title: Global analysis of phosphorylation of tau by the checkpoint kinases Chk1 and Chk2 in vitro.
abstract: Hyperphosphorylation of microtubule-associated protein tau is thought to contribute to Alzheimer's disease (AD) pathogenesis. We previously showed that DNA damage-activated cell cycle checkpoint kinases Chk1 and Chk2 phosphorylate tau at an AD-related site and enhance tau toxicity, suggesting potential roles of these kinases in AD. The purpose of this study is to systematically identify which sites in tau are directly phosphorylated by Chk1 and Chk2. Using recombinant human tau phosphorylated by Chk1 and Chk2 in vitro, we first analyzed tau phosphorylation at the AD-related sites by Western blot with phospho-tau-specific antibodies. Second, to globally identify phosphorylated sites in tau, liquid chromatography-tandem mass spectrometry (LC-MS(3)) was employed. These systematic analyses identified a total of 27 Ser/Thr residues as Chk1- or Chk2- target sites. None of them were proline-directed kinase targets. Many of these sites are located within the microtubule-binding domain and C-terminal domain, whose phosphorylation has been shown to reduce tau binding to microtubules and/or has been implicated in tau toxicity. Among these 27 sites, 13 sites have been identified to be phosphorylated in AD brains. Since DNA damage is accumulated in diseased brains, Chk1 and Chk2 may be involved in tau phosphorylation and toxicity in AD pathogenesis.
authors.affiliation: Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, 1425 Madison Avenue, Box 1498, New York, New York 10029, United States.
authors.forename: Jhoana
authors.initials: J
authors.lastname: Mendoza
authors.forename: Michiko
authors.initials: M
authors.lastname: Sekiya
authors.forename: Taizo
authors.initials: T
authors.lastname: Taniguchi
authors.forename: Koichi M
authors.initials: KM
authors.lastname: Iijima
authors.forename: Rong
authors.initials: R
authors.lastname: Wang
authors.forename: Kanae
authors.initials: K
authors.lastname: Ando
mesh_terms: D000544:Alzheimer Disease / Q000378:metabolism / Q000473:pathology; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2 / Q000378:metabolism*; D002853:Chromatography, Liquid; D004249:DNA Damage; D006801:Humans; D008870:Microtubules / Q000378:metabolism / Q000473:pathology; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D016875:tau Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D016875:tau Proteins; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1
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