microtubule-associated protein tau is phosphorylated by protein

T H E JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 267, No. 22, Issue of August 5, pp. 15721-15728,1992 (c) 1992 hy The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Microtubule-associated Protein Tau Is Phosphorylated by Protein Kinase C on Its Tubulin Binding Domain*

(Received for publication, October 31, 1991)

Isabel CorreasS, Javier Diaz-Nido, and Jesus Avila From the Centro de Biologia Molecular, Facultad de Ciencias, Uniuersidad Autonoma, 28049 Madrid, Spain

We have analyzed the in vitro phosphorylation of tau protein by Ca2+/calmodulin-dependent protein kinase, protein kinase C, CAMP-dependent protein kinase, casein kinase 11, and proline-directed serinelthreonine protein kinase. These kinases phosphorylate tau protein in sites localized in different regions of the molecule, as determined by peptide mapping analyses. Fo- cusing on the phosphorylation of tau by protein kinase C, it was calculated as an incorporation of 4 mol of phosphate/mol of tau. Limited proteolysis assays suggest that the phosphorylation sites could be located within the tubulin-binding domain. Direct phosphorylation of synthetic peptides corresponding to the cysteine-containing tubulin-binding region present in both fetal and adult tau isoforms demonstrates that serine 313 is modified by protein kinase C. Phos- phorylation of the synthetic peptide by protein kinase C diminishes its binding to tubulin, as compared with the unphosphorylated peptide.

Tau is a group of closely related brain microtubule-associated proteins with apparent molecular masses ranging from 55,000 to 62,000 (Cleveland et al., 1977), which arise from alternatively spliced transcripts (Goedert et al., 1988; Hi- mmler et al., 1989; Kosik et al., 1989; Lee et al., 1988) origi- nating from a single gene (Drubin et al., 1984; Himmler, 1989). The knowledge of the primary structure of tau isoforms, as identified from molecular cloning, has revealed that tau is a tripartite molecule composed of a highly conserved carboxyl- terminal domain with three or four repeated sequences which represent tubulin-binding motifs, a constant middle domain, and a variable amino-terminal domain with still unknown functions (Himmler et al., 1989). One putative in vivo role of tau is the stabilization of assembled microtubules (Drubin and Kirschner, 1986; Kanai et al., 1989), which may be achieved by interactions between the carboxyl-terminal regions of both tubulin (Maccioni et al., 1988; Serrano et al., 1985) and tau molecules (Himmler et al., 1989; Lee et al., 1989). Thus, in the normal nerve cell, tau is mainly associated with axonal microtubules (Binder et al., 1986), although the presence of tau within the somatodendritic compartment has also been reported (Papasozomenos and Binder, 1987). Recent evidence indicates that tau is also present in the somatodendritic compartment of certain degenerating nerve cells as a major component of the paired helical filaments, aberrant

* This work was supported by the Spanish Comisi6n Interminis- terial de Ciencia y Tecnologia. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ T o whom correspondence should be addressed. Fax: 341-397- 4799.

structures characteristic of the brains of patients with Alz- heimer’s disease or senile dementia of Alzheimer’s type (Goe- dert et al., 1988; Grundke-Iqbal et al., 1986b; Kosik et al., 1986; Nieto et al., 1988; Nukina and Ihara, 1986; Wischik et al., 1988; Wood et al., 1986).

The mechanisms leading to paired helical filament formation in Alzheimer’s disease remain unknown. However, several facts indicate that abnormal phosphorylation of tau protein might be a crucial step which precedes neurofibrillary tangle formation (Bancher et al., 1989; Flament et al., 1990). On the other hand, the in vitro phosphorylation of tau protein by different protein kinases reduces its ability to induce microtubule polymerization (Lindwall and Cole, 1984; Ya- mamoto et al, 1983). It has therefore been suggested that phosphorylation of tau may modulate its sorting between the somatodendritic and the axonal compartments of nerve cells (Kosik, 1990; Papasozomenos and Binder, 1987). Thus, a detailed knowledge of the main features of in vitro tau phosphorylation will help understand the role of tau phosphorylation in vivo under normal and pathological conditions.

Several in vitro studies on tau phosphorylation have indicated that Ca2+/calmodulin-dependent protein kinase (CaMK)’ (Baudier and Cole, 1987; Schulman, 1984; Yama- mot0 et al., 1983), Ca”/phospholipid-dependent protein kinase (PKC) (Baudier et al., 1987; Hoshi et al., 1987), cyclic AMP-dependent protein kinase (PKA) (Pierre and Nunez, 1983; Yamamoto et al., 1985), casein kinase type I (CKI) (Pierre and Nunez, 1983), casein kinase type I1 (CKII) (Cor- reas et al., 1991; Diaz-Nido et al., 1987), and an unidentified protein kinase (Ishiguro et al., 1988) all act on tau protein.

The identification of the CaMK phosphorylation site at the carboxyl-terminal end of the tau molecule has recently been reported (Steiner et al., 1990). It has been shown that this type of phosphorylation structurally modifies tau protein (Lichtenberg et al., 1988) and it has been hypothesized that the conformational change induced may influence the tubulin- binding domain on the tau molecule, thus reducing its affinity for microtubules (Steiner et al., 1990). No other phosphorylation sites have been located at present.

Here we report that the tubulin-binding domain of the tau protein can be phosphorylated by PKC at Ser-313, a serine residue which is conserved in all the repetitive tubulin-binding motifs. This might constitute a mechanism for weakening the

’ The abbreviations used are: CaMK, Ca2+/calmodulin-dependent protein kinase; PKC, Caz+/phospholipid-dependent protein kinase; PKA, cyclic AMP-dependent protein kinase; CKI and CKII, casein kinase types I and 11, respectively; PDPK, proline-directed serine/ threonine protein kinase; MES, 4-morpholineethanesulfonic acid; EGTA, [ethylenebis(oxyethylenenitrilo]tetraacetic acid PMSF, phenylmethylsulfonyl fluoride; MAP, microtubule-associatedprotein; SDS, sodium dodecyl sulfate; NTCB, 2-nitro-5-thiocyanobenzoic acid 1,5-IAEDANS, N-iodoacetyl-N”(5-sulfo-l-naphthyl)ethylene- diamine; HPLC, high performance liquid chromatography.

15721

15722 T a u Phosphorylation

interaction of tau with tubulin, thus destabilizing microtubules.

EXPERIMENTAL PROCEDURES

Protein Preparations-Tau was purified as described previously (Herzog and Weber, 1978) from bovine brain microtubule protein

by the procedure of Dedman et al. (1977). preparations. Calmodulin was purified from rat brain homogenates

Protein Kinases-The PKA holoenzyme was obtained from Sigma. PKC was purified from rat brain as indicated by Walsh et al. (1984). CaMK was isolated from rat brain following the procedure of Kelly c't al. (1987). CKII was purified from rat brain as described by Alcazar c r t al. (1988). PKC was purified about 200-fold to a specific activity of 1,200 units/mg, CaMK was purified 550-fold, and CKII about 2,000- fold to a specific activity of 1,400 units/mg. The isolation of proline- directed serine/threonine protein kinase (PDPK) from rat brain cytoskeletal protein preparations will be described.'

Protein Phosphorylation-The phosphorylation of t.au (20 pg) with PKA was performed in 0.1 M MES, 0.5 mM MgCl,, 2 mM EGTA, pH 6.8; (buffer A) supplemented with 5 mM MgCl,, 10 p~ CAMP, 1 mM PMSF, 0.05 units kinase/mg substrate (activity determined with MAP-2) and 10 p~ [r-"'PIATP. The Ca "/phospholipid-dependent phosphorylation of tau with purified PKC was performed in buffer A without EGTA and supplemented with 5 mM MgCl,, 1 mM CaC12, 80 pg/ml phosphatidyl serine, 0.150 pg/ml phorbol 12-myristate 13- acetate, 1 mM PMSF, 1 unit of kinase/mg of substrate (activity determined with MAP-2), and 10 p~ [Y-~'P]ATP. The Ca'+/calmodulin-dependent phosphorylation of tau with purified CaMK was carried out in buffer A without EGTA, supplemented with 20 pg/ml calmodulin, 5 mM MgC12, 1 mM CaC12, 1 mM PMSF, 0.7 unit of kinase/mg of substrate (activity determined with MAP-2), and 10 pM [r-:"P]ATP. Phosphorylation of tau with purified CKII was carried out in buffer A supplemented with 5 mM MgCl', 10 pg/ml poly-L- lysine, 1 mM PMSF, 0.5 unit of kinase/mg of substrate (activity determined with casein), and 10 pM [r-"PIATP. Finally, pbosphoryl- ation of tau with PDPK was performed in buffer A supplemented with 5 mM MgCl', 1 mM PMSF, and 10 p~ [y-"'PIATP. All reactions were performed in a final volume of 100 pl. Phosphorylation reactions were stopped by the addition of boiling SDS sample buffer. To determine the number of mol of phosphate incorporated into tau, the phosphorylation assays were performed at different concentrations of ATP.

Gel Electrophoresis-SDS-polyacrylamide gel electrophoresis was performed according to the procedure of Laemmli (1970). Phosphol- abeled proteins were detected by autoradiography of dried gels ex- posed to Kodak X-Omat films. The radioactivity associated with tau protein was determined by measuring the Cerenkov radiation of the excised phospholabeled bands in a liquid scintillation spectrometer (Beckman Instruments, Inc.).

Cysteine-specific Chemical Cleavage of Tau Protein by S-Cyanyla- tion-Phosphorylated tau bands were excised from the gels and equilibrated in 0.1 M Tris, pH 8.0, methanol (50%, v/v). Two milli- molar 2-nitro-5-thiocyanobenzoic acid (NTCB) dissolved in 7.5 M urea, 0.2 M Tris, 1 mM EDTA, pH 8.0, was added to the bands, and the reaction conditions were carried out as described previously (Jacobson et al., 1973).

Enzvmatic Cleavages of Tau Protein Phosohorylated by PKC-Tau Y .

protei;, phosphorylated by PKC as indicated above, was carboxy- methylated with N-iodoacetyl-N'-(5-sulfo-l-naphthyl)ethylenedia- mine (1,5-IAEDANS) as described by Olwin and Storm (1984). Tau was subsequently cleaved a t both glutamic and aspartic acid residues with Staphylococcus aureus V8 protease for 18 h at 37 "C in 50 mM sodium phosphate, pH 7.8, using an enzyme to substrate ratio of 1:30. The reaction was terminated by titrating the sample to pH 2.0 with trifluoroacetic acid. The sample was analyzed by reverse-phase chromatography on a NovaPak C18 columm equilibrated with 0.1% trifluoroacetic acid in water, using a Waters 501 apparatus. Peptides were eluted using a linear gradient of 0-80% acetonitrile in 0.1% trifluoroacetic acid and were detected by absorbance a t 215 nm and by aminonaphtholsulfonic acid fluorescence (excitation 342 nm and emission filter 418 nm). The radioactivityassociated with each sample was determined on a liquid scintillation spectrometer.

Additionally, both tau protein and peptide 369 (see below) were separately phosphorylated by PKC and subsequently digested with trypsin for 6h at 37 "C in 50 mM Tris-HC1, pH 8.0, using an enzyme

. "

'' I. Correas, J. Diaz-Nido, and J. Avila, manuscript in preparation.

to substrate ratio of 1:lOO. The tryptic peptides were fractionated by HPLC and the radioactivity associated with each fraction determined, as mentioned above.

Peptide Synthesis-The peptide KVTSKCGSLGNIHHKPGGG, which is part of the second repeated sequence in the tubulin binding domain present in the tau molecule (Goedert et al, 1988; Lee et al, 1988), was synthesized on an automatic solid phase peptide synthe- sizer (type 430A, Applied Biosystems) and purified by reverse-phase HPLC on a NovaPak C18 columm. The peptide was lyophilized and dissolved in 0.1% trifluoroacetic acid in water. For some experiments, the peptide KVTSKCGSLGNIHHKPGGG (peptide 369) and KVT- - AKCGSLGNIHHKPGGG (peptide 369~1~) were purchased from MedProbe A.S. (Oslo, Norway).

The in vitro phosphorylation assays of the synthetic peptides were carried out under the conditions described for tau protein phosphorylated with PKC, except that 3 h of incubation at 37 "C was used. Finally, peptide purification and radioactivity determinations were carried out as indicated above.

Tubulin Assembly Promotion Assay-Phosphocellulose purified tubulin (6 pg) was incubated with increasing concentrations of either the unphosphorylated peptide 369 or the peptide 369 phosphorylated by PKC in a final volume of 50 pl of buffer A in the presence of 1 mM GTP, 10 p~ taxol, and 1 mM dithiothreitol. The samples were loaded into Beckman Airfuge tubes and centrifuged for 5 min at 25 p.s.i. The pellets were washed with buffer A containing 1 mM GTP, and both supernatants and pellets were subjected to electrophoresis. The proportion of tubulin in each fraction was determined.

Tau Binding Competition Assay-Phosphocellulose-purified tubulin (6 pg) was incubated with PKC-phosphorylated tau (0.5 pg) in the presence of increasing amounts of either the unphosphorylated peptide 369 or the peptide 369 phosphorylated by PKC in a final volume of 50 pl of buffer A containing 1 mM GTP, 10 NM taxol, and 1 mM dithiothreitol. The samples were incubated for 20 min a t 37 "C, loaded into Beckman Airfuge tubes and centrifuged for 5 min a t 25 p.s.i. The pellets were washed with buffer A containing 1 mM GTP, and the radioactivity associated with the sedimented protein fraction was determined on a liquid scintillation spectrometer. In these experiments, tau protein was phosphorylated in the presence of 10 p M [y-"2P]ATP and peptide 369 in the presence of 1 mM ATP.

RESULTS

Distribution of Phosphorylation Sites on Tau Molecule after Incubation with Different Protein Kinases-The analysis of the primary structure of tau isoforms, as deduced by cDNA cloning studies, has revealed the existence of cysteine residues located only in some of the repeated sequences in the tubulin binding domain of tau molecules (Goedert et al., 1989a; Himmler et al., 1989; Kosik et al., 1989). Thus, when tau protein is treated with NTCB, a chemical reagent which specifically cleaves at cysteine residues (Jacobson et al., 1973), the molecule can be divided into two fragments: one containing the amino terminus and the other containing the carboxyl terminus (see Fig. 1). This allows the localization of tau regions phosphorylated in vitro by different protein kinases, after cleavage of phosphorylated tau molecules with NTCB.

In this study, bovine tau protein has been assayed with PKA, PKC, CaMK, CKII, and PDPK. These five kinases, all present in brain tissue, phosphorylate tau isoforms (Fig. 2). In our electrophoretic system, bovine tau is resolved as four intense and two weak bands designated, according to decreasing size, as T1 to T6 (see Fig. 2). When T2 to T5 bands of tau phosphorylated with PKA were cleaved with NTCB, it was observed that only the amino-terminal region of the molecule was radiolabeled (Fig. 3). Interestingly, T2 and T4 gave two phosphorylated amino-terminal fragments, suggesting the presence of 2 cysteine residues in each molecule. In contrast, T3 and T5 showed one phosphorylated amino-terminal fragment, suggesting the presence of a single cysteine residue in each molecule. Sequences of tau cDNA clones isolated from different organisms have indicated the existence of several isoforms of tau (Goedert et al., 1988; Himmler et al., 1989; Kosik et al., 1989; Lee et al., 1988). One class of

Tau Phosphorylation 15723

NTCB

SH SH COOH n

Hz? , , I , I , , ” I

PKA

- ] Uncleaved - -.. ] N-terminal

(I) - Uncleaved

- ( 2 - (1

FIG. 1. NTCB cleavage pattern of tau protein. Top, diagram representing an isoform of tau containing four repeated sequences in the tubulin binding domain (1, 2, 3, and 4). This type of tau isoform contains two cysteines ( S H ) located in the second and the third tuhulin binding repeats, respectively (Himmler et al., 1989; Goedert c,! a/ . , 1989a; Kosik et al., 1989). Specific NTCB cleavage sites at Cys residues are indicated. Partial cleavage can give rise to four major fragments: N, and N, (amino-terminal fragments) and C1 and C, (carboxyl-terminal fragments). Bottom, autoradiogram of iodinated \)ovine tau protein cleaved with NTCB. One of the tau bands (Mr- 56,000) was cleaved with NTCB as indicated under “Experimental Procedures” and electrophoresed in 10-20% slab gels. Five iodinated fragments were observed in the autoradiogram of the SDS-polyacrylamide gel: the uncleaved hand (M, - 56,000), N, (M, - 41,000), N, (M, - :36,000), C2 ( M , - 16,000), and C1 (M, - 13,000).

.

A

B ”

B

- 66

- 45

FIG. 2. Phosphorylation of bovine tau by different protein kinases. A, Coomassie Blue staining of tau phosphorylated by PKA, I’KC, CaMK, CKII, and PDPK. R, autoradiograms of tau phosphorylated by these kinases. The different tau bands are designated, according to decreasing size, as TI-T6. Numbers on the right indicate molecular mass markers in kDa.

these isoforms has three repeated sequences in the tubulin binding domain and a single cysteine residue which is located in the second repeat (Goedert et al., 1988; Lee et al., 1988). Another isoform class has four repeated sequences in the tubulin binding domain and 2 cysteine residues located in the original second repeat and in the additional inserted repeat (Goedert et al., 1989a; Himmler et al., 1989; Kosik et al., 1989). Therefore, our results suggest that T2 and T4 are isoforms containing four repeats, whereas T3 and T5 contain only

T2 T3 T 4 T5 FIG. 3. NTCB peptide mapping of tau phosphorylated by

PKA. T2 to T5 hands of‘ tau phosphorylated by I’KA were excised from the gel, cleaved with NTCR as indicated under “Experimental Procedures,” and electrophoresed in 10-20% slab gels. The autoradiograms are shown in the figure. Note that only amino-terminal fragments are phosphorylated. Both T2 and T4 give rise to two amino- terminal fragments of M, - 42,000 and 37,000 and of :37,000 and 32,000, respectively. Both T3 and T5 originate only one amino- terminal fragment of M, - 37,000 and 32,000, respectively. This indicates that T2 and T4 contain 2 Cys residues, whereas T:3 and T5 contain only one, suggesting that T2 and T4 probably are four-repeat- containing forms and that TB and T5 are three-repeat-containing forms.

three. Additional tau isoforms exist which contain 29 or 58 amino acid insertions in the amino-terminal region, in con- junction with three or four repeats (Goedert et al., 1989b; Himmler, 1989). The differences in electrophoretic mobility between T2 and T4 (four-repeat-containing forms) and between T3 and T5 (three-repeat-containing forms) may be because T2 and T3 contain the long amino-terminal insertion and T4 and T5 contain the short amino-terminal insertion.

NTCB cleavage of tau protein phosphorylated by CKII showed phospholabeled peptide patterns similar to those observed for PKA-phosphorylated tau (Fig. 4A). These results indicate that PKA and CKII modify residues located within the amino-terminal region of the tau molecule. Thus, residues located either in the sequence comprised between the 2 Cys residues or those in the carboxyl-terminal region after the 2nd Cys are not modified by these two kinases; otherwise, phosphorylated carboxyl-terminal fragments would be detected in their NTCB peptide maps.

Phosphorylation of tau protein by CaMK takes place at the carboxyl-terminal fragment of the molecule (Fig. 4R). Again, the fact that the large amino-terminal fragment was not phosphorylated indicates that the modified residue(s) is located neither within the amino acid sequence comprised between the 2 Cys residues nor at the amino-terminal region located before the 1st Cys residue. Indeed, a recent report has identified the phosphorylation site for CaMK near the carboxyl-terminal end of tau molecule (Steiner et al., 1990).

The NTCB peptide maps obtained for tau protein phosphorylated by PKC or PDPK show that both amino- and carboxyl-terminal fragments were phosphorylated (Fig. 4, C and D). These results suggest the existence of at least two phosphorylation sites located in different regions of the tau molecule and are also compatible with phosphorylation oc- curring within the tubulin binding domain a t residues located between the 2 cysteines.

Phosphorylation of the tau protein by different protein kinases, including CaMK and PKC, has been reported to diminish its ability to promote microtubule polymerization (Hoshi et al., 1987; Lindwall and Cole, 1984; Yamamoto et al., 1983). It would be of interest to determine which protein kinase phosphorylates tau protein in the tubulin binding

T a u Phosphorylation 15724

A CKII

Uncleaved [ m - N-terminal [ 1 -

T2 T3

C PKC

W.

Uncleaved [ 0

N-terminal [

0

6 CaMK "

W .) IUncleaved

IC-terminal

T 2 T3

D

PDPK 7 "

*- 1 Uncleaved

a. - ] N-terminal

T2 T3 T2 T3 FIG. 4. NTCB peptide map of tau phosphorylated by CKII,

CaMK, PKC, and PDPK. A, autoradiograms of CKII-phosphorylated T 2 and T3 bands cleaved with NTCR. Note that only amino- terminal fragments are phosphorylated. R, autoradiograms of CaMK- phosphorylated T2 and T3 bands cleaved with NTCR. Note that only carboxyl-terminal fragments are phosphorylated. C, autoradiograms of PKC-phsophorylated T2 and T3 bands cleaved with NTCR. Both amino-terminal and carboxyl-terminal fragments are phosphorylated. I ) , autoradiograms of PDPK-phosphorylated T 2 and T3 bands cleaved with NTCB. Both amino-terminal and carboxyl-terminal fragments are phosphorylated.

domain, thus directly affecting the association between tau and tubulin proteins. The results obtained in this study are compatible with PKC and PDPK as possible candidates. Since PDPK acts primarily on (K/R)S/TP-containing sequences (reviewed by Kemp and Pearson, 1990) and these are not present in the repeats, we have focused our attention on PKC.

Stoichiometry of the Phosphorylation of Tau by PKC-To determine the maximal incorporation of phosphate into tau by PKC, phosphorylation of tau was carried out a t increasing ATP concentrations. Fig. 5 shows a maximal incorporation of 4 mol of phosphate/mol of tau. Additionally, the phosphorylated residues were identified as serine (not shown), a result in agreement with that reported by Hoshi et al. (1988).

PKC Phosphorylates the Tubulin Binding Domain on the Tau Molecule-Taking advantage of the fact that Cys residues are located only within the tubulin binding domain (Goedert et al., 1989a; Himmler et al., 1989; Kosik et al., 1989), we have modified these Cys residues with 1,5-IAEDANS, a fluoro- phore-containing reagent, to detect whether or not residues phosphorylated by PKC are located close to the modified Cys

ATP ImM)

FIG. 5. Determination of the mol of phosphate incorporated into tau by PKC. Aliquots of 15 pI of purified tau (1 mg/ml) were incubated with PKC at different concentrations of ATP during 60 min a t 37 "C. After incubation, each aliquot was subjected to SDS- gel electrophoresis, and the region containing tau protein was excised from the gel. The radioactivity associated with tau was measured by Cerenkov radiation and converted to concentration of ATP.

I 0 1

I

Fraction Number

FIG. 6. Identification of phosphopeptides of tau containing Cys residues. Tau protein phosphorylated by PKC was modified a t Cys residues with 1,5-IAEDANS and digested with S. aureus V8 protease under the conditions indicated under "Experimental Procedures." HPLC elution profile of '"P-phosphorylated peptides (0- - -0). Fluorescence yield of eluted peptides (U). Absorb- ance a t 215 nm of eluted peptides (A- - -A).

residues. Thus, phosphorylated tau protein was modified with 1,5-IAEDANS, and, after exhaustive enzymatic cleavage with S. aurem V8 protease, the peptides were separated by reverse- phase HPLC (Fig. 6). The major fluorescence peak was associated with a radioactive peak indicating that there are phosphorylated tau peptides containing modified Cys residues. As pointed out above, this suggests that a phosphorylation site for PKC on the tau molecule may be located within the tubulin binding domain which contains Cys residues. A second radioactive peak does not appear to correlate with any fluorescence peak (Fig. 6), indicating that there are also phosphorylated tau peptides which do not contain modified Cys residues. To confirm this hypothesis further, we have located fragments with immunoreactivity against antibody 369 by dot blotting each HPLC fraction with this antibody. Antibody 369 was raised against the synthetic peptide KVTSKCGSLGNIHH- KPGGG (peptide 369), which is part of the cysteine-containing tubulin binding repeat present in both fetal and adult tau isoforms (Himmler et al., 1989; Kosik et al., 1989). The max- imum immunoreactivity peak corresponds to fractions 71-74 of the chromatogram (data not shown).


The results presented here suggest that the tubulin binding region mentioned above is phosphorylated by PKC. To show this directly, we have assayed the synthetic peptide with PKC; PDPK and CaMK have also been included in the assay. Neither PDPK nor, as expected from the NTCB peptide mapping results, CaMK was able to phosphorylate the synthetic peptide, whereas PKC phosphorylates this peptide under the same conditions (Fig. 7). However, peptide 369 has 2 serines, one at residue number 4 of the peptide (Ser-309 in the tau sequence reported by Himmler et al., 1989) and the other one at residue number 8 (Ser-313). The latter one is conserved in every tubulin binding repeat of both tau and MAP-2 proteins (Lewis et al., 1988). To test which serine is modified by PKC, the phosphorylation assay was repeated with peptide 369 and with a similar peptide in which serine number 4 (Ser-309) was substituted by an alanine residue (peptide 3 6 9 ~ 1 ~ ) . Table I shows that the degree of phosphate incorporated into each peptide is similar, indicating that serine 313 is the one modified by PKC.

To determine whether the tubulin binding domain of tau is a major phosphorylation target for PKC, tryptic peptides from PKC-phosphorylated tau were separated by reverse-phase HPLC and compared with triptic peptides from PKC-phosphorylated peptide 369. Fig. 8 shows that a tau phosphopeptide elutes at the same position in reverse-phase HPLC as a tryptic fragment of peptide 369. This tau phosphopeptide contains a high proportion of associated radioactivity and

A

B 260 -

200 -

m Q

B I 140-

80 -

20 -

minutes

FIG. 7. PKC phosphorylates a tubulin binding domain on the tau molecule. A , schematic representation of a four-repeat- containing isoform of tau showing the sequence of the synthetic peptide (peptide 369) used as substrate for PKC. E , the synthetic peptide (peptide 369) was incubated with PKC and purified by reverse-phase HPLC chromatography under the conditions described under “Experimental Procedures.” The elution profile of 32P-associated radioactivity is represented. Two peaks were observed, one corresponding to the void volume (nonincorporated [Y-~*P]ATP) and the other one to the phosphorylated peptide. No radioactivity was found associated with this latter peak when purified CaMK or PDPK were used instead of PKC. The inset shows the elution profile of the unphosphorylated synthetic peptide purified under the same conditions. The ordinate represents absorbance at 215 nm to determine the elution of the peptide.

TABLE I Modification of tau peptides by protein kinase C

Peptides 369 and 36gAls (a peptide with the same sequence that 369 except that the first serine was replaced by an alanine) were phosphorylated under the conditions indicated under “Experimental Pro- cedures.’’ The incorporation of phosphate into the peptides was determined.

Substrate ATP mol P/mol peptide mM

KVTSKCGSLGNIHHKPGGG 1.0 0.8 k 0.15 KVTAKCGSLGNIHHKPGGG 1.0 0.9 k 0.10

reacts with the antibody 369 (not shown). Additionally, amino acid analysis of the tryptic phosphopeptides shown in Fig. 8 0 indicated that the composition of the peptide eluting at fraction 37 is compatible with the sequence of the intact peptide 369, whereas that of the peptide eluting at fraction 42 is compatible with the sequence CGSLGNIHHKPGGG. This result is consistent with the previous one indicating that serine 313 is phosphorylated by PKC. It also suggests that the tubulin binding domain of tau is a major phosphorylation target for PKC.

Effect of PKC-catalyzed Phosphorylation of Peptide 369 on Its Ability to Induce Tubulin Polymerization-It has been indicated previously that peptides containing the sequence corresponding to the tubulin binding repeats of tau or MAP- 2 proteins could promote microtubule assembly (Joly et al., 1989; Ennulat et al., 1989, Maccioni et al., 1989) and that, therefore, these peptides could compite with tau or MAP-2 for the common tubulin binding site. An exhaustive analysis has been carried out with the second repeated sequence of MAP-2 (Joly and Purich, 1990), a sequence very similar to the third tubulin binding motif of the four-repeat tau isoform (Lewis et al., 1988).

As expected, peptide 369 is able to induce tubulin assembly (Fig. 9A). Phosphorylation of the peptide by PKC reduces its capacity to promote tubulin assembly (Fig. 9A). Moreover, unphosphorylated peptide 369 competes for tau binding to microtubules in a better way than the peptide modified by PKC (Fig. 9B). Thus, the interaction of tau with taxol- polymerized tubulin is reduced in the presence of peptide 369, preferentially when it is not phosphorylated by PKC.

DISCUSSION

In an attempt to identify phosphorylation sites on the tubulin binding domain of tau, we have phosphorylated this protein with five different kinases, including PKA, CKII, CaMK, PKC, and PDPK. All five kinases present in brain tissues phosphorylate tau protein. Peptides of the type R-X- X-S are good substrates for PKA, PKC, and CaMK (reviewed by Kemp and Pearson, 1990), and this motif is present several times on the tau molecule. However, the NTCB peptide maps obtained for tau protein phosphorylated by these three kinases show that PKA modifies residues located in the amino-terminal fragment of the tau molecule, CaMK modifies residues located in the carboxyl-terminal fragment, and PKC phosphorylates residues in both fragments. This indicates that these three kinases modify different residues in the tau molecule. An interesting finding observed in the NTCB peptide patterns is that three-repeat-containing tau forms can be distinguished from four-repeat-containing forms. This analysis suggests that the four major forms of bovine tau are constituted by isoforms containing four and three repeats with probably two amino-terminal insertions (T2 and T3, respectively) and by four and three repeats with probably one amino-terminal insertion (T4 and T5, respectively). Our re-


O i

a 5 0 1

0 I

A I C

I I I I I I i I 10 20 30 LO 50 6 0 70 80 10 15 20 2 5 30 35 LO L 5 SO 5 5 60 65 70

6

I I L o 10 20 30 40 SO 60 70 BO

rn .= x E 4

, I 32 r l 28

- 12

- 16

- 2 0

- 2 4

-

I l l I

D

Fractlon number Fractlon number

FIG. 8. The tubulin binding domain of tau molecule is a major phosphorylation target for PKC. A , tau, phosphorylated by PKC, was digested with trypsin at an enzyme to substrate ratio of 1:100, and the resulting peptides were fractionated by reverse-phase HPLC. The absorbance at 215 nm of eluted peptides is indicated. B, the synthetic peptide 369, phosphorylated by PKC, was digested with trypsin as in A , and the resulting peptides were purified by reverse-phase HPLC under the same conditions used in A (see “Experimental Procedures”). The absorbance at 215 nm of eluted peptides is shown. C, the 32P-associated radioactivity for fractions obtained in A was determined in a liquid scintillation spectrophotometer and is shown in the figure. D, the 32P-associated radioactivity for fractions obtained in B was determined in a liquid scintillation spectrophotometer and is shown in the figure. The arrows indicate the elution position of a tau phosphopeptide ( A and C) which co-migrates with a phosphorylated fragment of peptide 369 ( B and D) whose amino acid composition accounts for the sequence CGSLGNIHHKPGGG.

sults agree well with those of Himmler (1989). Good substrates for CKII are serines followed by acidic

residues (Kemp and Pearson, 1990), and several such sequences are present in the amino-terminal portion of the tau molecule. Substrates for PDPK are serines adjacent to pro- lines (Kemp and Pearson, 1990), and tau contains several such sequences, which could well account for the NTCB peptide map obtained.

Focusing on the kinase(s) responsible for phosphorylating tubulin binding sites on the tau molecule, PKC appeared to be the best candidate. In fact, PKC phosphorylates the synthetic peptide KVTSKCGSLGNIHHKPGGG, which is part of the repeat present in both adult and fetal tau isoforms (Himmler et al., 1989; Kosik et al., 1989) and is efficient in tubulin and in actin binding (Correas et al., 1990). This peptide contains two serines, both in a K-X-X-S sequence, which suggests that either could be good substrates for PKC. It is interesting to note that the second serine is highly conserved in all tau and MAP-2 repeated sequences (Kosik et al., 1989; Lewis et al., 1988; Wiche et al., 1991).

The use of a synthetic peptide lacking the first serine allowed us to determine that the second serine is the one modified by PKC. Since the stoichiometry of phosphorylation is 4 mol of phosphate/mol of tau, a result in agreement with that reported by Hoshi et al. (1987), and since the second serine is highly conserved in all tau repeated sequences (Kosik et al., 1989) it would be plausible to speculate that not only Ser-313, located in the third tubulin binding repeat, is modified but also the corresponding serines in the other tubulin binding repeats. However, the presence of other additional

phosphorylation sites on the tau molecule cannot be ruled out.

MAP-2, a neuronal-specific microtubule-associatedprotein, is also phosphorylated by PKC (Hoshi et al., 1988; Tsuyama et al., 1986). Phosphorylation takes place in the projection domain of the molecule as well as in its tubulin binding region (Diaz-Nido et al., 1990; Hernandez et al., 1987; Hoshi et al., 1988). The latter contains three repeated sequences highly homologous to those of tau (Lewis et al., 1988) and which, interestingly, contain the conserved serine residue present in tau repeats. It has been suggested that the tau-tubulin and MAP-2-tubulin interaction could be based on ionic interactions involving positive charges present in tau (or MAP-2) and negative charges present in the carboxyl-terminal end of tubulin (Avila, 1991); the introduction of one or more negative charges in the tubulin binding domain of tau (or MAP-2) by phosphorylation may therefore result in decreased interaction with tubulin. Indeed, Hoshi et al. (1988) have reported such effect for MAP-2 phosphorylated at its tubulin binding domain by PKC. Hoshi et al. (1987) have also analyzed tau phosphorylated with PKC and found that the degree of binding to tubulin is inversely correlated with the number of phosphates incorporated into the tau molecule. We have found that the phosphorylation of peptide 369 by PKC at serine number 8 (Ser-313 in the tau sequence reported by Himmler et al., 1989) reduces both its ability to promote tubulin assembly and to bind to microtubules. The possibility that phosphorylation of additional sites close to Ser-313 may be responsible for the greater inhibition of tubulin binding which is observed when the whole tau molecule is phosphoryl-


changes associated with learning and memory in the adult nervous system (Friedrich, 1990).

On the other hand, the abnormal and uncontrolled activation of PKC may lead to a hyperphosphorylation of tau and/ or MAP-2, with a subsequent destabilization of the microtubule cytoskeleton. Furthermore, the hyperphosphorylation of tau may be one of the posttranslational mechanisms driving the self-assembly (or the co-assembly with still undefined components) of tau into the paired helical filaments consti- tuting the NFTs characteristics of Alzheimer’s disease (Bancher et al., 1989; Flament et al., 1990; Grundke-Iqbal et al., 1986a; Ihara et al., 1986; Kosik, 1990). It is possible that multiple protein kinases participate in the aberrant hyperphosphorylation of tau protein in Alzheimer’s disease. The involvement of tau phosphorylation by CaMK has been dis- cussed previously (Baudier and Cole, 1987; Grundke-Iqbal et al., 1986a; Steiner et al., 1990). The involvement of other protein kinases not yet identified is also strongly suggested by the aberrant phosphorylation of other sites along the tau molecule, including the Tau-1 epitope (Kosik et al., 1988), the KESP motif (Iqbal et al., 1989) and the KSPV motif (Lee et al., 1991) in Alzheimer’s disease. A similar role could be proposed for PKC. Whether or not the tubulin binding domain of the tau molecule is actually phosphorylated by PKC in Alzheimer’s disease awaits further investigation.

A I

- 0.5 1.0

B mM peptide

25 -

I I I

0.5 1.0 m M peptide

FIG. 9. Effect of PKC phosphorylation of peptide 369 on tubulin assembly and the interaction of tau with microtubules. A , unmodified peptide 369 (0) or the peptide phosphorylated by PKC (0,6 mol of phosphate incorporated/mol peptide) (0) were incubated with tubulin as indicated under “Experimental Procedures.” Error bars represent the standard deviation from five experiments. B, increasing amounts of unmodified peptide 369 (o”--o) or peptide 369 modified by PKC (.”-.) were incubated with taxol-assembled tubulin (6 pg) and 3 Z P - t a ~ (0.5 Kg) as indicated under “Experimental Procedures.” After the incubation, the samples were loaded into Airfuge tubes and centrifuged for 5 min at 25 p.s.i. in a Beckman ultracentifuge. The radioactivity associated to the pelleted protein fraction was determined and indicated in the figure. 100% is the value for cpm associated to the pellet fraction in the absence of peptide. Error bars represent standard deviation from four experiments.

ated by PKC cannot be ruled out (Hoshi et al., 1987). It is tempting to speculate that the phosphorylation of the

tubulin binding domain in tau and MAP-2 may constitute one of the key events in neuronal plasticity. There is ample evidence supporting the hypothesis that neurotransmitters may regulate neuronal morphology (Mattson, 1988; Mattson et al., 1988; Lipton and Kater, 1989). It thus appears plausible that PKC activation constitutes one of the links between the binding of some neurotransmitters to their surface receptors and the resulting modifications of the cytoskeleton. Electron microscopic observations have revealed the localization of PKC immunoreactivity to microtubules within dendrites in hippocampal pyramidal neurons (Kose et al., 1990). In partic- ular, PKC activation would result in an augmented phosphorylation of tau and/or MAP-2. Phosphorylated tau and MAP-2 molecules would consequently bind more weakly to tubulin (Hoshi et al., 1987, 1988), thereby favoring localized and transient microtubule instability events. This would give rise to a relatively uncross-linked, malleable cystoskeleton which would allow cell shape changes. These structural changes may be as necessary for the rearrangements of neuronal connectivity which occur in the developing nervous system (Aoki and Siekevitz, 1985) as for the adaptative

Acknowledgments-We thank Dr. Carlos L6pez-0th for helping to identify the phosphorylated synthetic peptide by sequencing. We also acknowledge Drs. Javier Diez-Guerra, Rosario Armas, and Mar Garcia-Rocha for generous help. We thank Fundacion Ram6n Areces for the institutional support.

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