Proc. Nati. Acad. Sci. USAVol. 89, pp. 5384-5388, June 1992Biochemistry

Phosphorylation-dependent epitopes of neurofilament antibodies ontau protein and relationship with Alzheimer tau

(microtubule-associated proteins/Alzheimer paired helical filaments/protein engineering/Ser-pro-directed protein kns)

B. LICHTENBERG-KRAAG*, E.-M. MANDELKOW*, J. BIERNAT*, B. STEINER*, C. SCHROTER*, N. GUSTKE*,H. E. MEYERt, AND E. MANDELKOW***Max-Planck-Unit for Structural Molecular Biology, c/o DESY, Notkestrasse 85, D-2000 Hamburg 52, Federal Republic of Germany; and tInstitute forPhysiological Chemistry, Ruhr-Universitat, Universitatsstrasse 150, D-4630 Bochum, Federal Republic of Germany

Communicated by Hans J. Muller-Eberhard, January 29, 1992 (receivedfor review November 7, 1991)

ABSTRACT We have studied the phosphorylation of tauprotein from Alzheimer paired helical filaments, of tau fromnormal human brain, and of recombinant tau isoforms. As atool we used monoclonal antibodies against neurofiamentprotein [Sternberger, N., Sternberger, L. & Ulrich, J. (1985)Proc. Nad. Acad. Sci. USA 82, 4274-4276] that crossreact withtau in a phosphorylation-dependent manner. This allowed us todeduce the state of phosphorylation in normal and pathologicaltau, as well as antibody epitopes. The epitope of antibodySM133 is at the first Lys-Ser-Pro sequence motif (residues234-236) and requires an unphosphorylated Ser-235. AntibodySMI31 binds between Ser-396 (in the second Lys-Ser-Pro motif)and Ser-404, both of which must be phosphorylated. SM134has a conformational epitope that depends on the interactionbetween regions on either side of the microtubule-bindingregion; it also requires phosphorylation. The phosphorylatableserines detected by the SMI antibodies are part of Ser-Promotifs and can be phosphorylated by a protein kinase activitythat can be used to induce a paired helical filament-like state inhuman brain tau in vitro. The phosphates are incorporated inseveral stages that can be identified by antibody reactivity andgel shift. This suggests a role for the phosphorylation sites inAlzheimer disease, as well as the involvement of a Ser-Pro-directed protein kinase.

Alzheimer disease brains contain paired helical filaments(PHFs) whose nature has been a matter of debate (reviewedin refs. 1 and 2). Two main possibilities were considered,neurofilament protein and tau protein, based on antibodyreactivities. Sternberger et al. (3, 4) generated neurofilamentantibodies that crossreacted with PHFs and distinguisheddifferent states of phosphorylation. This was used to showthat neurofilaments were phosphorylated (4-7), in agreementwith other antibody work (e.g., refs. 8 and 9), and suggestedthat PHFs consisted of phosphorylated neurofilaments.However, antibodies against tau protein also reacted withPHFs in a phosphorylation-dependent manner, suggestingthat the PHFs consisted of tau protein in an abnormal stateof phosphorylation (10-13).

Later it was realized that neurofilament antibodies maycrossreact with tau (12, 14, 15). The major phosphorylationsites of neurofilament heavy chain occurred in Lys-Ser-Pro-Val (KSPV) motifs (9, 15), one of which exists in tau as well.Antibodies against a phosphorylated synthetic peptide re-acted with A68 protein from Alzheimer tangles (16). Thisconfirmed that A68 was a variant of tau and that PHFsconsisted of tau, in agreement with results of molecularcloning showing that normal and PHF tau occurred in severalisoforms arising from alternative splicing (17-22).

The data suggested that the neurofilament antibodies ofSternberger et al. (3) might be suitable for analyzing thephosphorylation of tau. In this study we have monitored thephosphorylation of tau in three ways: (i) sequencing ofphosphopeptides; (ii) blotting with phosphorylation-dependent antibodies, which yields information about abnor-mal states; and (iii) SDS/PAGE mobility shift induced bycertain types ofphosphorylation (23, 24), which provided oneof the early clues that PHF tau was abnormally phosphory-lated (11). These approaches were used to study how PHF taudiffers from normal or recombinant tau.

MATERIALS AND METHODSThe preparation of tau from normal human or bovine brainwas done as described (24). PHF tau was prepared accordingto ref. 18. Alzheimer brain tissue was obtained from theKathleen Price Bryan Brain Bank (Duke University). Phos-phorylation of tau was done with porcine brain extract. Theextract was prepared by homogenization in 10 mM Tris HCl,pH 7.2/5 mM EGTA/2 mM MgCl2/2 mM dithiothreitol/protease inhibitors (leupeptin, aprotinin, pepstatin A, a2-macroglobulin, phenylmethylsulfonyl fluoride) and centrifu-gation at 100,000 X g for 30 min at 40C. The supernatant wasused after addition of2mM ATP and 10 ,uM okadaic acid. Forphosphorylation, 1 ul of extract was added to 40 tkl of tauprotein solution (0.5-1 mg/ml) at 370C.Human tau cDNA clones were kindly provided by M.

Goedert (19, 20). The cDNA inserts were subcloned into theexpression vector pNG2, a derivative of pET-3b (25). Re-combinant plasmids were used to transform Escherichia coliBL21(DE3) cells (25). The protein was isolated on the basisof its heat stability and by Mono S (Pharmacia) FPLC (24).PCR amplifications were carried out with Taq polymerase(Perkin-Elmer/Cetus). Residue numbering in this paper re-fers to the sequence of htau40 (20).Monoclonal antibodies SMI31 (IgG), SM133 (IgM), SM134

(IgG), SM135 (IgG), and SMI310 (IgG) were obtained fromSternberger Monoclonals (Baltimore) and used for blottingaccording to the manufacturer's instructions (dilution, 1:300to 1:1000). After SDS/PAGE (gradient of 4-20%o or 7-15%acrylamide), proteins were transferred to Immobilon mem-branes (Millipore) for immunoblot analysis.

RESULTSNeurofilament Antibodies SM133 and SML34/SMI31 Rec-

ogize PHF tau in a Complementary Fashion. To analyzeAlzheimer-specific abnormalities we have to combine anagent causing the abnormality (a kinase activity), a substrate(tau protein), and a detector (monoclonal antibodies, gel

Abbreviation: PHF, paired helical filament.lTo whom reprint requests should be addressed.

5384

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 89 (1992) 5385

shift, etc.). Detectors used in this study were the neurofila-ment antibodies SMI31, SMI34, SMI35, or SMI310 (withphosphorylated epitopes) and SM133 [unphosphorylatedepitopes (3)]. The first step was to ascertain the antibodyreactivities with tau from different sources. Coomassie bluestaining of normal human tau in the "native" state ofphosphorylation (as isolated) shows several bands becausethere are several isoforms and phosphorylation states (Fig.la, lane 1). The bands of PHF tau are higher than those ofnormal tau, as if PHF tau were more highly phosphorylated(Fig. la, lanes 1 and 3).SMI33 recognizes normal human brain tau (Fig. lb, lane 1)

but not PHF tau, unless it is dephosphorylated (lane 4). Thissuggests that the epitope of SM133 is blocked by somephosphorylation in the PHF-like state. Both SMI31 andSM134 are complementary to SM133, recognizing tau in thePHF-like state (Fig. 1 c and d, lanes 3), but not when it isdephosphorylated (lanes 4), and not the normal tau control(lanes 1).

Alzheimer-Like Phosphorylation of tau Can Be Induced inVito. In contrast to the kinases we tested previously (27), thekinase activity used here can phosphorylate normal brain tau(bovine, porcine, or human) or recombinant tau so that itacquires PHF-like immunoreactivities (Fig. 1). The phos-phorylation can be seen by the shift in apparent size (Fig. 2a)and by autoradiography (Fig. 2 b). The gel shift reveals threemain stages of phosphorylation (Fig. 2a): Starting from theunphosphorylated protein (58 kDa for the recombinanthtau34), the first main stage is completed around 2-3 hr, whenmost of the protein is shifted up to 62 kDa. The second stageis reached after about 10 hr (64 kDa), and the third after about24 hr (66 kDa). The stages are well separated in time,suggesting that the phosphorylation proceeds sequentially(Fig. 2c). There are 1.5-2 Pi incorporated per stage, so thatafter 24 hr there are 5-6 Pi incorporated. Similar time coursesare obtained with other tau isoforms (data not shown).Next we asked how the antibodies react with different

phosphorylation states. SM133, whose epitope is masked bya PHF-specific phosphorylation (Fig. lb, lane 3), reacts withthe unphosphorylated protein (Fig. 3d, time 0); the reactivityremains through the first stage but is lost during the secondstage. Thus, the third or fourth Pi incorporated destroys theepitope of SMI33. Antibody SM131 has the opposite behavior(Fig. 3b, and compare Fig. 1c, lane 3): it requires a phos-

ht PHF-t SM 131

a- + + -

.I }t

- + +-_c

1 2 3 4 1 2 34

0 30t 6090' 2h 3h 6h 8h10h14h16h 20h24a PAGE

b AR

1 2 3 4 5 6 7 8 9 10 11 1213

c

0 5 10 1 I

time [h]

FIG. 2. Time course (0-24 hr) of phosphorylation of recombinanthuman tau (htau34). (a) SDS/PAGE (Coomassie blue stain) showingan increase of apparent molecular mass in three main stages: stage1, 2-3 hr; stage 2, 10 hr; and stage 3, 24 hr. (b) Autoradiogram (AR)of a. (c) Time course of intensity of bands in a. *, unshifted protein(0); o, stage 1; o, stage 2; *, stage 3.

phorylated epitope that is specific for PHFs. No reactivity isobserved up to the first stage, but the reactivity appearsduring the second stage and remains throughout the third.Similar time courses are found with antibodies SM134,SM135, and SMI310 (Fig. 3 c, g, and h). For comparison we

0'3076090* 2 h 3 h 6h 8n10h 14h 16h18h20h24h

PAGE a VW W*.ht23 -0 -"a

SM131 b

SM 134 C

-w._wwwW"

Uwwwwww"

SM 133 W 4 = la

eT- 1 ff1* s s0 .,

SM 1 33 SM1 34

b d

1 2 3 4 1 2 3 4

FIG. 1. SDS/PAGE and immunoblots of normal human (ht) tauand PHF tau (PHF-t). (a) Lane 1, SDS/PAGE of human brain tau,five to six bands of 55-65 kDa; lane 2, after phosphorylation, allbands shifted up; lane 3, blot of PHF tau with antibody 5E2, whichrecognizes tau independently of phosphorylation (26); lane 4, PHFtau after dephosphorylation (alkaline phosphatase), bands shifteddown. (b) Blot of a with SM133, which recognizes normal human tau(lane 1), and PHF tau after dephosphorylation (lane 4). (c and d)SMI31 and SM134 recognize normal human tau after phosphorylation(lanes 2) and PHF tau (lanes 3).

SM135ht34

t 8. _ t -

1 2 3 4 5 6 7 8 9 1011 12 1314

9

SM1310

0 30 6090 2h 3h 6h 8h10h 14h 16h 20h24h

FIG. 3. Time course of phosphorylation and immunoblots. (a)SDS/PAGE of recombinant htau23. (b-f) Blots of htau23 withSMI31, SM134, SM133, TAU1, and AT8; (g and h) blots of htau34with SM135 and SMI310.

AT-8

Biochemistry: Lichtenberg-Kraag et al.

W. .44

5386 Biochemistry: Lichtenberg-Kraag et al.

also include the blots with AT8 (Fig. 3f), an Alzheantibody (28), and TAU1, an antibody against dellated tau (10); both have epitopes in the region(29). The time course of the AT8 reaction resemlSMI31, SM134, SMI3S, and SM1310, while TAUto SM133, even though the epitopes differ (see bstriking feature is that in each case it is the staphorylation that determines the antibody responsthere is a precise relationship between gel shift, llation, and immunoreactivity.Antibody Epitopes. The major phosphorylati(

neurofilaments are motifs of the type KSP, whephosphate acceptor (8, 15). tau has two such moS235 and S396. They lie on either side ofthe repeatare conserved in all tau isoforms. By analog)suspect that these sites are involved in the reacti4SMI neurofilament antibodies. We tested this byof tryptic peptides, by point mutation of the serinmaking truncated tau constructs (Fig. 4).We first consider constructs K10, K17, and K1

response to phosphorylation (Fig. 5a). K10 and Igel shift, but not K19. K10 shows three shifindicating three phosphorylation sites in the C-tigion. K17 shows only one shifted band, so that tIone shift-inducing site in the region before the re5 b-d show the blots with SM133, SMI31, and SMIon SM135 and SM1310 (not shown) are similar tSMI31. Antibody SM133 reacts only with K17 in 1phorylated state but not with K10 and K19 (Fig.This suggests that the epitope is in a region before 1between S198 and Q244, outside the sequences 4the other constructs, and compatible with an epifirst KSP site. Antibody SMI31 reacts with phosK10 but not K17 or K19 (Fig. Sd), indicating thatis in the region T373-L441, compatible with the si

CN K44 A103|- -J LI

Q244 E372

I 1 1.4A->s

(M) S198

P47

P154 R221

L344K44 A103 _______ A426

D387L....4*.'I I Fe **Le,,

Q244 V30-,- ~~~K~f4 V337

V337Q244

LJ I . zA .... ....

V306 L3440244 gw ..n H388

L344 H388

. ---. .

imer tanglephosphory-S199-S202bles that of1 is similarelow). Thege 2 phos-;e, and thatphosphory-

Dn sites ofre S is theitifs aroundt region andV one mayon with thesequencingies, and by

SMI--34

Pi +

b

+ I

SMl I--33 d SMI--31

..1XrL t A 4 -:)

FIG. 5. (a) SDS/PAGE of tau constructs K10 (lanes 1 and 2), K17(lanes 3 and 4), and K19 (lanes 5 and 6) before and after (+)phosphorylation. All constructs except K19 show a shift uponphosphorylation. (b) Blot of a with SM133, which recognizes onlyK17 in the unphosphorylated form (lane 3). (c) SM134 recognizes K10and K17 in the phosphorylated form (only top bands, lanes 2 and 4),but not K19 (lanes 5 and 6). (d) SMI31 recognizes only the top bandof the phosphorylated K10 (lane 2).

L9 and their site. Finally, antibody SM134 labels K10 and K17 in their(17 show a phosphorylated form but not K19 (Fig. 5c). The lack of K19ted bands reactivity argues that the epitope is not in the repeat region.erminal re: The reactivity of K10 and K17 argues that the epitope is bothhere is only before and after the repeats, which seems mutually exclusive.-peats. Fig. Our interpretation is that SMI34 has a conformational epitope34; the data that depends on phosphorylated tails on either side of theto those on repeats, and that at least one tail must be present.the dephos- To find the phosphorylation sites directly, tryptic peptides5b, lane 3). of htau34 were identified by HPLC and protein sequencing,the repeats, and phosphorylated residues were determined (30). There arecovered by two major phosphorylated tryptic peptides in these regions:itope at the peptide 1 (T231-K240, TPPKS(pPSSAK) contains the firstiphorylated KSP motif, phosphorylated at S235; peptide 2 (T386-R406,the epitope TDHGAEIVYKS(p)PVVSGDTS(p)PR) contains the secondecond KSP KSP site, phosphorylated at S3% and S404. We then made

point mutants of the phosphorylatable residues 235 and 3%htau23 (Fig. 6) and analyzed them by gel shift and antibody reactivity

(Fig. 7). The parent protein htau40 and its KAP mutants haveK19 nearly identical apparent molecular masses, and they all shift

by the same amount after phosphorylation (Fig. 7a, lanesK1O 1-8). The reactivity of SM133 is strongly reduced when S235

is mutated to alanine (Fig. 7b, lanes 3 and 7) and obliteratedK17 after phosphorylation (Fig. 7b, lanes 2, 4, 6, and 8). This

K2 means that the epitope of SM133 is around the first KSP site,K2 but phosphorylation at other sites has an influence as well

(perhaps via a conformational change). The mutation at S3%K3M (second KSP site) has no influence on the SMI33 staining

(Fig. 7b, lane 5 and 6).K4

K5

K6K7

... I L 1;I

... .. r. L;.- i.

IAK13

K14:. .?.. .v .-:-..I--

K15

FIG. 4. Constructs of tau. Residue numbering is that of htau40[not shown (see ref. 20); 441 residues (res.) four C-terminal repeats,Q244-E372, and two N-terminal inserts, E45-T102]; htau23 (352res.) has three repeats and no inserts. K19 (99 res.) has only threerepeats. K10 (168 res.), K17 (145 res.), K2 (204 res.), and K3M (335res.) have leaders or tails as shown. K4 (270 res.), K5 (310 res.), K6(322 res.), and K7 (321 res.) have two repeats. K13 (291 res.), K14(279 res.), and K15 (278 res.) have one repeat.

i. ..i

.JLA

FIG. 6. Point mutants of htau40, htau34, and htau23, with Ser-235, -396, and/or -404 replaced by alanine.

Proc. NatL Acad Sci. USA 89 (1992)

. . E 1FA.

Proc. NatL. Acad. Sci. USA 89 (1992) 5387

ht40 235 396 2 35196c

rI

bSM 1-33

SMI1-31

SMI-34

_s M-Ow0.. w

d.q.w

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

FIG. 7. SDS/PAGE and immunoblots of htau4O and point mu-tants of Fig. 6. (a) Lanes 1-8, SDS gel ofhtau4O and its mutants A235,A396, and A235/396 in the unphosphorylated and phosphorylated(+, note shift) forms. (b) Blot of a with SM133. The antibodyresponse is strongly reduced when S235 is mutated, both in thedephosphorylated and phosphorylated state (lanes 3 and 4, 7 and 8).When S396 is mutated to alanine the behavior is similar to that of theparent molecule (lanes 1 and 2, 5 and 6). (c and d) SMI31 and SMI34recognize htau4O and all mutants in the phosphorylated form (lanes2, 4, 6, and 8).

As mentioned above, the epitope of SMI31 depends on thephosphorylation of sites behind the repeat region. When S3%is mutated to alanine the antibody still reacts in a phos-phorylation-dependent manner, indicating that this serine isnot responsible for the epitope by itself (Fig. 7c, lane 6).Mutating S404 to alanine yields the same result. However, ifboth serines are mutated, the antibody no longer reacts uponphosphorylation (data not shown), showing that the epitopeincludes these two phosphorylated serines. SMI31 also has aconformational component: Constructs with only one repeat(K13-K15) are not recognized (data not shown).SM134 shows the most complex behavior because it de-

pends on phosphorylation sites before and after the repeatregion. The antibody recognizes all KAP mutants, so thatS235 and S396 cannot play a major role. However, SM134recognizes phosphorylated K17 and K10 but not K19 (Fig.Sc), suggesting that the regions before or after the repeatsmust cooperate with the repeats to generate the epitope. Onepossibility is that the epitope is noncontiguous; another isthat it depends on the number and conformation of therepeats. To check these possibilities we made constructs withdifferent combinations of two repeats (K5, K6, K7) andconstructs with one repeat only (K13, K14, K15; Fig. 4). Allof these showed a shift upon phosphorylation, and all ofthemwere recognized by SM134. This means that the epitope ofSMI34 does not depend on the number of repeats. However,the nature of the region just before the repeats seems to beimportant for the conformation and is sensitive to charges.This can be deduced from constructs such as K2 or K3M,where charged sequences have been brought close to therepeat region, resulting in a loss of SM134 reactivity. Appar-ently the charged sequences can mask the epitope indepen-dently of the phosphorylation itself (data not shown).

DISCUSSIONWe have investigated the interaction between the SMI neu-rofilament antibodies and tau protein in different states ofphosphorylation (cf. refs. 3, 4, and 31). We have shown thatthey react with tau from normal human brain, recombinanthuman tau, and pathological tau from Alzheimer PHFs andthat they are sensitive to the state of tau phosphorylation.The dependence is the same as that of neurofilament protein;e.g., SMI33 recognizes a dephosphorylated epitope in bothproteins, and SMI31 and SM134 recognize phosphorylatedones. A similar complementarity was recently found for theantibodies TAU1 and AT8, which react with normal or

pathological tau in a phosphorylation-dependent manner(29).A combination of peptide sequencing, protein engineering,

and immunoblotting was used to localize the epitopes. Theepitope of SM133 is at the first KSP motif and requires anunphosphorylated S235 (Fig. 8a). SM133 recognizes normalhuman tau, indicating that S235 is normally not phosphory-lated, but does not react with PHF tau, except when it isdephosphorylated (Fig. lb, lane 3 and 4). This means thatphosphorylation of S235 is a factor contributing to thepathological state.SMI31 is an antibody against a major phosphorylated

epitope in neurofilament heavy chain. Since the main sites inthe heavy chain are the repeated four-residue motifs KSPV(9, 15), and since the four-residue motif KSPV occurs oncein tau, one would expect that S396 is crucial for the antibodyreaction. However, the point mutation of S396 to alaninedoes not eliminate the reactivity, suggesting that phosphor-ylation sites other than S396 contribute to the epitope. Thesame is true for S404; but when both S396 and S404 arechanged to alanine the reactivity of SMI31 is lost. Thecombination of these two serines is therefore part of theSMI31 epitope and phosphorylated in PHF tau, consistentwith studies based on tau peptides (16). The epitope appearsto have a conformational aspect, because mutants containingonly one microtubule-binding repeat are not recognized(K13-K15).The search for the SMI34 epitope leads to an apparent

contradiction. Elimination of the region before or after therepeats (as in K10 or K17) leaves the epitope intact, suggest-ing an epitope in the repeat region (Fig. 5). But this region (inthe form of K19) shows no reaction either. We hypothesizethat SMI34 is a conformational antibody whose epitope isformed by two or more noncontiguous parts of the chain,possibly in or near the repeat region (Fig. 8a). The "kinked"conformation is conducive to SM134 binding; it requires (i)phosphorylation, (ii) the presence of a tail before or after therepeats, or both (Fig. 8 b, d, and e). Without these conditions,the conformation becomes nonreactive with SM134 (Fig. 8 c

a1- ()- SM133/I

lS235

SM3 C MCSM134 's_ SM131

bc'Iz-

d

- N

Tau (+P)

- N

Tau (-P)

K17 (+P)

N

e<3 K10 (+P)

f <sK19 (+/- P)

9 000N

K2 (+/- P)<c

FIG. 8. (a) Antibody epitopes on tau (dashed ellipses). The repeatregion is represented as a kinked line (with three repeats). The SM133site is probably a contiguous one and includes the first KSP motif,with S235 in the unphosphorylated state. The SMI31 site is aroundresidues S3% and S404, both phosphorylated. The site of SM134requires phosphorylation and is probably near the repeat region butotherwise is not well defined. (b-g) Diagrams giving a possibleexplanation of the reactivity of SM134 in terms of chain conforma-tion. The repeat region is represented either in a kinked shape, whichallows the reaction with SM134, or in a round shape, which preventsthe interaction. P, phosphorylation.

Biochemistry: Lichtenberg-Kraag et al.

5388 Biochemistry: Lichtenberg-Kraag et al.

and f). One way of stabilizing the bonding conformationwould be by an interaction between different parts of thechain in an antiparallel fashion. The interaction could bewithin one molecule, as suggested in Fig. 8, or between twomolecules (in the form of antiparallel dimers). Evidence forboth types of structure has been obtained (33).There are special constructs whose reactivities might shed

light on the three-dimensional structure of tau. One exampleis K3M, which lacks residues 155-220, bringing the N-ter-minal 154 residues closer to the repeat region (Fig. 4). Thisnearly eliminates the reactions with all three antibodies,independently of phosphorylation at the SP motifs. A similarresult was obtained with construct K2 (Fig. 8g). The newregions bordering on the repeats have pronounced charges,suggesting that a charged neighborhood upstream of therepeats might cause a rearrangement of the whole structureand that the regions before and after the repeats mightinteract in a phosphorylation-dependent manner.

tau can be phosphorylated by several kinases. Some in-crease the apparent molecular size; the labeling of S416 byCa2+/calmodulin-dependent kinase II is a case in point (27).The mobility shift led to the idea that PHF tau is abnormallyphosphorylated (11). Evidence from antibodies against phos-phorylated peptides pointed to an abnormal site at S396 (16).In addition, a tubulin-dependent kinase was reported tophosphorylate residues S202, T205, S235, and S404 (32), anda neurofilament-dependent kinase was shown to phosphor-ylate tau, resulting in a gel shift (6). Here we show that akinase activity in brain can turn normal tau into a PHF-likestate that can be monitored by neurofilament antibodies. Thetransformation takes place in several stages, and the stagethat induces the largest gel shift precedes the stage where thePHF-like immunoreactivity sets in. Finally, we have identi-fied the antibody epitopes and several phosphorylation sitesinvolved in the transformation of tau. They are part ofSer-Pro motifs, implying that the kinase activity belongs tothe class of Ser-Pro-directed kinases.

Note Added in Proof. We have recently found that the Alzheimer-likephosphorylation of tau is induced by mitogen-activated proteinkinase, MAPK (34).

Thanks for excellent technical assistance are due to A. Malchertand U. Boning (cloning), S. Wahlandt (immunoblotting), H. Korte(peptide sequencing), and J. Muller (photography). Clones of humantau isoforms were kindly made available by M. Goedert (MedicalResearch Council, Laboratory of Molecular Biology, Cambridge,U.K.), Alzheimer brain tissue by K. Dole and B. Crain (KathleenPrice Bryan Brain Bank, Duke University), TAU1 antibody by L.Binder (University ofAlabama), 5E2 antibody by K. Kosik (HarvardMedical School), AT8 antibody by M. Mercken (Innogenetics S.A.,Ghent, Belgium), and expression vector pET-3b by W. F. Studier(Brookhaven National Laboratory). This project was supported bythe Bundesministerium ffir Forschung und Technologie and theDeutsche Forschungsgemeinschaft.

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Proc. Natl. Acad. Sci. USA 89 (1992)