK3beta in vitro, comparable with tau. Under the same conditions, casein kinase I{delta} did not phosphorylate LRP6-C3 (data not shown). Previous studies have shown that casein kinase I{alpha} phosphorylates beta-catenin (38). Further, to determine whether GSK3beta specifically phosphorylates the PPP(S/T)P motifs, we substituted all of the Ser/Thr in the PPP(S/T)P motifs to Ala (GST-mut-LRP6-C3), and the GST-mut-LRP6-C3 was incubated in the presence of recombinant GSK3beta and [32P]ATP under identical conditions (Fig. 5). The results show that the mut-LRP6-C3 is not phosphorylated by GSK3beta, which strongly indicates that GSK3beta is the kinase that specifically phosphorylates the Ser/Thr in the motif.


Figure 4
View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 4.
LRP6-C3 inhibits GSK3beta phosphorylation of a primed peptide and unprimed recombinant tau protein. A, CHO cells were transiently transfected with GFP or GFP-LRP6-C3, and cell lysates (5 µg) were immunoblotted with the GSK3beta antibody to detect endogenous GSK3beta, a Ser(P)9-GSK3beta antibody to determine the levels of Ser9-phosphorylated GSK3beta or the GFP antibody to determine the expression of the transfected constructs. B, cell lysates were immunoprecipitated (IP) with either the GSK3beta antibody to directly precipitate the endogenous GSK3beta or with the GFP antibody to immunoprecipitate LRP6-C3. The immunoprecipitates were then used in kinase assays. In order to get similar amounts of GSK3beta in the immunoprecipitates, 30 µg of lysate was used when GSK3beta was immunoprecipitated directly, and 150 µg of lysate was used when GFP-LRP6-C3 was immunoprecipitated and GSK3beta was a co-immunoprecipitating protein. Representative immunoblots show that the levels of GSK3beta co-immunoprecipitated with LRP6-C3 and the immunoprecipitated GSK3beta are similar. LRP6-C3 prefers binding to the unphosphorylated GSK3beta, since the levels of phospho-Ser9 immunoreactivity are low in the immunoprecipitates. The overexposed blot of GFP-LRP6-C3 shows that when GSK3beta is immunoprecipitated, GFP-LRP-C3 does co-precipitate, although proportionally only a small fraction of the GSK3beta is interacting with the GFP-LRP-C3. When GFP-LRP6-C3 was immunoprecipitated and therefore all of the GSK3beta in the assay was bound to LRP6-C3, GSK3beta phosphorylated both the primed peptide (C) and unprimed recombinant tau (E) to a significantly lesser extent compared with when GSK3beta was immunoprecipitated directly. Data were normalized to immunoprecipitated GSK3beta levels and expressed as a percentage of the values obtained with immunoprecipitated GSK3beta from GFP lysates. Data are present as mean ± S.E. of three separate experiments. **, p < 0.001 compared with GSK3beta from GFP lysates. D, representative autoradiograph of phosphorylated recombinant tau protein.

 


Figure 5
View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 5.
Recombinant LRP6-C3 is phosphorylated by recombinant GSK3beta. GSK3beta was incubated with either wild type GST-LRP6-C3 (lane 1), GST-mut-LRP6-C3 (lane 2), or recombinant tau (rtau) (lane 3) as a positive control. Samples were resolved using SDS-PAGE, the gels were Coomassie-stained, and phosphorylated proteins were visualized by autoradiography. LRP6-C3 was strongly phosphorylated by GSK3beta in vitro, whereas mut-LRP6-C3 was not phosphorylated.

 
LRP6-C3 Decreases GSK3beta Phosphorylation of Recombinant beta-Catenin in Vitro—In order to test the effects of the GSK3beta/LRP6-C3 interaction on GSK3 activity toward the Wnt pathway substrate beta-catenin, in vitro phosphorylation assays were performed using recombinant beta-catenin in the absence and presence of LRP6-C3 (Fig. 6). The addition of recombinant LRP6-C3 to the assay significantly attenuated GSK3beta-mediated phosphorylation of beta-catenin. These data are in agreement with our previous in situ findings that overexpression of LRP6-C3 in cells results in a decrease in phospho-beta-catenin levels (18).

The C Terminus of LRP6 Inhibits GSK3beta-mediated Phosphorylation of Tau at both Primed and Unprimed Sites in Situ—Previous studies have provided evidence that GSK3-binding proteins, such as FRAT-1 and FRAT-2, which are the proteins involved in the Wnt canonical pathway, affect GSK3beta-mediated tau phosphorylation (23, 37, 39). Since LRP6 is a GSK3-binding protein that attenuates its activity in in vitro assays, we next examined the effects of LRP6 on GSK3-mediated tau phosphorylation in situ. CHO cells were transiently transfected with tau in the absence or presence of GSK3beta and in the presence or absence of GFP or GFP-LRP6-C3, and tau phosphorylation was examined by immunoblotting with phospho-specific antibodies (Fig. 7A). As expected, exogenous expression of GSK3beta resulted in an increased phosphorylation at both the PHF-1 (phospho-Ser396/404) and AT180 (phospho-Thr231) epitopes. However, when GFP-LRP6-C3 was expressed with GSK3beta, GSK3beta phosphorylation of tau at the unprimed PHF-1 epitope significantly decreased compared with what was observed with GSK3beta alone, and GSK3beta phosphorylation of tau at the primed AT180 epitope was completely abolished. GFP alone has no effect on GSK3beta-mediated tau phosphorylation (Fig. 7A). When GFP-LRP6-C3 was expressed alone with tau, there was a significant decrease in the phosphorylation state of tau at the unprimed PHF-1 epitope (Fig. 7B), which indicates that GFP-LRP6-C3 also inhibits the phosphorylation of tau by endogenous GSK3beta. GFP-LRP6-C3 also attenuated GSK3{alpha}-mediated tau phosphorylation in a manner similar to what was observed for GSK3beta (data not shown).


Figure 6
View larger version (10K):
[in this window]
[in a new window]
  		FIGURE 6.
Phosphorylation of beta-catenin by recombinant GSK3beta is attenuated by recombinant LRP6-C3. GSK3beta preincubated in the absence or presence of 8 pmol of LRP6-C3 was used to phosphorylate GST-beta-catenin. Phosphor images were quantitated, and the incorporation of [32P]ATP was normalized to total beta-catenin protein levels as determined by Coomassie staining followed by scanning densitometry. Values are expressed as a ratio of these two values. Data are present as mean ± S.E. of three experiments. *, p = 0.02.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we show for the first time that the C terminus of LRP6 directly binds to GSK3 and that this interaction significantly attenuates the activity of GSK3. GSK3 is a key protein in the Wnt canonical signaling pathway and plays an essential role in regulating tau phosphorylation. Here we clearly demonstrate in vitro that the C terminus of LRP6 inhibits GSK3beta-mediated phosphorylation of beta-catenin. Furthermore, in situ studies show that LRP6-C3 inhibits GSK3beta-mediated phosphorylation of both primed and unprimed sites on tau. Finally, we demonstrate that in vitro, GSK3beta phosphorylates the PPP(S/T)P motifs in LRP6. These are novel and important findings, since they provide insight into the mechanisms by which LRP6 may regulate the phosphorylation of specific GSK3beta substrates in and out of the Wnt canonical pathway.

Wnt signaling through beta-catenin has been studied extensively; however, much still remains unknown about this pathway, especially at the cell membrane level. The membrane proteins LRP5/6 are required coreceptors in the Wnt canonical pathway (10–12). LRP5/6 are the only two proteins from the low density lipoprotein receptor family that have been shown to facilitate signaling through the Wnt canonical pathway, although a minireceptor of LRP1, another member of the LDL receptor family that interacts with the human Fz-1, has been shown to repress Wnt 3a signaling by disrupting the Fz receptor-LRP5 and -6 coreceptor complex (40). It is clear that the main function of canonical Wnts is to nucleate the formation of a physical complex between receptor and coreceptor (11); this was confirmed in a recent study by using a Dickkopf-1-Fz5 fusion protein (41). However, how this membrane complex transduces the signal into the cell is still not completely clear. Studies showed that the intracellular domain of LRP5 and -6 interacts with axin and that the addition of Wnt appears to stimulate the recruitment of axin to LRP5 and -6 at the membrane. Once at the membrane, axin is degraded, which subsequently results in increased beta-catenin levels and signaling (16, 17). Further, it was demonstrated that the PPP(S/T)P motif in the intracellular domain of LRP5/6 is required for signaling (42) and that phosphorylation of this motif creates a docking site for axin (16). Recently, another group showed that FRAT-1 binds to the cytoplasmic domain of LRP5 and that the recruitment of axin and FRAT-1 to the membrane by LRP5 leads to both axin degradation and FRAT-1-mediated inhibition of GSK3 (43). Here we clearly demonstrate that the C terminus of LRP6 also interacts directly with GSK3. This direct interaction inhibits GSK3-mediated phosphorylation of beta-catenin. Although it is apparent that LRP6 can bind all three proteins (axin, FRAT-1, and GSK3), it is unlikely that they all exist in the same complex due to the fact that axin and FRAT-1 competitively bind GSK3 (44). Recently, it was shown that although FRAT is strictly required for maternal Wnt signaling in Xenopus, FRAT is not an essential component of the canonical Wnt pathway in higher organisms (45). These data suggest that FRAT-1 binding to LRP5 is not necessary for LRP5 function in the pathway. All of these studies have suggested that LRP5 and -6 can function in the Wnt canonical pathway through multiple mechanisms.


Figure 7
View larger version (38K):
[in this window]
[in a new window]
  		FIGURE 7.
LRP6-C3 inhibits phosphorylation of tau by GSK3beta at both primed and unprimed sites in situ. A, representative immunoblots showing total tau levels (Tau5/5A6), and tau phosphorylated at the primed AT180 site (pThr231) and the unprimed PHF-1 (pSer396/404) site. CHO cells were transiently transfected with tau and the other constructs as indicated below the panels. Tau protein levels were equivalent in all conditions. Expression of exogenous GSK3beta resulted in a decrease in the electrophoretic mobility of tau, indicative of increased phosphorylation, and phosphorylation at both the PHF-1 and AT180 epitopes increased as expected. The presence of GFP-LRP6-C3 significantly decreased the GSK3beta-mediated phosphorylation of tau at the PHF-1 epitope and completely abolished the GSK3beta-mediated phosphorylation of tau at the AT180 epitope. GFP alone had no effect on the GSK3beta-mediated phosphorylation of tau. The expression levels of transfected GFP, GFP-LRP6-C3, and GSK3beta (by blotting for HA) are also shown. {alpha}-Tubulin was used as the loading control. B, a longer exposure of the PHF-1 blot shown in A to clarify the GFP-LRP6-C3-mediated inhibition of the phosphorylation of the 396/404 epitope of tau by endogenous GSK3beta.

 
In the C terminus of LRP6, Ser and Thr represent nearly 30% of the amino acids. Here we show that GSK3beta, but not casein kinase I{delta}, phosphorylates the C terminus of LRP6 in an in vitro assay. However, the GST-mut-LRP6-C3 cannot be phosphorylated by GSK3beta, which strongly suggests that GSK3beta contributes to the phosphorylation of the PPP(S/T)P motifs of LRP6 to supply a binding site for axin in situ. Therefore, it can be speculated that the interaction of LRP6 with GSK3beta and the subsequent attenuation of GSK3beta activity in combination with phosphorylation of LRP6 is responsible for at least part of the function of LRP6 in the canonical pathway. It has recently been shown that the C termini of LRP5 and LRP6, which are not membrane-anchored, can efficiently activate the Wnt canonical pathway by decreasing the phospho-beta-catenin level and increasing LEF-1 activity in situ (18). It thus can be hypothesized that the binding of the cytosolic termini of LRP5 and -6 to GSK3 in the axin complex attenuates GSK3 phosphorylation of beta-catenin, thus activating the Wnt canonical pathway without extracellular signaling. However, whether the C terminus of LRP6 alone is a physiologically relevant entity is currently unknown. It may occur in some pathological situations to function as an oncogene for constitutively activating the Wnt canonical pathway (46). It is also interesting to note that other low density lipoprotein receptor family members, such as LRP (47) and megalin (48), undergo ectodomain shedding followed by release of the C terminus. Moreover, whether and how other proteins from the axin complex can affect the interaction between LRP6 and GSK3, thus changing the activity of the Wnt signal, needs to be further studied.

Besides the proteins from the Wnt canonical pathway and its namesake substrate glycogen synthase, GSK3 can phosphorylate more than 40 proteins, including over a dozen transcription factors (2). GSK3 activity is precisely regulated by a combination of phosphorylation, localization, and interactions with other proteins. It should be noted that previous studies have shown that GSK3beta activity can be regulated by its interaction with other proteins. For example, binding of the transcription factor p53 (49) or FRAT-2 (37) increases GSK3beta activity, whereas binding by FRAT-1 attenuates activity (23, 39). In this study, we show that LRP6 is another GSK3-binding protein that inhibits GSK3 phosphorylation of both primed and unprimed substrates, both in and out of the Wnt canonical pathway. It suggests that LRP6 generally attenuates GSK3 activity. In our study, the in situ inhibitory function of LRP6 on GSK3 activity is much stronger than in vitro, which highly suggests that other proteins from the complex may play a role in facilitating the interaction and/or function. It is also worth noting that LRP6-C3 preferentially binds to the active form of GSK3beta, which efficiently phosphorylates LRP6-C3 in the complex. Thus, the inhibitory function of LRP6-C3 on GSK3beta-mediated phosphorylation of beta-catenin, tau, and glycogen synthase is through protein-protein interactions.

Tau is a well characterized substrate of GSK3 both in situ and in vitro. Up to 25 sites in tau have been shown to be modified by GSK3, including both unprimed and primed Ser/Thr-Pro motifs (2). Modulation of tau phosphorylation is an important regulating mechanism both during development and in the adult brain, and GSK3beta may play a key role (50–52). The importance of primed site (e.g. Thr231) phosphorylation is in regulating tau-microtubule interactions (19), and the unprimed phosphorylation of tau at specific sites (e.g. Ser396/Ser404) may be involved in the pathological formation of filaments (20), which occur in the tau-neurodegenerative diseases, such as Alzheimer disease. From our study, LRP6 inhibits GSK3-mediated phosphorylation of tau at both primed and unprimed sites, which indicates that LRP6 could modulate both physiological and pathological tau phosphorylation by GSK3beta.

In summary, we demonstrate for the first time that GSK3beta phosphorylates the PPP(S/T)P motifs in LRP6 and that C terminus of LRP6 directly interacts with GSK3. Further, we show that this interaction inhibits GSK3beta activity toward beta-catenin, tau, and glycogen synthase in vitro. In situ GSK3-mediated phosphorylation of both primed and unprimed sites on tau is also decreased by the C terminus of LRP6. The LRP6-modulated decrease in the GSK3-mediated phosphorylation of beta-catenin may be an essential step in the Wnt canonical signaling pathway. In the case of tau, the interaction of LRP6 with GSK3beta may inhibit physiological phosphorylation events that regulate tau-microtubule interactions and attenuate phosphorylation at unprimed sites, which have the potential to be of more pathological significance. Overall, LRP6 plays a unique and important role in the regulation of GSK3beta activity in and out of the Wnt canonical pathway.


   FOOTNOTES
 
* This work was supported by a grant from the Alzheimer's Association and National Institutes of Health Grant NS051279. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Psychiatry, 1720 7th Ave. S., SC1061, University of Alabama at Birmingham, Birmingham, AL 35294-0017. Tel.: 205-934-2465; Fax: 205-934-2500; E-mail: Gvwj{at}Uab.Edu.

2 The abbreviations used are: GSK3, glycogen synthase kinase 3; Fz, Frizzled; FRAT, frequently rearranged in advanced T-cell lymphoma; LDL, low density lipoprotein; LRP, LDL receptor-related protein; BSA, bovine serum albumin; CHO, Chinese hamster ovary; E3, ubiquitin-protein isopeptide ligase; GST, glutathione S-transferase; HA, hemagglutinin. Back


   ACKNOWLEDGMENTS
 
We thank Dr. P. Davies and Dr. L. Binder for providing tau antibodies, Dr. J. R. Woodgett for the GSK3beta-HA construct, and Dr. W. I. Weis for the GST-beta-catenin construct.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Grimes, C. A., and Jope, R. S. (2001) Prog. Neurobiol. 65, 391–426[CrossRef][Medline] [Order article via Infotrieve]
  2. Jope, R. S., and Johnson, G. V. (2004) Trends Biochem. Sci. 29, 95–102[CrossRef][Medline] [Order article via Infotrieve]
  3. Woodgett, J. R. (1990) EMBO J. 9, 2431–2438[Medline] [Order article via Infotrieve]
  4. Lau, K. F., Miller, C. C., Anderton, B. H., and Shaw, P. C. (1999) J. Pept. Res. 54, 85–91[CrossRef][Medline] [Order article via Infotrieve]
  5. Amit, S., Hatzubai, A., Birman, Y., Andersen, J. S., Ben-Shushan, E., Mann, M., Ben-Neriah, Y., and Alkalay, I. (2002) Genes Dev. 16, 1066–1076[Abstract/Free Full Text]
  6. Ikeda, S., Kishida, S., Yamamoto, H., Murai, H., Koyama, S., and Kikuchi, A. (1998) EMBO J. 17, 1371–1384[CrossRef][Medline] [Order article via Infotrieve]
  7. Hart, M., Concordet, J. P., Lassot, I., Albert, I., del los Santos, R., Durand, H., Perret, C., Rubinfeld, B., Margottin, F., Benarous, R., and Polakis, P. (1999) Curr. Biol. 9, 207–210[CrossRef][Medline] [Order article via Infotrieve]
  8. Li, L., Yuan, H., Weaver, C. D., Mao, J., Farr, G. H., III, Sussman, D. J., Jonkers, J., Kimelman, D., and Wu, D. (1999) EMBO J. 18, 4233–4240[CrossRef][Medline] [Order article via Infotrieve]
  9. Seidensticker, M. J., and Behrens, J. (2000) Biochim. Biophys. Acta. 1495, 168–182[Medline] [Order article via Infotrieve]
  10. Pinson, K. I., Brennan, J., Monkley, S., Avery, B. J., and Skarnes, W. C. (2000) Nature 407, 535–538[CrossRef][Medline] [Order article via Infotrieve]
  11. Tamai, K., Semenov, M., Kato, Y., Spokony, R., Liu, C., Katsuyama, Y., Hess, F., Saint-Jeannet, J. P., and He, X. (2000) Nature 407, 530–535[CrossRef][Medline] [Order article via Infotrieve]
  12. Wehrli, M., Dougan, S. T., Caldwell, K., O'Keefe, L., Schwartz, S., Vaizel-Ohayon, D., Schejter, E., Tomlinson, A., and DiNardo, S. (2000) Nature 407, 527–530[CrossRef][Medline] [Order article via Infotrieve]
  13. Veeman, M. T., Axelrod, J. D., and Moon, R. T. (2003) Dev. Cell 5, 367–377[CrossRef][Medline] [Order article via Infotrieve]
  14. Strutt, D. (2003) Development 130, 4501–4513[Abstract/Free Full Text]
  15. Semenov, M. V., Tamai, K., Brott, B. K., Kuhl, M., Sokol, S., and He, X. (2001) Curr. Biol. 11, 951–961[CrossRef][Medline] [Order article via Infotrieve]
  16. Tamai, K., Zeng, X., Liu, C., Zhang, X., Harada, Y., Chang, Z., and He, X. (2004) Mol. Cell 13, 149–156[CrossRef][Medline] [Order article via Infotrieve]
  17. Mao, J., Wang, J., Liu, B., Pan, W., Farr, G. H., III, Flynn, C., Yuan, H., Takada, S., Kimelman, D., Li, L., and Wu, D. (2001) Mol. Cell 7, 801–809[CrossRef][Medline] [Order article via Infotrieve]
  18. Mi, K., and Johnson, G. V. (2005) J. Cell. Biochem. 95, 328–338[Medline] [Order article via Infotrieve]
  19. Cho, J. H., and Johnson, G. V. (2004) J. Neurochem. 88, 349–358[CrossRef][Medline] [Order article via Infotrieve]
  20. Abraha, A., Ghoshal, N., Gamblin, T. C., Cryns, V., Berry, R. W., Kuret, J., and Binder, L. I. (2000) J. Cell Sci. 113, 3737–3745[Abstract]
  21. Stoothoff, W. H., Bailey, C. D., Mi, K., Lin, S. C., and Johnson, G. V. (2002) J. Neurochem. 83, 904–913[CrossRef][Medline] [Order article via Infotrieve]
  22. Mudher, A., Chapman, S., Richardson, J., Asuni, A., Gibb, G., Pollard, C., Killick, R., Iqbal, T., Raymond, L., Varndell, I., Sheppard, P., Makoff, A., Gower, E., Soden, P. E., Lewis, P., Murphy, M., Golde, T. E., Rupniak, H. T., Anderton, B. H., and Lovestone, S. (2001) J. Neurosci. 21, 4987–4995[Abstract/Free Full Text]
  23. Thomas, G. M., Frame, S., Goedert, M., Nathke, I., Polakis, P., and Cohen, P. (1999) FEBS Lett. 458, 247–251[CrossRef][Medline] [Order article via Infotrieve]
  24. Mao, B., Wu, W., Li, Y., Hoppe, D., Stannek, P., Glinka, A., and Niehrs, C. (2001) Nature 411, 321–325[CrossRef][Medline] [Order article via Infotrieve]
  25. Zorn, A. M. (2001) Curr. Biol. 11, R592–R595[CrossRef][Medline] [Order article via Infotrieve]
  26. Caricasole, A., Copani, A., Caraci, F., Aronica, E., Rozemuller, A. J., Caruso, A., Storto, M., Gaviraghi, G., Terstappen, G. C., and Nicoletti, F. (2004) J. Neurosci. 24, 6021–6027[Abstract/Free Full Text]
  27. Huber, A. H., Nelson, W. J., and Weis, W. I. (1997) Cell 90, 871–882[CrossRef][Medline] [Order article via Infotrieve]
  28. Gietz, R. D., and Woods, R. A. (2002) Methods Enzymol. 350, 87–96[CrossRef][Medline] [Order article via Infotrieve]
  29. Goedert, M., Jakes, R., Crowther, R. A., Cohen, P., Vanmechelen, E., Vandermeeren, M., and Cras, P. (1994) Biochem. J. 301, 871–877[Medline] [Order article via Infotrieve]
  30. Hoffmann, R., Lee, V. M., Leight, S., Varga, I., and Otvos, L., Jr. (1997) Biochemistry 36, 8114–8124[CrossRef][Medline] [Order article via Infotrieve]
  31. Goedert, M., Spillantini, M. G., Jakes, R., Rutherford, D., and Crowther, R. A. (1989) Neuron 3, 519–526[CrossRef][Medline] [Order article via Infotrieve]
  32. Greenberg, S. G., and Davies, P. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5827–5831[Abstract/Free Full Text]
  33. Otvos, L., Jr., Feiner, L., Lang, E., Szendrei, G. I., Goedert, M., and Lee, V. M. (1994) J. Neurosci. Res. 39, 669–673[CrossRef][Medline] [Order article via Infotrieve]
  34. Carmel, G., Mager, E. M., Binder, L. I., and Kuret, J. (1996) J. Biol. Chem. 271, 32789–32795[Abstract/Free Full Text]
  35. Johnson, G. V., Seubert, P., Cox, T. M., Motter, R., Brown, J. P., and Galasko, D. (1997) J. Neurochem. 68, 430–433[Medline] [Order article via Infotrieve]
  36. Bafico, A., Gazit, A., Wu-Morgan, S. S., Yaniv, A., and Aaronson, S. A. (1998) Oncogene 16, 2819–2825[CrossRef][Medline] [Order article via Infotrieve]
  37. Stoothoff, W. H., Cho, J. H., McDonald, R. P., and Johnson, G. V. (2005) J. Biol. Chem. 280, 270–276[Abstract/Free Full Text]
  38. Liu, C., Li, Y., Semenov, M., Han, C., Baeg, G. H., Tan, Y., Zhang, Z., Lin, X., and He, X. (2002) Cell 108, 837–847[CrossRef][Medline] [Order article via Infotrieve]
  39. Culbert, A. A., Brown, M. J., Frame, S., Hagen, T., Cross, D. A., Bax, B., and Reith, A. D. (2001) FEBS Lett. 507, 288–294[CrossRef][Medline] [Order article via Infotrieve]
  40. Zilberberg, A., Yaniv, A., and Gazit, A. (2004) J. Biol. Chem. 279, 17535–17542[Abstract/Free Full Text]
  41. Holmen, S. L., Robertson, S. A., Zylstra, C. R., and Williams, B. O. (2005) Biochem. Biophys. Res. Commun. 328, 533–539[CrossRef][Medline] [Order article via Infotrieve]
  42. Brennan, K., Gonzalez-Sancho, J. M., Castelo-Soccio, L. A., Howe, L. R., and Brown, A. M. (2004) Oncogene 23, 4873–4884[CrossRef][Medline] [Order article via Infotrieve]
  43. Hay, E., Faucheu, C., Suc-Royer, I., Touitou, R., Stiot, V., Vayssiere, B., Baron, R., Roman-Roman, S., and Rawadi, G. (2005) J. Biol. Chem. 280, 13616–13623[Abstract/Free Full Text]
  44. Ferkey, D. M., and Kimelman, D. (2002) J. Biol. Chem. 277, 16147–16152[Abstract/Free Full Text]
  45. van Amerongen, R., Nawijn, M., Franca-Koh, J., Zevenhoven, J., van der Gulden, H., Jonkers, J., and Berns, A. (2005) Genes Dev. 19, 425–430[Abstract/Free Full Text]
  46. Li, Y., Lu, W., He, X., Schwartz, A. L., and Bu, G. (2004) Oncogene 23, 9129–9135[CrossRef][Medline] [Order article via Infotrieve]
  47. May, P., Reddy, Y. K., and Herz, J. (2002) J. Biol. Chem. 277, 18736–18743[Abstract/Free Full Text]
  48. Zou, Z., Chung, B., Nguyen, T., Mentone, S., Thomson, B., and Biemesderfer, D. (2004) J. Biol. Chem. 279, 34302–34310[Abstract/Free Full Text]
  49. Watcharasit, P., Bijur, G. N., Zmijewski, J. W., Song, L., Zmijewska, A., Chen, X., Johnson, G. V., and Jope, R. S. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7951–7955[Abstract/Free Full Text]
  50. Johnson, G. V., and Jenkins, S. M. (1999) J. Alzheimers Dis. 1, 307–328[Medline] [Order article via Infotrieve]
  51. Maas, T., Eidenmuller, J., and Brandt, R. (2000) J. Biol. Chem. 275, 15733–15740[Abstract/Free Full Text]
  52. Billingsley, M. L., and Kincaid, R. L. (1997) Biochem. J. 323, 577–591[Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us