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Isomerase Pin1 Stimulates Dephosphorylation of Tau Protein at Cyclin-dependent Kinase (Cdk5)-dependent Alzheimer Phosphorylation Sites*

Open AccessPublished:January 28, 2013DOI:https://doi.org/10.1074/jbc.M112.433326
      Neurodegenerative diseases associated with the pathological aggregation of microtubule-associated protein Tau are classified as tauopathies. Alzheimer disease, the most common tauopathy, is characterized by neurofibrillary tangles that are mainly composed of abnormally phosphorylated Tau. Similar hyperphosphorylated Tau lesions are found in patients with frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) that is induced by mutations within the tau gene. To further understand the etiology of tauopathies, it will be important to elucidate the mechanism underlying Tau hyperphosphorylation. Tau phosphorylation occurs mainly at proline-directed Ser/Thr sites, which are targeted by protein kinases such as GSK3β and Cdk5. We reported previously that dephosphorylation of Tau at Cdk5-mediated sites was enhanced by Pin1, a peptidyl-prolyl isomerase that stimulates dephosphorylation at proline-directed sites by protein phosphatase 2A. Pin1 deficiency is suggested to cause Tau hyperphosphorylation in Alzheimer disease. Up to the present, Pin1 binding was only shown for two Tau phosphorylation sites (Thr-212 and Thr-231) despite the presence of many more hyperphosphorylated sites. Here, we analyzed the interaction of Pin1 with Tau phosphorylated by Cdk5-p25 using a GST pulldown assay and Biacore approach. We found that Pin1 binds and stimulates dephosphorylation of Tau at all Cdk5-mediated sites (Ser-202, Thr-205, Ser-235, and Ser-404). Furthermore, FTDP-17 mutant Tau (P301L or R406W) showed slightly weaker Pin1 binding than non-mutated Tau, suggesting that FTDP-17 mutations induce hyperphosphorylation by reducing the interaction between Pin1 and Tau. Together, these results indicate that Pin1 is generally involved in the regulation of Tau hyperphosphorylation and hence the etiology of tauopathies.

      Introduction

      The neuropathological hallmarks of Alzheimer disease (AD)
      The abbreviations used are: AD
      Alzheimer disease
      Cdk
      cyclin-dependent kinase
      FTDP-17
      frontotemporal dementia with parkinsonism linked to chromosome 17
      Pin1
      peptidyl-prolyl cis/trans isomerase, NIMA-interacting 1
      GSK3β
      glycogen synthase kinase 3β
      PDPK
      proline-directed protein kinase
      PP2A
      protein phosphatase 2A
      Req
      response at equilibrium
      juglone
      5-hydroxy-1,4-naphthoquinone
      CBB
      Coomassie Brilliant Blue.
      include neurofibrillary tangles, which are composed mainly of abnormally phosphorylated microtubule-associated protein Tau (
      • Ballatore C.
      • Lee V.M.
      • Trojanowski J.Q.
      Tau-mediated neurodegeneration in Alzheimer's disease and related disorders.
      ). Aggregates of hyperphosphorylated Tau are also found in other neurodegenerative diseases that are collectively called tauopathies including Pick disease, progressive supranuclear palsy, corticobasal degeneration, and frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) (
      • Goedert M.
      • Jakes R.
      Mutations causing neurodegenerative tauopathies.
      ). FTDP-17 is an inherited form of tauopathy that is caused by mutations within the tau gene and is characterized by lesions containing hyperphosphorylated Tau (
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      ,
      • Poorkaj P.
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      • Raskind M.
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      Tau is a candidate gene for chromosome 17 frontotemporal dementia.
      ,
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      ). Genetically modified mice featuring the tau mutations of FTDP-17 developed similar aggregates of hyperphosphorylated Tau and showed dementia-like memory impairments, indicating a causative role of the tau mutations (
      • Goedert M.
      • Jakes R.
      Mutations causing neurodegenerative tauopathies.
      ,
      • Allen B.
      • Ingram E.
      • Takao M.
      • Smith M.J.
      • Jakes R.
      • Virdee K.
      • Yoshida H.
      • Holzer M.
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      • Atzori C.
      • Migheli A.
      • Crowther R.A.
      • Ghetti B.
      • Spillantini M.G.
      • Goedert M.
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      ,
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      • Kawarai T.
      • Kawarabayashi T.
      • Matsubara E.
      • Murakami T.
      • Sasaki A.
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      • Harigaya Y.
      • Abe K.
      • Okamoto K.
      • St George-Hyslop P.
      • Westaway D.
      Accumulation of filamentous tau in the cerebral cortex of human tau R406W transgenic mice.
      ). However, it is not yet known why these Tau mutations induce Tau aggregation and neurodegeneration. Understanding the molecular mechanisms that induce Tau hyperphosphorylation and aggregation in AD and FTDP-17 may be critical to unravel the processes underlying the etiology of tauopathies.
      Tau in neurofibrillary tangles is phosphorylated at more than 30 sites with most of them being located in the flanking regions of the microtubule-binding repeats (
      • Hanger D.P.
      • Anderton B.H.
      • Noble W.
      Tau phosphorylation: the therapeutic challenge for neurodegenerative disease.
      ,
      • Morishima-Kawashima M.
      • Hasegawa M.
      • Takio K.
      • Suzuki M.
      • Yoshida H.
      • Watanabe A.
      • Titani K.
      • Ihara Y.
      Hyperphosphorylation of tau in PHF.
      ,
      • Johnson G.V.
      • Stoothoff W.H.
      Tau phosphorylation in neuronal cell function and dysfunction.
      ). Many protein kinases have been implicated in Tau phosphorylation. Proline-directed protein kinases (PDPKs) such as glycogen synthase kinase 3β (GSK3β) and cyclin-dependent kinase 5 (Cdk5) have been thought to be critically involved in abnormal Tau phosphorylation because many proline-directed sites are hyperphosphorylated in Tau (
      • Goedert M.
      • Jakes R.
      Mutations causing neurodegenerative tauopathies.
      ,
      • Hanger D.P.
      • Anderton B.H.
      • Noble W.
      Tau phosphorylation: the therapeutic challenge for neurodegenerative disease.
      ,
      • Johnson G.V.
      • Stoothoff W.H.
      Tau phosphorylation in neuronal cell function and dysfunction.
      ,
      • Imahori K.
      • Uchida T.
      Physiology and pathology of tau protein kinases in relation to Alzheimer's disease.
      ,
      • Plattner F.
      • Angelo M.
      • Giese K.P.
      The roles of cyclin-dependent kinase 5 and glycogen synthase kinase 3 in tau hyperphosphorylation.
      ).
      Cdk5, originally purified as Tau kinase II (
      • Ishiguro K.
      • Takamatsu M.
      • Tomizawa K.
      • Omori A.
      • Takahashi M.
      • Arioka M.
      • Uchida T.
      Tau protein kinase I converts normal tau protein into A68-like component of paired helical filaments.
      ), is a serine/threonine kinase with pleiotropic functions in postmitotic neurons (
      • Dhavan R.
      • Tsai L.H.
      A decade of CDK5.
      ,
      • Hisanaga S.
      • Endo R.
      Regulation and role of cyclin-dependent kinase activity in neuronal survival and death.
      ). Cdk5 requires binding of the activation subunit, p35, for activation. The active holoenzyme Cdk5-p35 is localized to the cell membrane via the myristoylation of p35 (
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • de la Monte S.
      • Dikkes P.
      • Tsai L.H.
      Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration.
      ,
      • Asada A.
      • Yamamoto N.
      • Gohda M.
      • Saito T.
      • Hayashi N.
      • Hisanaga S.
      Myristoylation of p39 and p35 is a determinant of cytoplasmic or nuclear localization of active cyclin-dependent kinase 5 complexes.
      ,
      • Asada A.
      • Saito T.
      • Hisanaga S.
      Phosphorylation of p35 and p39 by Cdk5 determines the subcellular location of the holokinase in a phosphorylation-site-specific manner.
      ). Membrane-associated Cdk5-p35 exhibits moderate kinase activity due to a short half-life of p35, which is degraded by the proteasome (
      • Minegishi S.
      • Asada A.
      • Miyauchi S.
      • Fuchigami T.
      • Saito T.
      • Hisanaga S.
      Membrane association facilitates degradation and cleavage of the cyclin-dependent kinase 5 activators p35 and p39.
      ). Alternatively, p35 can be cleaved to p25 by calpain, and the Cdk5-p25 holoenzyme can subsequently relocalize to the cytoplasm and/or nucleus (
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • de la Monte S.
      • Dikkes P.
      • Tsai L.H.
      Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration.
      ,
      • Kusakawa G.
      • Saito T.
      • Onuki R.
      • Ishiguro K.
      • Kishimoto T.
      • Hisanaga S.
      Calpain-dependent proteolytic cleavage of the p35 cyclin-dependent kinase 5 activator to p25.
      ,
      • Lee M.S.
      • Kwon Y.T.
      • Li M.
      • Peng J.
      • Friedlander R.M.
      • Tsai L.H.
      Neurotoxicity induces cleavage of p35 to p25 by calpain.
      ). The Cdk5 activator, p25, has a long half-life (
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • de la Monte S.
      • Dikkes P.
      • Tsai L.H.
      Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration.
      ,
      • Lee M.S.
      • Kwon Y.T.
      • Li M.
      • Peng J.
      • Friedlander R.M.
      • Tsai L.H.
      Neurotoxicity induces cleavage of p35 to p25 by calpain.
      ) and induces aberrant Cdk5 activity toward Tau (
      • Cruz J.C.
      • Tseng H.C.
      • Goldman J.A.
      • Shih H.
      • Tsai L.H.
      Aberrant Cdk5 activation by p25 triggers pathological events leading to neurodegeneration and neurofibrillary tangles.
      ,
      • Noble W.
      • Olm V.
      • Takata K.
      • Casey E.
      • Mary O.
      • Meyerson J.
      • Gaynor K.
      • LaFrancois J.
      • Wang L.
      • Kondo T.
      • Davies P.
      • Burns M.
      • Veeranna
      • Nixon R.
      • Dickson D.
      • Matsuoka Y.
      • Ahlijanian M.
      • Lau L.F.
      • Duff K.
      Cdk5 is a key factor in tau aggregation and tangle formation in vivo.
      ). Consistently, silencing of Cdk5 reduced the phosphorylation of Tau in primary neuronal cultures and in brain and decreased the number of neurofibrillary tangles in the hippocampi of transgenic Alzheimer disease mice (
      • Piedrahita D.
      • Hernández I.
      • López-Tobón A.
      • Fedorov D.
      • Obara B.
      • Manjunath B.S.
      • Boudreau R.L.
      • Davidson B.
      • Laferla F.
      • Gallego-Gómez J.C.
      • Kosik K.S.
      • Cardona-Gómez G.P.
      Silencing of CDK5 reduces neurofibrillary tangles in transgenic Alzheimer's mice.
      ). However, it is not clear how Cdk5-p25 causes Tau hyperphosphorylation and aggregation.
      In FTDP-17 patients and transgenic mouse models, Tau is hyperphosphorylated (
      • Goedert M.
      • Jakes R.
      Mutations causing neurodegenerative tauopathies.
      ,
      • Hanger D.P.
      • Anderton B.H.
      • Noble W.
      Tau phosphorylation: the therapeutic challenge for neurodegenerative disease.
      ,
      • Johnson G.V.
      • Stoothoff W.H.
      Tau phosphorylation in neuronal cell function and dysfunction.
      ,
      • Imahori K.
      • Uchida T.
      Physiology and pathology of tau protein kinases in relation to Alzheimer's disease.
      ,
      • Lambourne S.L.
      • Sellers L.A.
      • Bush T.G.
      • Choudhury S.K.
      • Emson P.C.
      • Suh Y.H.
      • Wilkinson L.S.
      Increased tau phosphorylation on mitogen-activated protein kinase consensus sites and cognitive decline in transgenic models for Alzheimer's disease and FTDP-17: evidence for distinct molecular processes underlying tau abnormalities.
      ). In contrast, FTDP-17 mutant Tau is less phosphorylated than wild-type (WT) Tau in vitro or in cell cultures (
      • Matsumura N.
      • Yamazaki T.
      • Ihara Y.
      Stable expression in Chinese hamster ovary cells of mutated tau genes causing frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17).
      ,
      • Pérez M.
      • Lim F.
      • Arrasate M.
      • Avila J.
      The FTDP-17-linked mutation R406W abolishes the interaction of phosphorylated tau with microtubules.
      ,
      • Connell J.W.
      • Gibb G.M.
      • Betts J.C.
      • Blackstock W.P.
      • Gallo J.
      • Lovestone S.
      • Hutton M.
      • Anderton B.H.
      Effects of FTDP-17 mutations on the in vitro phosphorylation of tau by glycogen synthase kinase 3β identified by mass spectrometry demonstrate certain mutations exert long-range conformational changes.
      ,
      • Sakaue F.
      • Saito T.
      • Sato Y.
      • Asada A.
      • Ishiguro K.
      • Hasegawa M.
      • Hisanaga S.
      Phosphorylation of FTDP-17 mutant tau by cyclin-dependent kinase 5 complexed with p35, p25, or p39.
      ). These studies suggest that disruption of dephosphorylation rather than increased phosphorylation contributes to the hyperphosphorylated state of Tau. Accordingly, protein phosphatase 2A (PP2A) activity is decreased in AD brains (
      • Wang J.Z.
      • Gong C.X
      • Zaidi T.
      • Grundke-Iqbal I.
      • Iqbal K.
      Dephosphorylation of Alzheimer paired helical filaments by protein phosphatase-2A and -2B.
      ,
      • Sontag E.
      • Nunbhakdi-Craig V.
      • Lee G.
      • Bloom G.S.
      • Mumby M.C.
      Regulation of the phosphorylation state and microtubule-binding activity of Tau by protein phosphatase 2A.
      ,
      • Liang Z.
      • Liu F.
      • Iqbal K.
      • Grundke-Iqbal I.
      • Wegiel J.
      • Gong C.X.
      Decrease of protein phosphatase 2A and its association with accumulation and hyperphosphorylation of tau in Down syndrome.
      ), and highly phosphorylated Tau in paired helical filament is relatively resistant to dephosphorylation by PP2A (
      • Yamamoto H.
      • Hasegawa M.
      • Ono T.
      • Tashima K.
      • Ihara Y.
      • Miyamoto E.
      Dephosphorylation of fetal-tau and paired helical filaments-tau by protein phosphatases 1 and 2A and calcineurin.
      ). Furthermore, PP2A preferentially dephosphorylated phospho-(Ser/Thr)-Pro motifs in trans conformation when synthetic phospho-Thr-231 Tau peptide was used as a substrate (
      • Zhou X.Z.
      • Kops O.
      • Werner A.
      • Lu P.J.
      • Shen M.
      • Stoller G.
      • Küllertz G.
      • Stark M.
      • Fischer G.
      • Lu K.P.
      Pin1-dependent prolyl isomerization regulates dephosphorylation of Cdc25C and tau proteins.
      ,
      • Nakamura K.
      • Greenwood A.
      • Binder L.
      • Bigio E.H.
      • Denial S.
      • Nicholson L.
      • Zhou X.Z.
      • Lu K.P.
      Proline isomer-specific antibodies reveal the early pathogenic tau conformation in Alzheimer's disease.
      ). Peptidyl-prolyl cis/trans isomerase, NIMA-interacting 1 (Pin1) is a peptidylprolyl isomerase composed of two functional domains, the N-terminal WW domain, which binds to phosphorylated Ser or Thr at proline-directed sites, and the C-terminal cis/trans isomerase domain (
      • Lippens G.
      • Landrieu I.
      • Smet C.
      Molecular mechanisms of the phospho-dependent prolyl cis/trans isomerase Pin1.
      ,
      • Liou Y.C.
      • Zhou X.Z.
      • Lu K.P.
      Prolyl isomerase Pin1 as a molecular switch to determine the fate of phosphoproteins.
      ). Pin1 is found in neurofibrillary tangles, and Tau hyperphosphorylation is reported in Pin1-deficient mice (
      • Liou Y.C.
      • Sun A.
      • Ryo A.
      • Zhou X.Z.
      • Yu Z.X.
      • Huang H.K.
      • Uchida T.
      • Bronson R.
      • Bing G.
      • Li X.
      • Hunter T.
      • Lu K.P.
      Role of the prolyl isomerase Pin1 in protecting against age-dependent neurodegeneration.
      ). Hence, Pin1 could be a critical regulator of Tau dephosphorylation to (i) restore physiological Tau function such as microtubule binding and (ii) suppress neurofibrillary tangle formation by enhancing dephosphorylation by PP2A. We reported recently that Pin1 stimulates dephosphorylation of Tau phosphorylated by Cdk5-p25, suggesting that there are more Pin1 binding motifs in Tau (
      • Yotsumoto K.
      • Saito T.
      • Asada A.
      • Oikawa T.
      • Kimura T.
      • Uchida C.
      • Ishiguro K.
      • Uchida T.
      • Hasegawa M.
      • Hisanaga S.
      Effect of Pin1 or microtubule binding on dephosphorylation of FTDP-17 mutant tau.
      ). The Pin1 binding sites in Tau were shown to be phospho-Thr-231 (
      • Zhou X.Z.
      • Kops O.
      • Werner A.
      • Lu P.J.
      • Shen M.
      • Stoller G.
      • Küllertz G.
      • Stark M.
      • Fischer G.
      • Lu K.P.
      Pin1-dependent prolyl isomerization regulates dephosphorylation of Cdc25C and tau proteins.
      ,
      • Lu P.J.
      • Wulf G.
      • Zhou X.Z.
      • Davies P.
      • Lu K.P.
      The prolyl isomerase Pin1 restores the function of Alzheimer-associated phosphorylated tau protein.
      ) and phospho-Thr-212 (
      • Smet C.
      • Sambo A.V.
      • Wieruszeski J.M.
      • Leroy A.
      • Landrieu I.
      • Buée L.
      • Lippens G.
      The peptidyl prolyl cis/trans-isomerase Pin1 recognizes the phospho-Thr212-Pro213 site on Tau.
      ). However, these two Pin1 binding sites alone cannot prevent abnormal Tau phosphorylation at all the other hyperphosphorylation sites. Therefore, we wanted to assess Pin1 binding at additional Tau phosphorylation sites.
      Here we analyzed interaction of Pin1 with Cdk5-mediated Tau phosphorylation sites using a GST pulldown assay and Biacore technique. We observed that Pin1 binds to Tau and stimulates its dephosphorylation at all Cdk5 phosphorylation sites including Ser-202, Thr-205, Ser-235, and Ser-404. Furthermore, Tau carrying the FTDP-17 mutation P301L or R406W showed slightly weaker binding to Pin1 than WT Tau, suggesting that FTDP-17 mutations induce Tau hyperphosphorylation by reducing its interaction with Pin1.

      DISCUSSION

      In this study, we investigated the interaction of Pin1 with Cdk5-phosphorylated Tau and the effect on dephosphorylation at Cdk5 phosphorylation sites. Pin1 binds to Tau phosphorylated by Cdk5-p25 at any of its major phosphorylation sites, Ser-202, Thr-205, Ser-235, and Ser-404. The binding was slightly stronger to phospho-Ser-202 and -Thr-205 than to phospho-Ser-235 and -Ser-404. Pin1 facilitated the dephosphorylation of Tau at any of these sites. These are all abnormal phosphorylation sites found in AD (
      • Ballatore C.
      • Lee V.M.
      • Trojanowski J.Q.
      Tau-mediated neurodegeneration in Alzheimer's disease and related disorders.
      ,
      • Goedert M.
      • Jakes R.
      Mutations causing neurodegenerative tauopathies.
      ,
      • Lee V.M.
      • Goedert M.
      • Trojanowski J.Q.
      Neurodegenerative tauopathies.
      ). The FTDP-17 mutant Tau, P301L or R406W, showed slightly weaker binding to Pin1 than did WT Tau. These results support the idea that reduced Pin1-dependent dephosphorylation may underlie Tau hyperphosphorylation in tauopathies.
      Phospho-Thr-212 and -Thr-231 were the only sites that had been previously mapped as Pin1-interacting sites in Tau (
      • Liou Y.C.
      • Sun A.
      • Ryo A.
      • Zhou X.Z.
      • Yu Z.X.
      • Huang H.K.
      • Uchida T.
      • Bronson R.
      • Bing G.
      • Li X.
      • Hunter T.
      • Lu K.P.
      Role of the prolyl isomerase Pin1 in protecting against age-dependent neurodegeneration.
      ,
      • Lu P.J.
      • Wulf G.
      • Zhou X.Z.
      • Davies P.
      • Lu K.P.
      The prolyl isomerase Pin1 restores the function of Alzheimer-associated phosphorylated tau protein.
      ,
      • Smet C.
      • Sambo A.V.
      • Wieruszeski J.M.
      • Leroy A.
      • Landrieu I.
      • Buée L.
      • Lippens G.
      The peptidyl prolyl cis/trans-isomerase Pin1 recognizes the phospho-Thr212-Pro213 site on Tau.
      ,
      • Smet C.
      • Duckert J.F.
      • Wieruszeski J.M.
      • Landrieu I.
      • Buée L.
      • Lippens G.
      • Déprez B.
      Control of protein-protein interactions: structure-based discovery of low molecular weight inhibitors of the interactions between Pin1 WW domain and phosphopeptides.
      ). Phospho-Thr-231 was first identified by an ELISA as a site among many synthetic phospho-Tau peptides (
      • Lu P.J.
      • Wulf G.
      • Zhou X.Z.
      • Davies P.
      • Lu K.P.
      The prolyl isomerase Pin1 restores the function of Alzheimer-associated phosphorylated tau protein.
      ). Phospho-Thr-212 was subsequently found to be another Pin1-binding site among GSK3β phosphorylation sites (
      • Smet C.
      • Sambo A.V.
      • Wieruszeski J.M.
      • Leroy A.
      • Landrieu I.
      • Buée L.
      • Lippens G.
      The peptidyl prolyl cis/trans-isomerase Pin1 recognizes the phospho-Thr212-Pro213 site on Tau.
      ). Although the latter authors suggested that Thr-212 and Thr-231 are not unique as Pin1-binding sites based on the observation that Tau mutants at Thr-212 and Thr-231 still bound to Pin1 after phosphorylation in COS-7 cells (
      • Smet C.
      • Sambo A.V.
      • Wieruszeski J.M.
      • Leroy A.
      • Landrieu I.
      • Buée L.
      • Lippens G.
      The peptidyl prolyl cis/trans-isomerase Pin1 recognizes the phospho-Thr212-Pro213 site on Tau.
      ), no other sites have been reported. We show here that there are at least four additional Pin1-binding phosphorylation sites in Tau at Ser-202, Thr-205, Ser-235, and Ser-404.
      The binding of Pin1 to Tau at the Alzheimer-related phosphorylation site AT8, whose epitope is generated by phosphorylation of Tau at sites including Ser-202 and Thr-205 (
      • Goedert M.
      • Jakes R.
      • Vanmechelen E.
      Monoclonal antibody AT8 recognises tau protein phosphorylated at both serine 202 and threonine 205.
      ,
      • Shahpasand K.
      • Uemura I.
      • Saito T.
      • Asano T.
      • Hata K.
      • Shibata K.
      • Toyoshima Y.
      • Hasegawa M.
      • Hisanaga S.
      Regulation of mitochondrial transport and inter-microtubule spacing by tau phosphorylation at the sites hyperphosphorylated in Alzheimer's disease.
      ), has been reported recently in rat cortical neurons (
      • Nykänen N.P.
      • Kysenius K.
      • Sakha P.
      • Tammela P.
      • Huttunen H.J.
      γ-Aminobutyric acid type A (GABAA) receptor activation modulates tau phosphorylation.
      ); this finding is consistent with our present results. However, we do not know why these sites were not detected as Pin1-binding sites in previous work. It might be simply that no study has focused on Cdk5 phosphorylation sites, but we think there might be some mismatching between the protein kinases used and the phosphorylation sites examined. There are 16 (Ser/Thr)-Pro sequences in the longest isoform of Tau (
      • Goedert M.
      • Jakes R.
      Mutations causing neurodegenerative tauopathies.
      ,
      • Hanger D.P.
      • Anderton B.H.
      • Noble W.
      Tau phosphorylation: the therapeutic challenge for neurodegenerative disease.
      ,
      • Johnson G.V.
      • Stoothoff W.H.
      Tau phosphorylation in neuronal cell function and dysfunction.
      ), many of which are phosphorylated distinctly and with an overlap by different PDPKs and are not necessarily phosphorylated stoichiometrically. Some of them are minor phosphorylation sites that can be detected only with highly sensitive phosphospecific antibodies. In the present study, we used Cdk5-p25, which has a substrate specificity similar to that of Cdk1 toward Tau (
      • Imahori K.
      • Uchida T.
      Physiology and pathology of tau protein kinases in relation to Alzheimer's disease.
      ,
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      The endogenous and cell cycle-dependent phosphorylation of tau protein in living cells: implications for Alzheimer's disease.
      ,
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      • Itoh T.J.
      • Uchida T.
      • Hotani H.
      • Saito T.
      • Kishimoto T.
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      Microtubule-stimulated phosphorylation of tau at Ser202 and Thr205 by cdk5 decreases its microtubule nucleation activity.
      ). Having determined all major Cdk5 phosphorylation sites by a combination of phosphopeptide mapping and site-specific mutagenesis, we have shown that all major Cdk5 phosphorylation sites are also newly identified Pin1-binding sites. Extending this approach to other (Ser/Thr)-Pro sites could identify more Pin1-binding sites.
      The dissociation constant Kd of Pin1 to Cdk5-phosphorylated full-length Tau was measured as 1.11 ± 0.14 × 10−4 m using Biacore technology. Two different Kd values have been reported for the binding between Pin1 and phosphorylated Tau or a phosphorylated Tau peptide: ∼40 nm with the phospho-Thr-231 peptide when measured by an ELISA (
      • Lu P.J.
      • Wulf G.
      • Zhou X.Z.
      • Davies P.
      • Lu K.P.
      The prolyl isomerase Pin1 restores the function of Alzheimer-associated phosphorylated tau protein.
      ) and 3.8 ± 1.0 × 10−4 m at phospho-Thr-231 or 1.0 ± 0.3 × 10−4 m at phospho-Thr-212 by NMR (
      • Smet C.
      • Sambo A.V.
      • Wieruszeski J.M.
      • Leroy A.
      • Landrieu I.
      • Buée L.
      • Lippens G.
      The peptidyl prolyl cis/trans-isomerase Pin1 recognizes the phospho-Thr212-Pro213 site on Tau.
      ). Our Kd value is close to the values measured by NMR. Furthermore, a similar Kd value, ∼1 × 10−4 m, was reported for Csc25, a well known Pin1-binding protein (
      • Wintjens R.
      • Wieruszeski J.M.
      • Drobecq H.
      • Rousselot-Pailley P.
      • Buée L.
      • Lippens G.
      • Landrieu I.
      1H NMR study on the binding of Pin1 Trp-Trp domain with phosphothreonine peptides.
      ). This Kd is relatively weak compared with many other protein-protein interactions. Nevertheless, GST pulldown assay worked well for both Cdc25 and Tau (
      • Crenshaw D.G.
      • Yang J.
      • Means A.R.
      • Kornbluth S.
      The mitotic peptidyl-prolyl isomerase, Pin1, interacts with Cdc25 and Plx1.
      ). The affinity of phospho-(Ser/Thr)-Pro sequences for Pin1 appears to be affected by various experimental conditions including the methods of measurement, the ionic strength of the solution, the number of phosphorylation sites, the use of phosphopeptides versus phosphorylated full-length proteins, and the WW domain of Pin1 versus full-length Pin1. We used phosphorylated full-length Tau instead of phosphopeptides, which may explain the low binding affinity. However, the weak affinity measured in vitro does not necessarily mean that the binding does not occur in cells. Tau is highly concentrated in the vicinity of microtubules. Furthermore, in vivo Tau, particularly in brains with tauopathy, is phosphorylated at multiple (Ser/Thr)-Pro sites by multiple PDPKs, which may increase the binding affinity for Pin1. When highly phosphorylated, Tau may become a better target for Pin1, which may act on highly phosphorylated Tau in predisease conditions to prevent further phosphorylation. In other words, decreased Pin1 activity could be a consequence of Tau hyperphosphorylation. It has been reported that Pin1 is reduced in AD brains (
      • Lu P.J.
      • Wulf G.
      • Zhou X.Z.
      • Davies P.
      • Lu K.P.
      The prolyl isomerase Pin1 restores the function of Alzheimer-associated phosphorylated tau protein.
      ). We also examined the Pin1 level in an FTDP-17 brain and P301L transgenic mouse brains but could not detect differences between normal and patient or transgenic mouse brains (Fig. 6A and supplemental Fig. 5). We think that there must be other unknown factors that reduce the Pin1 activity in FTDP-17 brains.
      The binding affinity also depends on the phospho-(Ser/Thr)-Pro sequences. For example, Smad2/3 has four Pin1-binding (Ser/Thr)-Pro sequences in the linker region, which can be phosphorylated by Cdk and MAPK family members but with strong binding at only one site (
      • Nakano A.
      • Koinuma D.
      • Miyazawa K.
      • Uchida T.
      • Saitoh M.
      • Kawabata M.
      • Hanai J.
      • Akiyama H.
      • Abe M.
      • Miyazono K.
      • Matsumoto T.
      • Imamura T.
      Pin1 down-regulates transforming growth factor-β (TGF-β) signaling by inducing degradation of Smad proteins.
      ,
      • Matsuura I.
      • Chiang K.N.
      • Lai C.Y.
      • He D.
      • Wang G.
      • Ramkumar R.
      • Uchida T.
      • Ryo A.
      • Lu K.
      • Liu F.
      Pin1 promotes transforming growth factor-β-induced migration and invasion.
      ). In the case of Tau, phospho-Thr-212 has a lower Kd than phospho-Thr-231 (
      • Smet C.
      • Sambo A.V.
      • Wieruszeski J.M.
      • Leroy A.
      • Landrieu I.
      • Buée L.
      • Lippens G.
      The peptidyl prolyl cis/trans-isomerase Pin1 recognizes the phospho-Thr212-Pro213 site on Tau.
      ,
      • Smet C.
      • Duckert J.F.
      • Wieruszeski J.M.
      • Landrieu I.
      • Buée L.
      • Lippens G.
      • Déprez B.
      Control of protein-protein interactions: structure-based discovery of low molecular weight inhibitors of the interactions between Pin1 WW domain and phosphopeptides.
      ). We also observed here that the Cdk5 phosphorylation sites phospho-Ser-202 and -Thr-205 showed slightly stronger binding to Pin1 than did phospho-Ser-235 and -Ser-404. However, the correlation between Pin1 binding and dephosphorylation is not clear. The phosphorylation site grasped by the WW domain can be attacked by peptidylprolyl isomerase activity in the same Pin1 molecule (
      • Lippens G.
      • Landrieu I.
      • Smet C.
      Molecular mechanisms of the phospho-dependent prolyl cis/trans isomerase Pin1.
      ,
      • Liou Y.C.
      • Zhou X.Z.
      • Lu K.P.
      Prolyl isomerase Pin1 as a molecular switch to determine the fate of phosphoproteins.
      ). Ser-202 with strong binding to Pin1 was dephosphorylated faster than was the weaker Ser-404 binding site. However, Ser-235, the weakest binding site, was dephosphorylated as fast as the stronger binding site Ser-202. By contrast, Pin1-binding sites are not always the sites whose dephosphorylation is stimulated by Pin1. It has been reported that peptidylprolyl isomerase catalyzes a different phosphorylation site close to the WW domain-binding site when the target protein is phosphorylated at multiple sites (
      • Daum S.
      • Fanghänel J.
      • Wildemann D.
      • Schiene-Fischer C.
      Thermodynamics of phosphopeptide binding to the human peptidyl prolyl cis/trans isomerase Pin1.
      ,
      • Smet C.
      • Duckert J.F.
      • Wieruszeski J.M.
      • Landrieu I.
      • Buée L.
      • Lippens G.
      • Déprez B.
      Control of protein-protein interactions: structure-based discovery of low molecular weight inhibitors of the interactions between Pin1 WW domain and phosphopeptides.
      ). Tau is a multiphosphorylated protein with many clusters of (Ser/Thr)-Pro sequences. Previous research has also identified hierarchical phosphorylation by which GSK3β phosphorylation is facilitated by priming the phosphorylation by Cdk5 or PKA (
      • Ishiguro K.
      • Takamatsu M.
      • Tomizawa K.
      • Omori A.
      • Takahashi M.
      • Arioka M.
      • Uchida T.
      Tau protein kinase I converts normal tau protein into A68-like component of paired helical filaments.
      ,
      • Ishiguro K.
      • Sato K.
      • Takamatsu M.
      • Park J.
      • Uchida T.
      • Imahori K.
      Analysis of phosphorylation of tau with antibodies specific for phosphorylation sites.
      ,
      • Pei J.J.
      • Grundke-Iqbal I.
      • Iqbal K.
      • Bogdanovic N.
      • Winblad B.
      • Cowburn R.F.
      Accumulation of cyclin-dependent kinase 5 (cdk5) in neurons with early stages of Alzheimer's disease neurofibrillary degeneration.
      ,
      • Michel G.
      • Mercken M.
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      • Noguchi K.
      • Ishiguro K.
      • Imahori K.
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      Characterization of tau phosphorylation in glycogen synthase kinase-3β and cyclin dependent kinase-5 activator (p23) transfected cells.
      ). For example, Ser-404 phosphorylation is thought to be a priming site for Ser-400 and Ser-396 phosphorylation by GSK3β. The binding of Pin1 to phospho-Ser-404 may stimulate isomerization and then dephosphorylation at the Ser-396 GSK3β phosphorylation site. A similar relationship has been observed between the Ser-235 Cdk5 site and the Thr-231 GSK3β site (
      • Ishiguro K.
      • Kobayashi S.
      • Omori A.
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      • Anzai K.
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      • Uchida T.
      Identification of the 23 kDa subunit of tau protein kinase II as a putative activator of cdk5 in bovine brain.
      ).
      FTDP-17 mutant Tau is hyperphosphorylated in brains of patients, but it is not known why. The phosphorylation levels of several FTDP-17 Tau mutants are similar to those of WT Tau when examined in cultured cells or in vitro (
      • Matsumura N.
      • Yamazaki T.
      • Ihara Y.
      Stable expression in Chinese hamster ovary cells of mutated tau genes causing frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17).
      ,
      • Pérez M.
      • Lim F.
      • Arrasate M.
      • Avila J.
      The FTDP-17-linked mutation R406W abolishes the interaction of phosphorylated tau with microtubules.
      ,
      • Connell J.W.
      • Gibb G.M.
      • Betts J.C.
      • Blackstock W.P.
      • Gallo J.
      • Lovestone S.
      • Hutton M.
      • Anderton B.H.
      Effects of FTDP-17 mutations on the in vitro phosphorylation of tau by glycogen synthase kinase 3β identified by mass spectrometry demonstrate certain mutations exert long-range conformational changes.
      ,
      • Sakaue F.
      • Saito T.
      • Sato Y.
      • Asada A.
      • Ishiguro K.
      • Hasegawa M.
      • Hisanaga S.
      Phosphorylation of FTDP-17 mutant tau by cyclin-dependent kinase 5 complexed with p35, p25, or p39.
      ,
      • DeTure M.
      • Ko L.W.
      • Easson C.
      • Yen S.H.
      Tau assembly in inducible transfectants expressing wild-type or FTDP-17 tau.
      ). R406W Tau shows even less phosphorylation by Cdk5 (
      • Sakaue F.
      • Saito T.
      • Sato Y.
      • Asada A.
      • Ishiguro K.
      • Hasegawa M.
      • Hisanaga S.
      Phosphorylation of FTDP-17 mutant tau by cyclin-dependent kinase 5 complexed with p35, p25, or p39.
      ) probably because of mutation of Arg-406 to Trp, which changes the site to an unfavorable Cdk5 target sequence, S404PWH from S404PRH. We showed previously that P301L and R406W mutants do not show Pin1 dependence in dephosphorylation at the Cdk5 phosphorylation sites (
      • Yotsumoto K.
      • Saito T.
      • Asada A.
      • Oikawa T.
      • Kimura T.
      • Uchida C.
      • Ishiguro K.
      • Uchida T.
      • Hasegawa M.
      • Hisanaga S.
      Effect of Pin1 or microtubule binding on dephosphorylation of FTDP-17 mutant tau.
      ). We thought that Pin1 might not recognize the P301L or R406W Tau mutant phosphorylated by Cdk5, but this turned out not to be the case. At a physiological salt concentration, Pin1 bound to Cdk5-phosphorylated P301L and R406W Tau to the same extent as did WT Tau. By contrast, the protein stability of P301L Tau, but not WT Tau, was decreased in Pin1 knockdown or knock-out neurons, suggesting that the P301L mutation affects the interaction of Tau with Pin1 (
      • Lim J.
      • Balastik M.
      • Lee T.H.
      • Nakamura K.
      • Liou Y.C.
      • Sun A.
      • Finn G.
      • Pastorino L.
      • Lee V.M.
      • Lu K.P.
      Pin1 has opposite effects on wild-type and P301L tau stability and tauopathy.
      ). The binding of Pin1 to both P301L and R406W Tau was slightly weaker than that to WT Tau when the binding was measured in the presence of a higher salt concentration. The slight difference in the binding affinity might be increased in the cellular condition by an unidentified factor. Further detailed biochemical studies are required to understand the Pin1-dependent regulation of Tau phosphorylation.
      We found that Pin1 binds to and stimulates dephosphorylation of Tau at Cdk5-mediated sites Ser-202, Thr-205, Ser-235, and Ser-404. All of these sites conform at least in part to well known Tau hyperphosphorylation epitopes in AD (Ser-202 and Thr-205 for AT8, Ser-235 for AT180, and Ser-404 for PHF1). An involvement of Pin1 in the Tau hyperphosphorylation in AD has been suggested (
      • Liou Y.C.
      • Sun A.
      • Ryo A.
      • Zhou X.Z.
      • Yu Z.X.
      • Huang H.K.
      • Uchida T.
      • Bronson R.
      • Bing G.
      • Li X.
      • Hunter T.
      • Lu K.P.
      Role of the prolyl isomerase Pin1 in protecting against age-dependent neurodegeneration.
      ), but the two Pin1-binding sites identified so far could not explain the many other abnormally phosphorylated Tau sites. Our results identify novel Pin1-binding phosphorylation sites in Tau and characterize them as Cdk5-specific sites. Therefore, thorough analysis of the remaining (Ser/Thr)-Pro sites phosphorylated by other PDPKs may indeed reveal to what extent Pin1 can contribute to abnormal Tau phosphorylation in tauopathies.

      Acknowledgments

      We thank Junichiro Suzuki and Yutaka Sato for statistical data analysis.

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