motifs upstream of the microtubule binding region

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

The switch of tau protein to an Alzheimer-like state includes the phosphorylation of two serine- proline motifs 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 Physiological Chemistry, Ruhruniversitat, Universitatsstrasse 150, Geb. MA2/143, D-4630 Bochum, FRG, 3Laboratory of Neuropathology and Neurobiology, 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 disease consist mainly of the microtubule-associated protein tau. PHF tau differs from normal human brain tau in that it has a higher Mr and a special state of phosphorylation. However, the protein kinase(s) involved, the phosphorylation sites on tau and the resulting conformational changes are only poorly understood. Here we show that a new monoclonal antibody, AT8, records the PHFlike state of tau in vitro, and we describe a kinase activity that turns normal tau into a PHF-like state. The epitope of AT8 is around residue 200, outside the region of internal repeats and requires the phosphorylation of serines 199 and/or 202. Both of these are followed by a proline, suggesting that the kinase activity belongs to the family of proline-directed kinases. The epitope of AT8 is nearly coincident with that of another phosphorylationdependent 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/monoclonal antibodies/paired helical filaments/phosphorylation/protein kinase/tau protein

Introduction The brains of Alzheimer patients contain two characteristic types of protein deposits, the plaques and tangles. Much of current Alzheimer research is aimed at determining the nature of these deposits and the factors that cause them. A prominent component of the tangles are the paired helical filaments, PHFs, which are largely made up of the microtubule-associated protein tau. The question therefore arises: in what way is PHF tau different from normal tau and what causes the difference? There are several isoforms of tau (six in human brain) that arise from alternative splicing of one gene (Goedert et al., 1988, 1989; Lee et al., 1988; Himmner et al., 1989). The main biochemical differences between Oxford University Press

normal and PHF tau are the following: (i) PHF tau is highly insoluble in contrast to normal tau; (ii) PHF tau reacts with certain antibodies in a phosphorylation-dependent manner, suggesting that the protein is in a special state of 'abnormal' phosphorylation; (iii) PHF tau has a lower electrophoretic mobility in SDS gels, suggesting a higher Mr value, this effect is also related to phosphorylation (Grundke-Iqbal et 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 novel antibody that is specific for PHFs and sensitive to a phosphorylated epitope; we have isolated a protein kinase activity that phosphorylates this epitope; we have determined the two phosphorylation sites on tau that are crucial to this epitope, and show that the kinase activity has the charteristics of a proline-directed kinase; we show that phosphorylation by the kinase shifts the Mr of tau. This means that normal tau, isolated either from brain tissue or expressed in Escherichia coli, can be transformed into an 'Alzheimer' state in a controlled and reversible fashion in vitro.

Results Extract phosphorylation mimicks an Alzheimer-like state of tau A series of antibodies made against PHFs from Alzheimer brain was evaluated (Mercken et al., 1992). One of them (AT8) was specific for PHF tau and was selected for further studies. Figure 1 shows its reactivity against different tau species. The antibody recognizes all isoforms from Alzheimer PHFs (Figure lb, lane 1), but none from normal bovine or human brain (in mixed states of phosphorylation, Figure Ib, lanes 2-5). The same is true of the six individual human isoforms expressed in E.coli (unphosphorylated, Figure la and b, lanes 6-11). We conclude that AT8 is indeed specific for Alzheimer tau. We therefore began a search for the kinase(s) that were responsible for this behaviour and for the phosphorylation sites. We had studied various kinases and their phosphorylation sites earlier (Steiner et al., 1990) but none of them caused a reaction with the new antibody. We then prepared a kinase activity from porcine brain extract (see Materials and methods) and phosporylated the six human isoforms expressed in E.coli. Figure 2 shows that each isoform has a strong Mr shift and a strong immunoreactivity with the AT8 antibody. These results show that the phosphorylation of tau by this kinase activity is analogous to that of the Alzheimer state and that the phosphorylation site(s) must be in a region conserved in all isoforms. The region of the Alzheimer-like AT8 epitope contains two phosphorylated SP pairs in tandem Two aspects of tau antibodies make them potentially useful

for Alzheimer diagnostics. One is the reactivity with Alzheimer tau per se [such as the Alz50 antibody, (Ksiezak-

1593

J.Biernat et al.

I,..=.

....

-0O im

,wow

140,

NOW

.--w

*SW

Fig. 1. SDS gel and immunoblot of tau isoforms and PHF tau. (a) SDS gel: lane 1, marker proteins; lane 2, Tau from bovine brain showing several isoforms in a mixed state of phosphorylation; lane 3, bovine brain tau after dephosphorylation with alkaline phosphatase. Note that all isoforms shift to a lower M,; lanes 4 and 5, Tau from normal human brain before and after dephosphorylation; lanes 6-11, bacterially expressed human tau isoforms htau23, 24, 37, 34, 39 and 40 (Goedert et al., 1989). These isoforms have either three or four internal repeats of 31 or 32 residues in the C-terminal half (three: htau23, 37 and 39; four: htau24, 34 and 40). Near the N-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 four bands in the range of 60-70 kDa, all of them react strongly with AT8; lanes 2-11, same preparations as in (a), none of the bovine or normal human tau isoforms show any reaction.

t t

b

Fig. 2. Phosphorylation of bacterially expressed human tau isoforms with the kinase activity from brain. (a) SDS gel and (b) immunoblot with AT8. (a) Lanes 1 and 2, SDS gel of htau23 before and after extract phosphorylation (note the upward shift in Mr). Lanes 3-10 show the same pairs for other isoforms (htau24, 34, 39 and 40). (b) Immunoblots of (a) with AT8 antibody. It reacts with all tau isoforms after phosphorylation (even lanes; the case of htau37 is not shown here).

Reding et al., 1988)]; the other is the sensitivity to tau phosphorylation [such as TAUl, (Binder et al., 1985)] which in turn could be related to the Alzheimer state. Our antibody AT8 appeared to combine both properties and we therefore became 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 then to determine it by direct sequencing. Figure 3 describes some of the mutants used, K19, KIO, K17 and K3M. Except for K19 (a construct that comprises just three repeats of 31 or 32 residues), all of these mutants show an upward Mr shift in the SDS gel upon phosphorylation (Figure 4a). This means that the major phosphorylation site(s) are oustide the region of the repeats and that phosphorylation in both regions can produce different Mr shifts. The antibody AT8 recognizes 1594

Fig. 3. Diagram of constructs K3M, KIO, K19 and K17. K19 (99 residues) contains the sequence Gln244-Glu372 of htau23 plus an N-terminal methionine. This comprises three of the repeats (repeats 1,

3 and 4; repeat 2 is absent in htau23). KIO (168 residues) is similar except that it extends to the C-terminus of htau23 (L441). K17 (145 residues) contains the sequence Serl98-Glu372 (assembly domain

starting at the chymotryptic cleavage site up to the end of the fourth repeat, but without the second repeat plus an N-terminal methionine). K3M (335 residues) contains the N-terminal 154 residues of bovine tau4, plus the sequence Arg221 -Leu441 of htau23 (without the second repeat). The location of peptide Serl98-Thr220 is indicated in K17. By comparison of the constructs the epitope of AT8 must be in this region (see Figure 4). none of the unphosphorylated forms (as expected); after phosphorylation it reacts only with the construct K17 (Figure 4b, lane 6), not with KlO or K3M (Figure 4b, lanes 4 and 8). In other words, K17 retains the epitope while KIO and K3M have lost it. By reference to Figure 3 we conclude that the epitope is not in the region of the pseudo-repeats or in the 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) betweer Serl98 and Thr220, which includes 10 potential phosphorylation sites (Ser and Thr). We then made a total tryptic digest of radioactively labellec htau34, an isoform with four internal repeats (Goedert ei al., 1989). The peptides were isolated by HPLC and sequenced. One of them was in the area of interest, Serl95-Arg2O9 (Figure 5). This peptide contained two phosphates at Serl99 and Ser2O2. Both are followed by a proline, suggesting that the enzyme active in the extract was a proline-directed kinase. This was tested by engineering a mutant of htau23 (three repeats, no N-terminal insert) where Serl99 and Ser202 were both changed to Asp. This choice was made not only in order to rule out the phosphorylation of these residues by a kinase, but also to mimick in part the 'phosphorylated' state in terms of negative charges. On SDS gels this mutant showed a small upward shift (Figure 6, lane 3). When it was incubated with the kinase activity there was an additional shift to higher Mr (Figure 6, lane 4), but on immunoblots it showed no reaction with the AT8 antibody (Figure 6, lane 8). We conclude that the epitope of AT8 is in the region Serl99 -Ser202 and depends on the phosphorylation of one or both of these two

serines.

Antibodies AT8 and TAUl share the same binding site, but are complementary to each other Among the widely used tau antibodies, TAUl is particularly interesting because it distinguishes certain forms of phosphorylated and non-phosphorylated tau protein (Binder

Phosphorylation of Alzheimer-like tau PAGE K10 K17

ht4o a

K3M

ht23

BLOT- f;T8 ht40 K17

SP2

-8

w

I IP

......

_

_

T- 1

I

1w I

2

1

3

4 5 6 7 8

Klg

AT- 8

1

3

1 2

1

3 4 5

6

7 8

4

Fig. 4. Phosphorylation of htau40 and (constructs KIO, K17, K3M and K19. (a) SDS gel; odd lanes, htau40, KIO, K17 and K3M before phosphorylation; even lanes, after phosj phorylation. Note the upward shift of-the-_aav bands after Dhosnhorvlation . .In lane .-.- 4 there_wavtvVs vare two bands ..rVos 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) or K3M (lane 8) although these constructs are also phosphorylated and show an Mr shift. (c) Construct K19 before and after incubation with the kinase activity: lanes 1 and 2, SDS gel, there ig no Mr shift; lanes 3 and 4, immunoblot with AT8 showing no reaction. This confirms that the epitope is not in the repeat region. -

Y

197 [':4

YI 9

SG YS S PG S PGTPGSR P1

2 1--[--'

I

htau 23

..I

htau 34

209

P'

Fig. 5. Diagram of tryptic peptide Serl95-Arg209 and its location in htau23 (three repeats) and htau34 (four repeats). The 15 residue peptide (containing five serines and one threonine) was labelled with two radioactive phosphates at Serl99 and Ser202, as determined by sequencing.

1985; Ksiezak-Reding et al., 1988; Lee et al., 1991). Previous studies had located the epitope roughly between Prol89 and Gly207 (Kosik et al., 1988) which overlaps with the AT8 epitope. We therefore asked if our kinase activity had an effect on TAU I reactivity. We performed the above experiment but blotted with TAUl (Figure 6, lanes 9-12). The antibody reacted with the dephosphorylated parent protein (htau23), but not with its phosphorylated form; it also failed to react with the mutants at residues 199 and 202, irrespective of whether other sites were phosphorylated or not (Figure 6, lanes 11 and 12). This proves two points: one is that the epitope of TAUl must be very close to that of AT8 (around Serl99-Ser2O2), the other is that TAUl shows the opposite behaviour, it requires Serl99 and/or Ser2O2 in a dephosphorylated state (the faint staining with TAUl in Figure 6, lane 10, shows that the protein is not 100% phosphorylated at the two serines, although it is already shifted). et al.,

5

6

7 8

9 10 11 12

Fig. 6. Phosphorylation and antibody reactions of the Asp mutant of htau23 (Serl99 and Ser2O2 changed into Asp): lanes 1 and 2, SDS gel

c

2

2 3 4

of htau23 before and after

extract

phosphorylation; lanes 3 and 4,

Asp-mutant before and after extract phosphorylation [note that the Asp-mutant runs slightly higher than htau 23 (lanes 1 and 3), but after phosphorylation both proteins have the same position in the gel (lanes 2 and 4)]; lanes 5-8, immunoblots of lanes 1-4 with AT8 [the antibody reacts only with the extract phosphorylated htau23 (lane 6), but neither with the unphosphorylated form (lane 5) nor with the Asp mutant (lanes 7 and 8), although it was phosphorylated as seen by the additional shift and autoradiography (not shown)]; lanes 9-12, immunoblots of lanes 1-4 with TAUl. This antibody reacts only with htau23 before phosphorylation (lane 9) but not with the phosphorylated form (lane 10) nor with the Asp mutant (lanes 11, 12). The aspartic acid apparently mimicks a phosphorylated serine and thus masks the epitope. The minor reaction of htau23 with TAUI in lane 10 shows that the protein is not completely phosphorylated.

T- 1 h-t PHF -

1 2

3

4 5C

AT- 8 h-t

7 8 9

PH F-t

1011 12

Fig. 7. Phosphorylation and antibody epitopes of tau from Alzheimer brains. Lanes 1-6, immunoblot with antibody TAUI; lanes 7-12, immunoblots with AT8. All tau isoforms from normal human brain react with antibody TAUI in the native state of phosphorylation (lane 1) after dephosphorylation (lane 2) but not after rephosphorylation with the kinase activity (lane 3). Antibody AT8 shows the opposite behaviour (lanes 7-9), i.e. it does not react with the native or dephosphorylated normal human tau (lanes 7 and 8), but does react after phosphorylation with the kinase activity (lane 9). Tau from PHFs (lane 4-6) reacts with TAUI only after dephosphorylation (lane 5) but not in the native state (lane 4) or after rephosphorylation (lane 6). Antibody AT8 reacts again in an opposite fashion (lanes 10-12); it recognizes 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 Alzheimer tau in the phosphorylated state, then TAUl should react with this 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, after dephosphorylation with alkaline phosphatase, and after rephosphorylation with the brain kinase activity (Figure 7, lanes 4-6). This treatment shifted the protein bands in the gel; the important point is, however, that AT8 recognizes the Alzheimer tau in its native state of phosphorylation, or after dephosphorylation and rephosphorylation with the kinase (Figure 7, lanes 10 and 12), whereas TAU 1 reacts only with the dephosphorylated form (Figure 7, lane 5). Normal human brain tau acquires Alzheimerlike properties upon phosphorylation with the brain kinase The corresponding result can be obtained with tau from normal human brain (Figure 7, lanes 1-3). This protein has a lower Mr than PHF tau although it is already in a mixed state of phosphorylation. The lack of reactivity with AT8 shows that it is not phosphorylated at residues Serl99 and/or Ser2O2 (Figure 7, lane 7). However, when this normal 1595

J.Biernat et al.

human brain tau is incubated with the kinase activity, the Mr 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 the

TAUI reactivity

upon phosphorylation (Figure 7, lane 3).

These experiments show that one can convert normal human brain tau into Alzheimer-like tau by phosphorylation, as judged by antibody staining and Mr shift.

Discussion In what way is PHF tau different from normal tau? We have studied 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 of tau and correlates with other indicators such as the Mr shift; (iii) protein engineering and site directed mutagenesis of defined tau variants that can be related to the PHF components; (iv) biochemical identification of the phosphorylation sites involved in the transition from the normal to the Alzheimer state of tau. In order to analyse an Alzheimer-like state of tau, one first needs a diagnostic tool, such as monoclonal antibodies. Two mAbs have been particularly useful, Alz5O and TAU1.

Alz5O was raised against Alzheimer brain homogenate.

Its

epitope is near the N-terminus of all tau isoforms (KsiezakReding et al., 1990; Goedert et al., 1991) and does not depend on phosphorylation. TAUI also recognizes all tau isoforms, but PHF tau reacts only after it is dephosphorylated, suggesting that it is abnormally phosphorylated (GrundkeIqbal et al., 1986). A second diagnostic tool is the Mr shift: PHF tau has a higher Mr than normal tau (GrundkeIqbal 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 certain kinases (such as CaMK), even when only a single phosphate is incorporated (Steiner et al., 1990). We consider AT8 superior to most other antibodies as a diagnostic tool: it recognizes all tau isoforms prepared from PHFs, but none of the isoforms from normal mammalian brain (human, porcine or bovine) in their mixed state of phosphorylation, nor any of the engineered tau constructs. However, all of these isoforms are recognized after phosphorylation with a kinase activity from brain. Thus the antibody is more specific for the Alzheimer state than Alz5O and it has the advantage of reporting on the state of phosphorylation. Using different tau constructs, protein sequencing and directed mutagenesis we found that the epitope includes the phosphorylated serines 199 and 202. At the same time we found that phosphorylation by the kinase activity also increased the Mr of all tau isoforms. The increase is larger than that we had found previously with CaMK so that the largest tau isoform, htau40, is shifted into a position indistinguishable from that of the Alzheimer protein A68 (see Figure 1). Part of this Mr shift can be mimicked by negative charges as in the Asp mutant of htau23 (Figure 6, lane 3). An unexpected by-product of this study was the localization of the phosphorylation-dependent epitope of TAU1 and its complementarity to AT8. Earlier studies (Kosik et at., 1988) had shown that the epitope was roughly in the region 189-207; we show that Serl99 and/orSer202 are the residues whose phosphorylation controls the binding of 1596

TAUl in a way opposite to AT8. Thus, PHF tau is recognized by AT8 but not TAUl, normal tau is recognized by TAU1 but not AT8 and normal tau phosphorylated at residues 199 and 202 behaves like PHF tau. One significant aspect of this work is that we have now found a kinase activity which is capable of transforming normal tau into an Alzheimer-like state. This kinase activity is present in mammalian brain extract. As shown in detail elsewhere, the kinase activity phosphorylates several residues in tau; this leads to the large M, shift which makes normal tau similar to PHF tau on SDS gels. Two of the phosphorylated residues are Ser199 and Ser202. We note that both of them are followed by a proline and none of them are in a consensus sequence typical of protein kinase A, protein kinase C, CaMK or casein kinase II. Thus, it is likely that the kinase belongs to the family of proline-directed kinases, members of which have recently been shown to have a variety of regulatory functions (reviewed in Kemp and Pearson, 1990). An example is the phosphorylation of neurofilament subunits by a neurofilament specific kinase which controls their assembly properties. In this case the major epitopes are of the form KSP (Geisler et al., 1987) and there are antibodies against the phosphorylated form of this epitope, some of which also cross-react with PHFs (Brion et al., 1991; Lee et al., 1991). The motif KSP occurs twice in the tau sequence (residues 234-236 and 395 -397); the second one is probably phosphorylated in PHF tau (Lee et al., 1991). Similarly, a tubulin-dependent kinase appears to phosphorylate a combination of SP and TP sites of tau (Ishiguro et al., 1991), although in this case the relationship to PHFs is not clear. In our study, we have found not only the phosphorylation site(s) but also the kinase activity that can be detected by an Alzheimer PHF tau specific antibody; this means that we can now mimic the transition from normal to Alzheimer-like tau in vitro.

Materials and methods Preparation of tau The preparation of tau from human, bovine, or porcine brain, dephosphorylation and rephosphorylation were done as described (Hagestedt et al., 1989). For PHF tau, human brain tissues from neuropathologically confirmed cases of Alzheimer's disease were obtained from the Born-Bunge Foundation, University of Antwerpen (Belgium). The autopsies were performed between 4.5 and 20 h post mortem. The brain tissue was kept frozen at -70°C. PHF tau was prepared according to Greenberg and Davies (1990). The monoclonal antibody TAUl was a generous gift from Dr L.Binder. Antibody AT8 against Alzheimer PHF-tau was prepared as described (Mercken et al., 1992). SDS-PAGE was done with a gradient of 4-20%. Immunoblotting was done on Immobilon membranes (Millipore). The bound antibody was detected by a peroxidase conjugated second antibody (anti-mouse IgG, Dakopatts).

Phosphorylation by brain kinase activity Extract from porcine brain was prepared by homogenizing the brain in 10 mM Tris-HCI, pH 7.2, 5 mM EGTA, 2 mM DTT and a cocktail of protease inhibitors (leupeptin, aprotinin, pepstatin A,a-macroglobulin and at The supernatant PMSF) and centrifuged at100 000 g for 30 min 4°C. was either precipitated with 40% ammonium sulfate and the pellet redissolved in 10 mM Tris-HCI, 2 mM each EGTA, DTT and MgSO4, pH 7.2, or it was used directly for the phosphorylation experiments after addition of 2 mM ATP and zM 10 okadaic acid. The phosphorylation was done by adding1 Al of extract to 40Ml of tau protein solution (0.5-1 mg/ml) at37°C.

Sequencing of phosphorylated peptides The procedures were essentially as described (Steiner et al., 1990). The protein was phosphorylated as described above and unbound nucleotide was removed by passage over a NAP-S gel filtration column (Pharmacia). The protein 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, TPCK treated) four times in 6 h intervals (this procedure was used to optimize digestion conditions). The digest was lyophilized once more and resuspended in 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 phase column (Vydac, 4.6 x 250 mm, flow rate1 ml/min at room temperature). Crude fractions were obtained by a linear gradient of 0-40% acetonitrile in 10 mM ammonium acetate. Radioactivity was determined by a scintillation counter (Hewlett Packard TriCarb 1900CA). The radioactive peaks were reapplied to the same column and eluted with a linear gradient of 0-40% acetonitrile in 0.1 % trifluoroacetic acid. The sequence analysis of the peptides was performed using a 477A pulsed liquid phase protein/ peptide sequencer and a 120A on-line PTH amino acid analyser (Applied Biosystems). Phosphoserines were identified by gas phase sequencing, making use of the formation of the dithiothreitol adduct of dehydroalanine from serine phosphate (Meyer et al., 1990).

Lee,G., Cowan,N. and Kirschner,M. (1988) Science, 239, 285-288.

Lee,V.M.Y., Balin,B.J., Otvos,L. and Trojanowski,J.Q. (1991) Science, 251, 675-678. Mercken,M., Vandermeeren,M., Lubke,U., Six,J., Boons,J., Vanmechelen,E., Van de Voorde,A. and Gheuens,J. (1992) J. Neurochem., in press. Meyer,H.E., Hoffmann-Posorske,E. and Heilmeyer,L.M.G. (1990) Methods Enzymol, 201, 169-185. Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual, second edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Steiner,B. et al. (1990) EMBO J., 9, 3539-3544. Studier,W.F., Rosenberg,A.H., Dunn,J.J. and Dubendorff,J.W. (1990) Methods Enzymol., 185, 60-89. Received on December 12, 1991; revised on January 21, 1992

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

Acknowledgements We thank A.Malchert and U.Boning for excellent technical assistance in protein expression and preparation and U.Boning for photography. We are grateful to L.Binder (University of Alabama) for a gift of TAU I antibody, W.Studier (Brookhaven National Laboratory) for the expression vector, and N.Gustke for construct K3M. This project was supported by the Bundesministerium fur Forschung und Technologie and the Deutsche

Forschungsgemeinschaft.

References Binder,L.I., Frankfurter,A. and Rebhun,L. (1985) J. Cell Biol., 101, 1371-1378. Brion,J., Hanger,D., Bruce,M., Couck,A., Flament-Durand,J. and Anderton,B. (1991) Biochem. J., 273, 127-133. Flament,S. and Delacourte,A. (1989) FEBS Lett., 247, 213-216. Geisler,N., Vendekerckhove,J. and Weber,K. (1987) FEBS Lett., 221, 403-407. Goedert,M., Wischik,C., Crowther,R., Walker,J. and Klug,A. (1988) Proc. Natl. Acad. Sci. USA, 85, 4051-4055. Goedert,M., Spillantini,M., Jakes,R., Rutherford,D. and Crowther,R.A. (1989) Neuron, 3, 519-526. Goedert,M., Spillantini,M.G. and Jakes,R. (1991) Neurosci. Lett., 126, 149-154. Greenberg,S.G. and Davies,P. (1990) Proc. Natl. Acad. Sci. USA, 87, 5827 -5831. Grundke-Iqbal,I., Iqbal,K., Tung,Y., Quinlan,M., Wisniewski,H. and Binder,L. (1986) Proc. Natl. Acad. Sci. USA, 83, 4913-4917. Hagestedt,T., Lichtenberg,B., Wille,H., Mandelkow,E.-M. and Mandelkow,E. (1989) J. Cell Biol., 109, 1643-1651. Himmler,A., Drechsel,D., Kirschner,M. and Martin,D. (1989) Mol. Cell. Biol., 9, 1381-1388. Ishiguro,K., Omori,A., Sato,K., Tomizawa,K., Imahori,K. and Uchida,T. (1991) Neurosci. Lett., 128, 195-198. Kemp,B.E. and Pearson,R.B. (1990) Trends Biochem. Sci., 15, 342-346. Kosik,K., Orecchio,L., Binder,L., Trojanowski,J., Lee,V. and Lee,G. (1988) Neuron, 1, 817-825. Ksiezak-Reding,H., Davies,P. and Yen,S.-H. (1988) J. Biol. Chem., 263, 7943-7947. Ksiezak-Reding,H., Chien,C.H., Lee,V.M.Y. and Yen,S.H. (1990) J. Neurosci. Res., 25, 412-419.

1597