14-3-3 Connects Glycogen Synthase Kinase-3β to Tau within a Brain Microtubule-associated Tau Phosphorylation Complex
2003; Elsevier BV; Volume: 278; Issue: 15 Linguagem: Inglês
10.1074/jbc.m211491200
ISSN1083-351X
AutoresAlka Agarwal-Mawal, Hamid Y. Qureshi, Patrick Cafferty, Zongfei Yuan, Dong Cho Han, Rongtian Lin, Hemant K. Paudel,
Tópico(s)Macrophage Migration Inhibitory Factor
ResumoIn a recent study, we reported that in bovine brain extract, glycogen synthase kinase-3β 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 14-3-3ζ co-elutes with the tau phosphorylation complex components tau and GSK3β. From gel filtration fractions containing the tau phosphorylation complex, 14-3-3ζ, GSK3β, and tau co-immunoprecipitate with each other. From extracts of bovine brain, COS-7 cells, and HEK-293 cells transfected with GSK3β, 14-3-3ζ co-precipitates with GSK3β, indicating that GSK3β binds to 14-3-3ζ. From HEK-293 cells transfected with tau, GSK3β, and 14-3-3ζ in different combinations, tau co-immunoprecipitates with GSK3β only in the presence of 14-3-3ζ. In vitro, ∼10-fold more tau binds to GSK3β in the presence of than in the absence of 14-3-3ζ. In transfected HEK-293 cells, 14-3-3ζ stimulates GSK3β-catalyzed tau phosphorylation in a dose-dependent manner. These data indicate that in brain, the 14-3-3ζ dimer simultaneously binds and bridges tau and GSK3β and stimulates GSK3β-catalyzed tau phosphorylation. In a recent study, we reported that in bovine brain extract, glycogen synthase kinase-3β 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 14-3-3ζ co-elutes with the tau phosphorylation complex components tau and GSK3β. From gel filtration fractions containing the tau phosphorylation complex, 14-3-3ζ, GSK3β, and tau co-immunoprecipitate with each other. From extracts of bovine brain, COS-7 cells, and HEK-293 cells transfected with GSK3β, 14-3-3ζ co-precipitates with GSK3β, indicating that GSK3β binds to 14-3-3ζ. From HEK-293 cells transfected with tau, GSK3β, and 14-3-3ζ in different combinations, tau co-immunoprecipitates with GSK3β only in the presence of 14-3-3ζ. In vitro, ∼10-fold more tau binds to GSK3β in the presence of than in the absence of 14-3-3ζ. In transfected HEK-293 cells, 14-3-3ζ stimulates GSK3β-catalyzed tau phosphorylation in a dose-dependent manner. These data indicate that in brain, the 14-3-3ζ dimer simultaneously binds and bridges tau and GSK3β and stimulates GSK3β-catalyzed tau phosphorylation. Alzheimer's disease fast protein liquid chromatography glycogen synthase kinase-3 glutathione S-transferase 4-morpholinepropanesulfonic acid 1,4-piperazinediethanesulfonic acid dithiothreitol hemagglutinin pellet supernatant Microtubules, the major cytoskeletal structures of eukaryotic cells, are dynamic structures, and their assembly and disassembly is regulated by microtubule-associated proteins (1Hirokawa N. Curr. Opin. Cell Biol. 1994; 6: 71-84Crossref Scopus (350) Google Scholar). In neurons, tau is one of the major microtubule-associated proteins and is mainly found in the axonal compartment (for reviews, see Refs. 1Hirokawa N. Curr. Opin. Cell Biol. 1994; 6: 71-84Crossref Scopus (350) Google Scholar, 2Lee V.M.-Y. Goedert M. Trojanowski J.Q. Annu. Rev. 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These isoforms are phosphorylated on multiple sites in the brain and display a characteristic retarded mobility on an SDS gel upon phosphorylation (2Lee V.M.-Y. Goedert M. Trojanowski J.Q. Annu. Rev. Neurosci. 2000; 24: 1121-1159Crossref Scopus (2162) Google Scholar, 3Goedert M. Crowther R.A. Garner C.C. Trends Neurosci. 1991; 14: 193-199Abstract Full Text PDF PubMed Scopus (257) Google Scholar). Tau phosphorylation reduces the affinity of tau for microtubules and is one of the mechanisms that control microtubule structure and dynamics in vivo (1Hirokawa N. Curr. Opin. Cell Biol. 1994; 6: 71-84Crossref Scopus (350) Google Scholar, 2Lee V.M.-Y. Goedert M. Trojanowski J.Q. Annu. Rev. Neurosci. 2000; 24: 1121-1159Crossref Scopus (2162) Google Scholar, 3Goedert M. Crowther R.A. Garner C.C. Trends Neurosci. 1991; 14: 193-199Abstract Full Text PDF PubMed Scopus (257) Google Scholar). In Alzheimer's disease (AD)1brain, abnormally hyperphosphorylated tau accumulates and forms paired helical filaments (4Morishima-Kawashima M. Hasegawa M. Takio K. Suzuki M. Yoshida H. Titani K. Ihara Y. J. Biol. Chem. 1995; 270: 823-829Abstract Full Text Full Text PDF PubMed Scopus (529) Google Scholar, 5Lee V.M.-Y. Balin B.J. Otvos L. Trojanowski J.Q. Science. 1991; 251: 675-678Crossref PubMed Scopus (1253) Google Scholar). 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 (2Lee V.M.-Y. Goedert M. Trojanowski J.Q. Annu. Rev. Neurosci. 2000; 24: 1121-1159Crossref Scopus (2162) Google Scholar, 3Goedert M. Crowther R.A. Garner C.C. Trends Neurosci. 1991; 14: 193-199Abstract Full Text PDF PubMed Scopus (257) Google Scholar). 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 (6Kim L. Kimmel A.R. Curr. Opin. Genet. Dev. 2000; 10: 508-514Crossref PubMed Scopus (211) Google Scholar, 7Grimes C.A. Jope R.S. Progress Neurobiol. (N. Y.). 2001; 65: 391-426Crossref PubMed Scopus (1319) Google Scholar, 8Planel E. Sun X. Takashima A. Drug Dev. 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These data indicate that GSK3β 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 GSK3β (20Sun W. Qureshi H.Y. Cafferty P.W. Sobue K. Agarwal-Mawal A. Neufield K.D. Paudel H.K. J. Biol. Chem. 2002; 277: 11933-11940Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). The identification of all the complex components and the determination of their functions within the complex are essential to understanding the mechanism by which GSK3β phosphorylates tau in the brain. 14-3-3 is a family of conserved acidic proteins that are widely expressed in all eukaryotic tissues (21Fu H. Subramanian R.R. Masters S.C. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 617-647Crossref PubMed Scopus (1334) Google Scholar, 22Van Hemert M.J. Yde Steensma H. van Heusden P.H. 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Harda H. Yang E. Jockel J. Korsmeyer S.T. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2257) Google Scholar, 36Hashiguchi M. Sobue K. Paudel H.K. J. Biol. Chem. 2000; 275: 25247-25254Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 37Tzivion G. Luo Z.-J. Avruch J. J. Biol. Chem. 2000; 275: 29772-29778Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). By binding to its targets, 14-3-3 regulates enzyme activity, stabilizes enzyme conformation, controls subcellular localization of proteins, and mediates protein-protein interaction (21Fu H. Subramanian R.R. Masters S.C. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 617-647Crossref PubMed Scopus (1334) Google Scholar, 22Van Hemert M.J. Yde Steensma H. van Heusden P.H. Bioessays. 2001; 23: 936-946Crossref PubMed Scopus (473) Google Scholar). 14-3-3 regulates diverse cellular processes including cell growth, cell differentiation, cell division, apoptosis, and neuronal function (21Fu H. Subramanian R.R. Masters S.C. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 617-647Crossref PubMed Scopus (1334) Google Scholar, 22Van Hemert M.J. Yde Steensma H. van Heusden P.H. Bioessays. 2001; 23: 936-946Crossref PubMed Scopus (473) Google Scholar). In the brain, ∼1% of soluble protein is 14-3-3 and has been suggested to be critical for brain function (21Fu H. Subramanian R.R. Masters S.C. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 617-647Crossref PubMed Scopus (1334) Google Scholar). From bovine brain extract, 14-3-3ζ co-immunoprecipitates with tau (36Hashiguchi M. Sobue K. Paudel H.K. J. Biol. Chem. 2000; 275: 25247-25254Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). In vitro, 14-3-3ζ binds and changes the tau conformation, thus making tau susceptible for kinase phosphorylation (36Hashiguchi M. Sobue K. Paudel H.K. J. Biol. Chem. 2000; 275: 25247-25254Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). More importantly, a substantial amount of 14-3-3ζ co-purifies with microtubules from the brain extract (36Hashiguchi M. Sobue K. Paudel H.K. J. Biol. Chem. 2000; 275: 25247-25254Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). These observations suggest that 14-3-3ζ 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 14-3-3ζ. In this study, we have further analyzed microtubule-associated 14-3-3ζ. Herein we report that brain microtubule-associated 14-3-3ζ is part of the tau phosphorylation complex containing GSK3β and tau. Our data indicate that 14-3-3ζ mediates GSK3β-tau interaction and facilitates tau phosphorylation by GSK3β within the complex. The longest human tau isoform in a pET-3a vector (38Sobue K. Agarwal-Mawal A. Li W. Sun W. Miura Y Paudel H.K. J. Biol. Chem. 2000; 275: 16673-16680Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) 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 (20Sun W. Qureshi H.Y. Cafferty P.W. Sobue K. Agarwal-Mawal A. Neufield K.D. Paudel H.K. J. Biol. Chem. 2002; 277: 11933-11940Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). After amplification, the insert was released and ligated into the EcoR1/BamHI cloning site of FLAG-pcDNA3.1 Zeo vector (Invitrogen, Madison, WI). Human 14-3-3ζ cDNA was subcloned into theBamH1/EcoR1 site of Xpress-pcDNA3.1 (Invitrogen) as described above using 14-3-3ζ-pGEX-6p (36Hashiguchi M. Sobue K. Paudel H.K. J. Biol. Chem. 2000; 275: 25247-25254Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar) 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 GSK3β was a gift from Dr. James R. Woodgett (The University of Toronto). Other expression vectors, GSK3β-pGEX-6p, 14-3-3ζ-pGEX-6p, and tau-pET-3a, are described previously (20Sun W. Qureshi H.Y. Cafferty P.W. Sobue K. Agarwal-Mawal A. Neufield K.D. Paudel H.K. J. Biol. Chem. 2002; 277: 11933-11940Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 36Hashiguchi M. Sobue K. Paudel H.K. J. Biol. Chem. 2000; 275: 25247-25254Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 38Sobue K. Agarwal-Mawal A. Li W. Sun W. Miura Y Paudel H.K. J. Biol. Chem. 2000; 275: 16673-16680Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). 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 (2.5m) 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. Recombinant tau was purified from bacterial extract overexpressing the longest human tau isoform (39Paudel H.K. J. Biol. Chem. 1997; 272: 28328-28334Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). GST-14-3-3ζ and GST-GSK3β were purified from the respective bacterial lysates overexpressing the respective proteins by glutathione-agarose chromatography, and the GST tag was removed as described previously (20Sun W. Qureshi H.Y. Cafferty P.W. Sobue K. Agarwal-Mawal A. Neufield K.D. Paudel H.K. J. Biol. Chem. 2002; 277: 11933-11940Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 40Hung K. Paudel H.K. Proc. Natl. Acad. Sci. (U. S. A.). 2000; 97: 5824-5829Crossref PubMed Scopus (46) Google Scholar). Polyclonal antibodies against tau, GSK3β, and 14-3-3ζ have been described (20Sun W. Qureshi H.Y. Cafferty P.W. Sobue K. Agarwal-Mawal A. Neufield K.D. Paudel H.K. J. Biol. Chem. 2002; 277: 11933-11940Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 36Hashiguchi M. Sobue K. Paudel H.K. J. Biol. Chem. 2000; 275: 25247-25254Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Monoclonal antibodies against tau and GSK3β 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 (20Sun W. Qureshi H.Y. Cafferty P.W. Sobue K. Agarwal-Mawal A. Neufield K.D. Paudel H.K. J. Biol. Chem. 2002; 277: 11933-11940Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 36Hashiguchi M. Sobue K. Paudel H.K. J. Biol. Chem. 2000; 275: 25247-25254Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). GSK3β activity assay was performed essentially as described (20Sun W. Qureshi H.Y. Cafferty P.W. Sobue K. Agarwal-Mawal A. Neufield K.D. Paudel H.K. J. Biol. Chem. 2002; 277: 11933-11940Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Purification of microtubules from a fresh bovine brain extract by the temperature-induced microtubule assembly/disassembly has been described previously (38Sobue K. Agarwal-Mawal A. Li W. Sun W. Miura Y Paudel H.K. J. Biol. Chem. 2000; 275: 16673-16680Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). 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 14-3-3ζ, 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 14-3-3ζ was eluted with 200 ml of NaCl gradient (0–1 m in PEM buffer). Effluent fractions were immunoblotted using anti-14-3-3ζ antibody, and those containing 14-3-3ζ were combined and dialyzed against Mops buffer (25 mm MOPS (pH 7.4), 50 mm β-glycerol phosphate, 0.1 mm EDTA, 1 mm DTT, 0.2m NaCl, 10 mm NaF, and 15 mmMgCl2) 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 14-3-3ζ 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 14-3-3ζ 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. Cells in each culture dish were suspended in 1 ml of lysis buffer (50 mmTris-HCl (pH 7.5), 150 mm NaCl, 25 mmβ-glycerol phosphate, 1 mm EDTA, 1 mm EGTA, 10 mm NaF, 10 mm MgCl2, 1% Nonidet P-40, 100 nm okadaic acid (Sigma), 50 pmcypermethrane (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 (20Sun W. Qureshi H.Y. Cafferty P.W. Sobue K. Agarwal-Mawal A. Neufield K.D. Paudel H.K. J. Biol. Chem. 2002; 277: 11933-11940Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). 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 mmEDTA, 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 mmNaCl, 1 mm EDTA, 1 mm DTT, 0.05% Tween 20, 0.3% bovine serum albumin, and 50 μg/ml tau). To examine microtubule-associated 14-3-3ζ, 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-14-3-3ζ antibody indicated that 14-3-3ζ was present in all the fractions in a manner similar to tubulin (Fig. 1C). By quantitating the intensities of various bands in Fig. 1, Band C, we determined that ∼6.6, ∼2.8, ∼0.96, and ∼0.3% of total 14-3-3ζ 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 14-3-3ζ 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 14-3-3ζ remained stably associated with microtubules during purification in a manner similar to tubulin. These observations indicated that a significant amount of 14-3-3ζ is stably bound to microtubules in the brain. To further characterize microtubule-associated 14-3-3ζ, we depolymerized P3 microtubules by cold incubation and then subjected them to a phosphocellulose chromatography. 14-3-3ζ 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 14-3-3ζ and chromatographed through an FPLC Superose 12 gel filtration column. Most of 14-3-3ζ eluted within fractions 40–46 with a size of ∼500-kDa (Fig.2B). Since the size of dimeric 14-3-3ζ is ∼60-kDa (21Fu H. Subramanian R.R. Masters S.C. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 617-647Crossref PubMed Scopus (1334) Google Scholar, 22Van Hemert M.J. Yde Steensma H. van Heusden P.H. Bioessays. 2001; 23: 936-946Crossref PubMed Scopus (473) Google Scholar), these data indicated that 14-3-3ζ is bound to another biological molecule within the 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 14-3-3ζ-bound protein (s). In a previous study, we found that 14-3-3ζ is associated with tau in bovine brain extract and binds to tau in vitro(36Hashiguchi M. Sobue K. Paudel H.K. J. Biol. Chem. 2000; 275: 25247-25254Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). In a recent study, we showed that within brain microtubules, GSK3β 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 (20Sun W. Qureshi H.Y. Cafferty P.W. Sobue K. Agarwal-Mawal A. Neufield K.D. Paudel H.K. J. Biol. Chem. 2002; 277: 11933-11940Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). We noted a very similar gel filtration behavior between the high molecular size 14-3-3ζ present within fractions 40–46 (Fig. 2A) and the tau phosphorylation complex described by us in a previous study (20Sun W. Qureshi H.Y. Cafferty P.W. Sobue K. Agarwal-Mawal A. Neufield K.D. Paudel H.K. J. Biol. Chem. 2002; 277: 11933-11940Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). We therefore analyzed various Fig. 2A column fractions for the presence of tau and GSK3β. As shown in Fig. 2, C
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