title: Quantitative and combinatory determination of in situ phosphorylation of tau and its FTDP-17 mutants.
abstract: Tau is hyperphosphorylated in the brains of patients with tauopathies, such as Alzheimer's disease and frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). However, neither the mechanism of hyperphosphorylation nor its contribution to pathogenesis is known. We applied Phos-tag SDS-PAGE, a phosphoaffinity electrophoresis, to the analysis of tau phosphorylation in vitro by Cdk5, in cultured cells and in mouse brain. Here, we found that Cdk5-p25 phosphorylated tau in vitro at Ser404, Ser235, Thr205 and Ser202 in this order. In contrast in cultured cells, Ser404 was preferentially phosphorylated by Cdk5-p35, whereas Thr205 was not phosphorylated. Ser202 and Ser235 were phosphorylated by endogenous kinases. Tau exhibited ~12 phosphorylation isotypes in COS-7 cells with different combinations of phosphorylation at Thr181, Ser202, Thr231, Ser235 and Ser404. These phosphorylation sites were similar to tau phosphorylated in mouse brains. FTDP-17 tau with a mutation in the C-terminal region had different banding patterns, indicating a different phosphorylation pattern. In particular, it was clear that the R406W mutation causes loss of Ser404 phosphorylation. These results demonstrate the usefulness of the Phos-tag technique in the quantitative analysis of site-specific in vivo phosphorylation of tau and provide detailed information on in situ combinatory phosphorylation of tau.
authors.affiliation: Laboratory of Molecular Neuroscience, Department of Biological Sciences, Tokyo Metropolitan University, Hachioji, Tokyo 192-0397, Japan.
authors.forename: Taeko
authors.initials: T
authors.lastname: Kimura
authors.affiliation: Laboratory of Molecular Neuroscience, Department of Biological Sciences, Tokyo Metropolitan University, Hachioji, Tokyo 192-0397, Japan.
authors.forename: Tomohisa
authors.initials: T
authors.lastname: Hosokawa
authors.affiliation: Department of Chemistry, Tokyo Metropolitan University, Hachioji, Tokyo 192-0397, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Taoka
authors.affiliation: Laboratory of Molecular Neuroscience, Department of Biological Sciences, Tokyo Metropolitan University, Hachioji, Tokyo 192-0397, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Tsutsumi
authors.affiliation: Laboratory of Molecular Neuroscience, Department of Biological Sciences, Tokyo Metropolitan University, Hachioji, Tokyo 192-0397, Japan.
authors.forename: Kanae
authors.initials: K
authors.lastname: Ando
authors.affiliation: Juntendo University, Tokyo, 113-0033, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Ishiguro
authors.affiliation: Tokyo Metropolitan Institute of Medical Science, Setagaya, Tokyo 156-8506, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Hosokawa
authors.affiliation: Tokyo Metropolitan Institute of Medical Science, Setagaya, Tokyo 156-8506, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Hasegawa
authors.affiliation: Laboratory of Molecular Neuroscience, Department of Biological Sciences, Tokyo Metropolitan University, Hachioji, Tokyo 192-0397, Japan.
authors.forename: Shin-Ichi
authors.initials: S
authors.lastname: Hisanaga
mesh_terms: D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism / Q000473:pathology; D019556:COS Cells; D002522:Chlorocebus aethiops; D051360:Cyclin-Dependent Kinase 5 / Q000378:metabolism; D057180:Frontotemporal Dementia / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D010766:Phosphorylation; D016875:tau Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D016875:tau Proteins; D051360:Cyclin-Dependent Kinase 5; C495905:CDK5 protein, human; D000409:Alanine