The EMBO Journal vol. 1 1 no.4 pp. 1 593 - 1 597, 1992

The switch of tau protein to an Alzheimer-like stateincludes the phosphorylation of two serine- prolinemotifs upstream of the microtubule binding region

J.Biernat1, E.-M.Mandelkowi, C.Schroterl,B.Lichtenberg-Kraagl, B.Steinerl, B.Berling',H.Meyer2, M.Mercken3'4, A.Vandermeeren4,M.Goedert5 and E.Mandelkow1'6

Max-Planck-Unit for Structural Molecular Biology, c/o DESY,Notkestrasse 85, D-2000 Hamburg 52, 21nstitute for PhysiologicalChemistry, Ruhruniversitat, Universitatsstrasse 150, Geb. MA2/143,D-4630 Bochum, FRG, 3Laboratory of Neuropathology andNeurobiology, Born-Bunge Foundation, University of Antwerpen,Universiteitsplein 1, B-2610 Antwerpen-Wilrijk, Belgium,41nnogenetics S.A., Industriepark Zwijnaarde 7, Box 4, B-9052 Gent,Belgium and 5MRC Laboratory of Molecular Biology, Hills Road,Cambridge CB2 2QH, UK

6Corresponding author

Communicated by K.C.Holmes

The paired helical filaments (PHFs) of Alzheimer's diseaseconsist mainly of the microtubule-associated protein tau.PHF tau differs from normal human brain tau in thatit has a higher Mr and a special state of phosphoryla-tion. However, the protein kinase(s) involved, thephosphorylation sites on tau and the resulting conforma-tional changes are only poorly understood. Here we showthat a new monoclonal antibody, AT8, records the PHF-like state of tau in vitro, and we describe a kinase activitythat turns normal tau into a PHF-like state. The epitopeof AT8 is around residue 200, outside the region ofinternal repeats and requires the phosphorylation ofserines 199 and/or 202. Both of these are followed by a

proline, suggesting that the kinase activity belongs to thefamily of proline-directed kinases. The epitope of AT8is nearly coincident with that of another phosphorylation-dependent antibody, TAUl [Binder,L.I., Frankfuter,A.and Rebhun,L. (1985) J. Cell Biol., 101, 1371-1378],but the two are complementary since TAUl requires a

dephosphorylated epitope.Key words: Alzheimer's disease/microtubules/monoclonalantibodies/paired helical filaments/phosphorylation/proteinkinase/tau protein

IntroductionThe brains of Alzheimer patients contain two characteristictypes of protein deposits, the plaques and tangles. Much ofcurrent Alzheimer research is aimed at determining thenature of these deposits and the factors that cause them. Aprominent component of the tangles are the paired helicalfilaments, PHFs, which are largely made up of the micro-tubule-associated protein tau. The question therefore arises:in what way is PHF tau different from normal tau and whatcauses the difference? There are several isoforms of tau (sixin human brain) that arise from alternative splicing of one

gene (Goedert et al., 1988, 1989; Lee et al., 1988; Himmneret al., 1989). The main biochemical differences between

Oxford University Press

normal and PHF tau are the following: (i) PHF tau is highlyinsoluble in contrast to normal tau; (ii) PHF tau reacts withcertain antibodies in a phosphorylation-dependent manner,suggesting that the protein is in a special state of 'abnormal'phosphorylation; (iii) PHF tau has a lower electrophoreticmobility in SDS gels, suggesting a higher Mr value, thiseffect is also related to phosphorylation (Grundke-Iqbalet al., 1986; Lee et al., 1991).

In this study we have attempted to define the 'Alzheimer'state of tau by a new approach. We have used a novelantibody that is specific for PHFs and sensitive to aphosphorylated epitope; we have isolated a protein kinaseactivity that phosphorylates this epitope; we have determinedthe two phosphorylation sites on tau that are crucial to thisepitope, and show that the kinase activity has the charteristicsof a proline-directed kinase; we show that phosphorylationby the kinase shifts the Mr of tau. This means that normaltau, isolated either from brain tissue or expressed inEscherichia coli, can be transformed into an 'Alzheimer'state in a controlled and reversible fashion in vitro.

ResultsExtract phosphorylation mimicks an Alzheimer-likestate of tauA series of antibodies made against PHFs from Alzheimerbrain was evaluated (Mercken et al., 1992). One of them(AT8) was specific for PHF tau and was selected for furtherstudies. Figure 1 shows its reactivity against different tauspecies. The antibody recognizes all isoforms fromAlzheimer PHFs (Figure lb, lane 1), but none from normalbovine or human brain (in mixed states of phosphorylation,Figure Ib, lanes 2-5). The same is true of the six individualhuman isoforms expressed in E.coli (unphosphorylated,Figure la and b, lanes 6-11). We conclude that AT8 isindeed specific for Alzheimer tau.We therefore began a search for the kinase(s) that were

responsible for this behaviour and for the phosphorylationsites. We had studied various kinases and their phosphoryla-tion sites earlier (Steiner et al., 1990) but none of themcaused a reaction with the new antibody. We then prepareda kinase activity from porcine brain extract (see Materialsand methods) and phosporylated the six human isoformsexpressed in E.coli. Figure 2 shows that each isoform hasa strong Mr shift and a strong immunoreactivity with theAT8 antibody. These results show that the phosphorylationof tau by this kinase activity is analogous to that of theAlzheimer state and that the phosphorylation site(s) must bein a region conserved in all isoforms.

The region of the Alzheimer-like AT8 epitope containstwo phosphorylated SP pairs in tandemTwo aspects of tau antibodies make them potentially usefulfor Alzheimer diagnostics. One is the reactivity withAlzheimer tau per se [such as the Alz50 antibody, (Ksiezak-

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I,..=.

Fig. 1. SDS gel and immunoblot of tau isoforms and PHF tau.(a) SDS gel: lane 1, marker proteins; lane 2, Tau from bovine brainshowing several isoforms in a mixed state of phosphorylation; lane3, bovine brain tau after dephosphorylation with alkaline phosphatase.Note that all isoforms shift to a lower M,; lanes 4 and 5, Tau fromnormal human brain before and after dephosphorylation; lanes 6-11,bacterially expressed human tau isoforms htau23, 24, 37, 34, 39 and40 (Goedert et al., 1989). These isoforms have either three or fourinternal repeats of 31 or 32 residues in the C-terminal half(three: htau23, 37 and 39; four: htau24, 34 and 40). Near theN-terminus there can be zero, one or two inserts of 29 residues(zero: htau23 and 24; one: htau37 and 34; two: htau39 and 40).(b) Immunoblot with the AT8 antibody: lane 1, PHF tau showing fourbands in the range of 60-70 kDa, all of them react strongly withAT8; lanes 2-11, same preparations as in (a), none of the bovine ornormal human tau isoforms show any reaction.

t

t

b

Fig. 2. Phosphorylation of bacterially expressed human tau isoformswith the kinase activity from brain. (a) SDS gel and (b) immunoblotwith AT8. (a) Lanes 1 and 2, SDS gel of htau23 before and afterextract phosphorylation (note the upward shift in Mr). Lanes 3-10show the same pairs for other isoforms (htau24, 34, 39 and 40).(b) Immunoblots of (a) with AT8 antibody. It reacts with all tauisoforms after phosphorylation (even lanes; the case of htau37 is notshown here).

Reding et al., 1988)]; the other is the sensitivity to tauphosphorylation [such as TAUl, (Binder et al., 1985)] whichin turn could be related to the Alzheimer state. Our antibodyAT8 appeared to combine both properties and we thereforebecame interested in locating its epitope.The strategy was first to use several engineered mutants

in order to narrow down the location of the epitope and thento determine it by direct sequencing. Figure 3 describes someof the mutants used, K19, KIO, K17 and K3M. Except forK19 (a construct that comprises just three repeats of 31 or32 residues), all of these mutants show an upward Mr shiftin the SDS gel upon phosphorylation (Figure 4a). This meansthat the major phosphorylation site(s) are oustide the regionof the repeats and that phosphorylation in both regions canproduce different Mr shifts. The antibody AT8 recognizes1594

Fig. 3. Diagram of constructs K3M, KIO, K19 and K17. K19 (99residues) contains the sequence Gln244-Glu372 of htau23 plus anN-terminal methionine. This comprises three of the repeats (repeats 1,3 and 4; repeat 2 is absent in htau23). KIO (168 residues) is similarexcept that it extends to the C-terminus of htau23 (L441). K17 (145residues) contains the sequence Serl98-Glu372 (assembly domainstarting at the chymotryptic cleavage site up to the end of the fourthrepeat, but without the second repeat plus an N-terminal methionine).K3M (335 residues) contains the N-terminal 154 residues of bovinetau4, plus the sequence Arg221-Leu441 of htau23 (without thesecond repeat). The location of peptide Serl98-Thr220 is indicated inK17. By comparison of the constructs the epitope of AT8 must be inthis region (see Figure 4).

none of the unphosphorylated forms (as expected); afterphosphorylation it reacts only with the construct K17 (Figure4b, lane 6), not with KlO or K3M (Figure 4b, lanes 4 and8). In other words, K17 retains the epitope while KIO andK3M have lost it. By reference to Figure 3 we conclude thatthe epitope is not in the region of the pseudo-repeats or inthe C-terminal tail where we found a calmodulin (CaM)kinase site previously (since K1O and K19 are non-reactive),but rather that it has to be in peptide P (Figure 3) betweerSerl98 and Thr220, which includes 10 potential phos-phorylation sites (Ser and Thr).We then made a total tryptic digest of radioactively labellec

htau34, an isoform with four internal repeats (Goedert eial., 1989). The peptides were isolated by HPLC andsequenced. One of them was in the area of interest,Serl95-Arg2O9 (Figure 5). This peptide contained twophosphates at Serl99 and Ser2O2. Both are followed by aproline, suggesting that the enzyme active in the extract wasa proline-directed kinase.

This was tested by engineering a mutant of htau23 (threerepeats, no N-terminal insert) where Serl99 and Ser202 wereboth changed to Asp. This choice was made not only in orderto rule out the phosphorylation of these residues by a kinase,but also to mimick in part the 'phosphorylated' state in termsof negative charges. On SDS gels this mutant showed a smallupward shift (Figure 6, lane 3). When it was incubated withthe kinase activity there was an additional shift to higher Mr(Figure 6, lane 4), but on immunoblots it showed no reactionwith the AT8 antibody (Figure 6, lane 8). We conclude thatthe epitope of AT8 is in the region Serl99-Ser202 anddepends on the phosphorylation of one or both of these twoserines.

Antibodies AT8 and TAUl share the same bindingsite, but are complementary to each otherAmong the widely used tau antibodies, TAUl is particularlyinteresting because it distinguishes certain forms ofphosphorylated and non-phosphorylated tau protein (Binder

....

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*SWNOW140,

Phosphorylation of Alzheimer-like tau

PAGE

ht4o K10 K17 K3Ma _

1w

BLOT- f;T8ht40 K17

1 2 3 4 5 6 7 8 9 10 11 12

1 2 3 4 5 6 7 8

Klg AT- 8c

1 2 3 4

Fig. 4. Phosphorylation of htau40 and (

K19. (a) SDS gel; odd lanes, htau40,phosphorylation; even lanes, after phosjshift of the bands after Dhosnhorvlation

Fig. 6. Phosphorylation and antibody reactions of the Asp mutant ofhtau23 (Serl99 and Ser2O2 changed into Asp): lanes 1 and 2, SDS gel

1 2 3 4 5 6 7 8 of htau23 before and after extract phosphorylation; lanes 3 and 4,Asp-mutant before and after extract phosphorylation [note that theAsp-mutant runs slightly higher than htau 23 (lanes 1 and 3), but afterphosphorylation both proteins have the same position in the gel (lanes2 and 4)]; lanes 5-8, immunoblots of lanes 1-4 with AT8 [theantibody reacts only with the extract phosphorylated htau23 (lane 6),but neither with the unphosphorylated form (lane 5) nor with the Aspmutant (lanes 7 and 8), although it was phosphorylated as seen by theadditional shift and autoradiography (not shown)]; lanes 9-12,immunoblots of lanes 1-4 with TAUl. This antibody reacts only withhtau23 before phosphorylation (lane 9) but not with the phosphorylated

constructs KIO, K17, K3M and form (lane 10) nor with the Asp mutant (lanes 11, 12). The asparticKIO, K17 and K3M before acid apparently mimicks a phosphorylated serine and thus masks thephorylation. Note the upward epitope. The minor reaction of htau23 with TAUI in lane 10 shows. In lane 4 there are two bands that the protein is not completely phosphorylated.v--- -_aav ..rVos. .-.- - _wavtvVs

because K1O is not completely phosphorylated. (b) Immunoblot of (a)with AT8. The antibody reacts only with htau40 (lane 2) and K17(lane 6) both in the phosphorylated state, but not with K10 (lane 4) orK3M (lane 8) although these constructs are also phosphorylated andshow an Mr shift. (c) Construct K19 before and after incubation withthe kinase activity: lanes 1 and 2, SDS gel, there ig no Mr shift; lanes3 and 4, immunoblot with AT8 showing no reaction. This confirmsthat the epitope is not in the repeat region.

T- 1

h-t PHF - h-tAT- 8

PH F-t

1 2 3 4 5 C 7 8 9 1011 12

Y 197 [':4 I htau 23

YI 9 2 1--[--' ..I htau 34

SGYS S PGSPGTPGSR209

P1 P'

Fig. 5. Diagram of tryptic peptide Serl95-Arg209 and its location inhtau23 (three repeats) and htau34 (four repeats). The 15 residuepeptide (containing five serines and one threonine) was labelled withtwo radioactive phosphates at Serl99 and Ser202, as determined bysequencing.

et al., 1985; Ksiezak-Reding et al., 1988; Lee et al., 1991).Previous studies had located the epitope roughly betweenProl89 and Gly207 (Kosik et al., 1988) which overlaps withthe AT8 epitope. We therefore asked if our kinase activityhad an effect on TAUI reactivity. We performed the aboveexperiment but blotted with TAUl (Figure 6, lanes 9-12).The antibody reacted with the dephosphorylated parentprotein (htau23), but not with its phosphorylated form; italso failed to react with the mutants at residues 199 and 202,irrespective of whether other sites were phosphorylated ornot (Figure 6, lanes 11 and 12). This proves two points:one is that the epitope of TAUl must be very close to thatof AT8 (around Serl99-Ser2O2), the other is that TAUlshows the opposite behaviour, it requires Serl99 and/orSer2O2 in a dephosphorylated state (the faint staining withTAUl in Figure 6, lane 10, shows that the protein is not100% phosphorylated at the two serines, although it isalready shifted).

Fig. 7. Phosphorylation and antibody epitopes of tau from Alzheimerbrains. Lanes 1-6, immunoblot with antibody TAUI; lanes 7-12,immunoblots with AT8. All tau isoforms from normal human brainreact with antibody TAUI in the native state of phosphorylation (lane1) after dephosphorylation (lane 2) but not after rephosphorylation withthe kinase activity (lane 3). Antibody AT8 shows the oppositebehaviour (lanes 7-9), i.e. it does not react with the native ordephosphorylated normal human tau (lanes 7 and 8), but does reactafter phosphorylation with the kinase activity (lane 9). Tau from PHFs(lane 4-6) reacts with TAUI only after dephosphorylation (lane 5) butnot in the native state (lane 4) or after rephosphorylation (lane 6).Antibody AT8 reacts again in an opposite fashion (lanes 10-12); itrecognizes native PHF tau (lane 10) not dephosphorylated PHF tau(lane 11) and again rephosphorylated PHF tau (lane 12).

Thus, one would expect that if AT8 reacts with Alzheimertau in the phosphorylated state, then TAUl should react withthis protein after dephosphorylation. This is indeed the case(Figure 7), in agreement with recent findings (Lee et al.,1991). We compared a PHF preparation as isolated, afterdephosphorylation with alkaline phosphatase, and afterrephosphorylation with the brain kinase activity (Figure 7,lanes 4-6). This treatment shifted the protein bands in thegel; the important point is, however, that AT8 recognizesthe Alzheimer tau in its native state of phosphorylation, orafter dephosphorylation and rephosphorylation with thekinase (Figure 7, lanes 10 and 12), whereas TAU1 reactsonly with the dephosphorylated form (Figure 7, lane 5).

Normal human brain tau acquires Alzheimerlikeproperties upon phosphorylation with the brain kinaseThe corresponding result can be obtained with tau fromnormal human brain (Figure 7, lanes 1-3). This protein hasa lower Mr than PHF tau although it is already in a mixedstate of phosphorylation. The lack of reactivity with AT8shows that it is not phosphorylated at residues Serl99 and/orSer2O2 (Figure 7, lane 7). However, when this normal

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IP_ ......I

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human brain tau is incubated with the kinase activity, theMr shifts up to the same positions as the PHF tau and on

immunoblots we find AT8 reactivity with all isoforms(Figure 7, lane 9). At the same time this protein loses theTAUI reactivity upon phosphorylation (Figure 7, lane 3).These experiments show that one can convert normal humanbrain tau into Alzheimer-like tau by phosphorylation, as

judged by antibody staining and Mr shift.

DiscussionIn what way is PHF tau different from normal tau? We havestudied this question by combining several new approaches.These include (i) a specific kinase activity which enables us

to convert normal tau into an Alzheimer-like state; (ii) a new

antibody, AT8, that is diagnostic for the Alzheimer-state oftau and correlates with other indicators such as the Mr shift;(iii) protein engineering and site directed mutagenesis ofdefined tau variants that can be related to the PHF com-

ponents; (iv) biochemical identification of the phosphoryla-tion sites involved in the transition from the normal to theAlzheimer state of tau.

In order to analyse an Alzheimer-like state of tau, one firstneeds a diagnostic tool, such as monoclonal antibodies. TwomAbs have been particularly useful, Alz5O and TAU1.Alz5O was raised against Alzheimer brain homogenate. Itsepitope is near the N-terminus of all tau isoforms (Ksiezak-Reding et al., 1990; Goedert et al., 1991) and does not

depend on phosphorylation. TAUI also recognizes all tauisoforms, but PHF tau reacts only after it is dephosphorylated,suggesting that it is abnormally phosphorylated (Grundke-Iqbal et al., 1986). A second diagnostic tool is the Mr shift:PHF tau has a higher Mr than normal tau (Grundke-Iqbal et al., 1986; Flament and Delacourte, 1989; Lee et al.,1991). This can also be taken as a sign of phosphorylation,especially since normal tau shows a similar effect with certainkinases (such as CaMK), even when only a single phosphateis incorporated (Steiner et al., 1990).We consider AT8 superior to most other antibodies as a

diagnostic tool: it recognizes all tau isoforms prepared fromPHFs, but none of the isoforms from normal mammalianbrain (human, porcine or bovine) in their mixed state ofphosphorylation, nor any of the engineered tau constructs.However, all of these isoforms are recognized afterphosphorylation with a kinase activity from brain. Thus theantibody is more specific for the Alzheimer state than Alz5Oand it has the advantage of reporting on the state ofphosphorylation. Using different tau constructs, proteinsequencing and directed mutagenesis we found that theepitope includes the phosphorylated serines 199 and 202.At the same time we found that phosphorylation by the kinaseactivity also increased the Mr of all tau isoforms. Theincrease is larger than that we had found previously withCaMK so that the largest tau isoform, htau40, is shifted intoa position indistinguishable from that of the Alzheimerprotein A68 (see Figure 1). Part of this Mr shift can bemimicked by negative charges as in the Asp mutant of htau23(Figure 6, lane 3).An unexpected by-product of this study was the localiza-

tion of the phosphorylation-dependent epitope of TAU1 andits complementarity to AT8. Earlier studies (Kosik et at.,1988) had shown that the epitope was roughly in the region189-207; we show that Serl99 and/orSer202 are theresidues whose phosphorylation controls the binding of

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TAUl in a way opposite to AT8. Thus, PHF tau is recognizedby AT8 but not TAUl, normal tau is recognized by TAU1but not AT8 and normal tau phosphorylated at residues 199and 202 behaves like PHF tau.One significant aspect of this work is that we have now

found a kinase activity which is capable of transformingnormal tau into an Alzheimer-like state. This kinase activityis present in mammalian brain extract. As shown in detailelsewhere, the kinase activity phosphorylates several residuesin tau; this leads to the large M, shift which makes normaltau similar to PHF tau on SDS gels. Two of the phosphoryl-ated residues are Ser199 and Ser202. We note that both ofthem are followed by a proline and none of them are in aconsensus sequence typical of protein kinase A, proteinkinase C, CaMK or casein kinase II. Thus, it is likely thatthe kinase belongs to the family of proline-directed kinases,members of which have recently been shown to have avariety of regulatory functions (reviewed in Kemp andPearson, 1990). An example is the phosphorylation ofneurofilament subunits by a neurofilament specific kinasewhich controls their assembly properties. In this case themajor epitopes are of the form KSP (Geisler et al., 1987)and there are antibodies against the phosphorylated form ofthis epitope, some of which also cross-react with PHFs(Brion et al., 1991; Lee et al., 1991). The motif KSP occurstwice in the tau sequence (residues 234-236 and 395 -397);the second one is probably phosphorylated in PHF tau (Leeet al., 1991). Similarly, a tubulin-dependent kinase appearsto phosphorylate a combination of SP and TP sites of tau(Ishiguro et al., 1991), although in this case the relation-ship to PHFs is not clear. In our study, we have found notonly the phosphorylation site(s) but also the kinase activitythat can be detected by an Alzheimer PHF tau specificantibody; this means that we can now mimic the transitionfrom normal to Alzheimer-like tau in vitro.

Materials and methodsPreparation of tauThe preparation of tau from human, bovine, or porcine brain, dephosphoryla-tion and rephosphorylation were done as described (Hagestedt et al., 1989).For PHF tau, human brain tissues from neuropathologically confirmed casesof Alzheimer's disease were obtained from the Born-Bunge Foundation,University of Antwerpen (Belgium). The autopsies were performed between4.5 and 20 h post mortem. The brain tissue was kept frozen at -70°C.PHF tau was prepared according to Greenberg and Davies (1990). Themonoclonal antibody TAUl was a generous gift from Dr L.Binder. AntibodyAT8 against Alzheimer PHF-tau was prepared as described (Mercken et al.,1992). SDS-PAGE was done with a gradient of 4-20%. Immunoblottingwas done onImmobilon membranes (Millipore). The bound antibody wasdetected by a peroxidase conjugated second antibody (anti-mouse IgG,Dakopatts).

Phosphorylation by brain kinase activityExtract from porcine brain was prepared by homogenizing the brain in10 mM Tris-HCI, pH 7.2, 5 mM EGTA, 2 mM DTT and a cocktail ofprotease inhibitors (leupeptin, aprotinin, pepstatin A,a-macroglobulin andPMSF) and centrifuged at100 000 g for 30 min at4°C. The supernatantwas either precipitated with 40% ammonium sulfate and the pellet redissolvedin 10 mM Tris-HCI, 2 mM each EGTA, DTT and MgSO4, pH 7.2, orit was used directly for the phosphorylation experiments after addition of2 mM ATP and 10zM okadaic acid. The phosphorylation was done byadding1 Al of extract to 40Ml of tau protein solution (0.5-1 mg/ml) at37°C.

Sequencing of phosphorylated peptidesThe procedures were essentially as described (Steiner et al., 1990). Theprotein was phosphorylated as described above and unbound nucleotide wasremoved by passage over a NAP-S gel filtration column (Pharmacia). Theprotein was lyophilized and then resuspended in 10 mM NH4HCO3,

Phosphorylation of Alzheimer-like tau

pH 8.0 and digested for 24 h by adding 1/50 (w/w) of trypsin (Sigma, TPCKtreated) four times in 6 h intervals (this procedure was used to optimizedigestion conditions). The digest was lyophilized once more and resuspendedin buffer A (10mM ammonium acetate pH 6.0) for the first HPLC gradient.Separation was done on a Beckman HPLC system with a C18 reverse phasecolumn (Vydac, 4.6 x 250 mm, flow rate 1 ml/min at room temperature).Crude fractions were obtained by a linear gradient of 0-40% acetonitrilein 10 mM ammonium acetate. Radioactivity was determined by a scintilla-tion counter (Hewlett Packard TriCarb 1900CA). The radioactive peakswere reapplied to the same column and eluted with a linear gradient of0-40% acetonitrile in 0.1 % trifluoroacetic acid. The sequence analysis ofthe peptides was performed using a 477A pulsed liquid phase protein/peptide sequencer and a 120A on-line PTH amino acid analyser (AppliedBiosystems). Phosphoserines were identified by gas phase sequencing,making use of the formation of the dithiothreitol adduct of dehydroalaninefrom serine phosphate (Meyer et al., 1990).

Plasmid preparations and cloning proceduresPlasmid preparations and cloning procedures were performed according toSambrook et al. (1989). PCR amplifications were carried out using Taqpolymerase as specified by the manufacturer (Perkin Elmer Cetus). Thetau cDNA clones and constructs thereof were subcloned into the expres-sion vector pNG2, a derivative of pET-3b (Studier et al., 1990), modifiedin our laboratory by removal of PstI, HindlIl, NheI and EcoRV restrictionsites for convenient engineering of the tau clones. For the expression weused the BL21 (DE3) E.coli strain (Studier et al., 1990). Most constructswere derived from the human isoform htau23 which contains 352 residuesand three internal repeats in the C-terminal microtubule binding region.The numbering of residues used here refers to the sequence of htau40, thelargest of the human isoforms [441 residues, (Goedert et al., 1989)].Modified tau proteins were obtained by cassette mutagenesis. For the isolationof the constructs we made use of the heat stability of the protein; theconstructs were separated by FPLC Mono S (Pharmacia) chromatography[for details see Hagestadt et al. (1989)].

AcknowledgementsWe thank A.Malchert and U.Boning for excellent technical assistance inprotein expression and preparation and U.Boning for photography. We aregrateful to L.Binder (University of Alabama) for a gift of TAU I antibody,W.Studier (Brookhaven National Laboratory) for the expression vector, andN.Gustke for construct K3M. This project was supported by theBundesministerium fur Forschung und Technologie and the DeutscheForschungsgemeinschaft.

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Received on December 12, 1991; revised on January 21, 1992

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