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calmodulin-dependent protein kinase II in its tubulin binding sites
ABB Archives of Biochemistry and Biophysics 408 (2002) 255–262 www.academicpress.com
Phosphorylation of microtubule-associated protein tau by Ca2þ /calmodulin-dependent protein kinase II in its tubulin binding sites Hideyuki Yamamoto,a,*,1 Emiko Yamauchi,b,1 Hisaaki Taniguchi,b Tsunehiko Ono,c and Eishichi Miyamotoa a
c
Department of Pharmacology, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto 860-0811, Japan b Institute for Enzyme Research, University of Tokushima, Tokushima 770-8503, Japan Department of Neuropsychiatry, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto 860-0811, Japan Received 2 August 2002, and in revised form 17 September 2002
Abstract The paired helical filaments (PHF) found in AlzheimerÕs disease (AD) brain are composed mainly of the hyperphosphorylated form of microtubule-associated protein tau (PHF-tau). It is well known that tau is a good in vitro substrate for Ca2þ /calmodulindependent protein kinase II (CaM kinase II). To establish the phosphorylation sites, the longest human tau (hTau40) was bacterially expressed and phosphorylated by CaM kinase II, followed by digestion with lysyl endoprotease. The digests were subjected to liquid chromatography/mass spectrometry. We found that 5 of 22 identified peptides were phosphorylated. From the tandem mass spectrometry, two phosphorylation sites (serines 262 and 356) were identified in the tubulin binding sites. When tau was phosphorylated by CaM kinase II, the binding of tau to taxol-stabilized microtubules was remarkably impaired. As both serines 262 and 356 are reportedly phosphorylated in PHF-tau, CaM kinase II may be involved in hyperphosphorylation of tau in AD brain. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: AlzheimerÕs disease; Calmodulin; Ca2þ /calmodulin-dependent protein kinase II; Paired helical filaments; Tau
Neurofibrillary tangles are one of the pathologic hallmarks of AlzheimerÕs disease (AD),2 and their abundance was reported to correlate with the clinical degree of dementia [1]. The unit fibrils of the neurofibrillary tangles are called paired helical filaments (PHF) and PHF consist of hyperphosphorylated tau (designated PHF-tau) [2–4]. Hyperphosphorylation of tau appears to precede PHF formation [5]. Tau is one of the microtubule-associated proteins and induces microtubule assembly and stabilizes microtubules in vitro [4].
*
Corresponding author. Fax: +81-96-373-5078. E-mail address: [email protected] (H. Yamamoto). 1 These authors contributed equally to this paper. 2 Abbreviations used: AD, AlzheimerÕs disease; CaM kinase II, Ca2þ / calmodulin-dependent protein kinase II; PHF, paired helical filaments; LC/MS, capillary reverse-phase liquid chromatography/electrospray mass spectrometry.
These functions are diminished by phosphorylation with several protein kinases [3]. Tau is the major microtubule-associated protein in axons and appears to play an important role in the stabilization of microtubules during interactions with other cytoskeletal components in axons [6]. Therefore, hyperphosphorylation of tau in AD may disrupt the axonal cytoskeleton and axonal flow, leading to neuronal degeneration [3]. Molecular cloning has identified six tau isoforms in human brain tissue that are produced from a single gene by alternative mRNA splicing [2]. They range from 352 to 441 amino acids in length and differ from each other by the presence of three inserts. Among the isoforms, the longest tau isoform (hTau40) has all three inserts. By direct sequencing of PHF-tau, at least 25 sites have been chemically identified as phosphorylation sites on PHFtau [7–9]. Among the 25 phosphorylation sites, serines 262 and 356, numbered according to hTau40 [10], were located in the tubulin binding sites. The tubulin binding
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sites of tau are 18-amino-acid motifs repeated three or four times in the carboxyl-terminal half of the molecule [11]. The phosphorylation of these sites may result in the impairment of tau functions in axons. SDS–PAGE analysis showed that PHF-tau migrated to a higher position than normal tau, and the reduced electrophoretic mobility of PHF-tau appears to be due to hyperphosphorylation of some phosphorylation sites, presumably due to the conformational alterations (for review, see [8,12]). In addition to AD brain, hyperphosphorylated tau is accumulated also in the brains of neurodegenerative diseases known as ‘‘tauopathies,’’ including frontotemporal dementia with parkinsonism linked to chromosome 17, amyotrophic lateral sclerosis/parkinsonism–dementia complex, and progressive supranuclear palsy [13,14]. In order to understand the pathophysiologies of tauopathies as well as AD, it is important to identify protein kinases that are involved in hyperphosphorylation of tau in the brains. Ca2þ /calmodulin-dependent protein kinase II (CaM kinase II) is widely distributed in neuronal tissues and may be involved in a variety of Ca2þ -mediated cellular processes, including regulation of cytoskeletal proteins, gene expression, and synaptic plasticity [15–18]. We previously reported that tau was a good in vitro substrate for CaM kinase II and that its phosphorylation inhibited microtubule assembly [19–21]. It was also reported that phosphorylation of tau by CaM kinase II decreased the electrophoretic mobility [22]. Developments in mass spectrometry made it possible to determine the molecular mass of large proteins with high precision and resolution [23,24]. We have applied the capillary reverse-phase liquid chromatography/electrospray mass spectrometry (LC/MS) method to the identification of phosphorylation sites in multiply phosphorylated proteins [25–27]. In the present work, we tried to identify unequivocally the phosphorylation sites in tau by CaM kinase II using LC/MS analysis for two reasons. First, we intended to examine the possible involvement of CaM kinase II in abnormal hyperphosphorylation of PHF-tau. Second, we wanted to elucidate the molecular mechanisms by which phosphorylation of tau resulted in a diminished capacity to induce microtubule assembly.
Materials and methods Materials [c-32 P]ATP and 125 I-protein A were purchased from NEN; phosphocellulose paper was from Whatman; CaM kinase II substrate peptide syntide-2 was from Bachem; rabbit anti-tau antibody (T-6402) was from Sigma; Microtubule/Tubulin Biochem kit was from Cytoskeleton
(BK015). Tau peptide(401–412) (GDTSPRHLSNVSC, 1372 Da) was synthesized by Fujiya Biolaboratory (Hatano, Japan). Other chemicals used were of analytical grade. Purification of proteins An expression clone of the longest human tau isoform, hTau40 [10], was a gift from Dr. M. Goedert (MRC Laboratory of Molecular Biology, Cambridge, UK). The cDNA of hTau40 was in the pRK172 vector and expressed in the BL21(DE3) Escherichia coli system [28]. Recombinant tau was purified from E. coli cells as described [29]. Tau [30] and calmodulin [31] were purified from bovine brain. CaM kinase II was purified from rat brain [32]. Assay for CaM kinase II The assay system for CaM kinase II contained, in a final volume of 25 ll, the following constituents: 50 mM Hepes, pH 7.5, 10 mM MgðCH3 COOÞ2 , 1 mM CaCl2 , 1.5 lM calmodulin, 0.1 mM [c-32 P]ATP (3000– 5000 cpm/pmol), 50 nM CaM kinase II, and 40 lg=ml recombinant tau or tau purified from bovine brain [33]. After incubation at 30 °C for 60 min, 15 ll of the samples was spotted on phosphocellulose paper squares and processed as described [34]. When the amount of phosphate incorporated into tau peptide(401–412) was determined, 20 ll of the sample was applied to an AG1-X8 column (2 ml) to separate the phosphorylated peptide from [c-32 P]ATP [35]. The phosphate incorporated into the peptide was measured by a liquid scintillation counter. Protease digestion of tau Recombinant tau was phosphorylated as described above except that cold ATP was used instead of [c32 P]ATP for 30 min at 30 °C. The reaction was terminated by heat treatment at 60 °C for 5 min and kept at )80 °C until use. Tau (10 lg) phosphorylated in the presence or absence of cold ATP was digested with 1 lg of lysyl endoprotease (WAKO Pure Chemical Industries, Osaka) in 100 mM Tris–HCl, pH 8.9, in the presence of 2 M urea at 37 °C for 15 h. After the reaction was stopped by addition of trifluoroacetic acid to a final concentration of 0.1%, the peptide mixtures were directly injected into the LC/MS apparatus. Mass spectrometry LC/MS and MS/MS analyses were carried out with an electrospray ionization/quadrupole mass
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spectrometer (PE Sciex API-III) as described previously [25–27].
described [38]. The radioactivity of 125 I was measured using a Bio-Imaging analyzer (FLA-2000; Fujifilm, Tokyo, Japan).
Microtubule cosedimentation assays
Other procedures
Recombinant tau was phosphorylated by 50 nM CaM kinase II for 30 min at 30 °C as described above except that cold ATP was used instead of [c-32 P]ATP. For the control, CaM kinase II was pretreated with heat at 60 °C for 5 min. The reaction was terminated by heat treatment at 60 °C for 5 min. Tubulin purified from bovine brain (5 mg/ml) was incubated in 80 mM Hepes, pH 6.8, 1 mM MgCl2 , 1 mM EGTA, 10% glycerol, and 2 mM GTP at 35 °C for 20 min. The microtubules were diluted to 1.5 mg/ml and taxol was added to a final concentration of 15 lM. Tau (34 lg=ml) was incubated with 0.4 mg/ml microtubules at 35 °C for 10 min in 40 mM Hepes, pH 6.8, 0.5 mM MgCl2 , 0.5 mM EGTA, 5% glycerol, 1 mM GTP, and 7.5 lM taxol, in a final volume of 100 ll. Tau bound to microtubules was separated from unbound tau by centrifugation at 105,000g for 30 min at 30 °C. The supernatant (100 ll) was heat treated in the presence of SDS–PAGE sample buffer [36] at 100 °C for 1.5 min. The pellet was suspended in 100 ll of H2 O and heat treated in the presence of SDS–PAGE sample buffer. Each sample (25 ll) was applied to SDS– PAGE [36], followed by Coomassie brilliant blue R-250 staining or immunoblot analysis. The protein amount of tau in each lane was quantified by scanning using NIH Image (version 1.61). Immunoblot analysis was done by the method of Towbin et al. [37] using 125 I-protein A, as
Protein concentration was determined by the method of Bradford [39] or using the BCA protein assay reagent (Pierce) with bovine serum albumin as a standard. Statistical evaluation Data are expressed as means SE values. The significance of the difference was calculated using StudentÕs t test and p values <0.05 were considered statistically significant.
Results Phosphorylation of tau by CaM kinase II Recombinant tau (hTau40) was phosphorylated by CaM kinase II in a Ca2þ /calmodulin-dependent manner. About 2.6 and 0.04 mol phosphate/mol of tau was phosphorylated by CaM kinase II in the presence and absence of Ca2þ /calmodulin, respectively. When tau purified from bovine brain was phosphorylated by CaM kinase II in the presence of Ca2þ /calmodulin, 7.7 mol phosphate/mol of tau was incorporated, under the same assay conditions. These results suggested that CaM kinase II phosphorylated bovine brain tau more strongly
Fig. 1. Amino acid sequence of tau and the peptides identified by LC/MS analysis. Amino acid sequence of hTau40 [10] is shown with the peptides identified from the LC/MS analysis. The arrows indicate the peptides identified by the LC/MS analysis. The bold arrows indicate phosphorylated peptides. Four tubulin binding sites are boxed and phosphorylated serines 262 and 356 are indicated. Residue numbers are at the end of each line as well as at the residues discussed in the text.
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than bacterially expressed tau, for unknown reasons. We phosphorylated recombinant tau under these conditions except that cold ATP was used instead of [c-32 P]ATP and then carried out the identification of phosphorylation sites.
LC/MS analysis of lysyl endoprotease digest Unphosphorylated and phosphorylated tau proteins digested with lysyl endoprotease were subjected to LC/ MS analysis. The masses of each peptide sequentially eluted from LC were detected online by the mass spectrometer and were compared with the theoretical ones calculated from the amino acid sequence deduced from the cDNA sequence of hTau40 [10]. The totals of 22 peptides were identified from the mass values, as summarized in Fig. 1. When phosphorylated tau was subjected to LC/MS analysis, at least five peptides were accompanied by peptides that were 80, 160, 240, or 320 Da larger than those of the parent peptides (Figs. 2, 4–6). Since transfer of phosphate from ATP to serine or threonine increases the mass of corresponding peptides by 80 Da, these peptides were likely to be phosphorylated. The extent of the phosphorylation estimated from the peak intensities of the deconvoluted mass spectra varied from 15 to 60%, depending on the peptide. In previous work, we demonstrated that the extent of the phosphorylation did not affect ionization efficiency [25]. Identification of phosphorylation sites in tubulin binding sites Among the phosphopeptides identified, we found that the two peptides, namely K23 and K38, originated from the first and fourth repeat, respectively, of tubulin binding sites (Figs. 1 and 2). To identify the exact phosphorylation sites of K23 and K38 peptides, we isolated each peptide by applying the protein digest on reversed-phase liquid chromatography, and fractions containing each peptide were subject to MS/MS analysis (Fig. 3). In the MS/MS spectra of the phosphorylated K23 peptide, the presence of a y-ion series from y1 to y5 corresponding to the masses of the unphosphorylated fragments and the difference between the y5 and the y6 ions that corresponded to a phosphoserine indicated that serine 262 was phosphorylated. The y6 and y7 ions whose masses show dissociation of a phosphate group further confirm the phosphorylation site. The phosphorylation of serine 262 was also confirmed by the difference between the b2 and the b3 ions, which also corresponds to the mass of the phosphoserine (Fig. 3). From these results, we concluded that serine 262 was the sole phosphorylation site in K23 peptide. K38 peptide also contained two possible phosphorylatable residues,
Fig. 2. Deconvoluted electrospray mass spectra of K23 and K38 peptides from unphosphorylated and phosphorylated tau. Tau was phosphorylated in the absence (top) or presence (bottom) of cold ATP as described under Materials and methods. Tau was digested by lysyl endoprotease, and the resulting peptide mixture was subjected to LC/ MS analysis. Among several peaks observed, the peptide 80 Da larger than K23 or K38 peptide is observed only in the bottom graphs.
serine 356 and threonine 361. In the MS/MS spectra of phosphorylated K38 peptide, fragment ions containing threonine 361 could not be obtained in the present study, mainly because the residue was apart from both termini of the peptide; the eighth and ninth residue from the N-terminus and C-terminus, respectively. However, the mass difference between b2 and b3 in the spectra corresponds to phosphoserine, suggesting that serine 356 was the phosphorylation site in this peptide (Fig. 3). The primary sequence around serine 356 (KIGSLDN) resembles that around serine 262 (KIGSTEN). Therefore, together with the results in Fig. 3, it was highly
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Fig. 3. Tandem mass spectra of K23 and K38 peptides. The singly charged ion of each peptide was subjected to the MS/MS analysis. Fragment ions observed are indicated above and below the peptide sequence. The phosphorylated serine residues are indicated.
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Fig. 4. Deconvoluted electrospray mass spectra of K2 peptide from unphosphorylated (top) and phosphorylated (bottom) tau. The lysyl endoprotease digests of tau were injected into the LC/MS apparatus. The peptide 80 Da larger than K2 peptide is observed only in the bottom graph.
possible that serine 356 was the sole phosphorylation site in K38 peptide. Phosphorylation of K2, K16, and K44 peptides In addition to K23 and K38 peptides, the LC/MS analysis indicated that the other three peptides were phosphorylated. Although exact phosphorylation sites of each peptide could not be identified in the present study, K2 peptide corresponded to residues 25–44 of tau, and a low amount of K2 peptide was singly phosphorylated (Fig. 4). As there were two threonines but no serine in the peptide, one of the threonine residues appeared to be phosphorylated. We have reported that CaM kinase II phosphorylates tau slightly at threonines in addition to serines by phosphoamino acid analysis [20]. It was detected that K16 peptide contained doubly phosphorylated species in addition to singly phosphorylated species (Fig. 5). Therefore, at least two residues are phosphorylated among eight serines and four threonines in K16 peptide. K44 peptide contained 10 serines and 3 threonines and was phosphorylated at several sites (Fig. 6). Pearson et al. [35] reported that a minimum specificity determinant for CaM kinase II was the sequence RXXS/TX (X
Fig. 5. Deconvoluted electrospray mass spectra of K16 peptide from unphosphorylated (top) and phosphorylated (bottom) tau. The lysyl endoprotease digests of tau were injected into the LC/MS apparatus. The peptides 80 and 160 Da larger than K16 peptide are observed only in the bottom graph.
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Fig. 7. Effects of phosphorylation of tau by CaM kinase II on the interaction with taxol-stabilized microtubules. After tau was phosphorylated in the presence of CaM kinase II (P-Tau) or heat-treated CaM kinase II (Tau), tau was incubated with polymerized tubulin, and the mixture was subjected to ultracentrifugation as described under Materials and methods. (A) The pellet was applied to SDS–PAGE, followed by protein staining. (B) Immunoblot analysis of the supernatant was carried out using anti-tau antibody. We repeated the same experiments at least three times with reproducible results, and representative results are shown. Fig. 6. Deconvoluted electrospray mass spectra of K44 peptide from unphosphorylated (top) and phosphorylated (bottom) tau. The lysyl endoprotease digests of tau were injected into the LC/MS apparatus. The peptide 320 Da larger than K44 peptide is observed in addition to the peptides 80, 160, and 240 Da larger only in the bottom graph.
is a hydrophobic amino acid). However, it was reported that serine 409, which was in the RXXS/TX motif, was not phosphorylated by CaM kinase II [40]. In order to confirm their report, we synthesized tau peptide(401– 412) and examined the phosphorylation. We found that tau peptide(401–412) was not phosphorylated at all. Together with the previous reports, these results suggested that serine 409 was not a phosphorylation site in K44 peptide. Inhibition of binding of tau to microtubules by phosphorylation To determine whether phosphorylation of tau by CaM kinase II inhibited the interaction with microtubules, a cosedimentation assay of recombinant tau and taxol-stabilized microtubules was carried out. When unphosphorylated tau was incubated with microtubules, about 90% of tau was cosedimented with microtubules (Fig. 7A). In contrast, when phosphorylated tau was used, the amount of tau in the pellet was remarkably reduced. Furthermore, the supernatant was subjected to immunoblot analysis using anti-tau antibody (Fig. 7B). The amount of tau in the supernatant was increased 3:0 0:5-fold (n ¼ 3, p < 0:01) by phosphorylation. It should be noted that the electrophoretic mobility of tau slightly but clearly decreased after phosphorylation by CaM kinase II as reported [22] (Fig. 7). When bovine brain tau was incubated with microtubules, about 65% of the tau was cosedimented with microtubules before phosphorylation by CaM kinase II. Immunoblot anal-
ysis indicated that the amount of bovine brain tau in the supernatant was increased 1:4 0:1-fold (n ¼ 3, p < 0:05) by phosphorylation. These results suggest that the effect of phosphorylation of bovine brain tau was weaker than that of recombinant tau.
Discussion It is well known that tau is phosphorylated in vivo at multiple sites [41,42]. Therefore, to analyze unequivocally the phosphorylation sites by CaM kinase II, it was necessary to use bacterially expressed tau instead of tau purified from brain. As expected, essentially no phosphate was incorporated into recombinant tau. However, recombinant tau was less efficiently phosphorylated by CaM kinase II than bovine brain tau. Quantitative comparisons of their tubulin binding affinity revealed that recombinant tau had higher affinity to microtubules than bovine brain tau and that the effect of phosphorylation of recombinant tau was stronger than that of bovine brain tau. The reasons for these differences between recombinant tau and bovine brain tau are not clear at present. The folding and/or other posttranslational modifications of bacterially expressed tau may be different from those of the native tau. Although 2.6 mol phosphate/mol of tau was detected to be incorporated by the assay system for CaM kinase II, LC/MS analysis revealed that more than five sites were phosphorylated. Considering the overall stoichiometry, the stoichiometry of phosphorylation of some sites must be low. These results suggest that the LC/MS analysis is very sensitive. It has been reported that CaM kinase II phosphorylates tau exclusively at serine 416 [40]. Our analysis clearly showed that other sites were phosphorylated by CaM kinase II. We identified serines 262 and 356, which
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were located in the first and fourth repeats, respectively, of tubulin binding sites, as being phosphorylated. As phosphorylation of these sites by other protein kinases reportedly inhibited the binding of tau to microtubules [43], it is reasonable that phosphorylation of tau by CaM kinase II strongly reduced its binding to microtubules (Fig. 7). The reduction may be the main molecular mechanism by which phosphorylation of tau by CaM kinase II inhibits the microtubule assembly [19–21]. In the first study on the primary sequence specificity, the minimum specificity determinant for CaM kinase II was reported to be the sequence RXXS/ TX [35]. From the analysis of phosphorylation sites on vimentin, it was noted that aspartic acid 84 in vimentin acted as an essential determinant for the serine 82 phosphorylation and that another minimum specificity determinant for CaM kinase II was the sequence S/TXD [44]. The study using the peptide library indicated that CaM kinase II preferred lysine as well as arginine at the )3 position and glutamic acid as well as aspartic acid at the +2 position [45]. Furthermore, the hydrophobic amino acids at the )2 position was reported to enhance the phosphorylation [46]. The primary sequences around serines 262 and 356 are consistent with these optimal substrate recognition motifs of CaM kinase II. In contrast, serines 293 and 324, located in the second and third repeats, respectively, of tubulin binding sites, were not phosphorylated by CaM kinase II. Lysine at the +1 position of serine 293 and glycine instead of the acidic amino acids at the +2 position of serine 324 may be the negative determinant for phosphorylation of each site. An immunochemical approach with a monoclonal antibody (12E8) to tau phosphorylated at serine 262 and/ or serine 356 revealed that serine 262 and/or serine 356 were phosphorylated by CaM kinase II [47]. The sites phosphorylated by cyclic AMP-dependent protein kinase (protein kinase A) and protein kinase C have been identified by protein chemical approaches [48,49]. Protein kinase A phosphorylated serines 214, 324, 356, 409, and 416 [49]. However, phosphorylation of serine 262 by protein kinase A was not reported. Protein kinase C phosphorylated serine 324, but phosphorylation of serines 262 and 356 was not detected [48]. As PHF-tau was phosphorylated at serines 262 and 356 but not serine 324 [8,9], it may well be that CaM kinase II is the most likely candidate for involvement in hyperphosphorylation of PHF-tau among these protein kinases. In this context, it is of interest that CaM kinase II immunoreactivity is reportedly increased in neurons of CA1 and subiculum in AD brain [50]. It was reported that tau became long and stiff after phosphorylation by CaM kinase II [51]. It is not clear at present whether phosphorylation of serines 262 and 356 is related to the conformational alterations. Mutational analysis suggested that phosphorylation of serine 262 and/or serine 356 did not alter the electrophoretic mo-
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bility [52]. In contrast, it was reported that conversion of serine 416 to alanine resulted in the loss of the decrease in electrophoretic mobility after phosphorylation by CaM kinase II [40]. Furthermore, conversion of serine 416 to aspartic acid resulted in decreased electrophoretic mobility [53]. These results suggested that the decrease in electrophoretic mobility after phosphorylation by CaM kinase II was due to phosphorylation at serine 416. Unfortunately, our LC/MS analysis failed to identify the phosphorylation of serine 416, because K44 peptide (residues 396–438) obtained by lysyl endoprotease digestion was long and serine 416 was too far from both termini of the peptide. It is important to elucidate whether hyperphosphorylation of serine 416 by CaM kinase II is related to the reduced electrophoretic mobility of PHF-tau for future studies.
Acknowledgments We thank Dr. M. Goedert in the MRC Laboratory of Molecular Biology (Cambridge, UK) for kindly providing the cDNA of hTau40 in the pRK172 vector. This study was supported in part by Grants-in-Aid for Scientific Research and for Scientific Research on Priority from the Ministry of Education, Science, Sports, and Culture of Japan and by a Research Grant from the Human Frontier Science Program (H.Y. and E.M.).
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Phosphorylation of microtubule-associated protein tau by Ca2þ /calmodulin-dependent protein kinase II in its tubulin binding sites Hideyuki Yamamoto,a,*,1 Emiko Yamauchi,b,1 Hisaaki Taniguchi,b Tsunehiko Ono,c and Eishichi Miyamotoa a
c
Department of Pharmacology, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto 860-0811, Japan b Institute for Enzyme Research, University of Tokushima, Tokushima 770-8503, Japan Department of Neuropsychiatry, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto 860-0811, Japan Received 2 August 2002, and in revised form 17 September 2002
Abstract The paired helical filaments (PHF) found in AlzheimerÕs disease (AD) brain are composed mainly of the hyperphosphorylated form of microtubule-associated protein tau (PHF-tau). It is well known that tau is a good in vitro substrate for Ca2þ /calmodulindependent protein kinase II (CaM kinase II). To establish the phosphorylation sites, the longest human tau (hTau40) was bacterially expressed and phosphorylated by CaM kinase II, followed by digestion with lysyl endoprotease. The digests were subjected to liquid chromatography/mass spectrometry. We found that 5 of 22 identified peptides were phosphorylated. From the tandem mass spectrometry, two phosphorylation sites (serines 262 and 356) were identified in the tubulin binding sites. When tau was phosphorylated by CaM kinase II, the binding of tau to taxol-stabilized microtubules was remarkably impaired. As both serines 262 and 356 are reportedly phosphorylated in PHF-tau, CaM kinase II may be involved in hyperphosphorylation of tau in AD brain. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: AlzheimerÕs disease; Calmodulin; Ca2þ /calmodulin-dependent protein kinase II; Paired helical filaments; Tau
Neurofibrillary tangles are one of the pathologic hallmarks of AlzheimerÕs disease (AD),2 and their abundance was reported to correlate with the clinical degree of dementia [1]. The unit fibrils of the neurofibrillary tangles are called paired helical filaments (PHF) and PHF consist of hyperphosphorylated tau (designated PHF-tau) [2–4]. Hyperphosphorylation of tau appears to precede PHF formation [5]. Tau is one of the microtubule-associated proteins and induces microtubule assembly and stabilizes microtubules in vitro [4].
*
Corresponding author. Fax: +81-96-373-5078. E-mail address: [email protected] (H. Yamamoto). 1 These authors contributed equally to this paper. 2 Abbreviations used: AD, AlzheimerÕs disease; CaM kinase II, Ca2þ / calmodulin-dependent protein kinase II; PHF, paired helical filaments; LC/MS, capillary reverse-phase liquid chromatography/electrospray mass spectrometry.
These functions are diminished by phosphorylation with several protein kinases [3]. Tau is the major microtubule-associated protein in axons and appears to play an important role in the stabilization of microtubules during interactions with other cytoskeletal components in axons [6]. Therefore, hyperphosphorylation of tau in AD may disrupt the axonal cytoskeleton and axonal flow, leading to neuronal degeneration [3]. Molecular cloning has identified six tau isoforms in human brain tissue that are produced from a single gene by alternative mRNA splicing [2]. They range from 352 to 441 amino acids in length and differ from each other by the presence of three inserts. Among the isoforms, the longest tau isoform (hTau40) has all three inserts. By direct sequencing of PHF-tau, at least 25 sites have been chemically identified as phosphorylation sites on PHFtau [7–9]. Among the 25 phosphorylation sites, serines 262 and 356, numbered according to hTau40 [10], were located in the tubulin binding sites. The tubulin binding
0003-9861/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 3 - 9 8 6 1 ( 0 2 ) 0 0 5 5 6 - 8
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sites of tau are 18-amino-acid motifs repeated three or four times in the carboxyl-terminal half of the molecule [11]. The phosphorylation of these sites may result in the impairment of tau functions in axons. SDS–PAGE analysis showed that PHF-tau migrated to a higher position than normal tau, and the reduced electrophoretic mobility of PHF-tau appears to be due to hyperphosphorylation of some phosphorylation sites, presumably due to the conformational alterations (for review, see [8,12]). In addition to AD brain, hyperphosphorylated tau is accumulated also in the brains of neurodegenerative diseases known as ‘‘tauopathies,’’ including frontotemporal dementia with parkinsonism linked to chromosome 17, amyotrophic lateral sclerosis/parkinsonism–dementia complex, and progressive supranuclear palsy [13,14]. In order to understand the pathophysiologies of tauopathies as well as AD, it is important to identify protein kinases that are involved in hyperphosphorylation of tau in the brains. Ca2þ /calmodulin-dependent protein kinase II (CaM kinase II) is widely distributed in neuronal tissues and may be involved in a variety of Ca2þ -mediated cellular processes, including regulation of cytoskeletal proteins, gene expression, and synaptic plasticity [15–18]. We previously reported that tau was a good in vitro substrate for CaM kinase II and that its phosphorylation inhibited microtubule assembly [19–21]. It was also reported that phosphorylation of tau by CaM kinase II decreased the electrophoretic mobility [22]. Developments in mass spectrometry made it possible to determine the molecular mass of large proteins with high precision and resolution [23,24]. We have applied the capillary reverse-phase liquid chromatography/electrospray mass spectrometry (LC/MS) method to the identification of phosphorylation sites in multiply phosphorylated proteins [25–27]. In the present work, we tried to identify unequivocally the phosphorylation sites in tau by CaM kinase II using LC/MS analysis for two reasons. First, we intended to examine the possible involvement of CaM kinase II in abnormal hyperphosphorylation of PHF-tau. Second, we wanted to elucidate the molecular mechanisms by which phosphorylation of tau resulted in a diminished capacity to induce microtubule assembly.
Materials and methods Materials [c-32 P]ATP and 125 I-protein A were purchased from NEN; phosphocellulose paper was from Whatman; CaM kinase II substrate peptide syntide-2 was from Bachem; rabbit anti-tau antibody (T-6402) was from Sigma; Microtubule/Tubulin Biochem kit was from Cytoskeleton
(BK015). Tau peptide(401–412) (GDTSPRHLSNVSC, 1372 Da) was synthesized by Fujiya Biolaboratory (Hatano, Japan). Other chemicals used were of analytical grade. Purification of proteins An expression clone of the longest human tau isoform, hTau40 [10], was a gift from Dr. M. Goedert (MRC Laboratory of Molecular Biology, Cambridge, UK). The cDNA of hTau40 was in the pRK172 vector and expressed in the BL21(DE3) Escherichia coli system [28]. Recombinant tau was purified from E. coli cells as described [29]. Tau [30] and calmodulin [31] were purified from bovine brain. CaM kinase II was purified from rat brain [32]. Assay for CaM kinase II The assay system for CaM kinase II contained, in a final volume of 25 ll, the following constituents: 50 mM Hepes, pH 7.5, 10 mM MgðCH3 COOÞ2 , 1 mM CaCl2 , 1.5 lM calmodulin, 0.1 mM [c-32 P]ATP (3000– 5000 cpm/pmol), 50 nM CaM kinase II, and 40 lg=ml recombinant tau or tau purified from bovine brain [33]. After incubation at 30 °C for 60 min, 15 ll of the samples was spotted on phosphocellulose paper squares and processed as described [34]. When the amount of phosphate incorporated into tau peptide(401–412) was determined, 20 ll of the sample was applied to an AG1-X8 column (2 ml) to separate the phosphorylated peptide from [c-32 P]ATP [35]. The phosphate incorporated into the peptide was measured by a liquid scintillation counter. Protease digestion of tau Recombinant tau was phosphorylated as described above except that cold ATP was used instead of [c32 P]ATP for 30 min at 30 °C. The reaction was terminated by heat treatment at 60 °C for 5 min and kept at )80 °C until use. Tau (10 lg) phosphorylated in the presence or absence of cold ATP was digested with 1 lg of lysyl endoprotease (WAKO Pure Chemical Industries, Osaka) in 100 mM Tris–HCl, pH 8.9, in the presence of 2 M urea at 37 °C for 15 h. After the reaction was stopped by addition of trifluoroacetic acid to a final concentration of 0.1%, the peptide mixtures were directly injected into the LC/MS apparatus. Mass spectrometry LC/MS and MS/MS analyses were carried out with an electrospray ionization/quadrupole mass
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spectrometer (PE Sciex API-III) as described previously [25–27].
described [38]. The radioactivity of 125 I was measured using a Bio-Imaging analyzer (FLA-2000; Fujifilm, Tokyo, Japan).
Microtubule cosedimentation assays
Other procedures
Recombinant tau was phosphorylated by 50 nM CaM kinase II for 30 min at 30 °C as described above except that cold ATP was used instead of [c-32 P]ATP. For the control, CaM kinase II was pretreated with heat at 60 °C for 5 min. The reaction was terminated by heat treatment at 60 °C for 5 min. Tubulin purified from bovine brain (5 mg/ml) was incubated in 80 mM Hepes, pH 6.8, 1 mM MgCl2 , 1 mM EGTA, 10% glycerol, and 2 mM GTP at 35 °C for 20 min. The microtubules were diluted to 1.5 mg/ml and taxol was added to a final concentration of 15 lM. Tau (34 lg=ml) was incubated with 0.4 mg/ml microtubules at 35 °C for 10 min in 40 mM Hepes, pH 6.8, 0.5 mM MgCl2 , 0.5 mM EGTA, 5% glycerol, 1 mM GTP, and 7.5 lM taxol, in a final volume of 100 ll. Tau bound to microtubules was separated from unbound tau by centrifugation at 105,000g for 30 min at 30 °C. The supernatant (100 ll) was heat treated in the presence of SDS–PAGE sample buffer [36] at 100 °C for 1.5 min. The pellet was suspended in 100 ll of H2 O and heat treated in the presence of SDS–PAGE sample buffer. Each sample (25 ll) was applied to SDS– PAGE [36], followed by Coomassie brilliant blue R-250 staining or immunoblot analysis. The protein amount of tau in each lane was quantified by scanning using NIH Image (version 1.61). Immunoblot analysis was done by the method of Towbin et al. [37] using 125 I-protein A, as
Protein concentration was determined by the method of Bradford [39] or using the BCA protein assay reagent (Pierce) with bovine serum albumin as a standard. Statistical evaluation Data are expressed as means SE values. The significance of the difference was calculated using StudentÕs t test and p values <0.05 were considered statistically significant.
Results Phosphorylation of tau by CaM kinase II Recombinant tau (hTau40) was phosphorylated by CaM kinase II in a Ca2þ /calmodulin-dependent manner. About 2.6 and 0.04 mol phosphate/mol of tau was phosphorylated by CaM kinase II in the presence and absence of Ca2þ /calmodulin, respectively. When tau purified from bovine brain was phosphorylated by CaM kinase II in the presence of Ca2þ /calmodulin, 7.7 mol phosphate/mol of tau was incorporated, under the same assay conditions. These results suggested that CaM kinase II phosphorylated bovine brain tau more strongly
Fig. 1. Amino acid sequence of tau and the peptides identified by LC/MS analysis. Amino acid sequence of hTau40 [10] is shown with the peptides identified from the LC/MS analysis. The arrows indicate the peptides identified by the LC/MS analysis. The bold arrows indicate phosphorylated peptides. Four tubulin binding sites are boxed and phosphorylated serines 262 and 356 are indicated. Residue numbers are at the end of each line as well as at the residues discussed in the text.
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than bacterially expressed tau, for unknown reasons. We phosphorylated recombinant tau under these conditions except that cold ATP was used instead of [c-32 P]ATP and then carried out the identification of phosphorylation sites.
LC/MS analysis of lysyl endoprotease digest Unphosphorylated and phosphorylated tau proteins digested with lysyl endoprotease were subjected to LC/ MS analysis. The masses of each peptide sequentially eluted from LC were detected online by the mass spectrometer and were compared with the theoretical ones calculated from the amino acid sequence deduced from the cDNA sequence of hTau40 [10]. The totals of 22 peptides were identified from the mass values, as summarized in Fig. 1. When phosphorylated tau was subjected to LC/MS analysis, at least five peptides were accompanied by peptides that were 80, 160, 240, or 320 Da larger than those of the parent peptides (Figs. 2, 4–6). Since transfer of phosphate from ATP to serine or threonine increases the mass of corresponding peptides by 80 Da, these peptides were likely to be phosphorylated. The extent of the phosphorylation estimated from the peak intensities of the deconvoluted mass spectra varied from 15 to 60%, depending on the peptide. In previous work, we demonstrated that the extent of the phosphorylation did not affect ionization efficiency [25]. Identification of phosphorylation sites in tubulin binding sites Among the phosphopeptides identified, we found that the two peptides, namely K23 and K38, originated from the first and fourth repeat, respectively, of tubulin binding sites (Figs. 1 and 2). To identify the exact phosphorylation sites of K23 and K38 peptides, we isolated each peptide by applying the protein digest on reversed-phase liquid chromatography, and fractions containing each peptide were subject to MS/MS analysis (Fig. 3). In the MS/MS spectra of the phosphorylated K23 peptide, the presence of a y-ion series from y1 to y5 corresponding to the masses of the unphosphorylated fragments and the difference between the y5 and the y6 ions that corresponded to a phosphoserine indicated that serine 262 was phosphorylated. The y6 and y7 ions whose masses show dissociation of a phosphate group further confirm the phosphorylation site. The phosphorylation of serine 262 was also confirmed by the difference between the b2 and the b3 ions, which also corresponds to the mass of the phosphoserine (Fig. 3). From these results, we concluded that serine 262 was the sole phosphorylation site in K23 peptide. K38 peptide also contained two possible phosphorylatable residues,
Fig. 2. Deconvoluted electrospray mass spectra of K23 and K38 peptides from unphosphorylated and phosphorylated tau. Tau was phosphorylated in the absence (top) or presence (bottom) of cold ATP as described under Materials and methods. Tau was digested by lysyl endoprotease, and the resulting peptide mixture was subjected to LC/ MS analysis. Among several peaks observed, the peptide 80 Da larger than K23 or K38 peptide is observed only in the bottom graphs.
serine 356 and threonine 361. In the MS/MS spectra of phosphorylated K38 peptide, fragment ions containing threonine 361 could not be obtained in the present study, mainly because the residue was apart from both termini of the peptide; the eighth and ninth residue from the N-terminus and C-terminus, respectively. However, the mass difference between b2 and b3 in the spectra corresponds to phosphoserine, suggesting that serine 356 was the phosphorylation site in this peptide (Fig. 3). The primary sequence around serine 356 (KIGSLDN) resembles that around serine 262 (KIGSTEN). Therefore, together with the results in Fig. 3, it was highly
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Fig. 3. Tandem mass spectra of K23 and K38 peptides. The singly charged ion of each peptide was subjected to the MS/MS analysis. Fragment ions observed are indicated above and below the peptide sequence. The phosphorylated serine residues are indicated.
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Fig. 4. Deconvoluted electrospray mass spectra of K2 peptide from unphosphorylated (top) and phosphorylated (bottom) tau. The lysyl endoprotease digests of tau were injected into the LC/MS apparatus. The peptide 80 Da larger than K2 peptide is observed only in the bottom graph.
possible that serine 356 was the sole phosphorylation site in K38 peptide. Phosphorylation of K2, K16, and K44 peptides In addition to K23 and K38 peptides, the LC/MS analysis indicated that the other three peptides were phosphorylated. Although exact phosphorylation sites of each peptide could not be identified in the present study, K2 peptide corresponded to residues 25–44 of tau, and a low amount of K2 peptide was singly phosphorylated (Fig. 4). As there were two threonines but no serine in the peptide, one of the threonine residues appeared to be phosphorylated. We have reported that CaM kinase II phosphorylates tau slightly at threonines in addition to serines by phosphoamino acid analysis [20]. It was detected that K16 peptide contained doubly phosphorylated species in addition to singly phosphorylated species (Fig. 5). Therefore, at least two residues are phosphorylated among eight serines and four threonines in K16 peptide. K44 peptide contained 10 serines and 3 threonines and was phosphorylated at several sites (Fig. 6). Pearson et al. [35] reported that a minimum specificity determinant for CaM kinase II was the sequence RXXS/TX (X
Fig. 5. Deconvoluted electrospray mass spectra of K16 peptide from unphosphorylated (top) and phosphorylated (bottom) tau. The lysyl endoprotease digests of tau were injected into the LC/MS apparatus. The peptides 80 and 160 Da larger than K16 peptide are observed only in the bottom graph.
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Fig. 7. Effects of phosphorylation of tau by CaM kinase II on the interaction with taxol-stabilized microtubules. After tau was phosphorylated in the presence of CaM kinase II (P-Tau) or heat-treated CaM kinase II (Tau), tau was incubated with polymerized tubulin, and the mixture was subjected to ultracentrifugation as described under Materials and methods. (A) The pellet was applied to SDS–PAGE, followed by protein staining. (B) Immunoblot analysis of the supernatant was carried out using anti-tau antibody. We repeated the same experiments at least three times with reproducible results, and representative results are shown. Fig. 6. Deconvoluted electrospray mass spectra of K44 peptide from unphosphorylated (top) and phosphorylated (bottom) tau. The lysyl endoprotease digests of tau were injected into the LC/MS apparatus. The peptide 320 Da larger than K44 peptide is observed in addition to the peptides 80, 160, and 240 Da larger only in the bottom graph.
is a hydrophobic amino acid). However, it was reported that serine 409, which was in the RXXS/TX motif, was not phosphorylated by CaM kinase II [40]. In order to confirm their report, we synthesized tau peptide(401– 412) and examined the phosphorylation. We found that tau peptide(401–412) was not phosphorylated at all. Together with the previous reports, these results suggested that serine 409 was not a phosphorylation site in K44 peptide. Inhibition of binding of tau to microtubules by phosphorylation To determine whether phosphorylation of tau by CaM kinase II inhibited the interaction with microtubules, a cosedimentation assay of recombinant tau and taxol-stabilized microtubules was carried out. When unphosphorylated tau was incubated with microtubules, about 90% of tau was cosedimented with microtubules (Fig. 7A). In contrast, when phosphorylated tau was used, the amount of tau in the pellet was remarkably reduced. Furthermore, the supernatant was subjected to immunoblot analysis using anti-tau antibody (Fig. 7B). The amount of tau in the supernatant was increased 3:0 0:5-fold (n ¼ 3, p < 0:01) by phosphorylation. It should be noted that the electrophoretic mobility of tau slightly but clearly decreased after phosphorylation by CaM kinase II as reported [22] (Fig. 7). When bovine brain tau was incubated with microtubules, about 65% of the tau was cosedimented with microtubules before phosphorylation by CaM kinase II. Immunoblot anal-
ysis indicated that the amount of bovine brain tau in the supernatant was increased 1:4 0:1-fold (n ¼ 3, p < 0:05) by phosphorylation. These results suggest that the effect of phosphorylation of bovine brain tau was weaker than that of recombinant tau.
Discussion It is well known that tau is phosphorylated in vivo at multiple sites [41,42]. Therefore, to analyze unequivocally the phosphorylation sites by CaM kinase II, it was necessary to use bacterially expressed tau instead of tau purified from brain. As expected, essentially no phosphate was incorporated into recombinant tau. However, recombinant tau was less efficiently phosphorylated by CaM kinase II than bovine brain tau. Quantitative comparisons of their tubulin binding affinity revealed that recombinant tau had higher affinity to microtubules than bovine brain tau and that the effect of phosphorylation of recombinant tau was stronger than that of bovine brain tau. The reasons for these differences between recombinant tau and bovine brain tau are not clear at present. The folding and/or other posttranslational modifications of bacterially expressed tau may be different from those of the native tau. Although 2.6 mol phosphate/mol of tau was detected to be incorporated by the assay system for CaM kinase II, LC/MS analysis revealed that more than five sites were phosphorylated. Considering the overall stoichiometry, the stoichiometry of phosphorylation of some sites must be low. These results suggest that the LC/MS analysis is very sensitive. It has been reported that CaM kinase II phosphorylates tau exclusively at serine 416 [40]. Our analysis clearly showed that other sites were phosphorylated by CaM kinase II. We identified serines 262 and 356, which
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were located in the first and fourth repeats, respectively, of tubulin binding sites, as being phosphorylated. As phosphorylation of these sites by other protein kinases reportedly inhibited the binding of tau to microtubules [43], it is reasonable that phosphorylation of tau by CaM kinase II strongly reduced its binding to microtubules (Fig. 7). The reduction may be the main molecular mechanism by which phosphorylation of tau by CaM kinase II inhibits the microtubule assembly [19–21]. In the first study on the primary sequence specificity, the minimum specificity determinant for CaM kinase II was reported to be the sequence RXXS/ TX [35]. From the analysis of phosphorylation sites on vimentin, it was noted that aspartic acid 84 in vimentin acted as an essential determinant for the serine 82 phosphorylation and that another minimum specificity determinant for CaM kinase II was the sequence S/TXD [44]. The study using the peptide library indicated that CaM kinase II preferred lysine as well as arginine at the )3 position and glutamic acid as well as aspartic acid at the +2 position [45]. Furthermore, the hydrophobic amino acids at the )2 position was reported to enhance the phosphorylation [46]. The primary sequences around serines 262 and 356 are consistent with these optimal substrate recognition motifs of CaM kinase II. In contrast, serines 293 and 324, located in the second and third repeats, respectively, of tubulin binding sites, were not phosphorylated by CaM kinase II. Lysine at the +1 position of serine 293 and glycine instead of the acidic amino acids at the +2 position of serine 324 may be the negative determinant for phosphorylation of each site. An immunochemical approach with a monoclonal antibody (12E8) to tau phosphorylated at serine 262 and/ or serine 356 revealed that serine 262 and/or serine 356 were phosphorylated by CaM kinase II [47]. The sites phosphorylated by cyclic AMP-dependent protein kinase (protein kinase A) and protein kinase C have been identified by protein chemical approaches [48,49]. Protein kinase A phosphorylated serines 214, 324, 356, 409, and 416 [49]. However, phosphorylation of serine 262 by protein kinase A was not reported. Protein kinase C phosphorylated serine 324, but phosphorylation of serines 262 and 356 was not detected [48]. As PHF-tau was phosphorylated at serines 262 and 356 but not serine 324 [8,9], it may well be that CaM kinase II is the most likely candidate for involvement in hyperphosphorylation of PHF-tau among these protein kinases. In this context, it is of interest that CaM kinase II immunoreactivity is reportedly increased in neurons of CA1 and subiculum in AD brain [50]. It was reported that tau became long and stiff after phosphorylation by CaM kinase II [51]. It is not clear at present whether phosphorylation of serines 262 and 356 is related to the conformational alterations. Mutational analysis suggested that phosphorylation of serine 262 and/or serine 356 did not alter the electrophoretic mo-
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bility [52]. In contrast, it was reported that conversion of serine 416 to alanine resulted in the loss of the decrease in electrophoretic mobility after phosphorylation by CaM kinase II [40]. Furthermore, conversion of serine 416 to aspartic acid resulted in decreased electrophoretic mobility [53]. These results suggested that the decrease in electrophoretic mobility after phosphorylation by CaM kinase II was due to phosphorylation at serine 416. Unfortunately, our LC/MS analysis failed to identify the phosphorylation of serine 416, because K44 peptide (residues 396–438) obtained by lysyl endoprotease digestion was long and serine 416 was too far from both termini of the peptide. It is important to elucidate whether hyperphosphorylation of serine 416 by CaM kinase II is related to the reduced electrophoretic mobility of PHF-tau for future studies.
Acknowledgments We thank Dr. M. Goedert in the MRC Laboratory of Molecular Biology (Cambridge, UK) for kindly providing the cDNA of hTau40 in the pRK172 vector. This study was supported in part by Grants-in-Aid for Scientific Research and for Scientific Research on Priority from the Ministry of Education, Science, Sports, and Culture of Japan and by a Research Grant from the Human Frontier Science Program (H.Y. and E.M.).
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