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Keywords:

  • Tau;
  • Phosphorylation;
  • Cyclic AMP-dependent protein kinase;
  • Conformation;
  • Alzheimer's disease

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Immunoaffinity purification of PHFs
  5. HPLC separations
  6. RESULTS
  7. PHF-associated kinase activity in degenerative disease states
  8. Identification of PHF kinase phosphorylation sites on tau
  9. DISCUSSION
  10. Acknowledgements

Abstract : Immunoaffinity-purified paired helical filaments (PHFs) from Alzheimer's disease (AD) brain homogenates contain an associated protein kinase activity that is able to induce the phosphorylation of PHF proteins on addition of exogenous MgCl2 and ATP. PHF kinase activity is shown to be present in immunoaffinity-purified PHFs from both sporadic and familial AD, Down's syndrome, and Pick's disease but not from normal brain homogenates. Although initial studies failed to show that the kinase was able to induce the phosphorylation of tau, additional studies presented in this article show that only cyclic AMP-dependent protein kinase-pretreated recombinant tau is a substrate for the PHF kinase activity. Deletional mutagenesis, phosphopeptide mapping, and site-directed mutagenesis have identified the PHF kinase phosphorylation sites as amino acids Thr361 and Ser412 in htau40. In addition, the cyclic AMP-dependent protein kinase phosphorylation sites that direct the PHF kinase have been mapped to amino acids Ser356 and Ser409 in htau40. Additional data demonstrate that these hierarchical phosphorylations in the extreme C terminus of tau allow for the incorporation of recombinant tau into exogenously added AD-derived PHFs, providing evidence that certain unique phosphorylations of tau may play a role in the pathogenesis of neurofibrillary pathology in AD.

Alzheimer's disease (AD), classified as a neurodegenerative disorder, is the most prevalent cause of dementia in the elderly population. The two main pathological hallmarks of AD are the intraneuronal neurofibrillary tangle, composed of paired helical filaments (PHFs), and the senile plaque, composed of abnormally processed aggregates of β-amyloid interwoven with dystrophic neurites containing PHFs (Khachaturian, 1985). The main constituent of PHF is the microtubule-associated protein tau, which is posttranslationally altered in AD (Wood et al., 1986 ; Kosik et al., 1988 ; Lee et al., 1991). As such, much research has focused on elucidating the pathological alterations in tau that may contribute to the formation of PHFs in AD. Tau in PHFs (PHF-tau) differs from normal adult tau in its state of phosphorylation, with PHF-tau being hyperphosphorylated at as many as 20 amino acid residues as compared with normal adult tau (Grundke-Iqbal et al., 1986 ; Wood et al., 1986 ; Lee et al., 1991 ; Goedert et al., 1992 ; Kanemaru et al., 1992 ; Morishima-Kawashima et al., 1995). Aberrant activation of certain neuronal kinases and inactivation of certain neuronal phosphatases have been postulated to be the proximal cause of PHF formation in AD (for review, see Trojanowski and Lee, 1994, 1995).

Recent evidence has suggested that the hyperphosphorylation of tau seen in AD is due to the inactivation of protein phosphatases that are normally functional and quite active in the postmortem interval and that many of the previously-thought-to-be-pathological phosphorylations of tau are present in biopsy-derived tissues from non-Alzheimer patients (Matsuo et al., 1994 ; Hasegawa et al., 1996). These discoveries have been the impetus for the renewed search for true Alzheimer-specific events. Several Alzheimer-unique phosphorylations have been identified in PHF-tau by either phosphopeptide mapping strategies or antibody detection (Hasegawa et al., 1992, 1996 ; Matsuo et al., 1994). It is likely that this new class of unique phosphorylation events plays a direct role in the pathological modifications of tau leading to the neurofibrillary degeneration characteristic of AD and requires further study.

Recently, a method for immunoaffinity-purifying PHF from AD brain homogenates has been developed using an isotype-switched form of the Alz-50 monoclonal antibody, p42, linked to Sepharose beads (Vincent and Davies, 1992). Standard methods of PHF purification from AD brain homogenates include the use of harsh denaturing agents that inactivate associated enzymatic activities (Gonzalez et al., 1992 ; Greenberg et al., 1992), but the methods used for immunoaffinity purifying PHFs are relatively mild and have been shown to preserve a PHF-associated kinase activity (Vincent and Davies, 1992). Immunoaffinity-purified PHFs appear to be quite soluble in standard buffers and presumably represent an early stage in the pathogenesis of PHF formation in AD (Vincent and Davies, 1992). Thus, enzymatic activities associated with these preparations may contribute to the pathogenetic mechanisms involved in PHF formation.

Analysis of immunoaffinity-purified PHFs has shown that a serine/threonine kinase activity (PHF kinase) is present in these preparations. This PHF kinase has been shown to be able to induce the phosphorylation of PHF constituents on addition of exogenously added Mg2+ and ATP (Vincent and Davies, 1992). In addition, the level of phosphate incorporation can be increased by prior incubation with alkaline phosphatase, suggesting that the PHF kinase phosphorylation sites are partially phosphorylated in PHFs and that this phosphorylation is at least partially responsible for the pathological modifications of PHF-tau seen in AD (Vincent and Davies, 1992). Many neuronal kinases and phosphatases have been implicated in the pathogenesis of PHF formation (for review, see Trojanowski and Lee, 1994, 1995) ; however, only the PHF kinase has been shown to copurify with this pathological hallmark of AD (Vincent and Davies, 1992).

Although many neuronal kinases, including cyclic AMP-dependent protein kinase (PKA) (Pierre and Nunez, 1983 ; Steiner et al., 1990 ; Litersky and Johnson, 1992 ; Scott et al., 1993 ; Singh et al., 1994), protein kinase C (Hoshi et al., 1987 ; Steiner et al., 1990 ; Correas et al., 1992 ; Singh et al., 1994), calcium/calmodulin-dependent protein kinase (Yamamoto et al., 1985 ; Steiner et al., 1990 ; Baudier and Cole, 1987), casein kinase II (Steiner et al., 1990 ; Singh et al., 1995a), mitogen-activated protein (MAP) kinase (Drewes et al., 1992 ; Ledesma et al., 1992 ; Lu et al., 1993 ; Trojanowski et al., 1993 ; Arendt et al., 1995), p34-cdc2 (Hall et al., 1990 ; Ledesma et al., 1992 ; Mawal-Dewan et al., 1992 ; Baumann et al., 1993 ; Wood et al., 1993), cdk5 (Baumann et al., 1993 ; Paudel et al., 1993 ; for review, see Lew and Wang, 1995), glycogen synthase kinase 3 (GSK-3) (Hanger et al., 1992 ; Mandelkow et al., 1992 ; Ishiguro et al., 1993 ; Yang et al., 1993a,b ; Moreno et al., 1995 ; Singh et al., 1995b), and p110mrk (Drewes et al., 1995), can phosphorylate recombinant tau in vitro, the PHF kinase is ineffective (Vincent et al., 1994 ; Kuret et al., 1997), suggesting that it is not one of the kinases that has been studied previously as being responsible for the hyperphosphorylation of tau seen in AD.

The present study demonstrates that (a) PHF kinase activity is present in immunoaffinity eluents from all PHF-associated diseases but not from normal individuals, (b) only PKA phosphorylation of tau on Ser356 and Ser409 allows the PHF kinase to phosphorylate sequentially recombinant tau in vitro, and (c) the PHF kinase induces the phosphorylation of Thr361 and Ser412 on PKA-pretreated recombinant tau in vitro. These data illustrate the specificity of the PHF kinase for AD and other PHF-associated diseases, provide further support for the hypothesis that the sequential actions of multiple protein kinases are required to produce the hyperphosphorylation of tau seen in AD, and positively identify the major PHF kinase-induced phosphorylation of tau (Ser412) as a previously identified AD-specific event (Hasegawa et al., 1992). They also raise the possibility that the phosphorylation of Thr361 in PHFs may have been overlooked in previous mapping studies of PHF-tau and demonstrate the unique substrate specificity of the PHF-associated protein kinase on tau, providing some insight into the potential functional consequences of these unique phosphorylations on tau and the role they may play in the pathogenesis of neurofibrillary pathology in AD.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Immunoaffinity purification of PHFs
  5. HPLC separations
  6. RESULTS
  7. PHF-associated kinase activity in degenerative disease states
  8. Identification of PHF kinase phosphorylation sites on tau
  9. DISCUSSION
  10. Acknowledgements

Antibodies and immunoaffinity columns

Alz-50 is an IgM monoclonal antibody raised to AD basal forebrain homogenates (Wolozin et al., 1986). An isotype switched form of the Alz-50 antibody, p42, was conjugated to agarose beads to create an Alz-50 immunoaffinity column (Vincent and Davies, 1992). The MC-1 monoclonal antibody was raised to p42-immunoaffinity-purified PHF and has been shown to recognize a similar pathological conformation of the tau molecule that is recognized by Alz-50 (Jicha et al., 1997). MC-1, however, appears to have greater affinity and an increased specificity for pathological conformations of tau than Alz-50 (authors' unpublished data). To exploit these advantages, MC-1 antibody was purified from serum-free Hybridoma Medium SPF (Fisher Scientific) on protein A and coupled to Affi-gel 10 (Bio-Rad Laboratories) in 50 mM sodium phosphate (pH 8.1) at a ratio of 2-5 mg of protein/ml of gel. These conditions afforded binding of >95% of the antibody to the gel. Columns that contained a ratio of <2 mg of protein/ml of gel did not bind appreciable concentrations of antigen.

Immunoaffinity purification of PHFs

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Immunoaffinity purification of PHFs
  5. HPLC separations
  6. RESULTS
  7. PHF-associated kinase activity in degenerative disease states
  8. Identification of PHF kinase phosphorylation sites on tau
  9. DISCUSSION
  10. Acknowledgements

Purified, soluble PHF preparations were obtained according to the procedures of Vincent and Davies (1992). The MC-1 antibody/gel mixture was poured into columns with a length to diameter ratio of at least 20 and washed with 50 volumes of Tris-buffered saline (TBS ; 10 mM Tris and 150 mM NaCl, pH 7.4). Before purification of antigen, the columns were washed with 2 volumes of 3 M KSCN, followed with 5 volumes of TBS. Brain extracts were made by homogenizing 1 g of dissected cortical gray matter in 10 ml of TBS containing 1 mM phenylmethylsulfonyl fluoride using a PowerGen Polytron (Fisher Scientific) on maximal setting. Homogenates were centrifuged at 27,000 g for 20 min, and the supernatants were decanted. Pellets were rehomogenized in TBS using half of the original volume and recentrifuged. The supernatants then were pooled and filtered through a 4-cm-high, 3.5-cm-diameter column of Sepharose 400 superfine to prevent particulate matter from accumulating at the top of the MC-1 columns. The final extracts were eluted through the MC-1 columns at a flow rate of ~25 ml/h for up to 36 h, after which the columns were washed with TBS at flow rates of >100 ml/h for at least 18 h. Bound antigen was eluted with 3 M KSCN, and fractions were assayed for protein by the method of Bradford (1976). Samples containing at least 50 μg of protein/ml were pooled and dialyzed against TBS. The MC-1 columns were reused at least six times before replacement of the gel and demonstrated no reduction in the capability of the column to bind antigen.

Recombinant tau constructs and protein expression

Clone htau40 was the generous gift of M. Goedert (Goedert et al., 1989). Clone 10 encoding for a fetal tau construct missing exons 4 and 10—an N-terminal insert and the second microtubule binding repeat, respectively—was cloned from a human fetal λgt11 expression library. Clone htau40 was digested with NdeI and EcoRI, blunted with Klenow enzyme, and ligated into SmaI-digested pQE-32 (Qiagen) to produce a histidine-tagged recombinant protein, TauW. The cDNA encoding TauW was digested with HindIII and religated to itself producing a construct encoding amino acids 1-342 of htau40, TauNW. Clone 10 and the cDNA encoding TauW were digested with PstI and ligated into PstI-digested pQE-31, to create cDNAs encoding for TauP and TauP + exon 10, respectively. A series of site-directed mutants were constructed by oligo-directed mutagenesis, including S356A (in TauP + exon 10), S409G (in TauP), S416G (in TauP), S412A (in TauP), S413A (in TauP), and S412A + S413A + T414A (triple mutant in TauP) individually. All constructs (Fig. 1) were chemically transformed into Escherichia coli strain MC-15 harboring the pREP4 plasmid. Cells were grown to an absorbance at 600 nm of 0.5, and protein expression was induced by addition of isopropyl β-d-thiogalactopyranoside (Boehringer-Mannheim) to 2 mM for 5 h. Cell cultures were then centrifuged at 5,000 g for 5 min, and the cell pellet was resuspended in 50 mM sodium phosphate and 300 mM NaCl (pH 8.0) and frozen overnight at -20°C. Cell suspensions were thawed on ice, sonicated three times each for 5 min to lyse cells, and spun at 5,000 g for 5 min. Supernatants were then boiled for 10 min at 100°C and recentrifuged for 5 min at 5,000 g. Supernatants were then applied to a column of Ni2+-conjugated agarose equilibrated in lysis buffer, washed with 50 column volumes of lysis buffer, washed with 200 column volumes of wash buffer (50 mM sodium phosphate, 300 mM NaCl, and 10% glycerol, pH 6.3), and eluted with wash buffer adjusted to pH 4.5. The eluted sample was then dialyzed overnight in three changes of TBS in preparation for phosphorylation reactions. Protein concentrations were assayed by the method of Bradford (1976), and concentrations were adjusted to 0.2 mg/ml.

image

Figure 1. Diagrammatic representation of tau constructs used in this study. TauW encodes for full-length htau40 with an N-terminal 6× histidine tag. TauNW encodes for amino acids 1-342 of htau40 with an N-terminal 6× histidine tag. TauP, a fetal tau construct, encodes for amino acids 242-440 minus exon 10 in the numbering system of htau 40 with an N-terminal 6× histidine tag. TauP + exon 10 encodes for amino acids 242-440 in the numbering system of htau 40 with an N-terminal 6× histidine tag. TauP was used to create the site-directed mutants S409G, S416G, S412A, S413A, and S-S-T[RIGHTWARDS ARROW]A412-A413-A414. TauP + exon 10 was used to create the site-directed mutant S356A. Previously identified PKA phosphorylation sites are shown on TauW (Scott et al., 1993).

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Phosphorylation reactions

Four micrograms of substrate was used in each reaction, and the total reaction volume was included in each subsequent step. PKA, protein kinase C, casein kinase II, p34-cdc2, MAP kinase (p44), and GSK-3 were all purchased from Upstate Biotechnology, and reactions were carried out according to the manufacturer's specifications. Activated cdk5 was the generous gift of Dr. J. Wang and was reacted as described previously (Paudel et al., 1993). All primary phosphorylation reactions were incubated for 16 h to allow for maximal phosphorylation and to aid in kinase inactivation in preparation for sequential phosphorylation reactions. PHF preparations containing the associated protein kinase activity were prepared and reacted as previously described (Vincent and Davies, 1992). In brief, PHF kinase reactions were carried out in TBS with addition of 5 mM MgCl2 and 200 μM ATP. PHF kinase phosphorylation reactions were carried out for 2 h at 25°C. Twenty microliters of PHF preparations was used for all lanes of Fig. 2. Six micrograms of casein was included in each sample to control for variability in substrate concentration in each individual PHF preparation. PHF preparations from sporadic AD cases containing 1 μg of total protein were used in all other phosphorylation reactions requiring the PHF kinase activity. All primary kinase reactions intended for use in sequential phosphorylation reactions were reacted with unlabeled ATP. Before PHF kinase sequential phosphorylation reactions, either kinases used for primary phosphorylations were inactivated by heating to 95°C for 10 min, or the phosphorylated tau was repurified on an Ni2+-conjugated agarose column. Inactivation of kinase activities used for primary phosphorylations was verified by the absence of labeled PHF proteins analyzed by autoradiography after addition of [γ-32P]ATP and MgCl2, incubation, and protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All other reactions were performed with [γ-32P]ATP in preparation for autoradiogram and scintillation detection.

image

Figure 2a. : Coomassie Blue-stained gel of AD brain extract (lane 1), MC-1 column flow through (lane 2), and unconcentrated column eluent (lane 3) demonstrates a typical MC-1 column purification. B : Coomassie Bluestained gel of 20× concentrated column eluents from sporadic AD (lane 1), Down's syndrome (lane 2), and familial AD with a PS1 mutation (lane 3) brain extracts demonstrates the protein composition of typical MC-1 column eluents. C : Autoradiogram demonstrates PHF-tau and exogenously added casein phosphorylation in the immunoaffinity eluents from sporadic AD (lane 1), presenilin AD (lanes 2 and 3), amyloid precursor protein AD (lanes 4 and 5), Down's syndrome (lane 6), and Pick's disease (lanes 8 and 9) but not from normal adult brain homogenates (lane 7).

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Western blots and autoradiograms

The entire sample of recombinant phosphorylated tau fragments from each reaction (0.4 μg) was solubilized by heating in sample buffer [40 mM Tris (pH 6.8), 1% sodium dodecyl sulfate, and 50% glycerol] at 95°C for 5 min. Prepared samples were separated by polyacrylamide gel electrophoresis on 10% acrylamide gels. Proteins were then transferred to nitrocellulose (pore size, 0.45 μm) in preparation for western blotting and autoradiogram protocols. Blots were exposed to film for 12 h, and the resulting autoradiograms were developed. Blots were incubated in TBS with 5% milk for 30 min to block nonspecific antibody binding. Blots were incubated with TG-5 (Jicha et al., 1997), a monoclonal antibody raised in our laboratory recognizing amino acids 220-240 of the primary sequence of htau40, or with 6HIS, a monoclonal antibody raised in our laboratory recognizing the N-terminal histidine tag present on all recombinant tau constructs used in this study, at a 1:10 dilution in TBS with milk for 16 h at 4°C. Blots were then washed three times each for 5 min with TBS and incubated with horseradish peroxidase-labeled anti-mouse IgG1 or IgG2B antibody for 2 h at 25°C. Blots were washed three times each for 5 min, and reactive proteins were visualized by reaction with 4-chloronaphthal in peroxide. After visualization of protein bands, individual lanes were isolated and counted for radioactivity by scintillation on a Beckman model LS6000 spectrophotometer to determine stoichiometries of phosphate incorporation in each sample.

Protease digestions

In preparation for peptide mapping studies, phosphorylated TauP was incubated with AP-1 protease at a 1:100 enzyme to substrate ratio for 16 h at 37°C in TBS with addition of 2 M urea as described previously (Masaki et al., 1981 ; Hasegawa et al., 1992). Trypsin and elastase digestions were performed in 10 mM Tris-base (pH 8.5) with addition of CaCl2 to 5 mM at a 1:100 by weight enzyme to substrate ratio for 16 h at 37°C.

HPLC separations

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Immunoaffinity purification of PHFs
  5. HPLC separations
  6. RESULTS
  7. PHF-associated kinase activity in degenerative disease states
  8. Identification of PHF kinase phosphorylation sites on tau
  9. DISCUSSION
  10. Acknowledgements

Separations were achieved on an HP1090 chromatograph using Vydac C8 2.1-mm columns. All detection was at 214 nm. HPLC procedures consisted of a 20-min isocratic gradient of 100% solution A, followed by a 1% addition of solution B per minute for 60 min. Solution A contained 100% water with 0.1% trifluoroacetic acid, and solution B contained 100% acetonitrile with 0.08% trifluoroacetic acid.

Edman sequencing

Amino terminal sequence analysis of the digested peptide fragments was performed by automated Edman degradation with a model 477A sequencer (Applied Biosystems) using the manufacturer's standard programming and chemicals.

Electron microscopy

Ten microliters of PKA-reacted TauW, PKA-reacted TauW + PHFs, or PKA-reacted TauW + PHFs + Mg2+ + ATP was applied to Formvar carbon-coated, glow-discharged grids. The grids were treated with TBS containing 1% bovine serum albumin and 5% normal goat serum (TBS+) for 10 min. 6HIS monoclonal antibody was diluted 1:50 in TBS+, and the grids were stained for 1 h at 25°C. After washing with TBS+, 10-nm-diameter gold particles conjugated to goat anti-mouse IgG2B (Goldmark Biologicals) were diluted 1:40 in TBS+, and the grids were stained for 1 h at 25°C. Grids were then washed and fixed for 5 min with 2% glutaraldehyde in TBS. After fixation, the grids were stained with 5% uranyl acetate in water for 30 min. A JEOL Jem 100CX scope was used to visualize PHF samples at 30K magnification.

PHF-associated kinase activity in degenerative disease states

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Immunoaffinity purification of PHFs
  5. HPLC separations
  6. RESULTS
  7. PHF-associated kinase activity in degenerative disease states
  8. Identification of PHF kinase phosphorylation sites on tau
  9. DISCUSSION
  10. Acknowledgements

To investigate the specificity of the PHF kinase for all AD cases, a series of immunoaffinity purifications were performed on both familial and sporadic AD, Pick's disease, and normal adult brain homogenates. After addition of [γ-32P]ATP, Mg2+, and casein, sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation and autoradiographic analysis demonstrated that immunoaffinity column eluents from sporadic AD, chromosome 14 familial AD (presenilin 1), chromosome 21 familial AD (amyloid precursor protein), Down's syndrome, and Pick's disease brain homogenates all contained the PHF kinase activity (Fig. 2). Immunoaffinity column eluents from a normal adult control brain homogenate were devoid of protein kinase activity (Fig. 2).

Hierarchical tau phosphorylation

Because the major phosphorylated species identified in Fig. 2, other than the exogenously added casein, resembles the PHF-tau pattern demonstrated by many anti-tau antibodies in AD cases, the PHF kinase, PKA, protein kinase C, casein kinase II, MAP kinase, GSK-3, p34-cdc2, and cdk5 were all assayed for activity against unmodified recombinant TauW. All kinases previously implicated in the hyperphosphorylation of tau seen in AD, including PKA, protein kinase C, casein kinase II, MAP kinase, GSK-3, p34-cdc2, and cdk5, were able to phosphorylate recombinant TauW in vitro, but the PHF kinase was not (Fig. 3A), suggesting that it is not one of the kinases previously implicated as being responsible for the hyperphosphorylation of tau seen in AD.

image

Figure 3a. : Autoradiogram and TG-5 western blot of unmodified TauW (4 μg per lane) reacted with PKA, protein kinase C (PKC), casein kinase II (CKII), cdk5, p34-cdc2, MAP kinase (MAPK ; p44erk), GSK-3, and the PHF kinase preparation. Only the PHF kinase is unable to induce the phosphorylation of the unmodified recombinant tau used in this study. B : Autoradiogram and TG-5 western blot of sequential PHF kinase-induced phosphorylations on TauW (4 μg per lane) previously reacted with PKA, PKC, CKII, cdk5, p34-cdc2, MAPK, and GSK-3. Inactivation of kinases used for primary phosphorylations was verified by autoradiogram (data not shown). Only PKA-pretreated TauW is shown to be a substrate for the PHF kinase. Also, it should be noted that the GSK-3 reaction conditions seemed to enhance the PHF kinase-induced phosphorylation of endogenous PHF proteins, which migrate slightly faster than the 6× histidine-tagged recombinant tau used in this study.

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Because previous studies have suggested that the hyperphosphorylation of tau seen in AD may be due to the sequential actions of multiple kinases acting in a synergistic fashion (Yang et al., 1993a,b ; Blanchard et al., 1994 ; Singh et al., 1995b), PHF kinase activity was assayed against recombinant tau previously phosphorylated in vitro by a panel of protein kinases. Prior phosphorylation by protein kinase C, casein kinase II, MAP kinase, GSK-3, p34-cdc2, and cdk5 did not allow the PHF kinase to phosphorylate TauW further. In contrast, PKA-phosphorylated TauW was shown to be a substrate for the PHF kinase (Fig. 3B).

Identification of PHF kinase phosphorylation sites on tau

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Immunoaffinity purification of PHFs
  5. HPLC separations
  6. RESULTS
  7. PHF-associated kinase activity in degenerative disease states
  8. Identification of PHF kinase phosphorylation sites on tau
  9. DISCUSSION
  10. Acknowledgements

To identify the region(s) of the tau molecule that are sequentially phosphorylated by the PHF kinase, TauNW (encoding for amino acids 1-342) and TauP (encoding for amino acids 242-440 but missing exon 10) were assayed for sequential phosphorylation by the PHF kinase after PKA pretreatment. TauNW and TauP were shown to be substrates for PKA phosphorylation (Fig. 4A), but only PKA-phosphorylated TauP was a substrate for the PHF kinase (Fig. 4B). These data show that the PHF kinase phosphorylation site(s) reside in the C-terminal 99 amino acids of tau, a region conserved in all six tau isoforms (Goedert et al., 1989).

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Figure 4. Autoradiograms and 6HIS western blots of PKA- and PHF kinase-induced phosphorylations on TauNW (amino acids 1-342 ; 4μg per lane) and TauP (amino acids 242-440 ; 4 μg per lane). A : PKA is shown to induce the phosphorylation of both tau constructs. B : Only PKA-pretreated TauP is a substrate for the PHF kinase. Controls demonstrate inactivation of PKA and verify that the phosphorylation of TauP is due to the sequential actions of the PHF kinase.

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To identify the exact PHF kinase phosphorylation sites and the PKA sites that direct these phosphorylations, PHF kinase sequentially phosphorylated TauP was digested with either AP-1 protease (cleaves C-terminal to lysine residues) (Fig. 5A) or AP-1 protease plus trypsin plus elastase and purified by HPLC separation on a C8 column. Peaks were collected and counted by scintillation for incorporation of the 32P label. The PHF kinase was shown to induce the phosphorylation of both Thr361 (a minor site) and additional site(s) in the C-terminal fragment consisting of residues 396-438 (Fig. 5).

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Figure 5. HPLC chromatograms of PKA-PHF kinase sequentially phosphorylated TauP digested with either (A) AP-1 or (B) a combination of AP-1, trypsin, and elastase and eluted on a C8 column to enhance recovery. A : Fragment 1 eluted at 47.3 min and contained 10,483 cpm of incorporated 32P. Edman degradation was carried out for 11 cycles and demonstrated that this fragment contained the N-terminal sequence SPVVSGDTSPR, which corresponds to the AP-1 fragment containing amino acids 396-438 of tau (SPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAK). B : Fragment 2 eluted at 43.2 min and contained 1,000 cpm of incorporated 32P. Peptide sequencing data of the N terminus (XHVPGGGNK) demonstrated that this fragment corresponds to an AP-1, trypsin, and elastase cleavage product containing amino acids 361-369 (THVPGGGNK) of tau. These data positively identify Thr361 as a phosphate acceptor for the PHF kinase (phenylhydantoin-phosphothreonine is not identified as a regular phenylhydantoin amino acid during Edman degradation). mAU, millabsorbance units.

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Because sufficient quantities of PHF kinase sequentially phosphorylated fragments were not recovered from the HPLC system to allow exact detection of the PHF kinase phosphorylation site(s) in the C terminus of tau, additional site-directed mutagenic strategies were used. Site-directed mutagenesis of the previously identified PKA phosphorylation sites (Scott et al., 1993) S356A, S409G, and S416G demonstrates that the major sequential phosphorylation of tau by the PHF kinase is directed by the PKA-induced phosphorylation of Ser409 (Fig. 6A). An additional site-directed mutant, S412A + S413A + T414A (triple mutant in TauP), positively identified the PHF kinase phosphorylation sites as amino acids Ser412, Ser413, or Thr414 in htau40 (Fig. 6B). Further analysis of the PHF kinase using S412A and S413A mutants demonstrates that the primary PHF kinase phosphorylation site is Ser412 in the numbering system of htau40 (Fig. 6B).

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Figure 6. A : Autoradiogram and 6HIS western blots of PKA (left half) and PHF kinase sequentially phosphorylated (right half) TauP, Ser356[RIGHTWARDS ARROW]Ala, Ser409[RIGHTWARDS ARROW]Gly, and Ser416[RIGHTWARDS ARROW]Gly site-directed mutants (4 μg per lane) demonstrate that the PHF kinase activity against recombinant tau is dependent on the PKA-induced prior phosphorylation of Ser409. Controls demonstrate inactivation of PKA and verify that the phosphorylation of TauP is due to the sequential actions of the PHF kinase. B : Autoradiogram and 6HIS western blots of PKA (left half) and PHF kinase sequentially phosphorylated (right half) TauP, Ser412,413,415[RIGHTWARDS ARROW]Ala, Ser412[RIGHTWARDS ARROW]Ala, and Ser413[RIGHTWARDS ARROW]Ala site-directed mutants (4 μg per lane) demonstrate that the C-terminal PHF kinase phosphorylation site is Ser412. Controls demonstrate inactivation of PKA and verify that the phosphorylation of TauP is due to the sequential actions of the PHF kinase.

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Stoichiometry of phosphate incorporation

Our calculation of PHF kinase-induced phosphate incorporation into PKA-pretreated tau is 0.18 mol of phosphate/mol of TauP (Table 1). Analysis of the S409G and S412A mutants demonstrated an overall reduction of 0.22 mol of phosphate/mol of tau for PKA phosphorylations (S409G mutant) and an overall reduction of 0.16 mol of phosphate/mol of tau for PHF kinase sequential phosphorylations (S409G and S412A mutants ; see Table 1), suggesting an overall stoichiometry for PHF kinase sequential phosphorylation at Ser412 of 0.73 mol of phosphate/mol of Ser409-phosphorylated TauP (using the equation [TauP — S412A]PHF kinase/[TauP — S409G]PKA). Analysis of the S356A mutant demonstrated an overall reduction of 0.30 mol of phosphate/mol of tau for PKA phosphorylations and an overall reduction of 0.03 mol of phosphate/mol of tau for PHF kinase sequential phosphorylations (Table 1), suggesting an overall stoichiometry for PHF kinase sequential phosphorylation at Thr361 of 0.1 mol of phosphate/mol of Ser356-phosphorylated TauP (using the equation [TauP — S356A]PHF kinase/[TauP — S356A]PKA). This analysis demonstrates that the optimal consensus sequence for the PHF kinase is Ser(P)-X-X-Ser (amino acids 409-412 in tau) but that the PHF kinase also recognizes the consensus sequence Ser(P)-X-X-X-X-Thr (amino acids 361-369), albeit to a lesser extent.

Table 1. Stoichiometry of phosphate incorporation for PKA, control, and PHF kinase sequential phosphorylations on TauP and the site-directed mutants fromFig. 6Data are in mol of phosphate/mol of tau construct.
Tau constructPKAPKA controlPKA/PHF kinase
TauP (average)1.350.030.21
A3561.050.030.18
G4091.130.020.05
G4161.160.020.22
A412,413,4141.240.010.04
A4121.350.020.05
A4131.390.020.23

Hierarchical phosphorylation-induced aggregation

TauP, PKA-phosphorylated TauP, and PHF kinase sequentially phosphorylated TauP were all purified by HPLC separation on a C8 column (Fig. 7A-C). Sequential phosphorylations of TauP lead to a decreased recovery of protein from the HPLC column (Fig. 7D), suggesting either (a) that the addition of phosphates to the tau molecule results in an increased exposure of hydrophobic residues that bound irreversibly to the hydrophobic matrix of the column or (b) that sequential phosphorylation leads to the formation of high-molecular-weight aggregates that are not recoverable from the HPLC system. To investigate the possibility that the PKA and PHF kinase sequential phosphorylations of tau facilitate the aggregation of recombinant tau, immunoelectron microscopy using the 6HIS antibody was performed on PKA-reacted TauW, PKA-reacted TauW + PHF, and PKA-reacted TauW + PHF + Mg2+ + ATP. Figure 8 demonstrates that PKA-reacted TauW was ineffective at inducing filament formation. Addition of PHF in the absence of Mg2+ and ATP allowed for a slight incorporation of PKA-reacted TauW into existing PHFs in what may be a nucleation or seeding event (Fig. 8). In contrast, PKA-reacted TauW + PHF + Mg2+ + ATP resulted in an enhanced aggregation of histidine-tagged recombinanut tau to existing PHFs, suggesting that enzymatic modifications of tau may accelerate the process of filament formation in AD.

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Figure 7. HPLC chromatograms of (A) TauP, (B) PKA-phosphorylated TauP, and (C) PHF kinase sequentially phosphorylated TauP. Because mass data could only be determined for the unphosphorylated TauP (attempts at mass determinations for both PKA and PHF kinase sequentially phosphorylated TauP identified only high-molecular-weight hypermasses that were uninterpretable), the identity of the monomeric and dimeric peaks was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis under both reducing and nonreducing conditions (data not shown). Between each sample elution, the column was taken through a blank elution to ensure that all protein had eluted in the initial gradient and was recalibrated using a known peptide standard. D : Diminished recovery of tau protein after both PKA and PHF kinase phosphorylation.

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image

Figure 8. Sequential phosphorylations by PKA and the PHF kinase allow for the rapid aggregation of TauW into existing PHFs. Immunoelectron microscopy was done with monoclonal antibody 6HIS. A : PKA-reacted TauW (Fig. 3A). B : PHF kinase-reacted unmodified TauW (Fig. 3A, PHF kinase lane). C : PKA/PHF kinase-reacted TauW (Fig. 3B, PKA lane). Bar = 100 nm.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Immunoaffinity purification of PHFs
  5. HPLC separations
  6. RESULTS
  7. PHF-associated kinase activity in degenerative disease states
  8. Identification of PHF kinase phosphorylation sites on tau
  9. DISCUSSION
  10. Acknowledgements

The recent development of a method to immunoaffinity purify conformationally altered (Jicha et al., 1997) pathological tau molecules under relatively mild conditions (Vincent and Davies, 1992) has allowed the opportunity not only to study the pathological alterations in tau seen in AD, but also to study proteins that are associated with pathological tau. Initial analysis of the immunoaffinity column eluent demonstrated that a serine/threonine kinase was present in the collected sample (Vincent and Davies, 1992). The PHF kinase was able to induce the phosphorylation of the PHF constituents found in the eluent on addition of exogenous ATP and Mg2+ (Vincent and Davies, 1992).

Because hyperphosphorylated tau has been shown to be the major proteinaceous constituent of PHF in AD (Grundke-Iqbal et al., 1986 ; Wood et al., 1986 ; Lee et al., 1991 ; Goedert et al., 1992 ; Kanemaru et al., 1992), there is a great deal of interest in identifying the kinases that may be responsible for the pathological hyperphosphorylation of tau seen in AD. Although many studies have shown that almost all kinase preparations are able to phosphorylate unmodified recombinant tau in vitro (for review, see Trojanowski and Lee, 1994 ; Pelech, 1995), the PHF kinase cannot, suggesting that it is not any of the protein kinases studied previously as being responsible for the hyperphosphorylation of tau seen in AD. Although additional studies have shown by immunohistochemical staining a close association of various protein kinases with neurofibrillary pathology in AD brains (Hanger et al., 1992 ; Baumann et al., 1993 ; Trojanowski et al., 1993 ; Wood et al., 1993 ; Arendt et al., 1995), none appears to be as tightly associated as the PHF kinase, as demonstrated by our immunoaffinity purification procedure (Vincent and Davies, 1992).

All of the kinases previously postulated to be responsible for the pathological phosphorylations of tau seen in AD, including PKA, protein kinase C, casein kinase II, MAP kinase, GSK-3, p34-cdc2, and cdk5, are able to phosphorylate the recombinant tau fragments used in this study. However, the PHF kinase is unable to induce the phosphorylation of unmodified recombinant tau in vitro, despite the observation that PHF constituents are an excellent substrate for this kinase (Vincent and Davies, 1992). These observations suggest that certain phosphorylations of the tau molecule found in PHFs may be required to create or expose the PHF kinase substrate recognition sites in tau. This phenomenon has been termed hierarchical phosphorylation (Roach, 1991) and is known to play a key role in the substrate specificity of both GSK-3 and casein kinase I, two kinases that have been widely implicated as responsible for the hyperphosphorylation of tau seen in AD.

Initial characterization demonstrated that the PHF kinase shares many similarities with the casein kinase I family of protein kinases (Vincent and Davies, 1992). A more recent study using immunoaffinity PHF preparations similar to ours has suggested that the PHF kinase activity can be attributed to 36- and 39-kDa isoforms of casein kinase I as determined by in-gel renaturation assays, western analysis, inhibitor selectivity, and synthetic peptide substrate specificity (Kuret et al., 1997). Additional experiments using a polyclonal antibody to casein kinase Iα (Santa Cruz Biotechnology) demonstrate that the 39-kDa isoform is present in our PHF preparation (data not shown) and is likely to be responsible for the PHF kinase activity characterized in this study. In the previous study by Kuret et al. (1997) identification of phosphorylated residues on tau was not performed but was assumed to include Ser396 and Ser404, which have previously been demonstrated to be phosphate acceptors for casein kinase I (Singh et al., 1995a,b). Although the present study did not identify Ser396 and Ser404 as phosphate acceptors for the PHF kinase activity, it does provide further support for the conclusion that the kinase activity associated with PHF is a member of the casein kinase I family of kinases. Additional experiments (data not shown) demonstrate that exogenous casein kinase I, but not the PHF kinase, is capable of eliciting enhanced reactivity of the PHF-1 monoclonal antibody (recognizes phospho-Ser396 and phospho-Ser404) on sequentially phosphorylated recombinant tau and PHF-tau. This discrepancy suggests that the substrate specificity of casein kinase I may differ between members of this family of kinases or that the kinase itself may be altered in AD, perhaps as a result of its tight association with endogenous PHF constituents. Further studies attempting to elucidate the factors responsible for the unique substrate specificity of the PHF kinase are currently underway.

Although phosphorylations on Ser356 and Thr361 have not previously been identified on PHF-tau, phosphorylations in this region of the tau molecule composing the fourth microtubule binding repeat have been shown to influence the association between tau and microtubule arrays (Goedert and Jakes, 1990 ; Drechsel et al., 1992 ; Raffaelli et al., 1992 ; Biernat et al., 1993 ; for review, see Lee, 1993). It is possible that phosphorylation of these sites exists in PHF-tau and have simply been overlooked in past studies. Previous studies identifying phosphorylation sites on PHF-tau have relied almost exclusively on phosphopeptide mapping strategies, which can be inherently limited as demonstrated by the lack of recovery and inability to determine the exact PHF kinase sites in the extreme C terminus of tau seen in this study. Alternate strategies aimed at identifying phosphorylation sites in PHF-tau have relied on the creation and empirical reactivity of phosphorylation-dependent antibodies raised against PHF-tau. Although this approach has yielded important information regarding certain immunodominant phosphorylations found in PHF-tau, the usefulness of antibodies recognizing epitopes in the microtubule binding regions of tau has been limited by the extremely low levels of reactivity seen against PHF-tau (Dickson et al., 1992). This lack of reactivity is assumed to be due to the compact nature of PHF-tau, the core of which is thought to be composed of the microtubule binding repeats of tau (Novak et al., 1993), and the limited access of antibodies to their antigenic counterparts in these regions (Dickson et al., 1992). Future studies will attempt to determine if Ser356 and/or Thr361 are phosphorylated in PHF-tau, or if their identification in this study is merely an artifact of the in vitro conditions used.

Ser409 in tau has previously been shown to be phosphorylated in all PHF preparations analyzed to date (Hasegawa et al., 1992). In vitro analysis of recombinant tau reacted with various kinases has shown that only PKA (Scott et al., 1993) and a 35/41-kDa kinase (Biernat et al., 1993) are capable of inducing this phosphorylation. Subsequent studies on the 35/41-kDa kinase identified the 41-kDa fragment as the catalytic subunit of PKA (Drewes et al., 1995), suggesting that only PKA is capable of inducing the phosphorylation of Ser409 in tau. This observation may explain why PHF constituents are such excellent substrates for the PHF kinase. Tau in PHF is already phosphorylated at Ser409, bypassing the need for PKA pretreatment that is required for PHF kinase sequential phosphorylations of recombinant tau. These data suggest that the phosphorylation of tau on Ser409, although not necessarily pathological in itself, may be one of the earliest modifications of tau leading to PHF formation in AD.

It is possible that the phosphorylation of Ser409 does not contribute directly to the pathogenesis of PHF formation in AD, but rather represents a ubiquitous phosphorylation of the tau molecule found in all tau-expressing cells, and that only those cells destined for PHF assembly pathologically express the PHF kinase. Although plausible, this possibility is highly unlikely in that the PHF kinase is completely ineffective in inducing the phosphorylation of normal brain-derived tau (Vincent and Davies, 1992). It is also possible that PKA-induced phosphorylation of Ser409 directly contributes to the pathogenesis of PHF formation in AD as has been reported previously (Pierre and Nunez, 1983 ; Steiner et al., 1990 ; Litersky and Johnson, 1992 ; Scott et al., 1993 ; Singh et al., 1994).

Phosphorylations of tau in the C-terminal tail by both PKA on Ser409 and Ser416 and Ca2+/calmodulin-dependent protein kinase on Ser416 impose conformational restraints on the tau molecule that significantly retards migration by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (Steiner et al., 1990 ; Scott et al., 1993). Although Scott et al. (1993) assumed that the PKA-induced phosphorylation of Ser416 was responsible for the retarded migration of tau seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses, our S409G and S416G site-directed mutants clearly demonstrate that this is not the case. PKA phosphorylation of the S416G mutant demonstrates the typical shift in electrophoretic mobility seen in nonmutated tau, but this shift is completely blocked in the S409G tau mutant. Also, an S[RIGHTWARDS ARROW]E409 tau mutant constitutively exhibits this apparent molecular weight shift regardless of phosphorylation state (data not shown). This same aberrant migration pattern is seen in all preparations of PHF-derived tau (Flament et al., 1989 ; Greenberg and Davies, 1990 ; Lee et al., 1991 ; Goedert et al., 1992), suggesting that C-terminal conformational restraints may be required for the pathogenesis of PHF assembly in AD.

The phosphorylation of Ser412 by the PHF kinase may play an important role in the pathogenesis of PHF formation in AD. To date, the phosphorylation of Ser412 has only been identified in PHF-tau, but not in normal adult or fetal brain-derived tau, suggesting that this phosphorylation is entirely specific for PHF-tau. Also, no known kinases have been shown to be able to induce the phosphorylation of Ser412 in vitro or in vivo. Most of the known phosphorylation sites in tau can be modified by multiple protein kinases, complicating the issue of which kinase(s) are responsible for these phosphorylations in vivo, but the PHF kinase is clearly responsible for the phosphorylation of Ser412 seen in PHF-tau.

Previous studies of tau phosphorylations in the amino acid 412 region have focused on the GSK-3-induced phosphorylation of Ser413 (Ishiguro et al., 1992, 1995). Although these studies have provided valuable information, the phsophorylation of Ser413 has also been shown to exist in normal adult and fetal tau, raising questions about the role this phosphorylation plays in the pathogenesis of PHF formation in AD (Ishiguro et al., 1992, 1995). It is interesting that the phosphorylation of Ser413 in these studies resulted in a lack of recovery from HPLC, precluding the use of phosphopeptide mapping strategies in its identification (Ishiguro et al., 1992, 1995). This same effect is seen in the present study where phosphopeptide mapping strategies were ineffective at elucidating the most C-terminal PHF kinase phosphorylation sites in recombinant tau. These observations suggest that phosphorylations of the tau molecule in this region may promote the formation of high-molecular-weight aggregates responsible for the lack of recovery from the HPLC. Also, attempts at mass determinations of PHF kinase sequentially phosphorylated C-terminal recombinant tau fragments identified only high-molecular-weight hypermasses that were not interpretable (data not shown).

Additionally, phosphorylations by both PKA and the PHF kinase resulted in a substantial loss of recovery from the HPLC column for both the monomeric and dimeric forms of TauP. Previous analyses of PHF-tau from AD brain demonstrated this same low recovery of C-terminal fragments of tau unless the PHF-tau was first subjected to alkaline phosphatase treatment (Hasegawa et al., 1992), suggesting that the PKA- and PHF kinase-induced phosphorylations play an important role in the transition of normal tau to the pathologically altered form of PHF-tau seen in AD. Immunoelectron microscopy further demonstrates the effects these unique phosphorylations have on the in vitro aggregation of recombinant tau molecules. Recent analyses of in vitro PHF formation have suggested that phosphorylation of tau is not a necessary prerequisite for PHF formation (Goedert et al., 1996), but the present data demonstrate that certain unique phosphorylations of the tau molecule can drastically alter the equilibrium between soluble and aggregated forms of tau. To our knowledge, this is the first demonstration that the hyperphosphorylation of tau seen in AD may play a functional role in the pathogenesis of PHF formation in AD. A panel of monoclonal antibodies is currently being raised against both the PKA and PHF kinase phosphorylations identified in this study with the hopes that they will provide further insights into the roles these unique phosphorylations play in the pathogenesis of neurofibrillary pathology in AD.

Although it is clear from recent genetic findings that the etiological factors responsible for AD can be quite varied, the eventual development of neurofibrillary pathology in all AD cases suggests that it may be a crucial point of convergence of multiple degenerative cascades that provides an ideal target for potential diagnostics and therapeutic interventions in AD. Analysis of the immunoaffinity column eluent from various forms of AD, other degenerative diseases, and normal brain homogenates demonstrates that the PHF kinase is entirely specific for all disease states associated with neurofibrillary degeneration. Because hyperphosphorylation of the tau molecule has been shown to dissociate tau from microtubules (Goedert and Jakes, 1990 ; Drechsel et al., 1992 ; Raffaelli et al., 1992 ; Biernat et al., 1993 ; for review, see Lee, 1993), leading to disruption of the neuronal cytoskeleton, interference with cellular transport mechanisms, and a reduction in its rate of degradation (Vincent et al., 1994), and to facilitate aggregation, the PHF kinase may play an important role in the development of neurofibrillary pathology and the resultant neuronal dysfunction and loss seen in all cases of AD.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Immunoaffinity purification of PHFs
  5. HPLC separations
  6. RESULTS
  7. PHF-associated kinase activity in degenerative disease states
  8. Identification of PHF kinase phosphorylation sites on tau
  9. DISCUSSION
  10. Acknowledgements

The authors thank M. Goedert for providing the htau40 construct, Yuan Shi for sequencing the 32P-labeled peptides, and I. J. Vincent for the many invaluable discussions and insights that have contributed to this work. This work was supported by grant 38623 from the National Institute of Mental Health and grant AG06803 and training grant T32GM07288 from the National Institutes of Health.

  • 1
    Arendt T., Holzer M., Grobmann A., Zedlick D., Bruckner M.K. (1995) Increased expression and subcellular translocation of the mitogen activated protein kinase kinase and mitogen-activated protein kinase in Alzheimer's disease.Neuroscience 68,518.DOI: 10.1016/0306-4522(95)00146-A
  • 2
    Baudier J. & Cole R.D. (1987) Phosphorylation of tau proteins to a state like that in Alzheimer's brain is catalyzed by a calcium/calmodulin-dependent kinase and modulated by phospholipids.J. Biol. Chem. 262,1757717583.
  • 3
    Baumann K., Mandelkow E., Biernat J., Piwnica-Worms H., Mandelkow E. (1993) Abnormal Alzheimer-like phosphorylation of tau-protein by cyclin-dependent kinases cdk2 and cdk5.FEBS Lett. 336,417424.
  • 4
    Biernat J., Gustke N., Drewes G., Mandelkow E., Mandelkow E. (1993) Phosphorylation of Ser262 strongly reduces binding of tau to microtubules : distinction between PHF-like immunoreactivity and microtubule binding.Neuron 11,153163.
  • 5
    Blanchard B.J., Raghunandan R., Roder H.M., Ingram V.M. (1994) Hyperphosphorylation of human TAU by brain kinase PK40erk beyond phosphorylation by cAMP-dependent PKA : relation to Alzheimer's disease.Biochem. Biophys. Res. Commun. 200,187194.
  • 6
    Bradford M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 72,248254.
  • 7
    Correas I., Diaz-Nido J., Avila J. (1992) Microtubule-associated protein tau is phosphorylated by protein kinase C on its tubulin binding domain.J. Biol. Chem. 267,1572115728.
  • 8
    Dickson D.W., Ksiezak-Reding H., Liu W., Davies P., Crowe A., Yen S. -H.C. (1992) Immunocytochemistry of neurofibrillary tangles with antibodies to subregions of tau protein : identification of hidden and cleaved tau epitopes and a new phosphorylation site.Acta Neuropathol. (Berl.) 84,596605.
  • 9
    Drechsel D.N., Hyman A.A., Cobb M.H., Kirschner M.W. (1992) Modulation of the dynamic instability of tubulin assembly by the microtubule-associated protein tau.Mol. Biol. Cell 3,11411154.
  • 10
    Drewes G., Lichtenberg-Kraag B., Doring F., Mandelkow E., Biernat J., Goris J., Doree M., Mandelkow E. (1992) Mitogen activated protein (MAP) kinase transforms tau protein into an Alzheimer-like state.EMBO J. 11,21312138.
  • 11
    Drewes G., Trinczek B., Illenberger S., Biernat J., Schmidt-Ulms G., Meyer H.E., Mandelkow E., Mandelkow E. (1995) Microtubule associated protein/microtubule affinity regulating kinase (p110mark). J. Biol. Chem. 270,76797688.
  • 12
    Flament S., Delacourte A., Hemon B., Defossez A. (1989) Characterization of two pathological tau protein, variants in Alzheimer brain cortices.J. Neurol. Sci. 92,133141.
  • 13
    Goedert M. & Jakes R. (1990) Expression of separate isoforms of human tau protein : correlation with the tau pattern in brain and effects on tubulin polymerization.EMBO J. 9,42254230.
  • 14
    Goedert M., Spillantini M.G., Jakes R., Rutherford D., Crowther R.A. (1989) Multiple isoforms of human microtubule-associated protein-tau : sequences and localization in neurofibrillary tangles of Alzheimer's disease.Neuron 3,519526.
  • 15
    Goedert M., Spillantini M.G., Cairns N.J., Crowther R.A. (1992) Tau proteins of Alzheimer paired helical filaments : abnormal phosphorylation of all six brain isoforms.Neuron 8,159168.
  • 16
    Goedert M., Jakes R., Spillantini M.G., Hasegawa M., Smith M.J., Crowther R.A. (1996) Assembly of microtubule-associated protein tau into Alzheimer-like filaments induced by sulphated glycosaminoglycans.Nature 383,550553.
  • 17
    Gonzalez P.J., Correas I., Avila J. (1992) Solubilization and fractionation of paired helical filaments.Neuroscience 50,491499.
  • 18
    Greenberg S.G. & Davies P. (1990) A preparation of Alzheimer paired helical filaments that display distinct tau proteins.Proc. Natl. Acad. Sci. USA 87,58275831.
  • 19
    Greenberg S.G., Davies P., Schein J.D., Binder L.I. (1992) Hydrofluoric acid-treated τ PHF proteins display the same biochemical properties as normal τPHF.J. Biol. Chem. 267,564569.
  • 20
    Grundke-Iqbal I., Iqbal K., Tung Y., Quinlan M., Wisniewski H.M., Binder L.I. (1986) Abnormal phosphorylation of the microtubule-associated protein tau in Alzheimer cytoskeletal pathology.Proc. Natl. Acad. Sci. USA 83,49134917.
  • 21
    Hall F.L., Braun R.K., Mitchell J.P., Vulliet R.P. (1990) Phosphorylation of cytoskeletal proteins by proline directed protein kinase.Proc. West. Pharmacol. Soc. 33,213217.
  • 22
    Hanger D.P., Hughes K., Woodgett J.R., Brion J., Anderton B.H. (1992) Glycogen synthase kinase-3 induces Alzheimer's disease-like phosphorylation of tau : generation of paired helical filament epitopes and neuronal localization of the kinase.Neurosci. Lett. 147,5862.
  • 23
    Hasegawa M., Morishima-Kawashima M., Takio K., Suzuki M., Titani K., Ihara Y. (1992) Protein sequence and mass spectrometric analyses of tau in the Alzheimer's disease brain.J. Biol. Chem. 267,1704717054.
  • 24
    Hasegawa M., Jakes R., Crowther R.A., Lee V.M., Ihara Y., Goedert M. (1996) Characterization of mAb AP422, a novel phosphorylation-dependent monoclonal antibody against tau protein.FEBS Lett. 384,2530.
  • 25
    Hoshi M., Nishida E., Miyata Y., Sakai H., Miyoshi T., Ogawara H., Akiyama T. (1987) Protein kinase C phosphorylates tau and induces its functional alterations.FEBS Lett. 217,237241.
  •  
    Ishiguro K., Ihara Y., Uchida T., and Imahori K. (1988) A novel tubulin-dependent protein kinase forming a paired helical filament epitope on tau.J. Biochem. (Tokyo) 104,319321.
  • 27
    Ishiguro K., Omori A., Takamatsu M., Sato K., Arioka M., Uchida T., Imahori K. (1992) Phosphorylation sites on tau by tau protein kinase I, a bovine derived kinase generating an epitope of paired helical filaments.Neurosci. Lett. 148,202206.
  • 28
    Ishiguro K., Shiratsuchi A., Sato S., Omori A., Arioka M., Kobayashi S., Uchida T., Imahori K. (1993) Glycogen synthase kinase 3B is identical to tau protein kinase I generating several epitopes of paired helical filaments.FEBS Lett. 325,167172.
  • 29
    Ishiguro K., Sato K., Takamatsu M., Park J., Uchida T., Imahori K. (1995) Analysis of phosphorylation of tau with antibodies specific for phosphorylation sites.Neurosci. Lett. 202,8184.
  • 30
    Jicha G.A., Bowser R., Kazam I.G., Davies P. (1997) Alz-50 and MC-1, a new monoclonal antibody raised to paired helical filaments, recognize conformational epitopes on recombinant tau.J. Neurosci. Res. 48,128132.
  • 31
    Kanemaru K., Takio K., Mirua R., Titani K., Ihara Y. (1992) Fetal-type phosphorylation of the τ in paired helical filaments.J. Neurochem. 58,16671675.
  • 32
    Khachaturian Z.S. (1985) Diagnosis of Alzheimer's disease.Arch. Neurol. 42,10971106.
  • 33
    Kosik K., Orecchio L.D., Binder L.I., Trojanowski J.Q., Lee V.M., Lee G. (1988) Epitopes that span the tau molecule are shared with paired helical filaments.Neuron 1,817825.
  • 34
    Kuret J., Johnson G.S., Cha D., Christenson E.R., DeMaggio A.J., Hoekstra M.F. (1997) Casein kinase I is tightly associated with paired-helical filaments from Alzheimer's disease brain.J. Neurochem. 69,25062515.
  • 35
    Ledesma M.D., Correas I., Avila J., Diaz-Nido J. (1992) Implication of brain cdc2 MAP2 kinases in the phosphorylation of tau protein in Alzheimer's disease.FEBS Lett. 308,218224.
  • 36
    Lee G. (1993) Non-motor microtubule-associated proteins.Curr. Opin. Cell Biol. 5,8894.
  • 37
    Lee V.M., Balin B.J., Otvos L.J, Trojanowski J.Q. (1991) A68 : a major subunit of the paired helical filaments and derivatized forms of normal tau.Science 251,675678.
  • 38
    Lew J. & Wang J.H. (1995) Neuronal cdc2-like kinase.Trends Biol. Sci. 20,3337.
  • 39
    Litersky J.M. & Johnson G.V.W. (1992) Phosphorylation by cAMP-dependent protein kinase inhibits the degradation of tau by calpain.J. Biol. Chem. 267,15631568.
  • 40
    Lu Q., Soria J.P., Wood J.G. (1993) p44mpk MAP kinase induces Alzheimer type alterations in tau function in primary hippocampal neurons.J. Neurosci. 35,439444.
  • 41
    Mandelkow E., Drewes G., Biernat J., Gustke N., Van Lint J., Vandenheede J.R., Delkow E. (1992) Glycogen synthase kinase-3 and the Alzheimer-like state of microtubule-associated protein tau.FEBS Lett. 314,314321.
  • 42
    Masaki T., Fujihashi T., Nakamura K., Soejima M. (1981) Studies on a new proteolytic enzyme from Achromobacter lyticus M497-1. II. Specificity and inhibition studies of Achromobacter protease 1.Biochim. Biophys. Acta 660,5155.
  • 43
    Matsuo E.S., Shin R., Billingsley M.L., Van deVorde A., O'Connor M., Trojanowski J.Q., Lee V.M. -Y. (1994) Biopsy-derived adult human tau is phosphorylated at many of the same sites as Alzheimer's disease paired helical filament tau.Neuron 13,9891002.
  • 44
    Mawal-Dewan M., Parimal C.S., Abdel-Ghany M., Shalloway D., Racker E. (1992) Phosphorylation of tau protein by purified p34 cdc2 and a related protein kinase from neurofilament.J. Biol. Chem. 267,1970519709.
  • 45
    Moreno F.J., Medina M., Perez M., Montejo de Garcini E., Avila J. (1995) Glycogen synthase kinase 3 phosphorylates recombinant human tau protein at serine-262 in the presence of heparin (or tubulin).FEBS Lett. 372,6568.
  • 46
    Morishima-Kawashima M., Hasegawa M., Takio K., Suzuki M., Yoshida H., Titani K., Ihara Y. (1995) Proline-directed and nonproline-directed phosphorylation of PHF-tau.J. Biol. Chem. 270,823829.
  • 47
    Novak M., Kabat J., Wischik C.M. (1993) Molecular characterization of the minimal protease resistant tau unit of the Alzheimer's disease paired helical filament.EMBO J. 12,365370.
  • 48
    Paudel H.K., Lew J., Ali Z., Wang J.H. (1993) Brain proline-directed kinase phosphorylates tau on sites that are abnormally phosphorylated in tau associated with Alzheimer's paired helical filaments.J. Biol. Chem. 268,2351223518.
  • 49
    Pelech S.L. (1995) Networking with proline directed kinases implicated in tau phosphorylation.Neurobiol. Aging 16,247261.
  • 50
    Pierre M. & Nunez J. (1983) Multisite phosphorylation of tau proteins from rat brain.Biochem. Biophys. Res. Commun. 115,212219.
  • 51
    Raffaelli N., Yamauchi P.S., Purich D.L. (1992) Microtubule-associated protein autophosphorylation alters in vitro microtubule dynamic instability.FEBS Lett. 296,2124.
  • 52
    Roach P.J. (1991) Multisite and hierarchical protein phosphorylation.J. Biol. Chem. 266,1413914142.
  • 53
    Roder H.M. and Ingram V.M. (1991) Two novel kinases phosphorylate tau and the KSP site of heavy neurofilament subunits in high stoichiometric ratios.J. Neurosci. 11,33253343.
  • 54
    Schweers O., Mandelkow E.-M., Biernat J., and Mandelkow E. (1995) Oxidation of cysteine-322 in the repeat domain of microtubule-associated protein tau controls the in vitro assembly of paired helical filaments.Proc. Natl. Acad. Sci. USA 92,84638467.
  • 55
    Scott C.W., Spreen R.C., Herman J.L., Chow F.P., Davison M.D., Young J., Caputo C.B. (1993) Phosphorylation of recombinant tau by cAMP-dependent protein kinase.J. Biol. Chem. 268,11661173.
  • 56
    Singh T.J., Grundke-Iqbal I., McDonald B., Iqbal K. (1994) Comparison of the phosphorylation of microtubule-associated protein tau by non-proline dependent protein kinases.Mol. Cell. Biochem. 131,181189.
  • 57
    Singh T.J., Grundke-Iqbal I., Iqbal K. (1995a) Phosphorylation of τ protein by casein kinase I converts it to an abnormal Alzheimer-like state.J. Neurochem. 64,14201423.
  • 58
    Singh T.J., Zaidi T., Grundke-Iqbal I., Iqbal K. (1995b) Modulation of GSK-3-catalyzed phosphorylation of microtubule-associated protein tau by non-proline directed kinases.FEBS Lett. 358,48.
  • 59
    Steiner B., Mandelkow E.M., Biernat J., Gustke N., Meyer H.E., Schmidt B., Mieskes G., Soling H.D., Dreschel D., Kirschner M.W., Goedert M., Mandelkow E. (1990) Phosphorylation of microtubule-associated protein tau : identification of the site for Ca2(+)-calmodulin dependent kinase and relationship with tau phosphorylation in Alzheimer tangles.EMBO J. 9,35393544.
  • 60
    Trojanowski J.Q. & Lee V.M. -Y. (1994) Paired helical filament τ in Alzheimer's disease : the kinase connection.Am. J. Pathol. 144,449453.
  • 61
    Trojanowski J.Q. & Lee V.M. -Y. (1995) Phosphorylation of paired helical filament tau in Alzheimer's disease neurofibrillary lesions : focusing on phosphatases.FASEB J. 9,15701576.
  • 62
    Trojanowski J.Q., Mawal-Dewan M., Schmidt M.L., Martin J., Lee V.M. -Y. (1993) Localization of the mitogen activated protein kinase ERK2 in Alzheimer's disease neurofibrillary tangles and senile plaque neurites.Brain Res. 618,333337.
  • 63
    Vincent I.J. & Davies P. (1992) A protein kinase associated with paired helical filaments in Alzheimer disease.Proc. Natl. Acad. Sci. USA 89,28782882.
  • 64
    Vincent I.J., Rosado M., Kim E., Davies P. (1994) Increased production of paired helical filament epitopes in a cell culture system reduces the turnover of τ. J. Neurochem. 62,715723.
  • 65
    Wolozin B.L., Pruchnicki A., Dickson D.W., Davies P. (1986) A neuronal antigen in the brains of Alzheimer patients.Science 232,648650.
  • 66
    Wood J.G., Mirra S., Pollock N.J., Binder L.I. (1986) Neurofibrillary tangles of Alzheimer's disease share antigenic determinants with the axonal microtubule-associated protein tau.Proc. Natl. Acad. Sci. USA 83,40404043.
  • 67
    Wood J.G., Lu Q., Reich C., Zinsmeister P. (1993) Proline-directed kinase systems in Alzheimer's disease pathology.Neurosci. Lett. 156,8386.
  • 68
    Yamamoto H., Fukunaga K., Goto S., Tanaka E., Miyamoto E. (1985) Ca2+, calmodulin-dependent regulation of microtubule formation via phosphorylation of microtubule-associated protein 2, τ factor, and tubulin, and comparison with the cyclic AMP-dependent phosphorylation.J. Neurochem. 44,759768.
  • 69
    Yang S., Song J., Yu J., Shiah S. -G. (1993a) Protein kinase FA/GSK-3 phosphorylates τ on Ser235-Pro and Ser404-Pro that are abnormally phosphorylated in Alzheimer's disease brain. J. Neurochem. 61,17421747.
  • 70
    Yang S., Song J., Liu W., Yen S. -H. (1993b) Synergistic control mechanism for abnormal site phosphorylation of Alzheimer's disease brain tau by kinase FA/GSK-3a.Biochem. Biophys. Res. Commun. 197,400406.