14-3-3 Connects Glycogen Synthase Kinase-3beta to Tau within a Brain Microtubule-associated Tau Phosphorylation Complex*

Alka Agarwal-MawalDagger , Hamid Y. QureshiDagger , Patrick W. CaffertyDagger , Zongfei YuanDagger , Dong HanDagger , Rongtian Lin§, and Hemant K. PaudelDagger ||

From the Dagger  Bloomfield Center for Research in Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital and the Departments of  Neurology and Neurosurgery and § Medicine, McGill University, Montreal, Quebec H3T 1E2, Canada

Received for publication, November 11, 2002, and in revised form, January 13, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In a recent study, we reported that in bovine brain extract, glycogen synthase kinase-3beta and tau are parts of an ~400-500 kDa microtubule-associated tau phosphorylation complex (Sun, W., Qureshi, H. Y., Cafferty, P. W., Sobue, K., Agarwal-Mawal, A., Neufield, K. D., and Paudel, H. K. (2002) J. Biol. Chem. 277, 11933-11940). In this study, we find that when purified brain microtubules are subjected to Superose 12 gel filtration column chromatography, the dimeric scaffold protein 14-3-3zeta co-elutes with the tau phosphorylation complex components tau and GSK3beta . From gel filtration fractions containing the tau phosphorylation complex, 14-3-3zeta , GSK3beta , and tau co-immunoprecipitate with each other. From extracts of bovine brain, COS-7 cells, and HEK-293 cells transfected with GSK3beta , 14-3-3zeta co-precipitates with GSK3beta , indicating that GSK3beta binds to 14-3-3zeta . From HEK-293 cells transfected with tau, GSK3beta , and 14-3-3zeta in different combinations, tau co-immunoprecipitates with GSK3beta only in the presence of 14-3-3zeta . In vitro, ~10-fold more tau binds to GSK3beta in the presence of than in the absence of 14-3-3zeta . In transfected HEK-293 cells, 14-3-3zeta stimulates GSK3beta -catalyzed tau phosphorylation in a dose-dependent manner. These data indicate that in brain, the 14-3-3zeta dimer simultaneously binds and bridges tau and GSK3beta and stimulates GSK3beta -catalyzed tau phosphorylation.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Microtubules, the major cytoskeletal structures of eukaryotic cells, are dynamic structures, and their assembly and disassembly is regulated by microtubule-associated proteins (1). In neurons, tau is one of the major microtubule-associated proteins and is mainly found in the axonal compartment (for reviews, see Refs. 1-3). Tau binds to microtubules and stabilizes microtubule structure. Studies suggest that tau regulates microtubule dynamics, axonal transport, and neuronal morphology by binding and stabilizing the microtubule structure (1-3). There are six tau isoforms, which migrate with sizes 45-65 kDa on an SDS-polyacrylamide gel. These isoforms are phosphorylated on multiple sites in the brain and display a characteristic retarded mobility on an SDS gel upon phosphorylation (2, 3). Tau phosphorylation reduces the affinity of tau for microtubules and is one of the mechanisms that control microtubule structure and dynamics in vivo (1-3).

In Alzheimer's disease (AD)1 brain, abnormally hyperphosphorylated tau accumulates and forms paired helical filaments (4, 5). Since abnormally phosphorylated tau does not bind to microtubules, abnormal tau phosphorylation in AD brain is thought to cause a loss of tau function, microtubule dysfunction, and neurodegeneration (2, 3). It is not understood how abnormally phosphorylated tau accumulates in AD brain, but a defect in the regulatory mechanism that controls tau phosphorylation/dephosphorylation is very likely to be involved. The elucidation of the regulatory mechanism that controls tau phosphorylation in normal brain and the determination of how this regulation fails in AD brain are essential steps in understanding disease ontogeny and developing therapeutic interventions.

Glycogen synthase kinase-3 (GSK3) is an important regulatory enzyme that phosphorylates numerous substrates and regulates diverse physiological processes such as glycogen metabolism, gene expression, apoptosis, signal transduction, and cell fate specification (6-8). There are two isoforms of GSK3 that are highly expressed in the brain: ~51-kDa GSK3alpha and ~47-kDa GSK3beta (9). In transfected cells and transgenic mice, enhanced expression of GSK3beta leads to tau phosphorylation and microtubule instability (10-15). In AD brain, GSK3beta is activated in pretangle neurons and accumulates in paired helical filaments (16, 17). These observations suggest that GSK3beta phosphorylates tau in both normal and AD brain. Previous studies have shown that a large amount of GSK3beta in brain is associated with microtubules (18-20), and microtubule-associated GSK3beta is part of an ~400-500-kDa multiprotein complex containing tau and GSK3beta (20). These data indicate that GSK3beta phosphorylates tau within a microtubule-associated multiprotein complex (hereon designated as tau phosphorylation complex). The enormity of the tau phosphorylation complex suggests that within the complex, there may be proteins other than tau and GSK3beta (20). The identification of all the complex components and the determination of their functions within the complex are essential to understanding the mechanism by which GSK3beta phosphorylates tau in the brain.

14-3-3 is a family of conserved acidic proteins that are widely expressed in all eukaryotic tissues (21, 22). There are seven 14-3-3 isoforms, which are products of distinct genes. 14-3-3 is a naturally dimeric scaffold protein with the size of the monomer being ~30 kDa (21). Within the 14-3-3 dimer, the ligand binding grooves of each monomer run in opposite directions, and hence a 14-3-3 dimer can interconnect and bring two different proteins together. 14-3-3 binds to diverse cellular proteins, and more than 100 14-3-3 binding proteins have been identified (21). 14-3-3 is a cofactor of bacterial toxin Pseudomonas (23). 14-3-3 binds to Raf kinase and regulates the mitogen-activated protein (MAP) kinase signaling pathway (24, 25). It also binds cdc25, polyoma virus middle tumor antigen, p53, protein kinase C, Bcr, PI3 kinase, insulin-like growth factor, BAD, and p53 (26-37). By binding to its targets, 14-3-3 regulates enzyme activity, stabilizes enzyme conformation, controls subcellular localization of proteins, and mediates protein-protein interaction (21, 22). 14-3-3 regulates diverse cellular processes including cell growth, cell differentiation, cell division, apoptosis, and neuronal function (21, 22).

In the brain, ~1% of soluble protein is 14-3-3 and has been suggested to be critical for brain function (21). From bovine brain extract, 14-3-3zeta co-immunoprecipitates with tau (36). In vitro, 14-3-3zeta binds and changes the tau conformation, thus making tau susceptible for kinase phosphorylation (36). More importantly, a substantial amount of 14-3-3zeta co-purifies with microtubules from the brain extract (36). These observations suggest that 14-3-3zeta is an integral part of brain microtubules and is involved in the regulation of tau phosphorylation and microtubule dynamics. However, very little information is available about microtubule-associated 14-3-3zeta . In this study, we have further analyzed microtubule-associated 14-3-3zeta . Herein we report that brain microtubule-associated 14-3-3zeta is part of the tau phosphorylation complex containing GSK3beta and tau. Our data indicate that 14-3-3zeta mediates GSK3beta -tau interaction and facilitates tau phosphorylation by GSK3beta within the complex.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

cDNA Cloning and Plasmids-- The longest human tau isoform in a pET-3a vector (38) was amplified by Pfu DNA polymerase-catalyzed PCR using the forward primer (5'-AAA AAA GAA TTC AAT GGC TGA GCC CCG C-3') containing EcoR1 (italicized) and reverse primer (5'-AAA AAA GGA TCC TCA CAA ACC CTG CTT G-3') containing BamHI (italicized) sites. Adenine overhangs were added to the PCR product by TaqDNA polymerase, which was then ligated into a pGEX-T Easy vector (Promega) for amplification (20). After amplification, the insert was released and ligated into the EcoR1/BamHI cloning site of FLAG-pcDNA3.1 Zeo vector (Invitrogen, Madison, WI). Human 14-3-3zeta cDNA was subcloned into the BamH1/EcoR1 site of Xpress-pcDNA3.1 (Invitrogen) as described above using 14-3-3zeta -pGEX-6p (36) as the template and forward primer (5'-G GAA TTC TAT GAC AAT GGA TAA AAG T-3') containing the EcoR1 (italicized) and reverse primer (5'-CG GGA TCC TTA ATT TTC CCC TCC TTC-3') containing BamHI sites. All cloning procedures were confirmed by DNA sequencing. pcDNA3.1 containing HA (hemagglutinin)-tagged human GSK3beta was a gift from Dr. James R. Woodgett (The University of Toronto). Other expression vectors, GSK3beta -pGEX-6p, 14-3-3zeta -pGEX-6p, and tau-pET-3a, are described previously (20, 36, 38).

Cell Culture and Transfection-- COS-7 and HEK-293 cells were maintained in Dulbecco's modified Eagle's medium (high glucose) medium (Invitrogen) supplemented with 10% fetal bovine serum. Cells were plated in 100-mm culture dishes, grown to ~80% confluency, and transfected by standard calcium phosphate method with various amounts of the appropriate plasmids. For each 100-mm dish, 5-10 µg of DNA was mixed with 50 µl of CaCl2 (2.5 M) to give a final volume of 500 µl with distilled water. The mixture of DNA and CaCl2 was added to 500 µl of 2× HEPES-buffered saline (1.63% NaCl, 1.188% Hepes, 0.02% Na2HPO4 (pH 7.2)), and the mixture was allowed to settle at 20 °C for 30 min. DNA mixture was added to the cells dropwise, and cells were allowed to grow for 12-18 h. The medium was then changed, and cells were incubated for 48-72 h.

Proteins and GSK3beta Activity Assay-- Recombinant tau was purified from bacterial extract overexpressing the longest human tau isoform (39). GST-14-3-3zeta and GST-GSK3beta were purified from the respective bacterial lysates overexpressing the respective proteins by glutathione-agarose chromatography, and the GST tag was removed as described previously (20, 40). Polyclonal antibodies against tau, GSK3beta , and 14-3-3zeta have been described (20, 36). Monoclonal antibodies against tau and GSK3beta were obtained from NeoMarker (Fremont, CA), and Transduction Laboratories (Lexington, KY), respectively. Monoclonal anti-HA and anti-FLAG antibodies were from Sigma. Anti-Xpress monoclonal antibody was purchased from Invitrogen. Tau phosphorylation-sensitive monoclonal antibodies, AT8, PHF-1, and 12E8, are described previously (20, 36). GSK3beta activity assay was performed essentially as described (20).

Microtubule Assembly/Disassembly and Partial Purification of 14-3-3zeta from Microtubule Fractions-- Purification of microtubules from a fresh bovine brain extract by the temperature-induced microtubule assembly/disassembly has been described previously (38). Microtubule pellet obtained by centrifugation after first, second, third, and fourth cycles of assembly/disassembly were designated as P1, P2, P3, and P4, and the supernatants were designated as S1, S2, S3, and S4, respectively.

For a partial purification of 14-3-3zeta , all procedures were carried out at 4 °C. Microtubule pellet P3 (~4 mg) was homogenized in ~10 ml of PEM buffer (0.1 M PIPES, 1 mM EGTA, 1 mM MgSO4, and 1 mM DTT) containing 0.1 mM GTP using a glass homogenizer and then incubated in ice for 30 min. After incubation, the sample was centrifuged at 27,000 × g for 20 min, and the supernatant (~12 ml) was loaded onto a phosphocellulose (Whatman) column (25 × 5 cm) equilibrated in PEM buffer. The column was washed extensively, and the column-bound 14-3-3zeta was eluted with 200 ml of NaCl gradient (0-1 M in PEM buffer). Effluent fractions were immunoblotted using anti-14-3-3zeta antibody, and those containing 14-3-3zeta were combined and dialyzed against Mops buffer (25 mM MOPS (pH 7.4), 50 mM beta -glycerol phosphate, 0.1 mM EDTA, 1 mM DTT, 0.2 M NaCl, 10 mM NaF, and 15 mM MgCl2) for 4 h. Dialyzed sample was concentrated by Aquacide III (Calbiochem) and centrifuged at 27,000 × g for 30 min. The supernatant (~8 ml) was loaded onto an FPLC Superose 12 (Amersham Biosciences) gel filtration column (2.6 × 50 cm), equilibrated, and eluted with Mops buffer. Effluent fractions (1 ml each) were collected, and those containing 14-3-3zeta were pooled and dialyzed against 15 mM MOPS (pH 7.4), 1 mM EDTA, 20 mM NaCl, and 1 mM DTT. The dialyzed sample was loaded onto an FPLC Mono S column (Amersham Biosciences) equilibrated in 25 mM MOPS (pH 7.4), 0.1 mM EDTA, and 0.1 mM DTT. The column was washed with the equilibration buffer and then eluted with an NaCl gradient (0-0.5 M) in the equilibration buffer. Fractions (200 µl each) were collected, and those containing 14-3-3zeta were combined (~1.5 ml) and chromatographed through a Sepharose 4B (Sigma) gel filtration column (2.5 × 60 cm) equilibrated and eluted with Mops buffer. Fractions (0.5 ml each) were collected.

Immunoprecipitation and GST Pull-down Assay-- Cells in each culture dish were suspended in 1 ml of lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 25 mM beta -glycerol phosphate, 1 mM EDTA, 1 mM EGTA, 10 mM NaF, 10 mM MgCl2, 1% Nonidet P-40, 100 nM okadaic acid (Sigma), 50 pM cypermethrane (Calbiochem), 1 mM phenylmethulsulfonyl fluoride, and 1 µg/ml each of pepstatin, leupeptin, aprotinin). The cell suspension was incubated in ice for 1 h and then centrifuged at 4 °C for 15 min. The supernatant was either used for immunoprecipitation or used for GST pull-down assay.

For immunoprecipitation, the supernatant (~200 µl) was precleared with ~50 µl of protein G-agarose beads (Sigma) equilibrated in lysis buffer. The precleared sample was mixed with 10 µg of indicated antibody, and the mixture was shaken end-over-end for 6 h at 4 °C. After shaking, 30 µl of protein G-agarose beads was added to the mixture, and the shaking was continued for another 5 h. The beads were then collected by centrifugation and washed three times (30 min each). The washed beads were dissolved in 50 µl of SDS-PAGE sample buffer, boiled, and centrifuged, and 20 µl of supernatant was analyzed by immunoblot analysis using the indicated antibody. The immunoprecipitation procedure for generating Fig. 4 is essentially as described (20).

To perform GST pull-down assay, ~50 µl of glutathione-agarose beads (Sigma) coated with the indicated protein was incubated with 200 µl of the cell or brain extract with end-over-end shaking for 14 h at 4 °C. After shaking, beads were washed three times with 50 mM Tris-HCl (pH 7.5), 0.5% Nonidet P-40, 1 mM EDTA, and 1 mM DTT. The washed beads were dissolved in 50 µl of SDS-PAGE sample buffer, boiled, and centrifuged, and 20 µl of the supernatant was analyzed by immunoblot analysis using the indicated antibody. To generate Fig. 7, the GST pull-down assay was carried out as described above, except the brain or cell extract was replaced by the tau sample (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.05% Tween 20, 0.3% bovine serum albumin, and 50 µg/ml tau).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Microtubule-associated 14-3-3zeta -- To examine microtubule-associated 14-3-3zeta , we purified microtubules from a fresh bovine brain extract using repeated cycles of temperature-induced microtubule assembly and disassembly. SDS-PAGE and an immunoblot analysis showed that microtubules were enriched during each cycle of assembly and disassembly (Fig. 1, A and B). An immunoblot analysis using an anti-14-3-3zeta antibody indicated that 14-3-3zeta was present in all the fractions in a manner similar to tubulin (Fig. 1C). By quantitating the intensities of various bands in Fig. 1, B and C, we determined that ~6.6, ~2.8, ~0.96, and ~0.3% of total 14-3-3zeta in brain extract remained associated with first (P1), second (P2), third (P3), and fourth (P4) microtubule pellets, respectively. The amount of tubulin was ~29.5, ~12.4, ~7.2, and ~3.0% of the total in P1, P2, P3, and P4, respectively (data not shown). More importantly, the ratio of the amount of 14-3-3zeta to the amount of tubulin in P1, P2, P3, and P4 was ~0.23, ~0.30, ~0.20, and ~0.16, respectively (Fig. 1D). Thus, a fraction of 14-3-3zeta remained stably associated with microtubules during purification in a manner similar to tubulin. These observations indicated that a significant amount of 14-3-3zeta is stably bound to microtubules in the brain.


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 1.   Co-purification of 14-3-3zeta with microtubules. Microtubules were purified from a fresh bovine brain extract (H) by repeated cycles of microtubule assembly and disassembly. Samples (10 ml each) were analyzed by SDS-PAGE or immunoblot (IB) analysis using the indicated antibodies. A, SDS-PAGE showing tubulin and other microtubule-associated proteins in indicated fractions. B and C, immunoblots; D, 14-3-3zeta /tubulin ratio. Blots B and C were scanned, and the band intensity values of 14-3-3zeta and tubulin in various fractions were obtained. The ratio for the indicated fraction was then determined by dividing the 14-3-3zeta band intensity value by the band intensity value of tubulin in that fraction. Values are the average of three independent determinations. P1, P2, P3, and P4 indicate pellets, whereas S1, S2, S3, and S4 indicate supernatants obtained after first, second, third, and fourth microtubule assembly/disassembly cycles, respectively.

Microtubule-associated 14-3-3zeta Is Part of a Large Molecular Complex-- To further characterize microtubule-associated 14-3-3zeta , we depolymerized P3 microtubules by cold incubation and then subjected them to a phosphocellulose chromatography. 14-3-3zeta was not recovered within the flow-through fractions and eluted from the column with an NaCl gradient along with the other microtubule-associated proteins (data not shown, but see "Materials and Methods"). We then combined the column fractions containing 14-3-3zeta and chromatographed through an FPLC Superose 12 gel filtration column. Most of 14-3-3zeta eluted within fractions 40-46 with a size of ~500-kDa (Fig. 2B). Since the size of dimeric 14-3-3zeta is ~60-kDa (21, 22), these data indicated that 14-3-3zeta is bound to another biological molecule within the brain microtubules.


View larger version (46K):
[in this window]
[in a new window]
 
Fig. 2.   FPLC gel filtration of microtubule-associated 14-3-3zeta . Microtubules purified by three cycles of assembly and disassembly were chromatographed through a phosphocellulose column. The effluent fractions containing 14-3-3zeta were then analyzed by an FPLC Superose 12 gel filtration column calibrated previously with the indicated molecular weight marker proteins. Fractions (1 ml each) were collected, and 20 µl from each indicated fraction was immunoblotted using the indicated antibody. A, gel filtration profile. BSA indicates bovine serum albumin. B, C, and D, immunoblots. IB indicates immunoblot.

Identification of Molecules Bound to 14-3-3zeta within Brain Microtubules-- A silver-stained SDS gel of various column fractions from Fig. 2A showed numerous protein bands of various sizes within fractions 40-46 (data not shown) and did not give us any indication as to the identification of the 14-3-3zeta -bound protein (s). In a previous study, we found that 14-3-3zeta is associated with tau in bovine brain extract and binds to tau in vitro (36). In a recent study, we showed that within brain microtubules, GSK3beta and tau are parts of a multiprotein complex that elutes from an FPLC gel filtration column used in this study to generate Fig. 2A with an ~400-500-kDa size (20). We noted a very similar gel filtration behavior between the high molecular size 14-3-3zeta present within fractions 40-46 (Fig. 2A) and the tau phosphorylation complex described by us in a previous study (20). We therefore analyzed various Fig. 2A column fractions for the presence of tau and GSK3beta . As shown in Fig. 2, C and D, tau and GSK3beta were indeed present within fractions 40-46, indicating that 14-3-3zeta has co-eluted with the tau phosphorylation complex from the gel filtration column. We pooled fractions 40-46 containing 14-3-3zeta and a portion of the pooled fraction chromatographed through an FPLC Mono S column. SDS-PAGE and immunoblot analyses of various effluent fractions indicated that 14-3-3zeta , tau, and GSK3beta had co-eluted from the column (data not shown). We then pooled column fractions containing 14-3-3zeta and chromatographed through a Sepharose 4B gel filtration column. Tau, GSK3beta , and 14-3-3zeta again co-eluted (data not shown).

An SDS-polyacrylamide gel of the peak Sepharose 4B column fraction containing tau, GSK3beta , and 14-3-3zeta showed at least 11 prominent protein bands that migrated with various sizes on the gel (Fig. 3A). To find out which of these bands may represent protein(s) bound to 14-3-3zeta in brain microtubules, we determined the intensity value of each prominent band on the gel and then calculated the molar ratio value (band intensity divided by molecular size) of each band (Fig. 3B). The ratios for the ~25-, ~35-, ~100-, ~150-, ~180-, and ~220-kDa bands were ~16, ~14, ~13, ~8, ~9, and ~4, respectively. The ratios for the ~30-, ~47-, ~50-, ~55-, and ~65-kDa bands were ~30, ~28, ~29, and ~28, respectively. Our immunoblot analysis indicated that the ~30-kDa band corresponds to 14-3-3zeta , the ~47-kDa band corresponds to GSK3beta , and the ~50-, ~55-, and ~65-kDa bands correspond to various tau isoforms. Thus, in the column fraction containing partially purified 14-3-3zeta , the molar ratios of ~14-3-3zeta , GSK3beta , and tau were similar to and higher than those of ~25-, ~35-, ~100-, ~150-, ~180-, and ~220-kDa proteins. These observations suggested that 14-3-3zeta may be bound to GSK3beta and/or tau within brain microtubules and may be a component of tau phosphorylation complex.


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 3.   SDS-PAGE of the fraction containing partially purified 14-3-3zeta . Fractions 40-46 from Fig. 2A were combined, and a portion of the combined fraction was chromatographed through an FPLC Mono S column followed by a Sepharose 4B gel filtration column. An aliquot (20 µl) from the peak effluent gel filtration column fraction containing 14-3-3zeta , tau, and GSK3beta was electrophoresed on a 10% SDS gel. The gel was stained with Coomassie Brilliant Blue for proteins and used to determine the molar ratios. A, protein-stained gel. B, molar ratio. The gel in panel A was scanned, and the band intensity values of various bands were obtained. The molar ratio for the indicated protein was then determined by dividing the band intensity value by the molecular weight of that protein.

To determine whether 14-3-3zeta is part of the tau phosphorylation complex, we immunoprecipitated 14-3-3zeta , tau, or GSK3beta from the rest of the above combined column fractions from Fig. 2A. Each resulting immune complex was then immunoblotted with anti-tau, anti-GSK3beta , or anti-14-3-3zeta antibody. Tau and GSK3beta co-immunoprecipitated with 14-3-3zeta (Fig. 4A). Similarly, GSK3beta and 14-3-3zeta co-immunoprecipitated with tau (Fig. 4B), and tau and 14-3-3zeta co-immunoprecipitated with GSK3beta (Fig. 4C). Thus, 14-3-3zeta , tau, and GSK3beta within Fig. 2A fractions 40-46 could not be separated from each other. Based on these data and our previous study (20), we concluded that 14-3-3zeta is very likely to be one of the components of tau phosphorylation complex.


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 4.   Co-immunoprecipitation. Combined fractions from Fig. 2A containing 14-3-3zeta (A), tau (B), and GSK3beta (C) were used for immunoprecipitation using the indicated antibodies. Each resulting immune complex was immunoblotted using the indicated antibody. Similar results were obtained in four different experiments. IP and IB indicate immunoprecipitation and immunoblot, respectively.

To gain more evidence in support of the above idea and to study the interactions of 14-3-3zeta , tau, and GSK3beta within the tau phosphorylation complex, we first asked whether or not 14-3-3zeta could bind to GSK3beta directly. When glutathione-agarose beads coated with GST-14-3-3zeta were incubated with a brain extract, GSK3beta specifically precipitated with the GST-14-3-3zeta beads (Fig. 5A). Although this observation indicated that 14-3-3zeta associates with GSK3beta in the brain extract, we could not rule out the possibility that tau, which can bind to both GSK3beta (20) and 14-3-3zeta in vitro (36), may have influenced observed 14-3-3zeta and GSK3beta association (Fig. 5A). Therefore, we performed a similar GST pull-down assay as described above by using COS-7 cells that express GSK3beta but not tau. As shown in Fig. 5B, GSK3beta again came down with GST-14-3-3zeta from the cell extract. To confirm that it was GSK3beta that came down with GST-14-3-3zeta and not any other protein of similar size that may be immunoreactive to our anti-GSK3beta antibody used to generate Fig. 5, A and B, we transfected HEK-293 cells with HA-GSK3beta . Transfected cells were lysed, and glutathione-agarose beads coated with GST-14-3-3zeta were incubated with the cell lysates. Incubated beads were washed and immunoblotted by using an anti-HA antibody to test 14-3-3zeta -GSK3beta binding. As expected, HA-GSK3beta bound to GST-14-3-3zeta (Fig. 5C). Based on these data, we concluded that 14-3-3zeta directly binds to GSK3beta .


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 5.   GST pull-down assay. Glutathione-agarose beads coated with GST-14-3-3zeta or GST were mixed with an extract from bovine brain, COS-7 cells, or HEK-293 cells transfected with HA-GSK3beta . Beads were washed and then immunoblotted with the indicated antibody to detect bead bound GSK3beta . These experiments were repeated three different times with similar results. IP and IB indicate immunoprecipitation and immunoblot, respectively. An aliquot (20 µl each) was used from extracts of brain, COS-7 cells, and HEK-293 cells transfected with HA-GSK3beta as control in A, B, and C, respectively.

There are three possible mechanisms by which tau, GSK3beta , and 14-3-3zeta can interact within the tau phosphorylation complex. First, because in vitro tau binds to GSK3beta (20) as well as 14-3-3zeta (36) and the respective binding sites do not overlap, tau may bridge GSK3beta and 14-3-3zeta within the complex. Second, 14-3-3zeta is a scaffold protein that can bind two ligands at a same time (21, 22), and it binds tau (36) and GSK3beta (Fig. 5). Therefore, 14-3-3zeta may anchor GSK3beta to tau within the phosphorylation complex. Third, GSK3beta can bind tau in vitro (20) and can bind 14-3-3zeta in vitro (Fig. 5). Thus, GSK3beta may be the central molecule that may hold 14-3-3zeta and tau simultaneously within the complex.

To discriminate between the above possibilities, we transfected HEK-293 cells with FLAG-tau, Xpress-14-3-3zeta , and HA-GSK3beta constructs in various combinations. Transfected cells were lysed, and GSK3beta was immunoprecipitated from each lysate using an anti-HA antibody. Each immune complex was then immunoblotted with anti-FLAG antibody to detect tau. FLAG-tau did not co-immunoprecipitate from cells overexpressing HA-GSK3beta and FLAG-tau (Fig. 6A, lane 6), indicating that GSK3beta does not bind to tau directly in vivo. This means that neither can tau bridge GSK3beta to 14-3-3zeta nor can GSK3beta simultaneously bind to tau and 14-3-3zeta within the tau phosphorylation complex. Therefore, 14-3-3 must be the central molecule that holds tau and GSK3beta within the complex. Indeed, FLAG-tau co-immunoprecipitated with HA-GSK3beta from cells overexpressing FLAG-tau and HA-GSK3beta only when these cells also overexpressed Xpress-14-3-3zeta (Fig. 6A, lanes 8 and 9), indicating that GSK3beta associates with tau only in the presence of 14-3-3zeta . As discussed above, 14-3-3zeta binds to tau (36) and GSK3beta (Fig. 5) directly. Taken together, these observations indicated that 14-3-3zeta connects GSK3beta to tau in vivo.


View larger version (48K):
[in this window]
[in a new window]
 
Fig. 6.   Co-immunoprecipitation of tau with GSK3beta in the presence and absence of 14-3-3zeta . HEK-293 cells transfected with the indicated constructs were lysed, and the cell lysates were either used for immunoprecipitation using anti-HA antibody or used for immunoblotting using the indicated antibody. A, immunoprecipitation. The resulting anti-HA immune complex was immunoblotted with anti-FLAG antibody to detect FLAG-tau. B-D, immunoblots. An aliquot (20 µl) from each cell lysate was immunoblotted with the indicated antibody to monitor the expressions of indicated gene. Lane 2 represents mock-transfected cells. IP and IB indicate immunoprecipitation and immunoblot, respectively. Similar results were obtained in three different experiments.

To further confirm the above finding, we performed an in vitro GST pull-down assay. Glutathione-agarose beads coated with GST-GSK3beta were mixed with bacterially expressed recombinant tau in the presence of a series of 14-3-3zeta concentrations. Beads were washed, and bead-bound tau was detected by immunoblot analysis using an anti-tau antibody. Comparatively very little tau bound to beads when GST-GSK3beta was incubated with tau alone (Fig. 7, lane 3). However, when an increasing amount of 14-3-3zeta was included in the assay mixture, the amount of tau binding to GST-GSK3beta increased progressively (Fig. 7, lanes 4-8). When the amount of 14-3-3zeta was 100 µg/ml in the assay mixture, ~10-fold more tau bound to GST-GSK3beta than in the absence of 14-3-3zeta (compare lanes 3 and 7). Based on these data, we concluded that 14-3-3zeta promotes in vitro GSK3beta -tau binding and is required for a stable association of tau and GSK3beta in vivo.


View larger version (41K):
[in this window]
[in a new window]
 
Fig. 7.   Binding of GSK3beta with tau in the presence of 14-3-3zeta . Glutathione-agarose beads coated with GST-GSK3beta or GST were incubated with tau solution in the presence of indicated amounts of 14-3-3zeta . After incubation, beads were washed and immunoblotted with anti-tau antibody. This experiment was repeated three times with similar results. IB indicates immunoblot.

14-3-3zeta Stimulates GSK3beta -catalyzed Tau Phosphorylation-- GSK3beta is one of the kinases implicated to phosphorylate tau in vivo (10-20). Since we find that 14-3-3zeta is required for a stable association between GSK3beta and tau, we examined the influence of 14-3-3zeta on GSK3beta -catalyzed tau phosphorylation in vivo. We transfected HEK-293 cells in various combinations with FLAG-tau, Xpress-14-3-3zeta , and HA-GSK3beta constructs. Transfected cells were lysed, and the cell lysates were analyzed for tau phosphorylation using various tau phosphorylation-sensitive antibodies: AT8, PHF1, and 12E8, which recognize tau phosphorylated on Ser199/Ser202, Ser396/Ser404, and Ser262, respectively (20, 36). As shown in Fig. 8, A-C, tau was slightly phosphorylated in cells transfected with FLAG-tau alone (lane 3). This phosphorylation increased in cells co-transfected with FLAG-tau and HA-GSK3beta as expected (lane 5). In cells that were co-transfected with fixed amounts of FLAG-tau and HA-GSK3beta but different amounts of Xpress-14-3-3zeta , FLAG-tau phosphorylation increased progressively with the increase in the amount of Xpress-14-3-3zeta (lanes 7-9). This increase was evident not only by an increased immunoreactivity against all tau phosphorylation-sensitive antibodies tested but also by a retarded mobility of FLAG-tau on the SDS gel, a characteristic feature of hyperphosphorylated tau (2, 3). Thus, 14-3-3zeta profoundly stimulated GSK3beta -catalyzed tau phosphorylation in vivo.


View larger version (61K):
[in this window]
[in a new window]
 
Fig. 8.   Effect of 14-3-3zeta on GSK3beta -catalyzed tau phosphorylation in vivo. HEK-293 cells transfected with the indicated constructs were lysed, and 20 µg protein from each lysate was immunoblotted using the indicated antibody. A-C, immunoblot analysis using tau phosphorylation-sensitive monoclonal antibodies, AT8, PHF1, and 12E8, which only cross-react with phosphorylated tau. D-F, immunoblots to show expression levels of FLAG-tau, HA-GSK3beta , and Xpress-14-3-3zeta . Lane 1 represents mock-transfected cells. Similar results were obtained in three different experiments. IB indicates immunoblot.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Recently, we reported the existence of a tau phosphorylation complex containing GSK3beta and tau within brain microtubules (20). Since the observed size of the complex is ~400-500 kDa and the sum of the molecular sizes of tau and GSK3beta is ~97 kDa, we suggested that other proteins may also be present within the complex (20). In this study, we find that microtubule-associated 14-3-3zeta co-elutes with the tau phosphorylation complex from a gel filtration column, indicating that the size of high molecular size microtubule-associated 14-3-3zeta is the same as that of tau phosphorylation complex (Fig. 2). 14-3-3zeta and the tau phosphorylation complex in the microtubule fraction cannot be separated from each other by phosphocellulose, gel filtration, and Mono S chromatographies. Tau, 14-3-3zeta , and GSK3beta co-immunoprecipitate with each other from column fractions containing the phosphorylation complex (Fig. 4). In vitro, 14-3-3zeta binds to tau (36) and GSK3beta (Fig. 5). These and other data presented in this study indicate that 14-3-3zeta is also a part of the microtubule-associated tau phosphorylation complex.

Tau and GSK3beta co-immunoprecipitate with each other from brain extracts (Fig. 4) (20). In contrast, tau does not co-immunoprecipitate with GSK3beta from HEK-293 cell extracts co-transfected with GSK3beta and tau (Fig. 6A, lane 6). These data indicate that in HEK-293 cells, the interaction of tau with GSK3beta is weak, whereas in the brain, GSK3beta stably associates with tau. This in turn suggests that brain contains a factor required for a stable association of GSK3beta with tau, and this factor may be missing in HEK-293 cells.

Our gel filtration data (Fig. 2) and co-immunoprecipitation analysis (Fig. 4) indicate that within the tau phosphorylation complex, tau, GSK3beta , and 14-3-3zeta are inseparable. Moreover, in HEK-293 cells, tau associates with GSK3beta only in the presence of 14-3-3zeta (Fig. 6). In vitro, ~10-fold more tau binds to GSK3beta in the presence than in the absence of 14-3-3zeta (Fig. 7). Since 14-3-3zeta can bind to tau (36) and GSK3beta (Fig. 5) independently, these data indicate that 14-3-3zeta is the factor that connects and mediates the association of GSK3beta with tau within the brain. However, as discussed above, tau does not associate with GSK3beta in HEK-293 cells transfected with only tau and GSK3beta , although 14-3-3 is known to be widely expressed in various cell lines including HEK-293 cells (21). It is possible that within HEK-293 cells, the endogenous 14-3-3zeta either is not sufficient or is not available to mediate the interaction of transfected GSK3beta and tau.

In a previous study, we reported that in vitro GSK3beta binds to the N-terminal region of tau (20). Consistent with that report, we find that tau comes down with GST-GSK3beta in a GST pull-down assay (Fig. 7, lane 3). However, tau does not co-immunoprecipitate with GSK3beta from lysates of HEK-293 cells co-transfected with GSK3beta and tau (Fig. 6A, lane 6). These observations suggest that in the absence of 14-3-3zeta , GSK3beta binds to tau with a low affinity. It thus appears that in the brain, GSK3beta interacts with tau in two different ways: one with low affinity that does not require 14-3-3zeta and the other with high affinity that requires 14-3-3zeta .

The substrate recognition by GSK3 is regulated by two mechanisms. The first mechanism requires a priming phosphorylation of the substrate (41, 42). For example, casein kinase 2 phosphorylates glycogen synthase first and generates a recognition motif for GSK3. GSK3 then phosphorylates casein kinase 2-phosphoprylated glycogen synthase (42). The second mechanism does not require priming phosphorylation. Instead, a scaffold protein bridges GSK3beta to its substrate within a multiprotein complex (42). In the Wnt signaling pathway, GSK3beta phosphorylates beta -catenin within a beta -catenin destruction complex. beta -catenin alone is not a good substrate of GSK3beta . The scaffold protein axin connects GSK3beta to beta -catenin and facilitates beta -catenin phosphorylation by GSK3beta within the complex (6, 42-44).

Biochemical analyses and studies involving transgenic mice and cultured mammalian cells have established that GSK3beta phosphorylates tau in the brain (10-20). The mechanism by which GSK3beta phosphorylates tau is not clear. Our recent study (20) and the results presented in this study indicate that GSK3beta , tau, and 14-3-3zeta are parts of a microtubule-associated tau phosphorylation complex. Within the complex, 14-3-3zeta binds to tau and GSK3beta simultaneously and assembles the complex. Thus, the role of 14-3-3zeta within the tau phosphorylation complex appears to be similar to that of axin within the beta -catenin destruction complex. Furthermore, 14-3-3zeta binds to tau and changes the tau conformation, making tau susceptible for hyperphosphorylation in vitro (36) and perhaps in vivo (Fig. 8). Since 14-3-3zeta stimulates tau phosphorylation on Ser199, Ser198 Ser202, Ser262, Ser396, and Ser404 (Fig. 8), it appears that 14-3-3zeta -induced conformational change occurs within a large part of the C-terminal tau region, which is the main area of in vivo phosphorylation (45). These observations suggest that 14-3-3zeta not only enhances association of tau and GSK3beta within the complex but also prepares tau for GSK3beta action.

We have found a unique multiprotein complex containing tau, GSK3beta , and 14-3-3zeta within brain microtubules. Thus, a pool of GSK3beta in the brain is targeted to microtubules through a stable association with tau and 14-3-3zeta . Because the function of this complex is to regulate tau phosphorylation and microtubule dynamics, we named this complex the tau phosphorylation complex. It should be noted that the size of the phosphorylation complex is 400-500-kDa, whereas the sum of the sizes of tau, GSK3beta , and 14-3-3zeta dimer is ~167-kDa. Therefore, it is possible that there may be proteins other than, tau, GSK3beta , and 14-3-3zeta within the tau phosphorylation complex. These proteins may play important roles in regulating tau phosphorylation and interactions between various phosphorylation complex components. Studies are ongoing in our laboratory to identify all of the phosphorylation complex components.

    ACKNOWLEDGEMENTS

We express our sincere appreciation to Dr. J. R. Woodgett for the gift of human brain GSK3beta cDNA. We also thank Dr. Peter Davies and Ruth Motter for gifts of the PHF-1 and 12E8 antibodies, respectively.

    FOOTNOTES

* This work was supported by grants from the Canadian Institute of Health Research, the American Health Assistance Foundation (U. S. A.) and the Alzheimer Society of Canada (to H. K. P.).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.

|| To whom correspondence should be addressed: Bloomfield Center for Research in Aging, Lady Davis Institute for Medical Research, 3755 Cote Ste-Catherine, Montreal, Quebec H3T 1E2 Canada. Tel.: 514-340-8222, Ext. 4866; Fax: 514-340-8295; E-mail: hemant.paudel@mcgill.ca.

Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M211491200

    ABBREVIATIONS

The abbreviations used are: AD, Alzheimer's disease; FPLC, fast protein liquid chromatography; GSK3, glycogen synthase kinase-3; GST, glutathione S-transferase; MOPS, 4-morpholinepropanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid; DTT, dithiothreitol; HA, hemagglutinin; P, pellet; S, supernatant.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Hirokawa, N. (1994) Curr. Opin. Cell Biol. 6, 71-84
2. Lee, V. M.-Y., Goedert, M., and Trojanowski, J. Q. (2000) Annu. Rev. Neurosci. 24, 1121-1159[CrossRef]
3. Goedert, M., Crowther, R. A., and Garner, C. C. (1991) Trends Neurosci. 14, 193-199[CrossRef][Medline] [Order article via Infotrieve]
4. Morishima-Kawashima, M., Hasegawa, M., Takio, K., Suzuki, M., Yoshida, H., Titani, K., and Ihara, Y. (1995) J. Biol. Chem. 270, 823-829[Abstract/Free Full Text]
5. Lee, V. M.-Y., Balin, B. J., Otvos, L., and Trojanowski, J. Q. (1991) Science 251, 675-678[Medline] [Order article via Infotrieve]
6. Kim, L., and Kimmel, A. R. (2000) Curr. Opin. Genet. Dev. 10, 508-514[CrossRef][Medline] [Order article via Infotrieve]
7. Grimes, C. A., and Jope, R. S. (2001) Progress Neurobiol. (N. Y.) 65, 391-426
8. Planel, E., Sun, X., and Takashima, A. (2002) Drug Dev. Res. 56, 491-510[CrossRef]
9. Woodgett, J. R. (1990) EMBO J. 9, 2431-2438[Abstract]
10. Wagner, U., Utton, M., Gallo, J. M., and Miller, C. J. (1996) J. Cell Sci. 109, 1537-1543[Abstract/Free Full Text]
11. Lovestone, S., Hartley, C. L., Pearce, J., and Anderton, B. H. (1996) Neuroscience 73, 1145-1157[CrossRef][Medline] [Order article via Infotrieve]
12. Lucas, J. J., Hernandez, F., Gomez-Ramos, P., Moran, M. A., Hen, R., and Avila, J. (2001) EMBO J. 20, 27-39[Abstract/Free Full Text]
13. Spittaels, K., Van den Haute, C., Van Dorpe, J., Geerts, H., Mercken, M., Bruynseels, K., Lasrado, R., Vandezaride, K., Laenen, I., Boon, T., Van Lint, J., Vandenheede, J., Moechars, D., Loos, R., and Van Leuven, F. (2000) J. Biol. Chem. 275, 41340-41349[Abstract/Free Full Text]
14. Hong, M., Chen, D. C. R., Klein, P. S., and Lee, V. M.-Y. (1997) J. Biol. Chem. 272, 25326-25332[Abstract/Free Full Text]
15. Monz-Montano, J. R., Moreno, F. J., Avila, J., and Diaz-Nido, J. (1997) FEBS Lett. 411, 183-188[CrossRef][Medline] [Order article via Infotrieve]
16. Shiruba, R. A., Ishiguro, K., Takahashi, M., Sato, K., Spooner, E. T., Mereken, M., Yoshida, R., Wheellock, T. R., Yanagawa, H., Imahori, K., and Nixon, R. A. (1998) Brain Res. 737, 119-132[CrossRef]
17. Pei, J. J., Braak, E., Braak, H., Grundke-Iqbal, I., Iqbal, K., Winblad, B., and Cowburn, R. F. (1999) J. Neuropathol. Exp. Neurol. 56, 70-78
18. Ishiguro, K., Takamatsu, M., Tomizawa, K., Omori, A., Takahashi, M., Arioka, M., Uchida, T., and Imahori, K. (1992) J. Biol. Chem. 267, 10897-10901[Abstract/Free Full Text]
19. Mandelkow, E.-M., Drewes, G., Biernat, J., Gustke, N., Lint, J. V., Vandenheede, J. R., and Mandelkow, E. (1992) FEBS Lett. 314, 315-321[CrossRef][Medline] [Order article via Infotrieve]
20. Sun, W., Qureshi, H. Y., Cafferty, P. W., Sobue, K., Agarwal-Mawal, A., Neufield, K. D., and Paudel, H. K. (2002) J. Biol. Chem. 277, 11933-11940[Abstract/Free Full Text]
21. Fu, H., Subramanian, R. R., and Masters, S. C. (2000) Annu. Rev. Pharmacol. Toxicol. 40, 617-647[CrossRef][Medline] [Order article via Infotrieve]
22. Van Hemert, M. J., Yde Steensma, H., and van Heusden, P. H. (2001) Bioessays 23, 936-946[CrossRef][Medline] [Order article via Infotrieve]
23. Zhang, L., Wang, H., Masters, S. C., Wang, B., Barbieri, J. T., and Fu, H. (1999) Biochemistry 38, 12159-12164[CrossRef][Medline] [Order article via Infotrieve]
24. Tzivion, G., Luo, Z., and Avruch, J. (1998) Nature 394, 88-92[CrossRef][Medline] [Order article via Infotrieve]
25. Fantl, W. J., Muslin, A. J., Kikuchi, A., Martin, J. A., MacNicol, A. M., Gross, R. W., and Williams, L. T. (1994) Nature 371, 612-624[CrossRef][Medline] [Order article via Infotrieve]
26. Petosa, C., Masters, S. C., Bankston, L. A., Pohl, J., Wang, B., Fu, H., and Liddington, R. C. (1998) J. Biol. Chem. 273, 16305-16310[Abstract/Free Full Text]
27. Toker, A., Ellis, C. A., Sellers, L. A., and Aitken, A. (1990) Eur. J. Biochem. 191, 421-429[Abstract]
28. Pallas, D. C., Fu, H., Haehnel, L. C., Weller, W., Collier, R. J., and Roberts, T. M. (1994) Science 265, 535-537[Medline] [Order article via Infotrieve]
29. Reuther, G. W., Fu, H., Cripe, L. D., Collier, R. J., and Pendergast, A. M. (1994) Science 266, 129-133[Medline] [Order article via Infotrieve]
30. Lin, Y. C., Elly, C., Yoshida, H., Bonnefoy-Berard, N., and Attman, A. (1996) J. Biol. Chem. 271, 14591-14595[Abstract/Free Full Text]
31. Conklin, D. S., Galaktionov, K., and Beach, D. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7892-7896[Abstract]
32. Bonnefoy-Berard, N., Liu, Y. C., Von Willebrand, M., Sung, A., Elly, C., Mustelin, T., Yoshida, H., Ishizaka, K., and Atman, A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 421-429
33. Peng, C. Y., Graves, P. R., Thoma, R. S., Wu, Z., Shaw, A. S., and Oiwnicworms, H. (1997) Science 277, 1501-1505[Abstract/Free Full Text]
34. Craparo, A., Freund, R., and Gustafson, T. A. (1997) J. Biol. Chem. 272, 11663-11669[Abstract/Free Full Text]
35. Zha, J., Harda, H., Yang, E., Jockel, J., and Korsmeyer, S. T. (1996) Cell 87, 619-628[Medline] [Order article via Infotrieve]
36. Hashiguchi, M., Sobue, K., and Paudel, H. K. (2000) J. Biol. Chem. 275, 25247-25254[Abstract/Free Full Text]
37. Tzivion, G., Luo, Z.-J., and Avruch, J. (2000) J. Biol. Chem. 275, 29772-29778[Abstract/Free Full Text]
38. Sobue, K., Agarwal-Mawal, A., Li, W., Sun, W., Miura, Y, and Paudel, H. K. (2000) J. Biol. Chem. 275, 16673-16680[Abstract/Free Full Text]
39. Paudel, H. K. (1997) J. Biol. Chem. 272, 28328-28334[Abstract/Free Full Text]
40. Hung, K., and Paudel, H. K. (2000) Proc. Natl. Acad. Sci. (U. S. A.) 97, 5824-5829[Abstract/Free Full Text]
41. Dajani, R., Fraser, E., Roe, S. M., Young, N., Good, V., Dale, T. C., and Pearl, L. H. (2001) Cell 105, 721-732[CrossRef][Medline] [Order article via Infotrieve]
42. Frame, S., Cohen, P., and Biondi, R. M. (2001) Cell 7, 1321-1327[CrossRef]
43. Ikeda, S., Kishida, S., Yamamoto, H., Koyama, S., and Kikuchi, A. (1998) EMBO J. 17, 1371-1384[Abstract/Free Full Text]
44. Ikeda, S., Kishida, S., Matsuura, Y., Usui, H., and Kikuchi, A. (2000) Oncogene 19, 537-543[CrossRef][Medline] [Order article via Infotrieve]
45. Watanabe, A., Hasegawa, M., Suzuki, M., Takio, K., Morishima-Kawashima, M., Titani, K., Arai, T., Kosik, K. S., and Ihara, Y. (1993) J. Biol. Chem. 268, 25712-25717[Abstract/Free Full Text]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.