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• W2058968970 abstract "Most individuals with Down syndrome show early onset of Alzheimer disease (AD), resulting from the extra copy of chromosome 21. Located on this chromosome is a gene that encodes the dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A). One of the pathological hallmarks in AD is the presence of neurofibrillary tangles (NFTs), which are insoluble deposits that consist of abnormally hyperphosphorylated Tau. Previously it was reported that Tau at the Thr-212 residue was phosphorylated by Dyrk1A in vitro. To determine the physiological significance of this phosphorylation, an analysis was made of the amount of phospho-Thr-212-Tau (pT212) in the brains of transgenic mice that overexpress the human DYRK1A protein (DYRK1A TG mice) that we recently generated. A significant increase in the amount of pT212 was found in the brains of DYRK1A transgenic mice when compared with age-matched littermate controls. We further examined whether Dyrk1A phosphorylates other Tau residues that are implicated in NFTs. We found that Dyrk1A also phosphorylates Tau at Ser-202 and Ser-404 in vitro. Phosphorylation by Dyrk1A strongly inhibited the ability of Tau to promote microtubule assembly. Following this, using mammalian cells and DYRK1A TG mouse brains, it was demonstrated that the amounts of phospho-Ser-202-Tau and phospho-Ser-404-Tau are enhanced when DYRK1A amounts are high. These results provide the first in vivo evidence for a physiological role of DYRK1A in the hyperphosphorylation of Tau and suggest that the extra copy of the DYRK1A gene contributes to the early onset of AD. Most individuals with Down syndrome show early onset of Alzheimer disease (AD), resulting from the extra copy of chromosome 21. Located on this chromosome is a gene that encodes the dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A). One of the pathological hallmarks in AD is the presence of neurofibrillary tangles (NFTs), which are insoluble deposits that consist of abnormally hyperphosphorylated Tau. Previously it was reported that Tau at the Thr-212 residue was phosphorylated by Dyrk1A in vitro. To determine the physiological significance of this phosphorylation, an analysis was made of the amount of phospho-Thr-212-Tau (pT212) in the brains of transgenic mice that overexpress the human DYRK1A protein (DYRK1A TG mice) that we recently generated. A significant increase in the amount of pT212 was found in the brains of DYRK1A transgenic mice when compared with age-matched littermate controls. We further examined whether Dyrk1A phosphorylates other Tau residues that are implicated in NFTs. We found that Dyrk1A also phosphorylates Tau at Ser-202 and Ser-404 in vitro. Phosphorylation by Dyrk1A strongly inhibited the ability of Tau to promote microtubule assembly. Following this, using mammalian cells and DYRK1A TG mouse brains, it was demonstrated that the amounts of phospho-Ser-202-Tau and phospho-Ser-404-Tau are enhanced when DYRK1A amounts are high. These results provide the first in vivo evidence for a physiological role of DYRK1A in the hyperphosphorylation of Tau and suggest that the extra copy of the DYRK1A gene contributes to the early onset of AD. Down syndrome (DS) 3The abbreviations used are: DS, Down syndrome; AD, Alzheimer disease; DYRK1A, dual specificity tyrosine phosphorylation-regulated kinase 1A; DYRK1A TG mice, transgenic mice that overexpress the human DYRK1A protein; NFTs, neurofibrillary tangles; PHFs, paired helical filaments; pS202, phospho-Ser-202-Tau; pT212, phospho-Thr-212-Tau; pS404, phospho-Ser-404-Tau; protein nomenclature, Dyrk1A for mouse protein and DYRK1A for human protein; WT, wild type; PBS, phosphate-buffered saline; MOPS, 4-morpholinepropanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid; siRNA, short interfering RNA; GFP, green fluorescent protein; JNK, c-Jun N-terminal kinase; APP, β-amyloid precursor protein; Aβ, β-amyloid. is the most common genetic disorder with a frequency of 1 in 800 live births, and it is caused by the presence of an extra copy of whole or part of human chromosome 21 (1Lejeune J. Gautier M. Turpin R. C. R. Hebd. Seances Acad. Sci. 1959; 248: 1721-1722PubMed Google Scholar, 2Jacobs P.A. Baikie A.G. Court Brown W.M. Strong J.A. Lancet. 1959; 1: 710Abstract PubMed Scopus (153) Google Scholar). DS patients suffer various symptoms, including congenital heart defects, immune and endocrine system defects, mental retardation, and early onset of Alzheimer disease (AD) (3Korenberg J.R. Chen X.N. Schipper R. Sun Z. Gonsky R. Gerwehr S. Carpenter N. Daumer C. Dignan P. Disteche C. Graham Jr., J.M. Hudgins L. McGillivray B. Miyazaki K. Ogasawara N. Park J.P. Pagon R. Pueschol S. Sack G. Say B. Schuffenhauer S. Soukup S. Yamanaka T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4997-5001Crossref PubMed Scopus (596) Google Scholar). Both DS and AD patients have pathological hall-marks, amyloid plaques and neurofibrillary tangles (NFTs) that are insoluble deposits made of proteins called β-amyloid (Aβ) and hyperphosphorylated Tau, respectively (4Grundke-Iqbal I. Iqbal K. Tung Y.C. Quinlan M. Wisniewski H.M. Binder L.I. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4913-4917Crossref PubMed Scopus (2877) Google Scholar, 5Masters C.L. Simms G. Weinman N.A. Multhaup G. McDonald B.L. Beyreuther K. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4245-4249Crossref PubMed Scopus (3668) Google Scholar, 6Burger P.C. Vogel F.S. Am. J. Pathol. 1973; 73: 457-476PubMed Google Scholar). Although an early onset AD in DS patients is not clearly understood, one potential mechanism is the presence of three chromosomal copies of β-amyloid precursor protein (APP) gene. However, the APP overexpression alone in mice does not show the endosome abnormalities observed in AD-like pathology (7Cataldo A.M. Petanceska S. Peterhoff C.M. Terio N.B. Epstein C.J. Villar A. Carlson E.J. Staufenbiel M. Nixon R.A. J. Neurosci. 2003; 23: 6788-6792Crossref PubMed Google Scholar), implying the necessity of additional genes on the chromosome 21 for a full spectrum of AD pathologies. NFTs found in AD are composed of paired helical filaments (PHFs), which are mainly composed of hyperphosphorylated Tau protein (8Selkoe D.J. Neuron. 1991; 6: 487-498Abstract Full Text PDF PubMed Scopus (2195) Google Scholar). To date, more than 30 phosphorylation sites and 7–10 mol of phosphates per mol of Tau have been observed in PHF-Tau (9Ksiezak-Reding H. Liu W.K. Yen S.H. Brain Res. 1992; 597: 209-219Crossref PubMed Scopus (191) Google Scholar, 10Kopke E. Tung Y.C. Shaikh S. Alonso A.C. Iqbal K. Grundke-Iqbal I. J. Biol. Chem. 1993; 268: 24374-24384Abstract Full Text PDF PubMed Google Scholar). Although Tau protein is phosphorylated in vitro by numerous kinases, it is unclear how many kinases actually phosphorylate Tau in vivo. Currently, only glycogen synthase kinase 3β (GSK3β), cyclin-dependent kinase 5, cAMP-dependent protein kinase A, and microtubule affinity-regulating kinase are known to modulate Tau phosphorylation in vivo at some level, either directly or indirectly (11Johnson G.V. Stoothoff W.H. J. Cell Sci. 2004; 117: 5721-5729Crossref PubMed Scopus (440) Google Scholar). The DYRK1A (dual specificity tyrosine (Y)-phosphorylation-regulated kinase 1A) gene is isolated from human chromosome 21 and encodes a protein kinase (12Kentrup H. Becker W. Heukelbach J. Wilmes A. Schurmann A. Huppertz C. Kainulainen H. Joost H.G. J. Biol. Chem. 1996; 271: 3488-3495Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 13Guimera J. Casas C. Pucharcos C. Solans A. Domenech A. Planas A.M. Ashley J. Lovett M. Estivill X. Pritchard M.A. Hum. Mol. Genet. 1996; 5: 1305-1310Crossref PubMed Scopus (186) Google Scholar, 14Shindoh N. Kudoh J. Maeda H. Yamaki A. Minoshima S. Shimizu Y. Shimizu N. Biochem. Biophys. Res. Commun. 1996; 225: 92-99Crossref PubMed Scopus (104) Google Scholar, 15Song W.J. Sternberg L.R. Kasten-Sportes C. Keuren M.L. Chung S.H. Slack A.C. Miller D.E. Glover T.W. Chiang P.W. Lou L. Kurnit D.M. Genomics. 1996; 38: 331-339Crossref PubMed Scopus (149) Google Scholar). DYRK1A is ubiquitously expressed with strong expression in brain and heart (13Guimera J. Casas C. Pucharcos C. Solans A. Domenech A. Planas A.M. Ashley J. Lovett M. Estivill X. Pritchard M.A. Hum. Mol. Genet. 1996; 5: 1305-1310Crossref PubMed Scopus (186) Google Scholar, 15Song W.J. Sternberg L.R. Kasten-Sportes C. Keuren M.L. Chung S.H. Slack A.C. Miller D.E. Glover T.W. Chiang P.W. Lou L. Kurnit D.M. Genomics. 1996; 38: 331-339Crossref PubMed Scopus (149) Google Scholar, 16Rahmani Z. Lopes C. Rachidi M. Delabar J.M. Biochem. Biophys. Res. Commun. 1998; 253: 514-518Crossref PubMed Scopus (41) Google Scholar, 17Hammerle B. Carnicero A. Elizalde C. Ceron J. Martinez S. Tejedor F.J. Eur. J. Neurosci. 2003; 17: 2277-2286Crossref PubMed Scopus (88) Google Scholar, 18Marti E. Altafaj X. Dierssen M. de la Luna S. Fotaki V. Alvarez M. Perez-Riba M. Ferrer I. Estivill X. Brain Res. 2003; 964: 250-263Crossref PubMed Scopus (97) Google Scholar), and mRNA in DS fetal brains has been shown to be overexpressed (19Guimera J. Casas C. Estivill X. Pritchard M. Genomics. 1999; 57: 407-418Crossref PubMed Scopus (145) Google Scholar). Dyrk1A has dual substrate specificities as follows: autophosphorylation on the tyrosine 321 residue for self-activation and phosphorylation of target proteins on serine/threonine residues with a preferred phosphorylation consensus motif of RPX(S/T)P (12Kentrup H. Becker W. Heukelbach J. Wilmes A. Schurmann A. Huppertz C. Kainulainen H. Joost H.G. J. Biol. Chem. 1996; 271: 3488-3495Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 20Himpel S. Tegge W. Frank R. Leder S. Joost H.G. Becker W. J. Biol. Chem. 2000; 275: 2431-2438Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 21Lochhead P.A. Sibbet G. Morrice N. Cleghon V. Cell. 2005; 121: 925-936Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). Dyrk1A potentially phosphorylates or interacts with several proteins, including transcription factors (NFATc, STAT3, Forkhead, CREB, and Gli1) and eukaryotic initiation factor 2Bϵ, Dynamin 1, cyclin L2, Hip-1, and α-synuclein, implying the multiple biological functions of DYRK1A (22Galceran J. de Graaf K. Tejedor F.J. Becker W. J. Neural Transm. Suppl. 2003; 67: 139-148Crossref PubMed Scopus (60) Google Scholar). Dyrk1A knock-out mice show a general delay in fetal development and are embryonic lethal, indicating the vital and nonredundant biological functions of Dyrk1A (23Fotaki V. Dierssen M. Alcantara S. Martinez S. Marti E. Casas C. Visa J. Soriano E. Estivill X. Arbones M.L. Mol. Cell. Biol. 2002; 22: 6636-6647Crossref PubMed Scopus (252) Google Scholar). We recently generated transgenic mice that overexpress human DYRK1A (DYRK1A TG mice) by introducing a bacterial artificial chromosome that only carries the human DYRK1A genomic DNA, including the endogenous promoter (24Ahn K.J. Jeong H.K. Choi H.S. Ryoo S.R. Kim Y.J. Goo J.S. Choi S.Y. Han J.S. Ha I. Song W.J. Neurobiol. Dis. 2006; 22: 463-472Crossref PubMed Scopus (172) Google Scholar). These DYRK1A TG mice show learning and memory deficits similar to what is seen in DS patients, a finding that is comparable with previous studies on the mouse Dyrk1A cDNA transgenic mice or the human genomic transgenic mice carrying a few chromosome 21 genes, including DYRK1A on a yeast artificial chromosome (24Ahn K.J. Jeong H.K. Choi H.S. Ryoo S.R. Kim Y.J. Goo J.S. Choi S.Y. Han J.S. Ha I. Song W.J. Neurobiol. Dis. 2006; 22: 463-472Crossref PubMed Scopus (172) Google Scholar, 25Altafaj X. Dierssen M. Baamonde C. Marti E. Visa J. Guimera J. Oset M. Gonzalez J.R. Florez J. Fillat C. Estivill X. Hum. Mol. Genet. 2001; 10: 1915-1923Crossref PubMed Scopus (327) Google Scholar, 26Smith D.J. Stevens M.E. Sudanagunta S.P. Bronson R.T. Makhinson M. Watabe A.M. O'Dell T.J. Fung J. Weier H.U. Cheng J.F. Rubin E.M. Nat. Genet. 1997; 16: 28-36Crossref PubMed Scopus (265) Google Scholar). Previously, it was reported that Dyrk1A phosphorylates Tau at Thr-212 in vitro, a residue that is phosphorylated in fetal Tau and hyperphosphorylated in filamentous Tau from AD brains (27Woods Y.L. Cohen P. Becker W. Jakes R. Goedert M. Wang X. Proud C.G. Biochem. J. 2001; 355: 609-615Crossref PubMed Scopus (256) Google Scholar). In this study, we investigated the physiological significance of this phosphorylation event using transgenic mice that overexpress human DYRK1A (DYRK1A TG mice). It was found that that Tau phosphorylation at Thr-212 is significantly increased in DYRK1A TG mice. Furthermore, Tau protein was also phosphorylated at Ser-202 and Ser-404 residues by Dyrk1A in vitro, and the levels of the phosphorylation at those residues were increased in DYRK1A TG mice. These results indicate that the overexpression of DYRK1A in DS brains may contribute to early onset of AD pathology through the hyperphosphorylation of Tau. Proteins and Antibodies—The full-length mouse Dyrk1A cDNAs (wild-type and Y321F kinase-inactive mutant) were cloned into pET-25b (Novagen), followed by isopropyl 1-thio-β-d-galactopyranoside induction in Escherichia coli BL21(DE3) codon plus RIL (Stratagene). Using its endogenous 13-histidine repeat, the Dyrk1A protein was purified with nickel-nitrilotriacetic acid resin as described by the manufacturer (Qiagen). Human recombinant Tau, nonglycosylated and nonphosphorylated form of 441-amino acid Tau splice variant, was obtained from Calbiochem. Bovine tubulin was purchased from Cytoskeleton, and JNK2 was from Upstate. Antibodies for Tau (Tau-5) and phospho-Tau (pS202, pT212, pS404) were obtained from BIOSOURCE. Phosphopeptides and non-phosphopeptides were purchased to perform the peptide competition assay for all three phospho-Tau antibodies (BIOSOURCE). The anti-Dyrk1A antibody was custom-made as described previously (24Ahn K.J. Jeong H.K. Choi H.S. Ryoo S.R. Kim Y.J. Goo J.S. Choi S.Y. Han J.S. Ha I. Song W.J. Neurobiol. Dis. 2006; 22: 463-472Crossref PubMed Scopus (172) Google Scholar). Immunohistochemistry—Mice were anesthetized with isoflurane, and perfused transcardially with phosphate-buffered saline (PBS, pH 7.4) followed by fixative (4% paraformaldehyde in PBS, pH 7.4). The brains were fixed at 4 °C overnight and were cryoprotected. Sagittal sections of mouse brains were prepared as described previously (24Ahn K.J. Jeong H.K. Choi H.S. Ryoo S.R. Kim Y.J. Goo J.S. Choi S.Y. Han J.S. Ha I. Song W.J. Neurobiol. Dis. 2006; 22: 463-472Crossref PubMed Scopus (172) Google Scholar) and incubated first with phospho-Tau antibodies (pS202, pT212, and pS404; 1:100 dilution), then with biotinylated anti-rabbit IgG (1:200 dilution), and finally with avidin biotinylated horseradish peroxidase (Vector Laboratories). After visualization with an SG reagent set (Vector Laboratories), signals were examined with a Zeiss Axioplan microscope using conventional optics. Preparation of Mouse Brain Lysates and Immunoblotting—DYRK1A TG mice that overexpressed the human DYRK1A gene, which was transcribed from the genomic DYRK1A DNA carried on a bacterial artificial chromosome, were produced and maintained as described previously (24Ahn K.J. Jeong H.K. Choi H.S. Ryoo S.R. Kim Y.J. Goo J.S. Choi S.Y. Han J.S. Ha I. Song W.J. Neurobiol. Dis. 2006; 22: 463-472Crossref PubMed Scopus (172) Google Scholar). Experiments were performed in accordance with guidelines set forth by the Inje University Council Directive for the proper care and use of laboratory animals. Mouse brain lysates were prepared by homogenization in a tissue grinder (Fisher) in RIPA buffer (150 mm NaCl, 1% Nonidet P-40, 0.1% SDS, 50 mm Tris, pH 8.0, 0.5% deoxycholic acid) containing a protease inhibitor mixture (Calbiochem), 20 μm sodium orthovanadate, and 1 mm phenylmethylsulfonyl fluoride. The protein concentration was determined using the BCA method (Sigma). Proteins were separated by SDS-PAGE and analyzed by immunoblot with phospho-Tau and Tau antibodies. Densitometric quantification of Western blotting signal bands was carried out with a Science Lab 2001 Image Gauge (version 4.0, Fuji Photo Film Co., Ltd.). In Vitro Tau Phosphorylation—Human recombinant Tau protein (1.1 μg) was incubated for 90 min at 30 °C with the purified Dyrk1A (300 ng) in kinase buffer (20 mm MOPS, pH 7.0, 10 mm MgCl2, 1 mm dithiothreitol, 20 μm sodium orthovanadate, and 100 μm ATP; 30-μl reaction volume). The reaction mixtures were separated on SDS-PAGE and analyzed by immunoblot with Tau and phospho-Tau antibodies. Microtubule Assembly—Tau protein (9.2 μm) was phosphorylated by either no kinase, JNK2 (0.83 μm), or Dyrk1A (0.90 μm) for 2 h as described above. Phosphorylated Tau proteins were subjected to microtubule assembly assay as described previously (28Yoshida H. Hastie C.J. McLauchlan H. Cohen P. Goedert M. J. Neurochem. 2004; 90: 352-358Crossref PubMed Scopus (140) Google Scholar). Briefly, nonphosphorylated or phosphorylated Tau (final concentration, 1.3 μm) was mixed with tubulin (20 μm) in polymerization buffer (80 mm Na-PIPES, pH 6.9, 10 mm MgCl2, 1 mm EGTA and 1 mm GTP) at 37 °C. Microtubule assembly was initiated by addition of glycerol (final concentration, 5%) and was monitored by measuring the absorbance at 340 nm over 20 min. Plasmids, siRNAs, and HEK293T Cell Transfection—A full-length wild-type Dyrk1A cDNA from a mouse brain and a full-length human Tau cDNA were cloned into pcDNA3.1 (Invitrogen). The Y321F kinase-inactive mutant Dyrk1A cDNA was generated by DpnI-mediated site-directed mutagenesis. For siRNA experiment, the mouse Dyrk1A-specific siRNA (5′-GGUAUGAAAUCGACUCCUU) with the TT overhang was designed and synthesized by Samchully Pharm. Co. (South Korea). For siRNA delivery, Dyrk1A-specific duplex siRNA (60 pmol for 6-well plate) was co-transfected into human embryonic kidney (HEK293T) cells (8 × 105 cells/well) with Dyrk1A and Tau plasmids using Lipofectamine 2000 (Invitrogen). Fluorescein isothiocyanate-conjugated GFP-specific siRNA (5′GUUCAGCGUGUCCGGCGAGTT) was used as a nonsilencing control. After 24 h of siRNA treatment, cell lysates were prepared for immunoblot analyses. Immunocytochemistry—COS-7 cells on PDL-coated coverslip (6-well) were transfected with Dyrk1A and Tau plasmids. After 24 h, cells were fixed with 4% (w/v) paraformaldehyde in PBS, pH 7.4, permeabilized with 0.1% Triton X-100 in PBS, and blocked with PBS containing 1% normal goat serum for 2 h. Cells were incubated with the primary antibodies (Dyrk1A, 1:200; Tau, 1:500) at room temperature for 2 h, and then with Alexa Fluor 488 goat anti-rabbit or Cy3 goat anti-mouse-coupled secondary antibodies (1:200) for 1 h. Technical support by examining the images using a confocal microscope (Carl Zeiss LSM 510) was provided by the Korea Basic Science Institute. Co-immunoprecipitation—Brain lysate (1 mg) from the DYRK1A TG mice was incubated with control IgG (R & D Systems), Tau (BIOSOURCE), or Dyrk1A antibody for overnight at 4 °C in RIPA buffer with protease inhibitors and 1 mm phenylmethylsulfonyl fluoride. The next day after a 1-h incubation with protein A beads (Pierce), the beads were gently washed with 1% Triton X-100 in RIPA buffer, and then the bound proteins were subjected to immunoblot analysis for the indicated antibodies. Increased Phospho-Thr-212-Tau in DYRK1A TG Mice—To investigate the link between DYRK1A and Tau phosphorylation at the Thr-212 residue in vivo, transgenic mice that overexpress human DYRK1A (DYRK1A TG mice) were analyzed. These DYRK1A TG mice contained two copies of the mouse Dyrk1A gene and one copy of the human DYRK1A genomic DNA, and the human DYRK1A protein expression was tightly controlled by the endogenous promoter. The overexpression of DYRK1A in these mice resulted in approximately a 70% increase in kinase activity. 4Ryoo, S.-R., Cho, H.-J., Lee, H.-W., Jeong, H. K., Radnaabazar, C., Kim, Y.-S., Kim, M.-J., Son, M.-Y., Seo, H., Chung, S.-H., and Song, W.-J., J. Neurochem., in press. Immunohistochemical analyses revealed that phospho-Thr-212-Tau (pT212) expression was markedly increased in the hippocampus and frontal cortex of DYRK1A TG mice compared with control littermates (Fig. 1, A and B). The increase in pT212 was observed in the pyramidal cell layer of the hippocampus and the polymorphic layer of the dentate gyrus where DYRK1A overexpression was observed previously (24Ahn K.J. Jeong H.K. Choi H.S. Ryoo S.R. Kim Y.J. Goo J.S. Choi S.Y. Han J.S. Ha I. Song W.J. Neurobiol. Dis. 2006; 22: 463-472Crossref PubMed Scopus (172) Google Scholar). The results were corroborated by Western analysis of brain lysates prepared from the hippocampus of DYRK1A TG mice and control littermates (Fig. 1C). After normalization with Tau, the amounts of pT212 in the DYRK1A TG mice were increased by 49 ± 18% (n = 8, p < 0.05), relative to control littermates. The fact that increased phosphorylation of Tau at Thr-212 was evident in both immunohistochemical and immunoblot experiments suggests that overexpression of DYRK1A is functionally linked to Tau phosphorylation in vivo. In Vitro Phosphorylation of Tau at Ser-202 and Ser-404 by Dyrk1A—Previously it was reported that Dyrk1A preferentially phosphorylates substrate proteins at serine and/or threonine residue(s) when proline is present at the +1 position, (S/T)P (20Himpel S. Tegge W. Frank R. Leder S. Joost H.G. Becker W. J. Biol. Chem. 2000; 275: 2431-2438Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). An examination of the amino acid sequence of Tau reveals the presence of multiple serine/threonine residues followed by proline. To further explore the role of Dyrk1A in Tau hyperphosphorylation, we examined whether this kinase phosphorylates Tau in vitro at other residues that are implicated in NFT formations in AD, such as Ser-202 and Ser-404 residues (29Augustinack J.C. Schneider A. Mandelkow E.M. Hyman B.T. Acta Neuropathol. 2002; 103: 26-35Crossref PubMed Scopus (725) Google Scholar). The full-length mouse Dyrk1A proteins (wild-type (WT) and Y321F kinase-inactive mutant (YF)) were expressed in E. coli and were purified as ∼90 kDa.4 The recombinant full-length human Tau was incubated in the presence or absence of Dyrk1A, and the reaction mixture was then subjected to immunoblotting analysis. Phosphorylation of Ser-202 and Ser-404 was detected with phospho-Ser-202-Tau (pS202)- and phospho-Ser-404-Tau (pS404)-specific antibodies, respectively. Only in the presence of both Tau and Dyrk1A were the pS202- and pS404-specific antibodies able to detect a protein band that corresponded to Tau on the immunoblotted membrane (Fig. 2A). This result indicates that Dyrk1A can phosphorylate the Ser-202 and Ser-404 residues of Tau directly in vitro. The phosphorylation of Tau by Dyrk1A did not result from any contaminating E. coli kinase activity during Dyrk1A purification, as the Tau protein was phosphorylated only by the WT Dyrk1A and not by the Y321F mutant (Fig. 2B). The Y321F mutant Dyrk1A showed slightly faster mobility on the SDS-PAGE, because of mutation of the Tyr-321 residue, the autophosphorylation site required for kinase activation (Fig. 2B and Fig. 4A). When the WT and Y321F proteins were treated with λ phosphatase for dephosphorylation, they showed the same mobility (data not shown).FIGURE 4Tau phosphorylation by Dyrk1A in mammalian cells. A, HEK293T cells were transfected with indicated plasmid(s) or a control pcDNA3.1. After 24 h, cell lysates were subjected to SDS-PAGE, followed by immunoblot analyses for the indicated phospho-Tau, Tau, and Dyrk1A. B, HEK293T cells were co-transfected with human Tau, mouse Dyrk1A, and Dyrk1A-specific siRNAs. The level of Dyrk1A, phospho-Tau, and Tau were examined by immunoblot analyses. Fluorescein isothiocyanate-conjugated GFP-specific siRNA was used as a nonsilencing siRNA control (Con). C, peptide competition assay for phospho-Tau antibodies. Mouse hippocampal lysates were analyzed by Western blotting with indicated phospho-specific Tau antibodies preincubated in the absence (N) or presence of non-phosphopeptides (NP), phosphopeptides (P), or unrelated phosphopeptides (UP). The pS404 phosphopeptide was used as an unrelated phosphopeptide control for the pS202 antibody competition assay and vice versa. The pS202 phosphopeptide was used as an unrelated phosphopeptide control for the pT212 antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Inhibition of Microtubule Assembly by Dyrk1A-mediated Tau Phosphorylation—It is known that hyperphosphorylated Tau inhibits microtubule assembly, whereas normal Tau stimulates assembly and stabilizes the microtubule. We examined the extent to which Tau phosphorylation by Dyrk1A affects the ability of Tau in promoting microtubule assembly. Phosphorylation of Tau by Dyrk1A strongly inhibited microtubule assembly (Fig. 3). After incubation for 20 min, assembly of the samples incubated with Tau phosphorylated by Dyrk1A was inhibited by 80 ± 7% (n = 4, p < 0.01) when compared with that of control (no kinase). The extent of Dyrk1A-mediated inhibition was comparable with that of JNK2, a known Tau kinase that phosphorylates several Tau residues (28Yoshida H. Hastie C.J. McLauchlan H. Cohen P. Goedert M. J. Neurochem. 2004; 90: 352-358Crossref PubMed Scopus (140) Google Scholar). The strong inhibition of the microtubule assembly in the presence of Dyrk1A supports that Dyrk1A potentially phosphorylates multiple residues in Tau. Tau Phosphorylation by the Dyrk1A in Mammalian Cells—To determine whether Dyrk1A phosphorylates full-length Tau on Ser-202, Ser-404, and Thr-212 in vivo, a Western analysis was performed with lysates of human embryonic kidney (HEK293T) cells that had been transiently transfected with a full-length Tau expression plasmid, either in the presence or absence of a plasmid encoding mouse Dyrk1A (WT or Y321F). Although the amounts of Tau were similar, the amounts of pS202, pT212, and pS404 were strongly increased in cells expressing Dyrk1A WT, but not Dyrk1A Y321F (Fig. 4A), indicating that full-length Tau is phosphorylated by Dyrk1A in cells. The Ser-202, Thr-212, and Ser-404 of Tau were weakly phosphorylated in the absence of exogenous mouse Dyrk1A, potentially on account of the presence of endogenous Dyrk1A and other Tau-phosphorylating kinases, including JNK (28Yoshida H. Hastie C.J. McLauchlan H. Cohen P. Goedert M. J. Neurochem. 2004; 90: 352-358Crossref PubMed Scopus (140) Google Scholar). Furthermore, Y321F mutant, a dominant negative kinase-inactive mutant of Dyrk1A, may inhibit kinase activity of Dyrk1A by competing with endogenous Dyrk1A in HEK293T cells, resulting in further inhibition of Dyrk1A-mediated Tau phosphorylation, compared with that of Tau-transfected control (Fig. 4A). To confirm the above finding, HEK293T cells were co-transfected with Dyrk1A siRNA along with plasmids encoding the full-length mouse Dyrk1A and human Tau. A nonsilencing fluorescein isothiocyanate-conjugated GFP-specific siRNA was used as a negative control. After 24 h of treatment, Dyrk1A siRNA strongly inhibited expression of Dyrk1A. Correspondingly, the levels of pS202, pT212, and pS404 were also reduced, whereas Dyrk1A siRNA did not affect Tau expression (Fig. 4B). This result supports that Dyrk1A phosphorylates Tau on Ser-202, Thr-212, and Ser-404 in vivo. Specificity of each phospho-specific antibody was demonstrated by the peptide competition assay. Fig. 4C showed that the specific band signal in an immunoblot of mouse brain lysate was blocked only when the membrane was preincubated with the corresponding phosphopeptide, but not with the non-phosphopeptide or the unrelated phosphopeptide. Increase in the Amounts of Phos-pho-Ser-202 and Phospho-Ser-404 in the Hippocampus and Frontal Cortex of DYRK1A TG Mice—To determine the physiological significance of Tau phosphorylation at the Ser-202 and Ser-404 residues, DYRK1A TG mice were analyzed with immunohistochemistry and immunoblotting. Immunohistochemical analyses revealed that pS202 and pS404 were increased in the hippocampus and frontal cortex of DYRK1A TG mice relative to control littermates (Fig. 5). Strong immunohistochemical signals were observed in the neuronal cell bodies of the hippocampus or apical dendrites of the frontal cortex in DYRK1A TG mice relative to those of control mice. Increases in pS202, pT212, and pS404 were also observed in scattered neurons in the hippocampus and axonal tract in the corpus callosum of DYRK1A TG mice (Fig. 1A and Fig. 5). The results were corroborated by Western analysis of brain lysates prepared from the hippocampus or frontal cortex of DYRK1A TG mice and control littermates. Immunoblot analyses showed that after normalization with Tau, the amounts of pS202 and pS404 in the hippocampal lysates of DYRK1A TG mice were increased by 26 ± 9% (n = 11, p < 0.01) and 29 ± 7% (n = 9, p < 0.05) relative to that of control littermates, respectively (Fig. 6A). In addition, after normalization with Tau, the amounts of pS202 and pS404 in the frontal cortex of DYRK1A TG mice were also increased by 32 ± 4% (n = 3, p < 0.05) and 74 ± 6% (n = 3, p < 0.01) relative to that of control littermates, respectively (Fig. 6B). Taken together, these results demonstrate that phosphorylation of Tau at Ser-202 and Ser-404 is enhanced when the amount of DYRK1A is increased suggesting that overexpression of DYRK1A is functionally linked to Tau hyperphosphorylation in vivo. Interaction of Dyrk1A with Tau—To determine whether Tau is phosphorylated through the physical interaction with Dyrk1A, the brain lysates of DYRK1A TG mice were immunoprecipitated with anti-Tau-antibody. As shown in Fig. 6C, immunoprecipitation of Tau led to co-immunoprecipit" @default.
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• W2058968970 title "DYRK1A-mediated Hyperphosphorylation of Tau" @default.
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