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Casein Kinase 1 Delta Phosphorylates Tau and Disrupts Its Binding to Microtubules*

  • Guibin Li
    Affiliations
    Center for Molecular Neurobiology, Ohio State University College of Medicine and Public Health, Columbus, Ohio 43210
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  • Haishan Yin
    Affiliations
    Ohio State Biochemistry Program, Ohio State University College of Medicine and Public Health, Columbus, Ohio 43210
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  • Jeff Kuret
    Correspondence
    To whom correspondence should be addressed: Center for Neurobiotechnology, 1060 Carmack Rd., Columbus, OH 43210. Tel.: 614-688-5899; Fax: 614-292-5379;
    Affiliations
    Center for Molecular Neurobiology, Ohio State University College of Medicine and Public Health, Columbus, Ohio 43210

    Ohio State Biochemistry Program, Ohio State University College of Medicine and Public Health, Columbus, Ohio 43210

    Department of Molecular and Cellular Biochemistry, Ohio State University College of Medicine and Public Health, Columbus, Ohio 43210
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grant AG14452 (to J. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:February 02, 2004DOI:https://doi.org/10.1074/jbc.M314116200
      Tau hyperphosphorylation precedes neuritic lesion formation in Alzheimer's disease, suggesting it participates in the tau fibrillization reaction pathway. Candidate tau protein kinases include members of the casein kinase 1 (CK1) family of phosphotransferases, which are highly overexpressed in Alzheimer's disease brain and colocalize with neuritic and granulovacuolar lesions. Here we characterized the contribution of one CK1 isoform, Ckiδ, to the phosphorylation of tau at residues Ser202/Thr205 and Ser396/Ser404 in human embryonic kidney 293 cells using immunodetection and fluorescence microscopy. Treatment of cells with membrane permeable CK1 inhibitor 3-[(2,3,6-trimethoxyphenyl)methylidenyl]-indolin-2-one (IC261) lowered occupancy of Ser396/Ser404 phosphorylation sites by >70% at saturation, suggesting that endogenous CK1 was the major source of basal phosphorylation activity at these sites. Overexpression of Ckiδ increased CK1 enzyme activity and further raised tau phosphorylation at residues Ser202/Thr205 and Ser396/Ser404in situ. Inhibitor IC261 reversed tau hyperphosphorylation induced by Ckiδ overexpression. Co-immunoprecipitation assays showed direct association of tau and Ckiδ in situ, consistent with tau being a Ckiδ substrate. Ckiδ overexpression also produced a decrease in the fraction of bulk tau bound to detergent-insoluble microtubules. These results suggest that Ckiδ phosphorylates tau at sites that modulate tau/microtubule binding, and that the expression pattern of Ckiδ in Alzheimer's disease is consistent with it playing an important role in tau aggregation.
      AD
      The abbreviations used are: AD, Alzheimer's disease; Cdk5, cyclin-dependent kinase 5; CK1, casein kinase 1; Ckiδ-Δ317, C-terminal truncated Ckiδ; GSK3β, glycogen synthase kinase-3β; His6, polyhistidine tag; HEK, human embryonic kidney; IC261, 3-[(2,3,6-trimethoxyphenyl)methylidenyl]-indolin-2-one; siRNA, small interfering RNA; PIPES, 1,4-piperazinediethanesulfonic acid.
      1The abbreviations used are: AD, Alzheimer's disease; Cdk5, cyclin-dependent kinase 5; CK1, casein kinase 1; Ckiδ-Δ317, C-terminal truncated Ckiδ; GSK3β, glycogen synthase kinase-3β; His6, polyhistidine tag; HEK, human embryonic kidney; IC261, 3-[(2,3,6-trimethoxyphenyl)methylidenyl]-indolin-2-one; siRNA, small interfering RNA; PIPES, 1,4-piperazinediethanesulfonic acid.
      is a progressive neurodegenerative disease characterized in part by the appearance of neurofibrillary tangles, neuritic plaques, and neurophil threads (
      • Buee L.
      • Bussiere T.
      • Buee-Scherrer V.
      • Delacourte A.
      • Hof P.R.
      ). Each manifestation of neuritic pathology is comprised of tau protein aggregated into filaments. Because the spatial distribution of tau fibrillization appears stereotypically, correlates with neuronal cell loss, and parallels cognitive decline, it is a useful marker of and may contribute to degeneration in AD and other dementias.
      Tau fibrillization is accompanied by extensive phosphorylation on over 25 distinct sites (
      • Hanger D.P.
      • Betts J.C.
      • Loviny T.L.
      • Blackstock W.P.
      • Anderton B.H.
      ,
      • Morishima-Kawashima M.
      • Hasegawa M.
      • Takio K.
      • Suzuki M.
      • Yoshida H.
      • Watanabe A.
      • Titani K.
      • Ihara Y.
      ), some of which function to modulate the ability of tau to bind and stabilize microtubules (
      • Bramblett G.T.
      • Goedert M.
      • Jakes R.
      • Merrick S.E.
      • Trojanowski J.Q.
      • Lee V.M.
      ,
      • Wagner U.
      • Utton M.
      • Gallo J.
      • Miller C.
      ). Candidate enzymes for catalyzing the hyperphosphorylation of tau protein in disease include members of the CK1 family of protein kinases, which in mammals consist of at least seven isoforms derived from distinct genes including Ckiα, β, γ1, γ2, γ3, δ, and ϵ (
      • Fish K.J.
      • Cegielska A.
      • Getman M.E.
      • Landes G.M.
      • Virshup D.M.
      ,
      • Graves P.
      • Haas D.
      • Hagedorn C.
      • DePaoli-Roach A.
      • Roach P.
      ,
      • Rowles J.
      • Slaughter C.
      • Moomaw C.
      • Hsu J.
      • Cobb M.H.
      ,
      • Zhai L.
      • Graves P.R.
      • Robinson L.C.
      • Italiano M.
      • Culbertson M.R.
      • Rowles J.
      • Cobb M.H.
      • DePaoli-Roach A.A.
      • Roach P.J.
      ,
      • Kitabayashi A.N.
      • Kusuda J.
      • Hirai M.
      • Hashimoto K.
      ). Each isoform shares a highly conserved N-terminal catalytic domain (
      • Xu R.M.
      • Carmel G.
      • Sweet R.M.
      • Kuret J.
      • Cheng X.
      ) with diverse C-terminal domains of variable lengths. CK1 family members phosphorylate cytoskeletal proteins such as spectrin, troponin, myosin, ankyrin, tau, and α-synuclein (
      • Simkowski K.
      • Tao M.
      ,
      • Singh T.
      • Grundke-Iqbal I.
      • Iqbal K.
      ,
      • Behrend L.
      • Milne D.M.
      • Stoter M.
      • Deppert W.
      • Campbell L.E.
      • Meek D.W.
      • Knippschild U.
      ,
      • Okochi M.
      • Walter J.
      • Koyama A.
      • Nakajo S.
      • Baba M.
      • Iwatsubo T.
      • Meijer L.
      • Kahle P.J.
      • Haass C.
      ,
      • Behrend L.
      • Stoter M.
      • Kurth M.
      • Rutter G.
      • Heukeshoven J.
      • Deppert W.
      • Knippschild U.
      ,
      • Singh T.J.
      • Zaidi T.
      • Grundke-Iqbal I.
      • Iqbal K.
      ), but also non-cytoskeletal proteins such as SV40 T antigen (
      • Cegielska A.
      • Gietzen K.F.
      • Rivers A.
      • Virshup D.M.
      ), p53 (
      • Behrend L.
      • Milne D.M.
      • Stoter M.
      • Deppert W.
      • Campbell L.E.
      • Meek D.W.
      • Knippschild U.
      ,
      • Knippschild U.
      • Milne D.M.
      • Campbell L.E.
      • DeMaggio A.J.
      • Christenson E.
      • Hoekstra M.F.
      • Meek D.W.
      ), and β-catenin (
      • Marin O.
      • Bustos V.H.
      • Cesaro L.
      • Meggio F.
      • Pagano M.A.
      • Antonelli M.
      • Allende C.C.
      • Pinna L.A.
      • Allende J.E.
      ). Phosphorylation of these substrates is thought to modulate diverse physiological functions such as vesicular trafficking, DNA repair, cell cycle kinetics, and cell division (
      • Gross S.
      • Simerly C.
      • Schatten G.
      • Anderson R.
      ).
      CK1 levels appear to be regulated within the cell. For example, Ckiδ levels are elevated in both AD brain (
      • Ghoshal N.
      • Smiley J.F.
      • DeMaggio A.J.
      • Hoekstra M.F.
      • Cochran E.J.
      • Binder L.I.
      • Kuret J.
      ,
      • Yasojima K.
      • Kuret J.
      • DeMaggio A.J.
      • McGeer E.
      • McGeer P.L.
      ) and the spinal cords of a murine model of amyotropic lateral sclerosis (
      • Hu J.H.
      • Chernoff K.
      • Pelech S.
      • Krieger C.
      ). Moreover, isoforms Ckiα and Ckiδ associate with the neurofibrillary lesions of AD (
      • Kuret J.
      • Johnson G.S.
      • Cha D.
      • Christenson E.R.
      • DeMaggio A.J.
      • Hoekstra M.F.
      ,
      • Schwab C.
      • DeMaggio A.J.
      • Ghoshal N.
      • Binder L.I.
      • Kuret J.
      • McGeer P.L.
      ), the pathological hallmarks in AD and other tauopathic neurofibrillary degenerations diseases. These observations led to the hypothesis that CK1 enzymes regulate tau phosphorylation in vivo, and that their up-regulation in level or activity contributes to tau hyperphosphorylation in disease (
      • Ghoshal N.
      • Smiley J.F.
      • DeMaggio A.J.
      • Hoekstra M.F.
      • Cochran E.J.
      • Binder L.I.
      • Kuret J.
      ).
      Here we test this hypothesis by examining the ability of one CK1 isoform, Ckiδ, to phosphorylate tau under basal and overexpression conditions in situ. The data suggests that CK1 activity makes a major contribution to basal levels of tau phosphorylation, and that Ckiδ can modulate microtubule stability by direct interaction with tau protein.

      EXPERIMENTAL PROCEDURES

      Materials—Polyclonal antibody against the C terminus of p35 (C-19) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), whereas monoclonal antibodies AT8, Tau5, PHF1, and 128A were obtained from Endogen (Woburn, MA), Dr. L. I. Binder, Northwestern University Medical School, Dr. Peter Davies, Einstein College of Medicine, and ICOS Corp. (Bothell, WA), respectively. Monoclonal anti-α-tubulin antibody (
      • Blose S.H.
      • Meltzer D.I.
      • Feramisco J.R.
      ), Taxol, and protease inhibitors (1× complete contained: 1 mm 4-(2-aminoethyl)-benezenesulfonyl fluoride, 0.8 μm aprotinin, 40 μm bestatin, 20 μm leupeptin, 15 μm pepstatin A, 14 μm l-transepoxysuccinyl-leucylamido-[4-guanidino]butane, and 26 μmN-acetyl-Leu-Leu-norleu-al) were obtained from Sigma. HEK-293 cells were purchased from ATCC (Manassas, VA), TransFast™ transfection reagent from Promega (Madison, WI), inhibitor IC261 from Calbiochem (San Diego, CA), and purified bovine brain tubulin from Cytoskeleton (Denver, CO).
      Recombinant Proteins—Recombinant htau40 containing a His6 tag was expressed and purified as described (
      • Carmel G.
      • Mager E.M.
      • Binder L.I.
      • Kuret J.
      ). Poly-His free recombinant htau40 was prepared the same way, except that Escherichia coli expression was from vector pET-14b (EMD Biosciences, San Diego, CA), and protein isolated from immobilized metal affinity chromatography was digested 20 h at 4 °C with thrombin prior to repetition of immobilized metal affinity chromatography and gel filtration chromatography.
      Human brain Ckiδ cDNA served as template for PCR amplification of C-terminal-truncated Ckiδ (Ckiδ-Δ317). The sense primer (5′-ACGGATCCCATATGGAGCTGAGAGTC) incorporated an NdeI site at the start codon, whereas the downstream primer (5′-CTCGAGTTAGTGTCTCCAGCCGCTC) contained a TTA stop codon downstream of the codon for His317 followed by an XhoI site. The amplified PCR product was subcloned directly into TOPO2.1 (Invitrogen, Carlsbad, CA) and sequenced in both directions for errors. The Ckiδ-Δ317-TOPO2.1 cDNA was digested with NdeI and XhoI, the approximate 0.9-kb Ckiδ-Δ317 fragment was gel purified and subcloned into E. coli expression vector PT7C (
      • Carmel G.
      • Mager E.M.
      • Binder L.I.
      • Kuret J.
      ). Recombinant Ckiδ-Δ317 was purified by immobilized metal affinity chromatography and gel filtration chromatography as described previously for tau protein (
      • Carmel G.
      • Mager E.M.
      • Binder L.I.
      • Kuret J.
      ).
      Cell Culture, Transfection, and Treatment—cDNAs encoding the longest human tau isoform (htau40), human Ckiδ, or p25 (an activator of Cdk5) were cloned into the mammalian cytomegalovirus expression vector pcDNA3.1 (Invitrogen). HEK-293 cells, which were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% bovine serum, 100 units/ml penicillin G, 250 ng/ml amphotericin B, and 100 μg/ml streptomycin (37 °C with 5% CO2), were transiently transfected with these plasmids (5 μg per 60-mm dish) using TransFast™ reagent according to the manufacturer's instructions.
      To generate a cell line stably expressing human htau40 (stable tau cells), 5 μg of tau-pcDNA3.1 plasmid was linearized with BglII and transfected into HEK-293 cells. Forty h after transfection, cells were cultured in selective medium containing 500 μg/ml G418.
      To assay CK1 inhibitor IC261, tau stable cells treated with different IC261 concentrations were harvested after 30, 60, 120, 240, and 480 min of incubation. IC261 was stored as a 50 mm stock solution in Me2SO and diluted to a working stock of 1 mm in H2O before treatment.
      To assess toxicity of various treatments, cells were stained with the DNA-specific fluorochrome Hoechst 33258 (Molecular Probes, Eugene, OR) and examined under fluorescence for chromatin condensation (
      • McGinty A.
      • Chang Y.W.
      • Sorokin A.
      • Bokemeyer D.
      • Dunn M.J.
      ). Cells with uniformly distributed chromatin were scored as “viable.”
      Immunoprecipitation—Cells were harvested and disrupted in Lysis buffer (20 mm Tris-HCl, pH 7.4, 140 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, 1 mm Na3NO4, 10 mm NaF, 0.5% (v/v) Nonidet-40, 1 mm EDTA, and 1× complete protease inhibitors), and cell debris was removed by centrifugation (10 min at 10,000 × g) at 4 °C. Protein concentrations were determined using the Coomassie Blue binding method with bovine serum albumin as standard (
      • Bradford M.M.
      ). For immunoprecipitation experiments, extracts (500 μg) were incubated with 2 μg of each monoclonal antibody examined (anti-Ckiδ, 128A; anti-tau, Tau5; anti-p25, C-19) for 2 h at 4 °C. Protein G-agarose beads (60 μl of 25% (w/v) slurry) were then added to the samples and incubation was continued for 1 h at 4 °C. The resultant immunocomplexes were collected by centrifugation, washed three times with Lysis buffer, and subjected to immunoblot or in vitro protein kinase assays as described below.
      Immunoblot Analysis—Aliquots of cell lysates (20–40 μg) or immunocomplexes derived from immunoprecipitation experiments were boiled in SDS-PAGE Loading buffer (1× buffer contained 48 mm Tris-HCl, pH 6.8, 7.5% glycerol, 1.8% SDS, and 3.8% 2-mercaptoethanol), separated by SDS-PAGE, and transferred to polyvinylidene difluoride membranes. After 1 h in Blocking buffer (10 mm Tris-Cl, pH 8.0, 150 mm NaCl, pH 8.0, 0.1% Tween 20, 5% nonfat milk), membranes were incubated with primary antibody for 2 h at room temperature followed by 1 h with secondary antibody (horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG). Immunoreactivity was detected by enhanced chemiluminescence, collected on x-ray film, and finally quantified on a Bio-Rad GS-800 calibrated laser densitometer.
      In Vitro Protein Kinase Assay—Purified protein kinase (Ckiδ-Δ317) or immunocomplexes derived from immunoprecipitation experiments were incubated with 2 μg of htau40 in 20 μl of Kinase buffer (50 mm Tris, 200 μm ATP, and 1 mm dithiothreitol) at 37 °C for up to 4 h. When necessary, reactions were supplemented with 1.25 μCi of [γ-32P]ATP. All reactions were stopped by addition of 10 μl of 4× SDS-PAGE Loading buffer. Following SDS-PAGE, gels were subjected to immunoblot analysis as described above or stained and dried for autoradiography using a Molecular Imager FX Pro Plus multi-imager system (Bio-Rad).
      RNA Interference—Sequence specific post-transcriptional silencing of selected genes was initiated by transfection of HEK-293 cells with double stranded RNA oligonucleotides (synthesized by Dharmacon, Lafayette, CO). Ckiδ/ϵ isoforms were silenced with RNA duplexes consisting of oligonucleotides 5′-CUGGGGAAGAAGGGCCdTdT and 5′-GGUUGCCCUUCCCCAGdTdT (
      • Eide E.J.
      • Vielhaber E.L.
      • Hinz W.A.
      • Virshup D.M.
      ). RNA duplexes targeting luciferase, a non-mammalian protein found in fireflies, were used to control for nonspecific effects of siRNA transfection. These oligonucleotides, termed GL2, consisted of sense (5′-CGUACGCGGAAUACUUCGAdTdT) and antisense (5′-UCGAAGUAUUCCGCGUACGdTdT) components (
      • Elbashir S.M.
      • Harborth J.
      • Lendeckel W.
      • Yalcin A.
      • Weber K.
      • Tuschl T.
      ). GL2 or Ckiδ/ϵ siRNA duplexes (5 μg) were transfected into stable tau cells using TransFast™ reagents for 48 h. Levels of Ckiδ, tau, and phospho-tau were then quantified by immunoblot analysis performed on cell lysates (30 μg of protein) as described above.
      Microtubule Binding Assay—To assay the ability of tau to bind microtubules in situ, HEK-293 cells transfected with pcDNA3.1/tau or Ckiδ/tau for 24 h were harvested in 500 μl of Microtubule-stabilizing buffer (80 mm PIPES, pH 6.8, 1 mm GTP, 1 mm MgCl2, 1 mm EGTA, 0.5% Triton X-100, 30% glycerol, and 1× complete protease inhibitors) in the presence of 10 μm Taxol at 37 °C. After cell debris was removed by centrifugation (10,000 × g for 10 min) at 37 °C, supernatant fractions were collected and centrifuged again at 100,000 × g for 1 h at 35 °C (
      • Wagner U.
      • Utton M.
      • Gallo J.
      • Miller C.
      ). The resultant pellets (P1) were washed twice with and resuspended (500 μl) in Microtubule-stabilizing buffer, sonicated for 10 s, and then subjected along with 100,000 × g supernatants (S1) to SDS-PAGE and immunoblot analysis as described above.
      To assay the ability of tau to bind microtubules in vitro, tubulin (1 mg/ml) was first assembled in Microtubule assembly buffer (80 mm PIPES, pH 6.8, 1 mm MgCl2, 1 mm EGTA, 1 mm GTP) containing 10 μm Taxol for 30 min at 30 °C. Resultant microtubules were then incubated (37 °C for 30 min) with supernatants (S1; 500 μg) from the in situ microtubule binding assays described above, and then harvested by centrifugation (100,000 × g for 1 h at 25 °C). Pellet fractions (P2) were resuspended in Microtubule-assembly buffer so that their final volumes equaled those of supernatant fractions (S2). Equal amounts of supernatant (S2) and pellet (P2) fractions were then analyzed by SDS-PAGE and the distribution of tau protein was examined by immunoblot analysis using monoclonal antibody Tau5.
      Cell Extraction and Immunofluorescence Staining—Wild-type HEK-293 cells were cultured on coverslips for 24 h and transfected with Ckiδ/tau or PcDNA3.1/tau for 24 h as described above. To extract soluble proteins, cells were washed with phosphate-buffered saline and incubated with Microtubule-stabilizing buffer containing 10 μm Taxol for 2 min at room temperature. Cells were washed once with Microtubule-stabilizing buffer without detergent. Both the extracted cells and non-extracted cells were fixed with methanol. Following the phosphate-buffered saline washes, cells were sequentially incubated with phosphate-buffered saline containing 3% bovine serum albumin for 1 h, Tau5 antibody (1:1000) overnight at 4 °C, and fluorescein-labeled goat anti-mouse IgG secondary antibody (1:1000) for 1 h. Immunostaining was visualized by fluorescence microscopy (Eclipse 800, Nikon, Japan), captured with a SPOT II digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI), and processed with Metamorph imaging software (Universal Imaging Corp., Downingtown, PA).
      Analytical Methods—Hyperbolic inhibition curves were fit to the rectangular hyperbola: y = ax/(b + x), where y is immunoreactivity determined at inhibitor concentration x, and constant b corresponds to x at 50% ymax (i.e. IC50).
      Probability values were determined by Student's t test for single comparison and one-way analysis of variance with Tukey's post-hoc test for multiple comparisons. All analyses were performed using the InStat Software program.

      RESULTS

      Ckiδ Phosphorylates Tau in Vitro—To determine whether Ckiδ could directly phosphorylate tau, a truncated form of the human enzyme (Ckiδ-Δ317) was expressed and purified as a His6 fusion protein and used to phosphorylate tau protein in vitro. Truncated enzyme was used because it was easily prepared after heterologous expression in E. coli while retaining the protein kinase activity of full-length protein (
      • Graves P.R.
      • Roach P.J.
      ). Incubation of htau40 with purified Ckiδ-Δ317 in the presence of radioactive nucleotide substrate led to detectable phosphate incorporation within 30 min (Fig. 1A). Detection of total tau protein on Western blots using monoclonal antibody Tau5, the reactivity of which is not phosphorylation dependent (
      • Carmel G.
      • Mager E.M.
      • Binder L.I.
      • Kuret J.
      ), revealed a substantial electrophoretic mobility shift by 4 h incubation (Fig. 1B). These slowly migrating tau species were strongly labeled by monoclonal antibodies AT8 and PHF1, which selectively bind phospho-tau residues Ser202/Thr205 (
      • Goedert M.
      • Jakes R.
      • Vanmechelen E.
      ) and Ser396/Ser404 (
      • Otvos Jr., L.
      • Feiner L.
      • Lang E.
      • Szendrei G.I.
      • Goedert M.
      • Lee V.M.
      ), respectively. These data show that the CK1 isoform Ckiδ could directly phosphorylate tau protein, and that monoclonal antibodies AT8 and PHF1 could be used as probes for Ckiδ-mediated phosphorylation reactions.
      Figure thumbnail gr1
      Fig. 1Ckiδ phosphorylates tau at Ser202, Thr205, Ser396, and Ser404in vitro. Recombinant His6-tagged htau40 (0.2 mg/ml) was incubated in the presence or absence of Ckiδ-Δ317 (50 ng) under phosphorylating conditions and subjected to SDS-PAGE and Western analysis. A, after 30 min at 37 °C in the presence of [γ-32P]ATP, phospho-tau could be detected by phosphorimaging. However, total tau as detected by Coomassie Blue (CB) staining of SDS gels shows essentially no band shift. The bottom panel shows Ckiδ-Δ317 detected with monoclonal antibody 128A antibody. B, after 4 h at 37 °C, total tau as detected with monoclonal antibody Tau5 shows a marked band shift. Phospho-tau species were detected by monoclonal antibodies PHF1 and AT8, suggesting that, at a minimum, Ckiδ-Δ317 phosphorylated sites Ser202, Thr205, Ser396, and Ser404 on htau40.
      Ckiδ Phosphorylates Tau in Situ—To determine whether Ckiδ could modulate tau phosphorylation in situ, HEK-293 cells were transiently transfected with plasmids encoding htau40 and Ckiδ, p25 (an activator of Cdk5), or empty pcDNA vector. Transfection times of 24 h were employed because cells remained viable during this time frame in all studies reported herein (data not shown). Cdk5 activator p25 was included because of its ability to activate endogenous Cdk5 in situ (
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • de la Monte S.
      • Dikkes P.
      • Tsai L.H.
      ) and therefore serve as an alternate source of tau hyperphosphorylation activity. Total tau protein was detected with monoclonal antibody Tau5, whereas phospho-tau was detected with antibodies AT8 and PHF1. All cells transfected with htau40 constructs contained detectable levels of tau protein (Fig. 2A). When these cells were co-transfected with empty vector (i.e. in the absence of exogenous protein kinase), a portion of total tau was PHF1 and AT8 positive, reflecting basal levels of tau phosphorylation. Basal levels of Ckiδ also were substantial in these cells as demonstrated by Western analysis using monoclonal antibody 128A. However, cotransfection with DNA constructs encoding Ckiδ significantly increased the amounts of both Ckiδ and phospho-tau detectable by both the AT8 and PHF1 antibodies (Fig. 1A). These data recapitulate the pattern seen in vitro (Fig. 1A) and suggest that Ckiδ overexpression significantly increased tau phosphorylation in situ (Fig. 2B). Expression of p25 also induced a large increase in tau phosphorylation, primarily because of the absence of endogenous p25 in non-transfected cells (Fig. 2B).
      Figure thumbnail gr2
      Fig. 2Ckiδ induces tau phosphorylation in situ. HEK-293 cells transiently co-transfected (24 h) with p25/tau, Ckiδ/tau, and empty pcDNA vector/tau were harvested, lysed, and subjected to immunoblot analysis using monoclonal antibodies AT8, PHF1, Tau5, 128A, or C-19. A, cells transfected with empty vector showed measurable basal levels of phospho-tau and Ckiδ but not of p25. Transfection of cells with Ckiδ or cdk5 activator p25 significantly increased phospho-tau over basal levels. B, total tau (Tau5 epitope) and phospho-tau (PHF1 epitope) levels were quantified densitometrically from three individual experiments. When normalized for total tau content, phospho-tau was found to increase significantly with both p25 and Ckiδ transfection. *, p < 0.01; **, p < 0.001 when compared with empty vector control.
      To confirm that the increased levels of Ckiδ observed upon overexpression were accompanied by increases in phosphotransferase activity, Ckiδ was immunoprecipitated and subjected to in vitro kinase assays using htau40 and [γ-32P]ATP as substrates 24 h after transient transfection with Ckiδ/tau or pcDNA3.1/tau constructs. Radioactive assay reaction products were separated by SDS-PAGE, stained with Coomassie Blue, and visualized on a phosphorimager. Although CK1 activity was found in both the Ckiδ/tau overexpressing and pcDNA3.1/tau control cells (Fig. 3A), quantitation of the data showed that Ckiδ overexpression led to ∼2-fold increases in recoverable phosphotransferase activity (Fig. 3B). These data confirmed that Ckiδ overexpression lead to increased Ckiδ activity within HEK-293 cells, but also showed that significant levels of Ckiδ activity were present under basal conditions.
      Figure thumbnail gr3
      Fig. 3In vitro kinase assay. HEK-293 cells transiently transfected (24 h) with Ckiδ/tau or empty vector/tau were harvested, lysed, and subjected to immunoprecipitation assays using anti-Ckiδ antibody 128A. Ckiδ immunocomplexes from Ckiδ/tau (lane 1) or from empty vector/tau (lane 2) were then incubated in vitro with His -htau40 and [γ-32P]ATP for 30 min and subjected to SDS-PAGE. A, 6phospho-tau detected by phosphorimaging shows that transfection with Ckiδ increased immunoprecipitable Ckiδ activity (32P), which correlated with levels of Ckiδ protein (128A). Bottom panel shows total tau detected by Coomassie Blue (CB) staining. B, quantitative data from three independent experiments presented as mean ± S.E. **, p < 0.001 compared with empty vector control.
      Ckiδ and Tau Directly Interact in Situ—The Ckiδ-mediated tau phosphorylation observed in HEK-293 cells could be direct or arise from an indirect protein phosphorylation cascade. To distinguish these alternatives, tau and Ckiδ were separately immunoprecipitated from stable tau cells and subjected to Western analysis using monoclonal antibodies 128A or Tau5. Results showed that tau and Ckiδ coimmunoprecipitated in both experimental paradigms (Fig. 4, C and D). These data suggest that tau associates with Ckiδ in situ, and therefore potentially serves as a direct substrate for Ckiδ-mediated phosphorylation reactions.
      Figure thumbnail gr4
      Fig. 4Ckiδ associates with tau in situ. Lysates (Lys) prepared from stable tau or wild-type HEK-293 cells were immunoprecipitated with anti-tau (Tau5) or anti-Ckiδ (128A) monoclonal antibodies as described under “Experimental Procedures” and subjected to immunoblot analysis using: A, 128A; or B, Tau5 as probes. A, both wild-type and stable tau cells contained Ckiδ that could be immunoprecipitated with 128A. B, in contrast, only stable tau cells contained detectable tau protein after immunoprecipitation with Tau5. Ckiδ and tau coimmunoprecipitated with either 128A or Tau5 antibodies in stable tau cells, suggesting direct association in situ. C, htau40 (1 μg/ml) without His6 tag incubated (4 °C overnight) with or without truncation mutant Ckiδ-Δ317 (1 μg/ml) in Lysis buffer was immunoprecipitated with antibodies Tau5 or 128A and subjected to immunoblot analysis with anti-tau (Tau5) and anti-Ckiδ (128A) antibodies. Tau and Ckiδ-Δ317 coimmunoprecipitated under these conditions, suggesting direct interaction between tau and the catalytic domain of Ckiδ in vitro.
      To determine the region of Ckiδ involved in binding tau protein, htau40 (non-His6 tagged) was incubated with or without the truncation mutant Ckiδ-Δ317 in vitro and immunoprecipitated with antibody 128A. Subsequent Western analysis with antibodies 128A and Tau5 confirmed direct association between truncated Ckiδ and htau40 (Fig. 4B). These data suggest that at least a portion of the amino acid sequences mediating direct binding of Ckiδ to tau protein are located in the protein kinase catalytic domain.
      CK1 Inhibitor IC261 Reverses Tau Hyperphosphorylation Induced by Ckiδ Overexpression—IC261 is a small molecule, membrane-permeable, ATP-competitive inhibitor of CK1 isoforms including Ckiδ (
      • Behrend L.
      • Milne D.M.
      • Stoter M.
      • Deppert W.
      • Campbell L.E.
      • Meek D.W.
      • Knippschild U.
      ,
      • Mashhoon N.
      • DeMaggio A.J.
      • Tereshko V.
      • Bergmeier S.C.
      • Egli M.
      • Hoekstra M.F.
      • Kuret J.
      ). To determine whether inhibitor treatment could selectively reverse Ckiδ-mediated tau hyperphosphorylation, HEK-293 cells were co-transfected with Ckiδ/tau, P25/tau, or pcDNA3.1/tau for 24 h, treated with and without IC261 (10 μm for 30 min), and then probed for phospho and total tau levels with antibodies PHF1, AT8, and Tau5. The resultant Western blots showed that IC261 inhibited tau phosphorylation induced by Ckiδ overexpression without affecting expression levels of Ckiδ (Fig. 5A). Inhibition was selective for CK1, as shown by the failure of IC261 to significantly modulate tau phosphorylation induced by p25 overexpression (Fig. 5B). These findings are consistent with the inhibitory selectivity of IC261 determined both in vitro and in situ (
      • Mashhoon N.
      • DeMaggio A.J.
      • Tereshko V.
      • Bergmeier S.C.
      • Egli M.
      • Hoekstra M.F.
      • Kuret J.
      ,
      • Liu F.
      • Ma X.-H.
      • Ule J.
      • Bibb J.A.
      • Nishi A.
      • DeMaggio A.J.
      • Yan Z.
      • Nairn A.C.
      • Greengard P.
      ), and confirm the utility of IC261 for inhibition of CK1 activity in culture. Moreover, they suggest that CK1-mediated tau phosphorylation is opposed by endogenous phosphoprotein phosphatase activity that can decrease occupancy of phosphorylation sites in response to IC261 over time courses as short as 30 min.
      Figure thumbnail gr5
      Fig. 5IC261 inhibits tau phosphorylation induced by Ckiδ. HEK-293 cells transiently transfected (24 h) with tau/p25, tau/Ckiδ, or empty vector/tau were treated with either 10 μm IC261(IC), or vehicle control (Con) for an additional 30 min. Cell lysates were then prepared and subjected to SDS-PAGE (30 μg/lane). A, representative immunoblots using monoclonal antibodies AT8, PHF1 Tau5, 128A, and C-19 show that IC261 inhibited basal and Ckiδ-induced tau phosphorylation without modulating the levels of total tau or Ckiδ. In contrast, neither p25 levels nor p25-induced tau phosphorylation was significantly affected by IC261. B, phospho-tau levels were quantified by densitometry from three independent experiments, normalized to levels of total tau, and plotted as percent phospho-tau in untreated, empty vector/tau-transfected cells. **, p < 0.01 compared with untreated empty vector/tau-transfected cells. ##, p < 0.01 compared with untreated Ckiδ/tau-transfected cells.
      Endogenous CK1 Contributes to Basal Levels of Tau Phosphorylation—The above data showed that basal levels of both Ckiδ and phospho-tau were substantial in HEK-293 cells expressing tau protein, and were consistent with previous studies showing that phosphorylation sites Ser396 and Ser202 were partially occupied in both stable and transiently transfected 3T3, Chinese hamster ovary, and SH-SY5Y cells (
      • Hamdane M.
      • Sambo A.V.
      • Delobel P.
      • Begard S.
      • Violleau A.
      • Delacourte A.
      • Bertrand P.
      • Benavides J.
      • Buee L.
      ,
      • Sato 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.
      ,
      • Sygowski L.A.
      • Fieles A.W.
      • Lo M.M.
      • Scott C.W.
      • Caputo C.B.
      ). To assess the contribution of endogenous Ckiδ activity to basal levels of tau phosphorylation, an HEK-293 cell line stably expressing htau40 was generated and employed in two experimental approaches. First, the dependence of tau phosphorylation on activity contributed by all CK1 isoforms was estimated by treating stable tau-HEK-293 cells with varying concentrations of IC261. In the absence of inhibitor, significant amounts of tau were detectable in this cell line, with a portion being PHF1 reactive (Fig. 6A). Although addition of IC261 for 30 min did not change total tau levels (i.e. detectable with antibody Tau5), it did result in large decreases in levels of phospho-tau detected by antibody PHF1 (Fig. 6A). Inhibition of tau phosphorylation was dose dependent, with an IC50 of 1.5 ± 0.5 μm and up to 71.9 ± 7.5% of PHF1 reactivity at saturation (Fig. 6B). In vitro, IC261 has been shown to inhibit purified recombinant Ckiδ with an IC50 of 1.1 ± 0.1 μm and 98.1 ± 3.2% of activity at saturation (Fig. 6B; data replotted from Fig. 1 of Ref.
      • Mashhoon N.
      • DeMaggio A.J.
      • Tereshko V.
      • Bergmeier S.C.
      • Egli M.
      • Hoekstra M.F.
      • Kuret J.
      ). These data suggest that CK1 activity makes a major contribution to basal levels of tau phosphorylation in HEK-293 cells.
      Figure thumbnail gr6
      Fig. 6CK1 inhibitor IC261 decreases tau phosphorylation.A, stable tau cells were grown to 80% confluence and then incubated with 0, 1, 3, or 10 μm inhibitor IC261 for 30 min. All reactions were controlled for Me2SO, the vehicle for IC261. Treated cells were harvested, lysed, and subjected to SDS-PAGE. A, representative immunoblots probed with monoclonal antibodies PHF1, Tau5, and 128A. IC261-mediated inhibition of phospho-tau levels (i.e. PHF1 immunoreactivity) was concentration dependent. B, phospho-tau levels were quantified by densitometry, normalized to levels of total tau, and plotted as percent inhibition, where 0% inhibition corresponds to PHF1 immunoreactivity in the absence of IC261. Each point represents mean ± S.E. of four independent experiments, whereas the line represents best fit of the data to a rectangular hyperbola (▪). IC261 inhibited tau phosphorylation (PHF1 epitope) with an IC50 of 1.5 ± 0.5 μm. This value is very similar to the IC50 determined in vitro for Ckiδ (□, 1.1 ± 0.1 μm, data from Ref.
      • Mashhoon N.
      • DeMaggio A.J.
      • Tereshko V.
      • Bergmeier S.C.
      • Egli M.
      • Hoekstra M.F.
      • Kuret J.
      ).
      As a second approach, the specific contribution of CK1 isoforms Ckiδ/ϵ to basal tau phosphorylation was assessed by siRNA-mediated down-regulation conducted 48 h after transfection. RNA interference promotes hydrolysis of targeted mRNA in a sequence-specific reaction (
      • Vickers T.A.
      • Koo S.
      • Bennett C.F.
      • Crooke S.T.
      • Dean N.M.
      • Baker B.F.
      ,
      • Elbashir S.M.
      • Lendeckel W.
      • Tuschl T.
      ). The levels of total tau, phosphorylated tau, and Ckiδ were then detected by immunoblot analysis as a negative control, siRNA oligonucleotides selective for luciferase (GL2) were employed. Compared with GL2 treatment, Ckiδ/ϵ siRNA lowered Ckiδ levels ∼30% 48 h after transfection and decreased phosphorylated tau about 25% as well (Fig. 7B). RNA interference did not change the expression levels of total tau (Fig. 7A). These data indicate that either down-regulating Ckiδ activity or its protein level decreases tau phosphorylation, and suggests that Ckiδ/ϵ contributes at least a portion of the basal CK1 activity detectable in HEK-293 cells.
      Figure thumbnail gr7
      Fig. 7SiRNA decreases tau phosphorylation. Stable tau cells transfected (48 h) with 5 μg of siRNA selective for Ckiδ or luciferase (GL2) were harvested, lysed, and subjected to SDS-PAGE (30 μg/lane). A, representative immunoblots probed with monoclonal antibodies PHF1, Tau5, and 128A. Treatment with Ckiδ siRNAs decreased levels of Ckiδ and phospho-tau (PHF1 epitope) but not total tau (Tau5 epitope) relative to GL2 control. B, phospho-tau and Ckiδ levels were quantified by densitometry and plotted as percent GL2 control. Each bar represents the mean ± S.E. of three independent experiments. *, p < 0.05, compared with GL2 control.
      Ckiδ-mediated Tau Phosphorylation Disrupts Microtubule Binding—The microtubule binding activity of tau protein is modulated by phosphorylation (
      • Bramblett G.T.
      • Goedert M.
      • Jakes R.
      • Merrick S.E.
      • Trojanowski J.Q.
      • Lee V.M.
      ,
      • Wagner U.
      • Utton M.
      • Gallo J.
      • Miller C.
      ). To assess the effect of Ckiδ-induced phosphorylation on tau/tubulin interactions, HEK-293 cells transiently co-transfected with Ckiδ/tau or pcDNA/tau constructs were examined by fluorescence microscopy after extraction with detergent in the presence of Taxol (a microtubule stabilizing agent). In the absence of extraction, total tau immunofluorescence in both Ckiδ/tau or pcDNA/tau transfections were similar, again demonstrating that Ckiδ overexpression did not markedly change tau levels over the time course of the experiment (Fig. 8A). But in detergent-extracted cells, which retained a detergent-insoluble cytoskeleton, levels of tau immunofluorescence were significantly lower in the presence of Ckiδ (Ckiδ/tau transfection) than in its absence (pcDNA/tau transfection). These data suggested that the fraction of detergent-stable (i.e. microtubule-associated) tau decreased in response to phosphorylation induced by Ckiδ overexpression.
      Figure thumbnail gr8
      Fig. 8Ckiδ-induced tau phosphorylation decreases tau binding to microtubule.A, HEK-293 cells transiently transfected (24 h) with either: a and c, empty vector/tau; or b and d, Ckiδ/tau were harvested and washed with either a and b, phosphate-buffered saline, or c and d, Microtubule-stabilizing buffer. All cells were then fixed with methanol and immunostained with monoclonal antibody Tau5. a and b, in the absence of detergent extraction, no change in tau immunofluorescence was observed with Ckiδ overexpression, showing that transfection of cells with Ckiδ did not change total tau levels. c and d, in detergent-extracted cells, however, expression of Ckiδ led to decreased tau association with detergent-insoluble cytoskeletons. These data are consistent with a migration of tau from detergent-insoluble to detergent-soluble fractions of the cell in response to Ckiδ overexpression. B, HEK-293 cells transfected (24 h) with pcDNA/tau or Ckiδ/tau were harvested, lysed, and separated into soluble (S1) and pellet (P1) fractions by centrifugation as described under “Experimental Procedures.” Total (Tau5 epitope), phospho-tau (PHF1 epitope), and α-tubulin levels were then detected by immunoblot analysis. In pcDNA/tau cells, tau was found distributed between soluble and particulate fractions. In Ckiδ/tau, however, tau distribution shifted toward the soluble pool, consistent with a decrease in microtubule association. C, total tau levels in particulate and soluble fractions were quantified by densitometry. Each bar represents the mean ± S.E. of four independent experiments (**, p < 0.01 compared with empty vector/tau control). D, microtubule binding assays were performed using Taxol-stabilized microtubules prepared in vitro and equal volumes of the soluble, cell-derived fractions (S1) described above. After centrifugation, the resultant pellet (P2; containing microtubules) and supernatant (S2) fractions were analyzed for tubulin by SDS-PAGE (Coomassie Blue) and total tau (Tau5) by immunoblotting. Whereas a portion of soluble tau prepared from cells transfected with pcDNA/tau was capable of binding microtubules, soluble tau prepared from Ckiδ/tau cells was almost devoid of microtubule binding activity.
      To confirm these findings, HEK-293 cells transiently co-transfected with Ckiδ/tau or pcDNA/tau for 24 h were detergent extracted, fractionated into soluble (S1) and particulate (P1) fractions (the latter containing microtubules), and subjected to Western analysis using antibodies Tau5 and PHF1. Phospho-tau (detected by PHF1) was found exclusively in the soluble fraction, with greater amounts recovered from cells transfected with Ckiδ/tau than with pcDNA/tau (Fig. 8B). In contrast, significant amounts of total tau (detected by Tau5) were recovered in both particulate and soluble fractions (Fig. 8B). However, the subcellular distribution of total tau shifted toward the soluble pool in cells transfected with Ckiδ/tau compared with pcDNA/tau cells (Fig. 8C).
      Together these data suggest that the microtubule binding activity of tau decreases in response to Ckiδ-mediated phosphorylation. To test this hypothesis, soluble tau prepared from each of the cell populations described above (fraction S1) was subjected to in vitro microtubule binding assays. Results showed that a portion of soluble tau from pcDNA/tau cells retained an ability to associate with synthetic microtubules (Fig. 8D), consistent with tau overexpression leading to saturation of endogenous microtubules. In contrast, soluble tau prepared from Ckiδ/tau cells was almost devoid of microtubule binding activity (Fig. 8D), despite accumulating to higher levels than in control extracts (Fig. 8, C and D). These data suggest that the accumulation of soluble tau observed in situ upon Ckiδ overpression results from phosphorylation of tau protein at sites that directly modulate microtubule binding.

      DISCUSSION

      CK1 Isozymes and Tau Phosphorylation—Because of its natively unfolded structure and high complement of hydroxyamino acids, tau is an efficient substrate for many protein kinases in vitro (
      • Mandelkow E.M.
      • Biernat J.
      • Drewes G.
      • Gustke N.
      • Trinczek B.
      • Mandelkow E.
      ), including tissue-derived CK1 activity (
      • Singh T.
      • Grundke-Iqbal I.
      • Iqbal K.
      ,
      • Behrend L.
      • Stoter M.
      • Kurth M.
      • Rutter G.
      • Heukeshoven J.
      • Deppert W.
      • Knippschild U.
      ). Here it was found that CK1 serves as a tau protein kinase in situ as well. First, studies with the selective inhibitor IC261 showed that over 70% of basal tau phosphorylation at Ser396/Ser404 sites in HEK-293 cells stems from CK1 activity. Supporting this conclusion, the IC50 for IC261 in situ was only slightly higher than the value determined in vitro with purified Ckiδ. Moreover, the IC261 inhibition isotherm was consistent with a single class of binding site. Because IC261 inhibits most CK1 isoforms with similar potency (
      • Mashhoon N.
      • DeMaggio A.J.
      • Tereshko V.
      • Bergmeier S.C.
      • Egli M.
      • Hoekstra M.F.
      • Kuret J.
      ), basal levels of tau phosphorylation could potentially result from the activity of multiple CK1 isoforms. But the decrease in phospho-tau accompanying down-regulation of Ckiδ/ϵ using RNA interference suggests that these specific isoforms compose at least a portion of IC261-sensitive tau phosphorylation activity under basal conditions. Indeed, Ckiδ and tau could be co-immunoprecipitated from stable tau cells containing only basal levels of Ckiδ. These data demonstrate that tau and Ckiδ directly associate in situ, and are consistent with their colocalization to microtubules (
      • Behrend L.
      • Stoter M.
      • Kurth M.
      • Rutter G.
      • Heukeshoven J.
      • Deppert W.
      • Knippschild U.
      ).
      Second, overexpression of Ckiδ increased tau phosphorylation at sites it phosphorylated in vitro. Again, IC261 totally reversed the tau hyperphosphorylation induced by Ckiδ overexpression. However, the recognition sequence mediating the substrate selectivity of Ckiδ is not understood in the context of tau phosphorylation. On the basis of studies with short peptides, CK1 isoforms are phosphotropic kinases that recognize the motif S/T(P)XXS/T (
      • Flotow H.
      • Graves P.R.
      • Wang A.Q.
      • Fiol C.J.
      • Roeske R.W.
      • Roach P.J.
      ,
      • Flotow H.
      • Roach P.J.
      ). Nonetheless, priming of substrates with phosphate is not a strict requirement, and motifs consisting of acidic residues N- or C-terminal to the phosphorylatable residue also are phosphorylated by CK1 (
      • Marin O.
      • Bustos V.H.
      • Cesaro L.
      • Meggio F.
      • Pagano M.A.
      • Antonelli M.
      • Allende C.C.
      • Pinna L.A.
      • Allende J.E.
      ,
      • Marin O.
      • Meggio F.
      • Pinna L.A.
      ,
      • Pulgar V.
      • Marin O.
      • Meggio F.
      • Allende C.C.
      • Allende J.E.
      • Pinna L.A.
      ). Although our in vitro data showing that Ckiδ phosphorylated recombinant tau at Ser202/Thr205 and Ser396/Ser404 is consistent with a previous study showing that tissue-derived CK1 activity phosphorylates tau at Thr231, Ser396/Ser404 (
      • Singh T.J.
      • Zaidi T.
      • Grundke-Iqbal I.
      • Iqbal K.
      ), the relationship between these sites and the motifs summarized above is weakly apparent only for Ser396/Ser404. It appears that recognition of full-length tau as substrate differs from that of short peptides. In any event, phosphorylation of tau at Ser202/Ser205 and Ser396/Ser404 by Ckiδ does not require priming by other protein kinases.
      Consequences of CK1-mediated Tau Phosphorylation—Tau protein binds and stabilizes microtubules (
      • Goedert M.
      ). However, this microtubule stabilizing function is regulated by its phosphorylation state. For example, hyperphosphorylated tau binds to microtubules weakly, but strong binding can be restored after dephosphorylation (
      • Bramblett G.T.
      • Goedert M.
      • Jakes R.
      • Merrick S.E.
      • Trojanowski J.Q.
      • Lee V.M.
      ,
      • Busciglio J.
      • Lorenzo A.
      • Yeh J.
      • Yankner B.A.
      ,
      • Sontag E.
      • Nunbhakdi-Craig V.
      • Bloom G.S.
      • Mumby M.C.
      ). Thus, the stability of the axonal cytoskeleton may be influenced the equilibrium between tau phosphorylation and dephosphorylation. Phosphorylation sites that mediate this activity include Ser199, Ser202, Ser231, Thr205, Ser396, and Ser404 (
      • Wagner U.
      • Utton M.
      • Gallo J.
      • Miller C.
      ,
      • Sontag E.
      • Nunbhakdi-Craig V.
      • Lee G.
      • Bloom G.S.
      • Mumby M.C.
      ). Ckiδ phosphorylates most of these sites in situ, and as a result tau/microtubule equilibrium shifts markedly toward free tau in response to Ckiδ overexpression. Similar shifts have been observed following Cdk5/p25, GSK3β, or protein kinase A-induced phosphorylation of tau (
      • Wagner U.
      • Utton M.
      • Gallo J.
      • Miller C.
      ,
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • de la Monte S.
      • Dikkes P.
      • Tsai L.H.
      ,
      • Wada Y.
      • Ishiguro K.
      • Itoh T.J.
      • Uchida T.
      • Hotani H.
      • Saito T.
      • Kishimoto T.
      • Hisanaga S.
      ,
      • Evans D.B.
      • Rank K.B.
      • Bhattacharya K.
      • Thomsen D.R.
      • Gurney M.E.
      • Sharma S.K.
      ,
      • Utton M.A.
      • Vandecandelaere A.
      • Wagner U.
      • Reynolds C.H.
      • Gibb G.M.
      • Miller C.C.
      • Bayley P.M.
      • Anderton B.H.
      ,
      • Scott C.
      • Spreen R.
      • Herman J.
      • Chow F.
      • Davison M.
      • Young J.
      • Caputo C.
      ). Moreover, Cdk5/p39-induced tau phosphorylation reduces its affinity for microtubules during development in a transgenic mouse model (
      • Takahashi S.
      • Saito T.
      • Hisanaga S.-i.
      • Pant H.C.
      • Kulkarni A.B.
      ). These data suggest that control of microtubule stability may involve common phosphorylation sites that are differentially regulated through the action of multiple protein kinases.
      Role of Tau Phosphorylation in Disease—The pathway through which tau fibrillizes in disease is not entirely clear but appears to begin with amorphous aggregation of tau protein into non-fibrillar deposits (
      • Bancher C.
      • Brunner C.
      • Lassmann H.
      • Budka H.
      • Jellinger K.
      • Wiche G.
      • Seitelberger F.
      • Grundke-Iqbal I.
      • Iqbal K.
      • Wisniewski H.M.
      ). Phosphorylation can play a major role in this process, by modulating the equilibrium between microtubule-bound and free populations as discussed above. In vitro, elevated concentrations of phospho-tau promotes amorphous aggregation (
      • Alonso A.C.
      • Grundke-Iqbal I.
      • Iqbal K.
      ). In vivo, amorphous deposits, frequently in conjunction with intracellular membranes, are where the earliest β-sheet enriched species are detectable using fluorescent dyes (
      • Galvan M.
      • David J.P.
      • Delacourte A.
      • Luna J.
      • Mena R.
      ). Studies with synthetic inducers of tau fibrillization have revealed the existence of a partially folded, thioflavin S-positive intermediate in the reaction pathway, the formation of which appears to be essential for fibrillization (
      • Chirita C.N.
      • Kuret J.
      ). Therefore, the key roles of tau phosphorylation in the early stage disease appear to be modulation of the tau/microtubule equilibrium to raise intracellular concentrations of free tau, and promotion of amorphous aggregation from which assembly competent intermediates can form. Consistent with this model, extensive hyperphosphorylation of tau in COS cells leads to thioflavin-S positive deposits that are not fibrillar in appearance and contain only modest amounts of tau filaments (
      • Sato 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.
      ).
      CK1 in Disease—The properties of CK1 isoforms are consistent with a role in disease. First, as shown here, CK1 composes the bulk of basal tau phosphorylation activity in HEK-293 cells, and phosphorylates tau on sites that modulate tau function. These observations are consistent with Ckiδ and tau being resident on microtubules (
      • Behrend L.
      • Stoter M.
      • Kurth M.
      • Rutter G.
      • Heukeshoven J.
      • Deppert W.
      • Knippschild U.
      ). Second, CK1 isoforms correlate spatially with neurofibrillary lesions in both AD (
      • Ghoshal N.
      • Smiley J.F.
      • DeMaggio A.J.
      • Hoekstra M.F.
      • Cochran E.J.
      • Binder L.I.
      • Kuret J.
      ) and other tauopathies (
      • Schwab C.
      • DeMaggio A.J.
      • Ghoshal N.
      • Binder L.I.
      • Kuret J.
      • McGeer P.L.
      ), and appear as major components in preparations of authentic, disease-derived filaments (
      • Kuret J.
      • Johnson G.S.
      • Cha D.
      • Christenson E.R.
      • DeMaggio A.J.
      • Hoekstra M.F.
      ). One isoform, Ckiδ, is a particularly robust marker of granulovacuolar degeneration bodies (
      • Ghoshal N.
      • Smiley J.F.
      • DeMaggio A.J.
      • Hoekstra M.F.
      • Cochran E.J.
      • Binder L.I.
      • Kuret J.
      ). Thus CK1 is associated with more than one lesion in AD. Third, the appearance of CK1-positive lesions correlates temporally with memory decline in longitudinal studies of AD, suggesting that CK1 is as powerful a marker of AD progression as is tau (
      • Ghoshal N.
      • Garcia-Sierra F.
      • Wuu J.
      • Leurgans S.
      • Bennett D.A.
      • Berry R.W.
      • Binder L.I.
      ). Finally, levels of at least one CK1 isoform, Ckiδ, are greatly increased in postmortem AD tissue (
      • Ghoshal N.
      • Smiley J.F.
      • DeMaggio A.J.
      • Hoekstra M.F.
      • Cochran E.J.
      • Binder L.I.
      • Kuret J.
      ). Unlike tau protein, overexpression of Ckiδ is observable at the mRNA level (
      • Yasojima K.
      • Kuret J.
      • DeMaggio A.J.
      • McGeer E.
      • McGeer P.L.
      ), suggesting that accumulation does not result solely from sequestration with insoluble proteinaceous inclusions. In situ, Ckiδ expression increases in response to drugor γ-irradiation-induced genotoxic stress suggesting DNA damage as a potential mechanism for the elevated levels seen in AD (
      • Knippschild U.
      • Milne D.M.
      • Campbell L.E.
      • DeMaggio A.J.
      • Christenson E.
      • Hoekstra M.F.
      • Meek D.W.
      ,
      • Knippschild U.
      • Milne D.
      • Campbell L.
      • Meek D.
      ). Further study of Ckiδ expression in AD will be required to clarify this issue.
      Despite these properties, CK1 is but one of several protein kinases capable of phosphorylating tau in situ. GSK3β and Cdk5 also physically associate with tau within cells (
      • Sun W.
      • Qureshi H.Y.
      • Cafferty P.W.
      • Sobue K.
      • Agarwal-Mawal A.
      • Neufield K.D.
      • Paudel H.K.
      ) and can modulate tau phosphorylation in transgenic animal models (
      • Lucas J.J.
      • Hernandez F.
      • Gomez-Ramos P.
      • Moran M.A.
      • Hen R.
      • Avila J.
      ,
      • Spittaels K.
      • Van den Haute C.
      • Van Dorpe J.
      • Geerts H.
      • Mercken M.
      • Bruynseels K.
      • Lasrado R.
      • Vandezande K.
      • Laenen I.
      • Boon T.
      • Van Lint J.
      • Vandenheede J.
      • Moechars D.
      • Loos R.
      • Van Leuven F.
      ). GSK3β most prominently catalyzes phosphorylation at Ser202, Ser235, Ser396, and Ser404 (
      • Utton M.A.
      • Vandecandelaere A.
      • Wagner U.
      • Reynolds C.H.
      • Gibb G.M.
      • Miller C.C.
      • Bayley P.M.
      • Anderton B.H.
      ), whereas Cdk5/p25 has been shown to phosphorylate tau at Thr181, Ser202, Thr205, Thr212, Thr217, Ser396, and Ser404 (
      • Evans D.B.
      • Rank K.B.
      • Bhattacharya K.
      • Thomsen D.R.
      • Gurney M.E.
      • Sharma S.K.
      ,
      • Lund E.T.
      • McKenna R.
      • Evans D.B.
      • Sharma S.K.
      • Mathews W.R.
      ,
      • Hashiguchi M.
      • Sobue K.
      • Paudel H.K.
      ). Thus GSK3β and Cdk5/p25 potentially join CK1 in modulating tau/microtubule equilibrium (
      • Cho J.H.
      • Johnson G.V.
      ). Although the potential interplay among these enzymes is unknown, it is interesting to note that GSK3β functions primarily as a phosphate-directed protein kinase dependent on priming of substrates by other protein kinases (). Because of its strong contribution to basal tau phosphorylation state and inducibility in response to stress, CK1 could potentially provide priming activity as it does with β-catenin phosphorylation (
      • Amit S.
      • Hatzubai A.
      • Birman Y.
      • Andersen J.S.
      • Ben-Shushan E.
      • Mann M.
      • Ben-Neriah Y.
      • Alkalay I.
      ). Yet CK1 also recognizes primed substrates as in the case of p53 (
      • Sakaguchi K.
      • Saito S.
      • Higashimoto Y.
      • Roy S.
      • Anderson C.W.
      • Appella E.
      ), and in this substrate recognition mode could potentially further amplify disease-associated phosphorylation of sites found tightly clustered around the microtubule repeat region (
      • Hanger D.P.
      • Betts J.C.
      • Loviny T.L.
      • Blackstock W.P.
      • Anderton B.H.
      ,
      • Morishima-Kawashima M.
      • Hasegawa M.
      • Takio K.
      • Suzuki M.
      • Yoshida H.
      • Watanabe A.
      • Titani K.
      • Ihara Y.
      ).
      Finally, we note that CK1 phosphorylates proteins in addition to tau in vivo. In the cases of p53 (
      • Sakaguchi K.
      • Saito S.
      • Higashimoto Y.
      • Roy S.
      • Anderson C.W.
      • Appella E.
      ), β-catenin (
      • Gao Z.H.
      • Seeling J.M.
      • Hill V.
      • Yochum A.
      • Virshup D.M.
      ), multidrug transporter Pdr5p (
      • Decottignies A.
      • Owsianik G.
      • Ghislain M.
      ), Hedgehog signaling effector Cubitus interruptus (
      • Price M.A.
      • Kalderon D.
      ), and proteins involved in circadian rhythm control (
      • Kloss B.
      • Price J.L.
      • Saez L.
      • Blau J.
      • Rothenfluh A.
      • Wesley C.S.
      • Young M.W.
      ), CK1-mediated phosphorylation modulates protein turnover. This function may be part of the neuronal response to stress, accounting for both the induction of CK1 in AD and its presence in granulovacuolar degeneration bodies. Thus CK1-mediated tau phosphorylation may result from an attempt by the cell to modulate protein turnover in response to cellular distress.

      Acknowledgments

      We thank Dr. Rick Dobrowsky, University of Kansas, for providing the p25-pCDNA construct, Dr. Peter Davies, Albert Einstein College of Medicine, for providing PHF1 antibody, and Dr. Lester I. Binder for providing monoclonal antibody Tau5.

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