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Microtubule-associated Protein/Microtubule Affinity-regulating Kinase (p110mark)

A NOVEL PROTEIN KINASE THAT REGULATES TAU-MICROTUBULE INTERACTIONS AND DYNAMIC INSTABILITY BY PHOSPHORYLATION AT THE ALZHEIMER-SPECIFIC SITE SERINE 262 (∗)
Open AccessPublished:March 31, 1995DOI:https://doi.org/10.1074/jbc.270.13.7679
      Aberrant phosphorylation of the microtubule-associated protein tau is one of the pathological features of neuronal degeneration in Alzheimer's disease. The phosphorylation of Ser-262 within the microtubule binding region of tau is of particular interest because so far it is observed only in Alzheimer's disease (Hasegawa, M., Morishima-Kawashima, M., Takio, K., Suzuki, M., Titani, K., and Ihara, Y.(1992) J. Biol. Chem. 26, 17047-17054) and because phosphorylation of this site alone dramatically reduces the affinity for microtubules in vitro (Biernat, J., Gustke, N., Drewes, G., Mandelkow, E.-M., and Mandelkow, E.(1993) Neuron 11, 153-163). Here we describe the purification and characterization of a protein-serine kinase from brain tissue with an apparent molecular mass of 110 kDa on SDS gels. This kinase specifically phosphorylates tau on its KIGS or KCGS motifs in the repeat domain, whereas no significant phosphorylation outside this region was detected. Phosphorylation occurs mainly on Ser-262 located in the first repeat. This largely abolishes tau's binding to microtubules and makes them dynamically unstable, in contrast to other protein kinases that phosphorylate tau at or near the repeat domain. The data suggest a role for this novel kinase in cellular events involving rearrangement of the microtubule-associated proteins/microtubule arrays and their pathological degeneration in Alzheimer's disease.

      INTRODUCTION

      Microtubule-associated proteins (MAPs)
      The abbreviations used are: MAP
      microtubule-associated protein
      PHF
      paired helical filament
      p110mark
      MAP/microtubule affinity regulating kinase
      DTT
      dithiothreitol
      HPLC
      high performance liquid chromatography
      MES
      4-morpholineethanesulfonic acid
      Pipes
      1,4-piperazinediethanesulfonic acid.
      regulate the extensive dynamics and rearrangement of the microtubule network, which is thought to drive neurite outgrowth (recently reviewed by Hirokawa(1994) and Kosik and McConlogue(1994)). Several lines of evidence suggest that the phosphorylation state of MAPs, balanced by protein kinases and phosphatases in a hitherto unknown way, plays a pivotal role in the modulation of these events. Tau protein, a class of MAPs stabilizing microtubules in mammalian brain (Cleveland et al., 1977; Drubin and Kirschner, 1986), is phosphorylated on several sites in vivo (Butler and Shelanski 1986; Watanabe et al., 1993) and is a substrate for many protein kinases in vitro (for review, see Lee(1993), Goedert(1993), Mandelkow and Mandelkow(1993), and Anderton(1993)). During neuronal degeneration in Alzheimer's disease, tau protein aggregates into paired helical filaments (PHFs), the principal fibrous components of the characteristic neurofibrillary lesions (for review, see Lee and Trojanowski(1992)). Tau isolated from these aggregates displays some biochemical alterations, of which hyperphosphorylation is the most striking (Grundke-Iqbal et al., 1986; Brion et al., 1991; Ksiezak-Reding et al., 1992; Goedert et al., 1992). Most of the reported aberrant phosphorylation sites are Ser/Thr-Pro sequences (Lee et al., 1991; Biernat et al., 1992; Lichtenberg-Kraag et al., 1992; Watanabe et al., 1993), suggesting a dysregulation of proline-directed kinases (Ishiguro et al., 1991; Drewes et al., 1992; Mandelkow et al., 1992; Hanger et al., 1992; Vulliet et al., 1992; Baumann et al., 1993; Paudel et al., 1993, Kobayashi et al., 1993) or the corresponding phosphatases (Drewes et al., 1993; Gong et al., 1994). Phosphorylation-dependent antibodies, which discriminate between “normal” tau and the hyperphosphorylated, “pathological” forms, were prepared by several laboratories (Kondo et al., 1988; Lee et al., 1991; Mercken et al., 1992; Greenberg et al., 1992). All of these antibodies were shown to be directed against epitopes of the Ser/Thr-Pro type (Biernat et al., 1992; Lichtenberg-Kraag et al., 1992; Lang et al., 1992; Watanabe et al., 1993).
      The microtubule binding region of tau (Fig. 1) includes three or four pseudorepeats of 31 residues each (Ennulat et al., 1989, Lee et al., 1989), depending on isoform type (Goedert et al., 1989; Himmler et al., 1989). This region probably forms the building block of the paired helical filaments (Kondo et al., 1988; Wischik et al., 1988; Ksiezak-Reding and Yen, 1991; Wille et al., 1992). It does not contain any of the 14-16 Ser/Thr-Pro motifs, which cluster in the regions flanking the repeats. However, it contains a conserved serine residue (Ser-262) within the sequence KIGS in the first repeat, which we found to be one of the predominant sites phosphorylated by a tissue extract from brain (Gustke et al., 1992). This site is also found to be phosphorylated in Alzheimer PHF-tau, but not in normal tau or fetal tau (Hasegawa et al., 1992). So far, it is the only pathological phosphorylation site found within the repeat domain of tau.
      Figure thumbnail gr1
      Figure 1Bar diagram of human tau (isoform htau40, the largest one in central nervous tissue (Goedert et al., 1989), construct K18 containing the four repeats and several sites phosphorylated by the brain extract (Gustke et al., 1992). The hatched boxes near the N terminus are inserts that may be absent because of differential splicing, the boxes labeled 1-4 represent the four repeats, of which repeat 2 may be absent. Most phosphorylated sites are in Ser-Pro or Thr-Pro motifs outside the repeats, but the brain kinase activity also phosphorylates two sites within the repeats, Ser-262 and Ser-356.
      Recently, we used a site-directed mutagenesis approach to show that phosphorylation of tau at this site strongly decreases its microtubule binding capacity, whereas the phosphorylation on Ser/Thr-Pro motifs had only a minor effect (Biernat et al., 1993). This initiated a search for protein kinases in neuronal tissue with the ability to phosphorylate tau at Ser-262. In our previous work, we had partially purified a kinase activity consisting of two components with apparent molecular masses of 35 and 41 kDa. Here we report the characterization of a novel kinase of 110 kDa, termed p110mark (for MAP/microtubule affinity-regulating kinase). This kinase phosphorylates all four KXGS motifs in the repeat domain of tau, particularly the first one containing Ser-262, and thus efficiently causes the detachment of tau from microtubules and the subsequent destabilization of microtubules, as measured by their higher dynamic instability. Because of this, p110mark is a good candidate for regulating the dynamics and rearrangements of microtubules in cells via the phosphorylation of tau or other MAPs.

      MATERIALS AND METHODS

      Proteins

      Human tau cDNA clones were kindly provided by M. Goedert (Goedert et al., 1989) and were expressed in Escherichia coli using variants of the pET vector (Studier et al., 1990), and the proteins were purified making use of heat stability, Mono S fast protein liquid chromatography (Hagestedt et al., 1989) and gel filtration. Construct K18 is derived from the four-repeat tau isoform and comprises the repeats region, residues 244-372 (Biernat et al., 1993). The numbering used here always refers to the longest human isoform (clone htau40). “tau-A262” is also based on htau40 and bears a single mutation, Ser-262 → Ala. Phosphocellulose-purified tubulin was prepared from porcine brain following Mandelkow et al.(1985). Protein kinase A catalytic subunit (from bovine heart, activity 27 milliunits/μl based on kemptide, 100 microunits/μl based on casein, 1 unit transfers 1 μmol of phosphate/min at 30°C, in 40 mM Tris-HCl, pH 7.4, 20 mM MgCl2, 0.2 mM ATP) was obtained from Promega. Protein kinase C (from rat brain, activity 80 microunits/μl based on histone H1) was from Boehringer Mannheim.

      Purification of p110mark

      All operations were performed at 4°C. Fresh porcine brains (∼1 kg) were obtained at the local slaughterhouse and homogenized into 1 liter of buffer A (50 mM Tris, pH 8.6, containing 5 mM EGTA, 100 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 1 mM Na3VO4, 1 mM DTT, 0.1% Brij 35). The homogenate was transported to the laboratory on ice within 20 min and centrifuged at 30,000 × g for 1 h. The supernatant was cleared by ultracentrifugation (50,000 × g, 30 min) (the pH was adjusted to 6.8) and loaded carefully onto a Büchner funnel containing 150 ml of Whatman P-11 equilibrated with buffer B (50 mM MES, pH 6.8, 2 mM EGTA, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 1 mM Na3VO4), 1 mM DTT, 0.1% Brij 35), by applying a slight vacuum. The phosphocellulose was washed with 500 ml of buffer B and eluted stepwise with 150 ml each of buffer B containing 0.15-1 M NaCl. Fractions were screened for activity by phosphorylation of a tau construct (K18) consisting of the four microtubule binding repeats. Active fractions were fractionated by ammonium sulfate precipitation. The precipitate obtained between 30 and 50% saturation was dialyzed against buffer A overnight on ice. The dialysate (∼50 ml) was cleared by ultracentrifugation and loaded onto an anion-exchange column (Q-Sepharose HR, Pharmacia Biotech Inc., 80 × 16 mm). The column was washed with 100 ml of buffer A and eluted with a stepwise gradient from 0 to 0.5 M NaCl (flow rate 5 ml/min, fraction size 7 ml). Active fractions (40 ml) were dialyzed against buffer B and loaded onto a cation-exchange column (SP-Sepharose HR, Pharmacia, 60 × 16 mm, flow rate 4 ml/min, fraction size 7 ml). After elution with 0-0.5 M NaCl, active fractions (∼40 ml) were pooled, and the buffer was exchanged for buffer A on a Sephadex G-25 column (300 × 26 mm), loaded onto a Mono Q HR 5/5 column (Pharmacia), and eluted with a steep NaCl gradient (flow rate, 0.5 ml/min; fraction size, 1 ml). Active fractions (2-3 ml) were concentrated 2-fold in a Centricon 30 microconcentrator (Amicon) and loaded onto a gel filtration column (Superdex 200, Pharmacia, 300 × 16 mm) equilibrated and eluted with buffer A (pH 7.8, containing 150 mM NaCl and 10% glycerol). The flow rate was 0.2 ml/min, and the fraction size was 2 ml. The column had previously been calibrated with a marker protein kit (Pharmacia). Active fractions were pooled, and the buffer was exchanged for buffer C (40 mM β-glycerophosphate, 10 mM MgCl2, 2 mM EGTA, 1 mM benzamidine, 0.2 mM DTT, 0.1% Brij 35) on a Sephadex G-25 column (100 × 16 mm). The protein pool from the G-25 column (10-15 ml) was loaded at 0.1 ml/min onto an ATP-Sepharose column (Upstate Biotechnology Inc., Lake Placid, 15 × 5 mm). The column was washed with 5 ml of buffer C and eluted with 2 ml of buffer C containing 5 mM MgATP. The eluate was concentrated and freed from ATP and buffer substances on a Mono Q 1.6/5 column (Smart system, Pharmacia), eluted with 25 mM Tris-HCl, pH 7.4, containing 250 mM NaCl, 1 mM EGTA, 0.2 mM DTT, 1 mM benzamidine, and 0.03% Brij 35. Active fractions were mixed with 50% (v/v) glycerol and stored at −20°C. Under these conditions, activity was preserved for at least 1 month.

      Phosphorylation Reactions

      Phosphorylation reactions were carried out in 40 mM Hepes, pH 7.2, containing 50 μM tau or K18, 1 mM [γ-32P]ATP (10-100 cpm/pmol), 5 mM MgCl2, 2 mM EGTA, 1 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, 0.03% Brij 35. When crude fractions were screened, 50 mM NaF and 1 μM okadaic acid (LC Services, Woburn, MA) were included. Phosphorylation was assayed in SDS gels (Steiner et al., 1990) or on phosphocellulose paper discs (Life Technologies, Inc.) (Casnellie, 1991). In-gel phosphorylation assays were performed according to Geahlen et al.(1986).

      Microtubule Binding Studies

      Binding studies were performed by measuring co-sedimentation of taxol-stabilized microtubules (30 μM) and tau by ultracentrifugation (Beckman TL 100) of 30-μl samples. Aliquots of the pellet and supernatant were assayed using SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining. Scanner densitometry of dried gels was used for quantification of protein (for details, see Gustke et al. (1992)).

      Assay of Microtubule Length Distribution by Video Microscopy

      Video microscopy of microtubules nucleated on axonemes was done essentially as described previously (Trinczek et al., 1993). Briefly, 5 μM phosphocellulose-purified tubulin, 2.5 μM tau (unphosphorylated or phosphorylated), and low amounts of sea urchin sperm axonemes (10-100 fM) were mixed in 50 mM Na-Pipes, pH 6.9, containing 3 mM MgCl2, 2 mM EGTA, 1 mM GTP, and 1 mM DTT. 1.0 μl of the samples was put on a slide, covered with 18 × 18 mm coverslips, sealed, and warmed up to 37°C in a temperature-controlled air flow within 5 s. The axoneme-nucleated microtubules were recorded at time 2.5, 5, 10, 15, 20, 25, and 30 min after the temperature shift. For each condition and time, three to five axonemes of a sample and 10-20 experiments were analyzed, and the lengths of 500-600 microtubule plus ends were measured. Only those microtubules that were clearly located within the focal plane were taken into account. The depth of solution was 3-4 μm, and the focal depth was 1-2 μm.

      Phosphopeptide Mapping by Thin Layer Electrophoresis/Chromatography and HPLC

      Following phosphorylation reactions, kinase proteins were removed by boiling of the samples in 0.5 M NaCl, 10 mM DTT and centrifugation. Tau remains in the supernatant and was precipitated by 15% trichloroacetic acid. Cysteine residues were modified by performic acid (Hirs, 1967). The protein was digested overnight with trypsin (Promega, sequencing grade) in the presence of 0.1 mM CaCl2, using two additions of the enzyme in a ratio of 1:10-1:20 (w/w). Two-dimensional phosphopeptide mapping on thin layer cellulose plates (Macherey and Nagel, Düren, FRG) was performed according to Boyle et al., 1991. First dimension electrophoresis was carried out at pH 1.9 in formic acid (88%)/acetic acid/water (50:156:1794), second dimension chromatography in n-butyl alcohol/pyridine/acetic acid/water (150:100:30:120). For the mapping of phosphorylation sites by sequencing, 200 μg of recombinant human tau were phosphorylated with p110mark and [γ-32P]ATP (100 Ci/mol) for 2 h. The phosphorylation was terminated by trichloroacetic acid precipitation. After performic acid treatment and dialysis against 10 mM ammonium bicarbonate, the protein was lyophilized and digested with trypsin (1:20) in the presence of 0.1 mM CaCl2. Separation of peptides was performed by HPLC on a Vydac 218TP52 column using a gradient of 0% acetonitrile, 0.075% trifluoroacetic acid to 50% acetonitrile, 0.05% trifluoroacetic acid in 150 min with a flow rate of 80 μl/min (Smart system, Pharmacia). Radioactive fractions were rechromatographed on a μRPC C2/C18 SC 2.1/10 column (Pharmacia), using a gradient of 0% acetonitrile, 0.075% trifluoroacetic acid to 50% acetonitrile, 0.05% trifluoroacetic acid in 60 min with a flow rate of 0.1 ml/min. Sequence analysis of peptides was performed using a 477-A pulsed liquid phase sequencer and a 120-A online phenylthiohydantoin-derivative analyzer (Applied Biosystems). Phosphoserines were identified as the dithiothreitol adduct of dehydroalanine by gas phase sequencing (Meyer et al., 1991, 1993).

      Phosphoamino Acid Analysis

      Aliquots of digestion samples were partially hydrolyzed in 6 N HCl (110°C, 60 min) and analyzed by two-dimensional electrophoresis at pH 1.9 and pH 3.5 (Boyle et al., 1991).

      RESULTS

      Purification and Characterization of the Protein Kinase p110mark

      In earlier studies on tau phosphorylation, we had been concerned mainly with phosphorylated motifs of the Ser-Pro or Thr-Pro type because some of these react with antibodies against PHFs and thus are of diagnostic value (Lichtenberg-Kraag et al., 1992). When searching for the protein kinase responsible for this type of phosphorylation, we found a neuronal MAP kinase (Drewes et al., 1992), but subsequently also two other proline-directed kinases in brain that phosphorylate tau in vitro (glycogen synthase kinase-3, Cdk5) (Mandelkow et al., 1992; Baumann et al., 1993). Whereas the sites of these kinases are generally outside the repeat domain, there are several KXGS motifs within the microtubule binding region of tau that can be phosphorylated by other kinases in vitro (protein kinase A, PK35, protein kinase C) (Steiner, 1993; Biernat et al., 1993; Correas et al., 1992). We therefore wanted to answer the question of what protein kinase is responsible for this type of phosphorylation in brain tissue.
      Six chromatographic steps were used to achieve a ∼=10,000 fold purification of a Ser-262-phosphorylating activity from porcine brain. As shown in detail in Fig. 2, we employed phosphocellulose (A), ion exchange chromatography on Q- and SP-Sepharose and Mono Q (B-D), gel filtration (E), and, finally, affinity chromatography using immobilized ATP. When using tau construct K18 as a substrate (comprising only the repeat region), the activity of kinase(s) in the tissue extract was approximately 0.2 milliunits/mg of protein, the activity of the affinity-purified kinase was approximately 2 units/mg (1 unit transfers 1 μmol of phosphate/min at 37°C, measured with 0.5 mM substrate and 1 mM ATP). The apparent molecular weight of the enzyme was around 90-100 kDa by gel filtration, but the activity peak was broad and showed pronounced tailing (Fig. 2E). On SDS gels, the apparent molecular mass was approximately 110 kDa (Fig. 3). The enzyme could be renatured in the gel; if tau was polymerized into the gel matrix as a substrate and the gel was incubated with [γ-32P]ATP, the 110 kDa band became prominent upon autoradiography (Fig. 3, lanes 4-6), whereas some minor contaminations observed in the silver-stained gel had no detectable activity. After phosphorylation with the 110-kDa kinase, both whole tau and construct K18 showed small but distinct mobility change in SDS-polyacrylamide gel electrophoresis (Fig. 4, lanes 1-4). The final amount of incorporated phosphate is approximately 1.8-2.5 mol/mol of tau, depending somewhat on enzyme concentration and activity; this level of phosphorylation could be achieved after approximately 2 h.
      Figure thumbnail gr2
      Figure 2Isolation of p110mark from porcine brain. A, the tissue extract was loaded onto phosphocellulose and eluted stepwise with 0.15-1 M NaCl. The shaded bars show the total protein concentration of the eluted material; open bars show the activity as measured with tau construct K18 as substrate. B, the material eluted with 0.35-0.5 M NaCl was submitted to ammonium sulfate precipitation, and the precipitate was dialyzed and loaded onto a Q-Sepharose column. The closed symbols show the protein concentration, and open symbols show the activity profile. The gradient composition is indicated on the right axis. C, fractions 8-15 from Q-Sepharose were dialyzed and loaded onto a SP-Sepharose column. D, fractions 12-16 from SP-Sepharose were dialyzed and loaded onto a Mono Q HR 5/5 column. E, fractions 9-11 from Mono Q were loaded onto a Superdex 200 gel filtration column. The elution positions of molecular weight markers are indicated on the right axis.
      Figure thumbnail gr3
      Figure 3Final purification of p110mark by affinity chromatography on ATP-Sepharose and characterization by in-gel phosphorylation. The most active fractions from the gel filtration column (lane 1) were loaded onto an ATP affinity column. The kinase was eluted specifically with 5 mM ATP (lanes 2 and 3). The silver-stained gel shows a fuzzy band with an apparent molecular mass of approximately 110 kDa and a second, sharp band of approximately 95 kDa. Lanes 4-6 show autoradiograms of the in-gel phosphorylation of the samples in lanes 1-3. As a substrate, tau (5 μM) was polymerized into a 8.5% acrylamide gel matrix. After renaturation and incubation with [γ-32P]ATP, it is clearly shown that only the 110 kDa band displays kinase activity toward tau.
      Figure thumbnail gr4
      Figure 4Phosphorylation of wild-type tau and construct K18 (microtubule binding domain) by p110mark. Htau40 (10 μM, lanes 1 and 2) and K18 (20 μM, lanes 3 and 4) were phosphorylated with 5 milliunits/ml of p110mark and 2 mM [γ-32P]ATP at 37°C for 2 h. Aliquots were electrophoresed on a 7-20% SDS gradient gel. Lanes 1 and 2, htau40 before and after phosphorylation; lanes 3 and 4, K18 before and after phosphorylation. Note the small molecular weight shift upon phosphorylation in lanes 2 and 4. The right panel shows an autoradiograph of the same gel; phosphorylated htau40 and K18 are seen in lanes 2 and 4.
      The specificity of the novel kinase for tau was examined by tryptic digestion of phosphorylated protein and subsequent two-dimensional high voltage thin layer electrophoresis/thin layer chromatography (Fig. 5, A-D). If one compares the phosphorylation patterns obtained from recombinant full-length 4-repeat tau (Fig. 5A) and the 4-repeat fragment K18 (Fig. 5B), it is apparent that most phosphorylated peptides are generated from the repeat domain. This was confirmed by analysis of a mixture of both samples (Fig. 5D). In a second approach, the tryptic digest was resolved by HPLC (Fig. 5E). This yielded several labeled peptides that were analyzed by phosphopeptide sequencing. The results are compiled in Table 1. Most of the radioactivity was found in a peptide containing phosphorylated Ser-262. Ser-356 (in the KIGS motif of the fourth repeat) and Ser-324 (from the KCGS motif of the third repeat) were also found. Two-dimensional analysis of these purified peptides lead to the identification of spots shown in Fig. 5C. This clearly shows that Ser-262 (spot 1) is the main target site of p110mark on tau, followed by Ser-356 (spot 2). Spot 3 was identified as the peptide containing Ser-305, spot 4 as Ser-324 (in the KCGS motif of the third repeat), and spot 5 as Ser-293 (in the KCGS motif of the second repeat).
      Figure thumbnail gr5
      Figure 5Tryptic phosphopeptide maps obtained by two-dimensional thin layer electrophoresis/thin layer chromatography of wild-type tau and construct K18 phosphorylated with p110mark. 30 μg of tau were phosphorylated with 0.5 milliunits of p110mark for 2 h at 37°C. A, full-length 4-repeat tau (htau40); B, construct K18 (MT binding region, residues 244-372 of full-length tau); C, diagram of the more prominent spots (spot 1 on upper left contains Ser-262, spot 2 on upper right Ser-356, spot 3 (below 1) Ser-305, spot 4 (always part of an overlapping doublet) contained Ser-324, spot 5 Ser-293; D, mixture of identical amounts of counts (10,000 cpm) derived from phosphopeptides shown in A and B; E, HPLC run of the tryptic digest. Incorporated radioactivity is shown below the elution profile, and the phosphorylated residues obtained by sequencing are indicated. The identification of phosphorylation sites shown in C was performed by two-dimensional analysis of the HPLC-purified and sequenced peptides (). 10,000 cpm of the peptides each were mixed with a 5000-cpm aliquot of the digest shown in A and analyzed by thin layer electrophoresis/thin layer chromatography in order to allow unambiguous identification.

      Tau-Microtubule Binding and Dynamic Instability

      In a previous paper (Biernat et al., 1993), we had shown that the phosphorylation of Ser-262 strongly decreased the interaction between tau and microtubules; that is, not only the dissociation constant increased, but also the stoichiometry decreased. Confirming these observations, a similar but even more pronounced effect on tau binding was observed after phosphorylation by the 110-kDa kinase. In fact, Fig. 6A shows that microtubule binding is completely abolished within the concentration range accessible, and consequently it was no longer possible to estimate values for the dissociation constant and the stoichiometry. Thus, the novel kinase was termed p110mark (MAP/microtubule-affinity regulating kinase) after its dramatic effect on tau-microtubule binding.
      Figure thumbnail gr6
      Figure 6Phosphorylation of Ser-262 abolishes the binding of tau to microtubules. A, binding of tau to taxol-stabilized microtubules (30 μM) was measured in a cosedimentation assay as described under “Materials and Methods.” Full-length wild-type tau (wt, htau40) and a Ser-262 → Ala mutant (A262) (10 μM) were previously phosphorylated with p110mark (final concentration 8.5 milliunits/ml) for 2 h at 37°C. Curves were obtained by nonlinear regression (Biernat et al., 1993). The binding of wild-type tau is completely abolished by phosphorylation (closed circles), whereas the A262 mutant still binds, although with lower affinity (triangles). For comparison, the binding of unphosphorylated tau is also shown (open circles). B, microtubule-bound tau comes off during phosphorylation by p110mark. htau 40 (10 μM) was incubated with taxol-stabilized microtubules (30 μM). At t = 0, p110mark was added to a final concentration of 10 milliunits/ml, and aliquots were withdrawn at time intervals from 1 to 20 h and pelleted. Tau was measured in the pellets and supernatants by densitometry of the SDS gels (closed circles). Incorporated phosphate was measured by Cerenkov counting of gel pieces (open circles) and is indicated on the right axis. Phosphate incorporation in tau without microtubules is shown to proceed faster (squares).
      In order to verify this result, Ser-262 was mutated into Ala. In this case, incubation with p110mark for 2 h left the microtubule binding capacity largely intact, although there was some decrease in affinity and stoichiometry (∼25%, Fig. 6A), probably due to phosphorylation of Ser-356 (see Fig. 5). This confirms two points of our previous study, (i) phosphorylation of Ser-262 is the major switch controlling tau's affinity for microtubules, (ii) other sites phosphorylated by the kinase have a small but measurable effect on the binding (i.e. mainly the equivalent Ser-356 in repeat 4).
      Our next question was do microtubules protect tau from being phosphorylated by p110mark? If this were the case, then tau (once bound to microtubules) might retain its high affinity for microtubules. To answer this point, taxol-stabilized microtubules were first saturated with tau and then incubated with p110mark. As illustrated in Fig. 6B, tau gradually dissociates from microtubules, concomitant with phosphorylation. Thus microtubules retard phosphorylation of tau by the kinase but cannot prevent it.
      One important function of tau is to stabilize microtubules and suppress their dynamic instability (Drechsel et al., 1992). Thus, if tau loses its binding to microtubules, one would expect stable microtubules to become dynamic. This effect can be illustrated by video dark-field microscopy of individual microtubules seeded onto flagellar axonemes (Fig. 7). In the experiment of Fig. 8A, the concentration of tubulin (5 μM) was chosen such that microtubules did not assemble by themselves but grew upon the addition of (unphosphorylated) tau (Fig. 8A, open circles). Tau phosphorylated with p110mark did not support growth, whereas the phosphorylated Ser-262 → Ala mutant did. Even more dramatic is the conversion of microtubules from undynamic to dynamic behavior under the influence of the kinase. In the experiment of Fig. 8B, we allowed microtubules to grow off axonemes in the presence of tau and recorded their mean length, which increased to approximately 50 μm over 20 min (similar to Fig. 8A, open circles). When p110mark and ATP were added together with tau, the mean length increased only to 20 μm and then dropped again, due to the gradual phosphorylation of tau and concomitant increase in microtubule dynamics (filled circles). When we used the point mutant, tau-A262, microtubules grew normally even when the kinase and ATP were present (triangles). Only after prolonged incubation with the kinase or with prephosphorylated tau-A262 (as in Fig. 8A), a small increase in dynamics is observed, comparable with the small decrease in binding, which is seen with A262-tau in cosedimentation assays (see Fig. 6A). Both can be explained by an increase with time of phosphorylation at other KXGS motifs. These results are summarized in the length histograms of Fig. 8, C-D. At early times after initiation of assembly, microtubules are short and rather homogeneous in length (peaks of open circles at 5 min), at later times of uninterrupted growth the microtubules become long and show a broad length distribution (Fig. 8, C and E, filled circles). However, when p110mark is allowed to phosphorylate tau at Ser-262, microtubules remain short (Fig. 8D, filled circles).
      Figure thumbnail gr7
      Figure 7Dark-field video microscopy of microtubules and effect of Ser-262 phosphorylation on tau. Microtubules (5 μM tubulin) were nucleated on sea urchin sperm axonemes in the presence of 2.5 μM tau (isoform htau40) and 10 milliunits/ml of p110mark. a, 20 min without ATP; b with ATP. In a, the microtubules grow continuously; in b, Ser-262 can be phosphorylated, leading to a destabilization and shortening of microtubules. Bar, 10 μm.
      Figure thumbnail gr8
      Figure 8Effect of the unphosphorylated and p110mark-phosphorylated tau on the length of axoneme nucleated microtubules measured by dark-field microscopy. For each condition 500-600 microtubule plus ends were measured; the mean length was plotted against time. For half-widths of length distribution, see C-E. Tubulin concentration was 5 μM; note that without added tau, no microtubules are observed at this concentration. Tau was 2.5 μM in all cases. A, microtubule assembly is initiated by the addition of unphosphorylated wild-type tau (open circles). Tau pre-phosphorylated by p110mark for 2 h does not promote microtubule growth (filled circles), but the prephosphorylated point mutant tau-A262 does (triangles), in accordance with time resolved binding assay in B. B, tubulin and wild-type tau (circles) or tau-A262 (triangles) were mixed at 4°C with 10 milliunits/ml of p110mark and 2 mM MgATP (final concentrations). At t = 0, the temperature was raised to 37°C. With wild-type tau, initial growth but subsequent shrinkage of microtubules is observed, whereas with the point mutant Ser-262 → Ala, microtubules grew continuously. C-E, microtubule length histograms at 5 min and 30 min of the corresponding curves in B. Each sample shows a pronounced peak around 20 μm after 5 min (empty circles). If MgATP was omitted (C) or Ser-262 was mutated into Ala (E), the distribution became broader and shifted to greater lengths at 30 min. By contrast, phosphorylation of tau successfully decreased the mean microtubule length within 30 min of incubation (D).

      Other Kinases Phosphorylating the Repeat Domain of Tau

      Tau can be phosphorylated in vitro by many kinases that can be classified by several criteria, depending on function, targets, or others. Certain proline-directed kinases phosphorylate the regions flanking the repeats, but appear to have little influence on tau-microtubule binding. Conversely, one would expect that kinases phosphorylating the repeat region have an influence on microtubule binding because the repeats of tau are involved in this function, and this is in fact borne out by the results with p110mark described so far. The question therefore arises how this kinase compares with other kinases phosphorylating tau in the repeat domain, such as protein kinase A, protein kinase C, or the 35/41-kDa kinase. To improve the quantitation, we used phosphopeptide mapping to analyze the effects of the kinases on 4-repeat tau and the repeat domain (construct K18). The proteins were phosphorylated with brain extract, p110mark, protein kinase C, and protein kinase A. Tryptic phosphopeptides were analyzed by two-dimensional mapping on thin layer chromatography plates, which gives a clear representation of the relative amount of phosphorylation at different sites. The results are shown in Fig. 9, where the phosphopeptides derived from K18 are labeled according to Fig. 5. Phosphopeptide spots generated by the other kinases were identified by running each sample along with the K18 sample phosphorylated with p110mark (data not shown).
      Figure thumbnail gr9
      Figure 9Tryptic phosphopeptide maps of wild-type tau (htau 40) and construct K18 phosphorylated with (A) brain extract, (B) p110mark, (C) protein kinase C, or (D) protein kinase A, respectively. The numbering of the spots is analogous to (spot 1, Ser-262; spot 2, Ser-356; spot 3, Ser-305; spot 4, Ser-324; spot 5, Ser-293). The panels on the right show the corresponding two-dimensional phosphoamino acid analysis of full-length tau for each kinase.
      The patterns shown in Fig. 9A were obtained by phosphorylating full-length tau and K18 with brain extract. With full-length tau, only spot 1 (Ser-262) is clearly seen; spot 2 (Ser-356) is barely visible. This is even more prominent in the phosphorylation pattern of K18. The data are in agreement with our earlier results (Gustke et al., 1992).
      When we examine the phosphorylation of K18 by p110mark, we find a peptide pattern similar to that generated by the brain extract (compare Fig. 9, A and B); the most prominent spots are again 1 and 2, containing Ser-262 and Ser-356. This confirms the role of p110mark as the major Ser-262 kinase in brain extracts. By contrast, reinvestigation of PK35 has so far yielded inhomogeneous results. Although it phosphorylates the same serines as p110mark, the weighting is different, and the activity of the kinase in brain extracts is at least 10-fold lower (data not shown). This explains why even prolonged incubations of tau with this kinase activity lead to only partial suppression of tau's binding to microtubules, as described earlier.
      As seen in Fig. 9C, protein kinase C only phosphorylated Ser-305 (spot 3), Ser-324 (spot 4) and Ser-293 (spot 5) to a significant extent in K18, but not Ser-262, which is in agreement with Correas et al.(1992). The smear and outermost spot to the left (arrow) are not phosphopeptides derived from tau since they also occurred in control experiments where no tau had been added (not shown). The remaining two spots could not be identified; the spot on the upper right (ast erisk) did not colocalize with either Ser-262 (spot 1) or Ser-356 (spot 2). Comparison of this pattern with one obtained from full-length tau revealed that the major phosphorylation sites of protein kinase C are outside the repeat domain. Only Ser-305 (spot 3) was faintly visible in this pattern (note that the spot on the upper right does not correspond to the upper right spot from K18 (asterisk), as confirmed by control experiments (not shown)).
      When using purified protein kinase A to phosphorylate full-length tau and construct K18 (Fig. 9D), we find mainly Ser-356 (spot 2), Ser-305 (spot 3), Ser-324 (spot 4), and Ser-293 (spot 5). Spot 1 is present but barely visible, showing that Ser-262 is only a very minor phosphorylation site. Phosphorylation of full-length tau (Fig. 9D, left panel) yielded similar spots, plus additional sites outside of the repeat region of tau. These result are in general agreement with earlier data (Scott et al., 1993; Steiner, 1993). Some of these sites had also been seen with the 35/41-kDa kinases (Biernat et al., 1993). In subsequent experiments, we have now determined that the 41-kDa component is the catalytic subunit of protein kinase A (using an antibody against protein kinase A obtained from H. Hilz, Hamburg, data not shown); this explains in part the overlap in the data.

      DISCUSSION

      Neurons affected by Alzheimer's disease are characterized by a decrease in the number of microtubules and an increased amount of tau protein in a hyperphosphorylated and aggregated state (paired helical filaments). In order to establish the relationship between these observations, it is necessary to identify the phosphorylation sites on tau, the kinases and phosphatases acting on them, and the functional consequences of phosphorylation, such as microtubule binding or aggregation. A number of phosphorylation sites have now been mapped. They include Ser/Thr-Pro motifs that are of diagnostic value because their phosphorylation state can be monitored by antibodies that discriminate between normal and Alzheimer tau (Lee et al., 1991; Lichtenberg-Kraag et al., 1992). These sites are the targets of certain proline-directed kinases and can be cleared by at least two phosphatases (PP-2A and PP-2B) (Drewes et al., 1993; Gong et al., 1994); they are phosphorylated not only in Alzheimer PHFs but also to some extent in fetal tau (Kanemaru et al., 1992; Bramblett et al., 1993; Watanabe et al., 1993). However, from a functional point of view, the importance of these sites is less clear since a major effect on tau-microtubule binding is not observed (Biernat et al., 1993). Since these sites lie mostly in the regions flanking the repeat domains, this would be consistent with the view that the repeats (but not the flanking regions) determine the interactions with microtubules (but see discussion below).
      Perhaps the most interesting phosphorylation sites are those that strongly affect tau-microtubule interactions. Thus far, only one such site is known, Ser-262 (Biernat et al., 1993). The significance of this finding is enhanced by the fact that Ser-262 is also uniquely phosphorylated in Alzheimer tau, as shown by Hasegawa et al.(1992) and confirmed in our laboratory (Gross et al., 1994). This prompted our search for kinases and phosphatases that regulate the phosphorylation at Ser-262. The phosphatase part of this search turned out to be relatively simple. Both PP-2A and PP-2B are capable of clearing in vitro all phosphorylation sites of tau we have studied so far, including Ser-262 (Drewes et al., 1993). However, the kinase part of the search was more protracted.
      First, it was clear from the beginning that the kinase(s) must be present and active in extracts obtained from normal mammalian brain. When tau is phosphorylated by the extract in the presence of phosphatase inhibitors (such as okadaic acid), the prominent target sites are several Ser/Thr-Pro motifs and the two KIGS motifs in repeats 1 and 4 (Ser-262 and Ser-356; Gustke et al., 1992). Second, some of these motifs or the corresponding KCGS motifs in repeats 2 and 3 can be phosphorylated by protein kinase A or protein kinase C, respectively (Scott et al., 1993; Correas et al., 1992; Steiner, 1993). However, the efficiency of phosphorylation at these sites is low and thus, not surprisingly, the effect of these kinases on tau-microtubule interactions is only minor. Another surprising feature was that in spite of the similarity of these four motifs, only the first one (Ser-262) has the pronounced effect on microtubule interactions (Biernat et al., 1993), a result that we have now confirmed in the present study.
      Our previous purification of the Ser-262 kinase had yielded a doublet of proteins at 35 and 41 kDa. We now know that the 41-kDa component is the catalytic subunit of protein kinase A. It phosphorylates Ser-214 (in the N-terminal flanking region); Ser-293, Ser-324, and Ser-356 (the KXGS motifs in repeats 2-4); Ser-409 and Ser416 (in the C-terminal flanking region; Ser-416 is also the main target of CaM kinase); however, Ser-262 is only a minor site (Table 2).
      During the continued efforts to improve the purification procedure and to analyze phosphorylation sites, we detected a novel kinase of higher Mr, now termed p110mark, which effectively phosphorylated the microtubule binding domain of tau and turned out to be of much higher activity toward Ser-262. While the 35/41-kDa kinases required a prolonged incubation time (16 h) to get appreciable phosphorylation, p110mark affected tau binding already after incubations of 30 min to 2 h. Because of the low yield of purification, it has not yet been possible to characterize the 35-kDa component in detail; we note, however, that the Mr is just slightly larger than the kinase catalytical domain (Hanks and Quinn. 1991), leaving the possibility that it is a degradation product of p110mark, which is, in fact, easily degraded during the purification procedure (data not shown). p110mark phosphorylates all four KXGS motifs, the first and fourth (Ser-262 and Ser-356) being the most pronounced sites. In this regard, the kinase mimics our earlier observations with the brain tissue extract (Gustke et al. (1992) and see Fig. 9). The most dramatic effects of the kinase are that it virtually eliminates tau's binding to microtubules (Fig. 6B), it causes the release of tau from microtubules, and it turns stable microtubules into dynamically unstable ones, as seen by video microscopy. These effects are mainly dependent on the phosphorylation of Ser-262, as shown by the point mutant Ser-262 → Ala. These features make p110mark a candidate enzyme for controlling the state of assembly of microtubules in neurons. They are also consistent with the “Tau Hypothesis of Alzheimer's Disease,” which assumes that tau's failure to bind to and stabilize microtubules leads to their breakdown and cessation of axonal transport. This could occur either by the detachment of tau from microtubules or by the inhibition of newly synthesized tau to bind to microtubules, in both cases resulting from phosphorylation. According to this scheme, an intervention that would slow down p110mark or turn off its potential activating cascade would be suitable for a treatment of Alzheimer's disease.
      p110mark can be renatured in SDS gels, showing that its active form is a single polypeptide with an molecular mass of approximately 110 kDa, which is relatively large for a protein kinase. Its preference for KXGS motifs is reminiscent of protein kinase C or calcium/calmodulin-dependent kinase II, whose consensus sequence is (K/R)XXS, and protein kinase A (consensus RRXS) (Kemp and Pierson, 1990). However, tau possesses several consensus motifs that do not get phosphorylated by these kinases and other nonconsensus motifs that do (a case in point being calcium/calmodulin-dependent kinase II, see Steiner et al. (1990)), so that predictions based on consensus motifs seem of little value here. We also note that the motif KXGS is conserved not only within the tau repeats but also within other MAPs such as the neuronal MAP2 and the ubiquitous MAP4 (for review, see Chapin and Bulinski(1992)). It is therefore possible that p110mark has a more general role, affecting different MAPs and/or other substrates. Preliminary experiments point in this direction.
      We conclude by commenting on two problems that are currently not resolved, the questions of microtubule binding and tau aggregation. The repeat domain of tau is usually considered as the microtubule binding domain (Butner and Kirschner, 1991; Goode and Feinstein, 1994); however, it is also known that the repeats alone bind poorly to microtubules (Ennulat et al., 1989; Joly and Purich, 1990), and several reports pointed to the importance of the flanking regions (e.g. Lee and Rook(1992) and Chen et al. (1992)). We have recently studied a set of tau constructs with different combinations of domains and confirmed that the repeats alone (such as construct K18) bind rather poorly, whereas repeatless tau binds strongly, albeit nonproductively in terms of microtubule polymerization (Gustke et al., 1994). These results can be rationalized by considering the flanking regions as “jaws” that position tau in the proper binding conformation. In this context it is surprising that the many phosphorylatable Ser/Thr-Pro motifs that lie in the strongly binding flanking regions of the repeats have such a weak influence on microtubule binding or assembly, whereas the single Ser-262, which lies in the weakly binding repeat domain, has a uniquely strong influence on microtubule binding. It is possible that phosphorylated Ser-262 prevents the approach of tau into its binding site on the microtubule surface (as illustrated in Fig. 10). Alternatively, Ser-262 might control a conformation of tau that is important for binding, and is acquired only upon docking onto the microtubule surface. This would be consistent with the fact that certain “conformation-sensitive” antibodies require the repeat domain plus phosphorylation on either one of the two flanking regions (Lichtenberg-Kraag et al., 1992).
      Figure thumbnail gr10
      Figure 10Diagram representing the influence of different phosphorylation sites on tau-microtubule interactions. The majority of Ser/Thr-Pro motifs are in the flanking regions of the repeat domain, they have only a small influence on the binding of tau. The repeat domain contains several phosphorylatable non-Ser-Pro sites, especially the four KXGS motifs. Of these, Ser-262 in the first KIGS motif has by far the greatest influence on microtubule binding.
      The second comment is that tau has an ill-defined structure, unlike a typical folded protein but comparable to a denatured random coil (Schweers et al., 1994). This makes it difficult to imagine how tau could bind specifically to microtubules, and how tau aggregates into the periodic PHF fibers. In particular, the self-assembly of tau into PHF-like fibers can be achieved in vitro with the repeat domain of tau alone (Wille et al., 1992), consistent with the fact that the Pronase-resistant core of PHFs also consists of the repeats (Kondo et al., 1988; Wischik et al., 1988; Ksiezak-Reding and Yen, 1991). PHFs containing full-length tau are hyperphosphorylated, whereas the selfassembly of the repeat domain in vitro does not require phosphorylation. It is therefore currently not possible to relate tau's self-assembly to its phosphorylation (in contrast to the clear relationship between tau's microtubule binding and phosphorylation described here). It is even possible that the hyperphosphorylation of tau observed in PHFs is only of secondary importance for the pathological process. This question can only be solved by testing the self-assembly of tau domains in defined states of phosphorylation.

      ACKNOWLEDGEMENTS

      We thank M. Barche (Hamburg) and H. Korte (Bochum) for excellent technical assistance. Antibodies against protein kinase A were generously made available by H. Hilz (Hamburg), and clones of human tau were supplied by M. Goedert (Cambridge, United Kingdom).