Mitotic-like Tau Phosphorylation by p25-Cdk5 Kinase Complex
2003; Elsevier BV; Volume: 278; Issue: 36 Linguagem: Inglês
10.1074/jbc.m302872200
ISSN1083-351X
AutoresMalika Hamdane, Anne‐Véronique Sambo, Patrice Delobel, Séverine Bégard, Anne Violleau, André Delacourte, Philìppe Bertrand, Jesús Bénavidès, Luc Buée,
Tópico(s)Microtubule and mitosis dynamics
ResumoAmong tau phosphorylation sites, some phosphoepitopes referred to as abnormal ones are exclusively found on tau aggregated into filaments in Alzheimer's disease. Recent data suggested that molecular mechanisms similar to those encountered during mitosis may play a role in abnormal tau phosphorylation. In particular, TG-3 phosphoepitope is associated with early stages of neurofibrillary tangles (NFTs). In this study, we reported a suitable cell model consisting of SH-SY5Y cells stably transfected with an inducible p25 expression vector. It allows investigation of tau phosphorylation by p25-Cdk5 kinase complex in a neuronal context and avoiding p25-induced cytotoxicity. Immunoblotting analyses showed that p25-Cdk5 strongly phosphorylates tau protein not only at the AT8 epitope but also at the AT180 epitope and at the Alzheimer's mitotic epitope TG-3. Further biochemical analyses showed that abnormal phosphorylated tau accumulated in cytosol as a microtubule-free form, suggesting its impact on tau biological activity. Since tau abnormal phosphorylation occurred in dividing cells, TG-3 immunoreactivity was also investigated in differentiated neuronal ones, and both TG-3-immunoreactive tau and nucleolin, another early marker for NFT, were also generated. These data suggest that p25-Cdk5 is responsible for the mitotic-like phosphoepitopes present in NFT and argue for a critical role of Cdk5 in neurodegenerative mechanisms. Among tau phosphorylation sites, some phosphoepitopes referred to as abnormal ones are exclusively found on tau aggregated into filaments in Alzheimer's disease. Recent data suggested that molecular mechanisms similar to those encountered during mitosis may play a role in abnormal tau phosphorylation. In particular, TG-3 phosphoepitope is associated with early stages of neurofibrillary tangles (NFTs). In this study, we reported a suitable cell model consisting of SH-SY5Y cells stably transfected with an inducible p25 expression vector. It allows investigation of tau phosphorylation by p25-Cdk5 kinase complex in a neuronal context and avoiding p25-induced cytotoxicity. Immunoblotting analyses showed that p25-Cdk5 strongly phosphorylates tau protein not only at the AT8 epitope but also at the AT180 epitope and at the Alzheimer's mitotic epitope TG-3. Further biochemical analyses showed that abnormal phosphorylated tau accumulated in cytosol as a microtubule-free form, suggesting its impact on tau biological activity. Since tau abnormal phosphorylation occurred in dividing cells, TG-3 immunoreactivity was also investigated in differentiated neuronal ones, and both TG-3-immunoreactive tau and nucleolin, another early marker for NFT, were also generated. These data suggest that p25-Cdk5 is responsible for the mitotic-like phosphoepitopes present in NFT and argue for a critical role of Cdk5 in neurodegenerative mechanisms. Tau aggregation is a common feature among neurodegenerative disorders referred to as tauopathies. Mechanisms leading to tau aggregation and neurofibrillary degeneration are poorly understood. However, abnormal phosphorylation seems to be involved in tau conformational changes and aggregation (1Alonso A. Zaidi T. Novac M. Grundke-Iqbal I. Iqbal K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6923-6928Crossref PubMed Scopus (731) Google Scholar, 2Sato S. Tatebayashi Y. Akagi T. Chui D.H. Murayama M. Miyasaka T. Planel E. Tanemura K. Sun. X. Hashikawa T. Yoshioka K. Ishiguro K. Takashima A. J. Biol. Chem. 2002; 277: 42060-42065Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Phosphorylation is a key post-translational modification involved in the regulation of tau function regarding microtubule polymerization and stability. Whereas many phosphorylation sites are common between tau aggregated into filaments (tau-PHF) in AD 1The abbreviations used are: AD, Alzheimer's desease; NFT, neurofibrillary tangle; NGF, nerve growth factor; Pipes, 1,4-piperazinediethanesulfonic acid.1The abbreviations used are: AD, Alzheimer's desease; NFT, neurofibrillary tangle; NGF, nerve growth factor; Pipes, 1,4-piperazinediethanesulfonic acid. and normal tau from biopsy-derived material, some phosphoepitopes referred to as abnormal ones are exclusively found on tau-PHF (for a review, see Ref. 3Buée L. Bussière T. Buée-Scherrer V. Delacourte A. Hof P.R. Brain Res. Rev. 2000; 33: 95-130Crossref PubMed Scopus (1522) Google Scholar). Our recent data suggest that molecular mechanisms similar to those encountered during mitosis may play a role in the formation of abnormal tau phosphoepitopes (4Delobel P. Flament S. Hamdane M. Mailliot C. Sambo A.V. Bégard S. Sergeant N. Delacourte A. Vilain J.P. Buée L. J. Neurochem. 2002; 83: 412-420Crossref PubMed Scopus (60) Google Scholar). Understanding the role of abnormal tau phosphorylation in NFT formation requires identification of kinases leading to these specific phosphoepitopes. A large number of kinases can phosphorylate tau on specific Thr or Ser residues (for a review, see Ref. 3Buée L. Bussière T. Buée-Scherrer V. Delacourte A. Hof P.R. Brain Res. Rev. 2000; 33: 95-130Crossref PubMed Scopus (1522) Google Scholar). It was found that many kinases, belonging to the Pro-directed protein kinase family, have an enzymatic activity for one of the 17 Ser/Thr-Pro motifs in full-length tau. Other kinases, however, such as protein kinase A were found to phosphorylate Ser or Thr residues that are not followed by a Pro, whereas GSK-3β can modify both Ser/Thr residues that are or not followed by a Pro (for a review, see Ref. 5Planel E. Sun X. Takashima A. Drug Dev. Res. 2002; 56: 491-510Crossref Scopus (74) Google Scholar). A number of phosphorylation-dependent antibodies were used to monitor tau phosphorylation. For instance, AD2 recognizes phosphorylated residues Ser396 and Ser404 (6Buée-Scherrer V. Condamines E. Mourton-Gilles C. Jakes R. Goedert M. Pau B. Delacourte A. Mol. Brain Res. 1996; 39: 79-88Crossref PubMed Scopus (146) Google Scholar), whereas AT8 and AT180 recognize phospho-Ser202/Ser205 and phospho-Thr231, respectively (7Goedert M. Jakes R. Vanmechelen E. Neurosci. Lett. 1995; 189: 167-170Crossref PubMed Scopus (469) Google Scholar, 8Goedert M. Jakes R. Crowther R.A. Cohen P. Vanmechelen E. Vandermeeren M. Cras P. Biochem. J. 1994; 301: 871-877Crossref PubMed Scopus (346) Google Scholar). Among abnormal tau phosphoepitopes, TG-3 recognizes phospho-Thr231 in a conformation-dependent manner. This phosphoepitope is exclusively found in mitotic cells (9Vincent I. Rosado M. Davis P. J. Cell Biol. 1996; 132: 413-425Crossref PubMed Scopus (351) Google Scholar) with only the exception of degenerating neurons of AD and seems to be associated with early stages of the disease (10Vincent I. Zheng J.H. Dickson D.W. Kress Y. Davis P. Neurobiol. Aging. 1998; 19: 287-296Crossref PubMed Scopus (161) Google Scholar, 11Augustinack J.C. Schneider A. Mandelkow E.M. Hyman B.T. Acta. Neuropathol. 2002; 103: 26-35Crossref PubMed Scopus (713) Google Scholar). The Cdc2 kinase probably generates TG-3 epitope in mitotic cells, but the kinase(s) responsible for this phosphorylation in neurons remain unknown (9Vincent I. Rosado M. Davis P. J. Cell Biol. 1996; 132: 413-425Crossref PubMed Scopus (351) Google Scholar, 12Dranovsky A. Vincent I. Gregori L. Schwarzman A. Colflesh D. Enghild J. Strittmatter W. Davis P. Goldgaber D. Neurobiol. Aging. 2001; 22: 517-528Crossref PubMed Scopus (74) Google Scholar, 13Husseman J.W. Nochlin D. Vincent I. Neurobiol. Aging. 2000; 21: 815-828Crossref PubMed Scopus (168) Google Scholar). For instance, it was reported that Thr231 could be phosphorylated in vitro by four kinases (GSK3β, p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinase 2) (14Reynolds C.H. Betts J.C. Blackstock W.P. Nebreda A.R. Anderton B.H. J. Neurochem. 2000; 74: 1587-1595Crossref PubMed Scopus (309) Google Scholar). Nevertheless, one of the most evident candidates is Cdk5, the neuronal Cdc2-like kinase. Several data support the idea of Cdk5 involvement in AD (15Lee K.Y. Clark A.W. Rosales J.L. Chapman K. Fung T. Johnston R.N. Neurosci. Res. 1999; 34: 21-29Crossref PubMed Scopus (133) Google Scholar, 16Patrick G.N. Zukerberg L. Nikolic M. de la Monte S. Dikkes P. Tsai L.H. Nature. 1999; 402: 615-622Crossref PubMed Scopus (1307) Google Scholar, 17Pei J.J. Grundke-Iqbal I. Iqbal K. Bogdanovic N. Winblad B. Cowburn R.F. Brain Res. 1998; 797: 267-277Crossref PubMed Scopus (198) Google Scholar). Cdk5 activity is regulated in neuronal cells by its binding with its activating partner p35, p39, and the p35 proteolytic fragment, p25 protein (for a review, see Ref. 18Dahavan R. Tsai L.H. Mol. Cell. Biol. 2001; 2: 749-759Google Scholar). p35 proteolysis into p25 occurs in response to diverse insults that probably trigger calpain activation (19Kusakawa G. Saito T. Ohuki R. Ishiguro K. Kishimoto T. Hisanaga S. J. Biol. Chem. 2000; 275: 17166-17172Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 20Lee M. Kwon Y.T. Li M. Peng J. Friedlander R.M. Tsai L.H. Nature. 2000; 405: 360-364Crossref PubMed Scopus (901) Google Scholar, 21Nath R. Davis M. Probert A.W. Kupina N.C. Ren X. Schielke G.P. Wang K.K.W. Biochem. Biophys. Res. Commun. 2000; 274: 16-21Crossref PubMed Scopus (149) Google Scholar). p25-Cdk5 complex leads to a deregulated kinase activity that has been linked to tau hyperphosphorylation and neurotoxicity (16Patrick G.N. Zukerberg L. Nikolic M. de la Monte S. Dikkes P. Tsai L.H. Nature. 1999; 402: 615-622Crossref PubMed Scopus (1307) Google Scholar, 22Paudel H.K. Lew J. Ali Z. Wang J.H. J. Biol. Chem. 1993; 268: 23512-23518Abstract Full Text PDF PubMed Google Scholar, 23Alvarez A. Munoz J.P. Maccioni R.B. Exp. Cell Res. 2001; 264: 266-274Crossref PubMed Scopus (120) Google Scholar). Tau phosphorylation has been well characterized in vitro (24Lund E.T. McKenna R. Evans D.B. Sharma S.K. Mathews W.R. J. Neurochem. 2001; 76: 1221-1232Crossref PubMed Scopus (61) Google Scholar), but data on Cdk5-induced tau phosphorylation in vivo and in cellular models are lacking. In the present study, biochemical studies have been undertaken to investigate tau phosphorylation by p25-Cdk5 complex. The toxicity linked with p25 expression was avoided by using an appropriate cell model that expresses inducible p25-Cdk5 kinase activity.EXPERIMENTAL PROCEDURESCell Culture and TransfectionsSH-SY5Y human neuroblastoma cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mml-glutamine, 1 mm nonessential amino acids, and 50 units/ml penicillin/streptomycin (Invitrogen) in a 5% CO2 humidified incubator at 37 °C. Tau-SY5Y cells were previously described (4Delobel P. Flament S. Hamdane M. Mailliot C. Sambo A.V. Bégard S. Sergeant N. Delacourte A. Vilain J.P. Buée L. J. Neurochem. 2002; 83: 412-420Crossref PubMed Scopus (60) Google Scholar, 25Mailliot C. Bussière T. Hamdane M. Sergeant N. Caillet M.L. Delacourte A. Buée L. Ann. N. Y. Acad. Sci. 2000; 920: 107-114Crossref PubMed Scopus (24) Google Scholar). They constitutively express the tau isoform with three microtubule-binding domains (2+3–10–).Tau-SY5Y and SH-SY5Y cells were used as the basis for a tetracycline-regulated mammalian expression T-Rex system (Invitrogen). p25 cDNA was cloned into pcDNA4/TO vector (Invitrogen). First, stable cell lines that constitutively express tetracycline repressor were generated by transfection of pcDNA6/TR vector (Invitrogen) using ExGen500 (Euromedex, France) according to the manufacturer's instructions. Isolated clones, maintained in medium with 5 μg/ml blasticidin, were then transfected with inducible expression vector alone (mock) or with p25 cDNA. Individual stable clones were generated following Zeocin selection (100 μg/ml), and those that exhibited the weakest basal expression of p25 were selected. For induction of p25 expression, cells were maintained in medium with tetracycline at 1 μg/ml.NGF-Cell DifferentiationCells were differentiated for 7 days in Dulbecco's modified Eagle's medium/F-12 medium supplemented with 2 mml-glutamine, 50 units/ml penicillin/streptomycin, 7 μg/ml progesterone, 1% insulin/transferrin/selenium (Invitrogen), and 10 ng/ml NGF 2.5 S (Sigma). Medium was replenished every 2 days.Measurement of Cell ToxicityCells were seeded in 96-well plates (2000 cells/well). 24 h later, they were replenished with medium either containing or not containing tetracycline and 2 μm roscovitine (Calbiochem) (day 0). Treatments were renewed at day 3, and cytotoxicity assays were conducted at day 6 by measurement of lactate dehydrogenase release according to the manufacturer's instructions (Cyto Tox 96; Promega). For each experiment, measurements were carried out in triplicate.AntibodiesAnti-tau—The numbering of the tau epitopes is given according to the longest human 441 tau isoform. Phosphorylation-dependent monoclonal antibodies are directed against specific phosphorylated Ser/Thr-Pro sites (for a review, see Ref. 3Buée L. Bussière T. Buée-Scherrer V. Delacourte A. Hof P.R. Brain Res. Rev. 2000; 33: 95-130Crossref PubMed Scopus (1522) Google Scholar). They included AD2 (anti-phospho-Ser396–404; dilution 1:20,000) (6Buée-Scherrer V. Condamines E. Mourton-Gilles C. Jakes R. Goedert M. Pau B. Delacourte A. Mol. Brain Res. 1996; 39: 79-88Crossref PubMed Scopus (146) Google Scholar), AT8 (Innogenetics, Ghent, Belgium) (anti-phospho-Ser202 and -Thr205; dilution 0.5 μg/ml) (7Goedert M. Jakes R. Vanmechelen E. Neurosci. Lett. 1995; 189: 167-170Crossref PubMed Scopus (469) Google Scholar), AT180 (Innogenetics) (anti-phospho-Thr231; dilution 0.4 μg/ml) (8Goedert M. Jakes R. Crowther R.A. Cohen P. Vanmechelen E. Vandermeeren M. Cras P. Biochem. J. 1994; 301: 871-877Crossref PubMed Scopus (346) Google Scholar), and TG-3 (a gift of P. Davies; a marker of PHF and M phase of eukaryotic cells that recognizes a specific conformation of phospho-Thr231) (26Jicha G.A. Lane E. Vincent I. Otvos Jr., J. Hoffmann R. Davis P. J. Neurochem. 1997; 69: 2087-2095Crossref PubMed Scopus (216) Google Scholar) (dilution 1:20). Phosphorylation-independent antibodies M19G and tau-C-ter are well characterized antisera, directed against the first 19 amino acids (4Delobel P. Flament S. Hamdane M. Mailliot C. Sambo A.V. Bégard S. Sergeant N. Delacourte A. Vilain J.P. Buée L. J. Neurochem. 2002; 83: 412-420Crossref PubMed Scopus (60) Google Scholar, 6Buée-Scherrer V. Condamines E. Mourton-Gilles C. Jakes R. Goedert M. Pau B. Delacourte A. Mol. Brain Res. 1996; 39: 79-88Crossref PubMed Scopus (146) Google Scholar) and the last 15 amino acids of tau sequence (27Sergeant N. Sablonniere B. Schraen-Maschke S. Ghestem A. Maurage C.A. Wattez A. Vermersch P. Delacourte A. Hum. Mol. Genet. 2001; 10: 2143-2155Crossref PubMed Scopus (230) Google Scholar), respectively (dilution 1:20,000).Other Antibodies—Cdk5 monoclonal antibody (J-3; Santa Cruz Biotechnology, TEBU, France) (dilution 1:1000), p35-C-ter polyclonal antibody (C-19; Santa Cruz Biotechnology) (dilution 1:1000), neuronal specific γ-enolase antibody (Santa Cruz Biotechnology) (dilution 1:50,000), nucleolin monoclonal antibody (3G4B2; from Upstate Biotechnology, OZYME, France) (dilution 1:2000), tubulin polyclonal antibody (28Delobel P. Flament S. Hamdane M. Delacourte A. Vilain J.P. Buée L. FEBS Lett. 2002; 516: 151-155Crossref PubMed Scopus (20) Google Scholar) (dilution 1:2000), acetylated tubulin monoclonal antibody (6-11B-1; Sigma) (dilution 1:2000), α-synuclein (gift of Innogenetics) (dilution 1:2000), and poly(ADP-ribose) polymerase polyclonal antibody (Roche Applied Science) (dilution 1:2000).Western BlottingCells were harvested in ice-cold radioimmune precipitation assay modified buffer (50 mm Tris, pH 7.4, 1% Nonidet P-40, 1% Triton X-100, 150 mm NaCl, 1 mm EDTA) with protease inhibitors (Complete Mini; Roche Applied Science) and a 125 nm concentration of the phosphatase inhibitor okadaic acid (Sigma), sonicated, and stirred 1 h at 4 °C. Cell lysate was recovered in supernatant after centrifugation at 12,000 × g at 4 °C for 20 min. Protein concentration was determined by the BCA protein assay kit (Pierce). Samples were mixed with an equal volume of 2× Laemmli buffer and dithiothreitol and heated for 5 min at 100 °C, and then 10–20 μg were loaded onto SDS-PAGE gel. After transfer, membranes were blocked in TNT buffer (Tris-buffered saline, pH 8, 0.05% Tween 20), depending on the antibody without (TG-3 and AT anti-tau antibodies) or with 5% skim milk (the remaining antibodies), and incubated with primary antibody. Horseradish peroxidase-conjugated antibody (Sigma) was used as secondary antibody, and horseradish peroxidase activity was detected with the ECL detection kit (Amersham Biosciences). The IMAGE-MASTER 1D ELITE software (Amersham Biosciences) was used to quantify signals.Immunoprecipitationp25-overexpressing cells (24-h tetracycline treatment) were harvested in ice-cold lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 0.1% Nonidet P-40) supplemented with protease inhibitors, and cell lysate was prepared as described under “Western Blotting.” Protein concentration was determined by the BCA protein assay kit. For the immunoprecipitation experiments, cell lysates (400 μg at 2 mg/ml) were incubated with either anti-Cdk5 or anti-p35-C-ter antibodies (dilution 1/100) overnight at 4 °C and then incubated with 20 μl of protein A/G-agarose beads (Pierce) for 1 h at 4 °C. Immunoprecipitated complexes were washed four times in lysis buffer (centrifugation at 2000 × g at 4 °C for 5 min), recovered in Laemmli buffer, boiled for 5 min, and then analyzed by immunoblotting.In Vitro Kinase AssayThe longest human tau (441 residues) cloned in pET15b was produced in the BL21(DE3) Star Escherichia coli strain (Invitrogen). In vitro phosphorylation was performed by incubating TALON resin (BD Clontech) saturated with His-tagged tau 441 in the presence of lysates from either mock- or p25-tetracycline-treated cells prepared (as described under “Western Blotting”) in lysis buffer: 40 mm Tris-HCl, pH 7.2, 125 nm okadaic acid, 4 mm β-mercaptoethanol, protease inhibitors (EDTA-free Complete Mini; Roche Applied Science); 2 mm MgCl2, and 7.5 mm ATP with gentle shaking at 37 °C for 4 h. Immobilized recombinant tau proteins were then washed and eluted, and their phosphorylation state was analyzed by immunoblotting.When kinase inhibitors (LiCl (Sigma), PD98059 (Calbiochem), roscovitine (Calbiochem), and SB203580 (Calbiochem)) were used, they were mixed with cell lysates before adding the phosphorylation ATP buffer.For in vitro kinase assay by immunopurified p25-Cdk5 complex, Immunoprecipitation was performed from p25-expressing cells with anti-p35-C-ter antibody. Immunoprecipitated complex was washed in kinase buffer and then used to phosphorylate 100 μg of His-tagged tau 441, as described above. Reaction was stopped by adding Laemmli buffer and boiling for 5 min.Cell Fractionation into Cytosol and Microtubule FractionsAn equivalent number of mock and p25-expressing cells was recovered in equal volumes of warmed lysis buffer (80 mm Pipes, pH 6.8, 1 mm MgCl2,2mm EGTA, 30% glycerol, 0.1% Triton X-100) with protease inhibitors and okadaic acid. After ultracentrifugation (100,000 × g at 21 °C) for 30 min, supernatants were collected as cytosolic fractions. The remaining pellets were resuspended by sonication in ice-cold modified radioimmune precipitation assay buffer with protease inhibitors and okadaic acid in a volume equal to that of cytosolic fractions and then centrifuged at 12,000 × g (4 °C) for 20 min. Supernatants were recovered as microtubule fractions. Samples were then mixed to an equal volume of 2× Laemmli buffer and boiled at 100 °C for 5 min; equivalent volumes were then loaded on SDS-PAGE and analyzed by immunoblotting.Detergent Tau SolubilizationFor separation of soluble and insoluble tau proteins, an equivalent number of mock and p25-expressing cells was recovered in equal volumes of ice-cold saline buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, protease inhibitors, okadaic acid) with 1% SDS and then ultracentrifuged (100,000 × g at 4 °C) for 30 min. Supernatants were recovered as the soluble fractions and mixed (v/v) with 2× Laemmli buffer. The remaining pellets were solubilized in 70% formic acid, dried, and resuspended in Laemmli buffer (insoluble fractions). Samples were then subjected to immunoblotting analyses (soluble and insoluble fractions were loaded with the proportion of 1:6, with reference to cell number).For differential detergent tau solubility, an equivalent number of cells was recovered in equal volumes of ice-cold saline buffer and centrifuged as above to collect supernatants referred to as S1 fractions. The remaining pellets were successively homogenized by sonication in equivalent volumes of subsequent lysis buffers containing increased concentrations of Triton X-100 (0.01, 0.025, 0.05, 0.1, 0.25, and 1%), centrifuged as above to collect supernatants referred to as fractions (S2, S3, S4, S5, S6, and S7, respectively). Samples were mixed with an equal volume of 2× Laemmli buffer; equivalent volumes were then loaded on SDS-PAGE and analyzed by immunoblotting.RESULTSCharacterization of Stable Tau-SY5Y Cells Inducibly Expressing p25 Protein—Stable transfection of SH-SY5Y with the p25-inducible expression vector was unsuccessful, probably because of the toxicity of the low basal level expression of p25 protein (see “Discussion”). However, viable stable cell lines were obtained using SH-SY5Y cells that constitutively express tau protein (tau-SY5Y) (25Mailliot C. Bussière T. Hamdane M. Sergeant N. Caillet M.L. Delacourte A. Buée L. Ann. N. Y. Acad. Sci. 2000; 920: 107-114Crossref PubMed Scopus (24) Google Scholar). As shown in Fig. 1A, p25 was not found in mock tau-SY5Y cells. Conversely, p25 noninduced cells displayed a low basal expression of transgene protein, whereas tetracycline treatment induced a high p25 expression without affecting the endogenous level of its catalytic subunit Cdk5. Furthermore, co-immunoprecipitation analysis has been performed to ascertain the formation of p25-Cdk5 complex in p25-overexpressing cells (Fig. 1B).To investigate kinase activity in lysates from p25-overexpressing cells, we used recombinant tau protein as substrate. Western blotting with specific phosphorylation-dependent antibodies (Fig. 2) showed that lysate from p25-overexpressing cells but not that from mock ones allowed tau phosphorylation at the reported Cdk5 kinase sites in vitro including AD2, AT8, and AT180 epitopes (24Lund E.T. McKenna R. Evans D.B. Sharma S.K. Mathews W.R. J. Neurochem. 2001; 76: 1221-1232Crossref PubMed Scopus (61) Google Scholar). Thus, tetracycline treatment of p25-inducible cells allowed a substantial expression of p25 protein and its direct association with Cdk5 to allow kinase activity.Fig. 2In vitro tau phosphorylation. Recombinant tau was subjected to in vitro kinase assay by incubation with lysates from either mock or p25-expressing cells (24-h tetracycline treatment) and then analyzed by immunoblotting using phosphorylation-dependent anti-tau antibodies (AD2, AT8, and AT180) and a phosphorylation-independent antibody, M19G.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Tau Phosphorylation in p25-expressing Cells—P25 overexpression has been reported to be toxic in mammalian cells (16Patrick G.N. Zukerberg L. Nikolic M. de la Monte S. Dikkes P. Tsai L.H. Nature. 1999; 402: 615-622Crossref PubMed Scopus (1307) Google Scholar). Thus, prior to tau phosphorylation analyses, we monitored the effect of time course p25-induced overexpression on cell death. Measurement of lactate dehydrogenase release showed that the toxic effect of p25 on tau-SY5Y cells is detectable from at least 3 days up of tetracycline treatment (Fig. 3). Moreover, p25 toxicity is attenuated by roscovitine, known as a preferential inhibitor of Cdk5 activity (Fig. 3). No evidence of apoptosis was observed at 24 h following p25 induction, since poly(ADP-ribose) polymerase proteolysis and carboxyl-terminal tau cleavage were not observed by Western blotting (data not shown).Fig. 3Lactate dehydrogenase measurement of p25-induced cytotoxicity. Histograms represent lactate dehydrogenase release as percentages of maximal lactate dehydrogenase release by detergent lysis. Values of tetracycline-treated cells have been normalized to those of tetracycline-untreated cells. In mock cells, similar data were observed at 3 and 6 days of tetracycline treatment.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To investigate tau phophorylation in p25-inducible cells independently of any p25 toxic effect, experiments have been performed from 24-h tetracycline-induced p25 overexpression. Immunoblots were carried out using four phosphorylation-dependent anti-tau antibodies (AT8, AD2, AT180, and TG-3) and the phosphorylation-independent antibody M19G, used to normalize for the amount of tau proteins (Fig. 4A). Tau phosphorylation was quantified as a ratio of each phosphorylation-dependent antibody immunoreactivity to that of M19G (Fig. 4B). This ratio was compared between tetracycline-treated (+Tet) and -untreated (–Tet) cells.Fig. 4Tau phosphorylation in p25 expressing cells. A, immunoblotting analyses. Lysates from mock and p25-inducible cells, treated (+) or not (–) with tetracycline (Tet) and roscovitine (RSV), were analyzed by phosphorylation-dependent tau antibodies: AD2, AT8, AT180, and TG-3. The total amount of loaded tau proteins is visualized by M19G antibody. B, quantification of tau phosphorylation in mock and p25-expressing cells. Ratios of densitometric values of each phospho-tau antibody to those of M19G antibody are presented. These ratios are normalized to those obtained from mock cells with neither tetracycline nor roscovitine treatment ((–)Tet; arbitrary value = 1).View Large Image Figure ViewerDownload Hi-res image Download (PPT)As expected, tau proteins were found phosphorylated in mock cells and –Tet p25 cells. However, p25 overexpression in +Tet cells induced tau phosphorylation at the AT8 epitope, the major phosphorylation site of Cdk5 reported in vivo (29Ahlijanian M.K. Barrezueta N.X. Williams R.D. Jakowski A. Kowsz K.P. McCarthy S. Coskran T. Carlo A. Seymour P.A. Burkhadt J.E. Nelson R.B. McNeish J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2910-2915Crossref PubMed Scopus (304) Google Scholar, 30Takashima A. Murayama M. Yazutake K. Takahashi H. Yàkoyama M. Ishiguro K. Neurosci. Lett. 2001; 306: 37-40Crossref PubMed Scopus (74) Google Scholar), whereas no significant increase in AD2 immunoreactivity was observed. Interestingly, a robust increase in tau phosphorylation was observed at the AT180 epitope and more strongly at the TG-3 epitope, suggesting that phospho-Thr231 could be a major phosphorylation site of p25-Cdk5 in vivo. No significant increase in phosphorylation was detected in p25-overexpressing cells following roscovitine treatment (+RSV), suggesting a specific action of the complex p25-Cdk5. However, the direct tau phosphorylation by p25-Cdk5 has to be clearly established, since this kinase complex may also activate other kinases. In fact, Thr231 is a residue that can be phosphorylated by a large number of kinases other than Cdk, including c-Jun N-terminal kinase, p38, extracellular signal-regulated kinase 2, and GSK3β (14Reynolds C.H. Betts J.C. Blackstock W.P. Nebreda A.R. Anderton B.H. J. Neurochem. 2000; 74: 1587-1595Crossref PubMed Scopus (309) Google Scholar).Direct Phosphorylation of Thr231 by the Complex p25-Cdk5—To investigate whether p25-Cdk5 directly phosphorylates Thr231, we first performed an in vitro tau phosphorylation using the p25-overexpressing cell lysate in the presence of well recognized kinase inhibitors including PD98059 (MEK1 inhibitor), SB203580 (p38 inhibitor), LiCl (GSK3β inhibitor), and roscovitine (Cdk inhibitor) (31Davies S.P. Reddy H. Caivano M. Cohen P. Biochem. J. 2000; 351: 95-105Crossref PubMed Scopus (3919) Google Scholar). Tau phosphorylation at Thr231 was monitored by Western blotting (Fig. 5A). Whereas LiCl slightly decreased phosphorylation at Thr231, roscovitine completely abolished the signal. Other inhibitors did not have any effect on Thr231 phosphorylation. Altogether, these data strongly suggested that in vivo tau phosphorylation at Thr231 resulted from a direct effect of p25-Cdk5 kinase. To definitely confirm this direct phosphorylation, the p25-Cdk5 was immunoprecipitated, using the anti-p25 antibody from the lysate. An in vitro tau phosphorylation was performed using the immunoprecipitated complex. Phosphorylation at Thr231 was visualized using the AT180 antibody, indicating that the complex p25-Cdk5 can directly phosphorylate tau at Thr231 (Fig. 5B).Fig. 5Direct tau phosphorylation at Thr231 by p25-Cdk5 kinase. Recombinant tau was subjected to in vitro kinase assay by incubation with the following. A, crude extract from p25-expressing cells (24 h of tetracycline), alone and in the presence of kinase inhibitors: roscovitine (10 μm), LiCl (20 mm), PD98059 (50 μm), and SB203580 (10 μm). B, immunopurified p25-Cdk5 complex from mock cells (control) or p25-expressing cells. Tau phosphorylation was analyzed by immunoblotting (WB) with AT180. The anti-tau M19G was used as a control antibody.View Large
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