©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Proline-directed and Non-proline-directed Phosphorylation of PHF-tau (*)

(Received for publication, August 26, 1994; and in revised form, October 21, 1994)

Maho Morishima-Kawashima (1) Masato Hasegawa (1) Koji Takio (2) Masami Suzuki (3) Hirotaka Yoshida (1) Koiti Titani (3) Yasuo Ihara (1)(§)

From the  (1)Department of Neuropathology, Institute for Brain Research, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan, the (2)Division of Biomolecular Characterization and Biodesign Research Group, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-01, Japan, and the (3)Division of Biomedical Polymer Science, Institute for Comprehensive Medical Science, School of Medicine, Fujita Health University, Toyoake, Aichi 470-11, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To gain insight into the abnormal phosphorylation of PHF-tau, we have determined the phosphorylation sites by identifying phosphopeptides by means of ion spray mass spectrometry followed by sequencing of ethanethiol-modified peptides. Nineteen sites have been identified; all but Ser-262 are localized to the amino- and carboxyl-terminal flanking regions of the microtubule-binding domain. Eleven sites correspond to fetal type sites. Unexpectedly, 10 are non-proline-directed, whereas the others are proline-directed. Thus, the abnormal phosphorylation of PHF-tau can be considered to consist of fetal type phosphorylation and additional proline-directed and non-proline-directed phosphorylation. This non-fetal type phosphorylation may provide PHF-tau with the unusual characteristics.


INTRODUCTION

Abundant neurofibrillary tangles throughout the cortex are one of the hallmarks of Alzheimer's disease (AD). (^1)Their abundance is reported to be correlated with the clinical degree of dementia (1) and appears to be closely related to the extent of neuronal loss found in AD brain(2, 3) . In contrast to beta-amyloid that is currently believed to be directly involved in the pathogenesis of AD (for reviews see Refs. 4 and 5), the tangle formation is a late event (6) and presumably a final common pathway to neuronal death in subsets of neurons.

Paired helical filaments (PHF), unit fibrils of the neurofibrillary tangle, have an unusual morphology. They are apparently composed of two 10-nm filaments wound around each other, giving a half-periodicity of 80 nm. This kind of unusual fiber has never been observed in other mammals, including nonhuman primates, and is unique to humans. PHF also show unusual biochemical characteristics: insolubility and remarkable resistance to proteases, which prevented their extensive biochemical analysis for a long time. It was only 6 years ago that SDS-insoluble PHF were clearly shown to be composed of tau, a microtubule-associated protein (7, 8) and ubiquitin, an essential element in the cytosolic ATP-dependent protein degradation system(9, 10) . The former, tau, was originally isolated as a tubulin assembly-promoting factor(11, 12) .

PHF undergo chemical evolution in situ; PHF at their late stage are SDS-insoluble and composed of the carboxyl third of tau and ubiquitin(7, 13) , whereas PHF at their early stage, which are found to be SDS-soluble, consist only of hyperphosphorylated full-length tau (14, 15) , called A68 (15, 16) or PHF-tau(17) . PHF-tau is characterized by (i) an unusually slow mobility on SDS-PAGE, (ii) loss of the tubulin assembly-promoting activity, and (iii) normalization of these two parameters by dephosphorylation(18, 19, 20) . Consistent with this, we were unable to detect any abnormal modification in PHF-tau other than phosphorylation; N-acetylation at the amino terminus and deamidation at some asparaginyl residues found in PHF-tau are also present in normal tau(21) .

One possible scenario in the PHF formation is as follows. A certain protein kinase(s) activated or up-regulated during AD phosphorylates tau to such an extent that it loses the affinity for microtubules, which in turn are destabilized(22) . Because of its negligible affinity for tubulin, PHF-tau molecules would interact with each other, probably at the microtubule-binding domain to form dimers and finally PHF(17, 23, 24) . According to this scheme, identification of the involved protein kinase(s) and also its counteracting protein phosphatase(s) (25, 26, 27) is of primary importance, because this perturbation in the phosphorylation cascade initiates successive biochemical events leading to PHF formation and simultaneously brings about the microenvironmental alterations that cause tangle-bearing neurons to degenerate.

For this reason, many investigators have been concerned about the nature of PHF-tau phosphorylation and thus the potentially involved protein kinases and/or protein phosphatases. Although the unambiguous determination of the in vivo phosphorylation sites should be the first step toward addressing the above issue, to date only a few sites have been definitely identified as abnormal phosphorylation sites: Thr-231, Ser-235, and Ser-262, with the last being only partially phosphorylated (21) (the numbering is according to a 441-residue human tau isoform; (28) ). We previously found that PHF-tau is highly phosphorylated in the tau 1 portion (29) and carboxyl-terminal portion outside the binding domain but failed to determine the exact phosphorylation sites(21) . These two portions were not effectively subjected to protein chemical analyses because (i) both, relatively large peptides, provide no significant mass spectral signals, unless dephosphorylated; (ii) they are unusually resistant to proteases; and (iii) smaller phosphopeptides, even if generated, are not or are only poorly recovered from HPLC columns (see (21) ). Consequently, several likely phosphorylation sites have been postulated solely on an immunochemical basis using phosphorylation-dependent PHF monoclonals and mutated recombinant tau(30, 31, 32) . However, this approach invariably leaves some ambiguity regarding the exact sites, and furthermore, entirely depends on the available phosphorylation-dependent PHF monoclonals; it is quite possible that there exist some/many abnormal phosphorylation sites left unrecognized by a panel of PHF monoclonals. Because of this, we have been attempting to overcome the difficulties in applying the protein chemical procedures to the two highly phosphorylated portions in PHF-tau. Here we show that the two portions of PHF-tau are phosphorylated on non-proline-directed sites as well as proline-directed sites.


EXPERIMENTAL PROCEDURES

Purification of PHF-tau from AD Brain

PHF-tau was purified essentially as described previously(21) . The Sarkosyl pellets were suspended in a small volume of 50 mM Tris-HCl (pH 7.6) and dissolved with 6 M guanidine HCl for further purification. The guanidine HCl suspension was centrifuged at 450,000 times g for 30 min in a CS-120 microcentrifuge (Hitachi). The supernatants were treated with iodoacetate after reduction and fractionated on TSK gel G-3000 SWXL columns (7.8 times 300 mm times 2; Tosoh) arrayed in tandem equilibrated with 6 M guanidine HCl in 10 mM phosphate buffer (pH 6.0) at a flow rate of 0.5 ml/min. The TSK fractions containing PHF-tau were further purified on an Aquapore RP-300 column (2.1 times 30 mm, Applied Biosystems) by HPLC (Hewlett-Packard model 1090M), which was developed with a linear gradient of 20-40% acetonitrile in 0.1% aqueous trifluoroacetic acid in 10 min at a flow rate of 0.2 ml/min.

On several occasions, the Sarkosyl pellets were prepared in the presence of protein phosphatase inhibitors including 50 mM tetrasodium pyrophosphate, 50 mM KF, 50 mM imidazole, 50 mM beta-glycerophosphate, 100 mM sodium orthovanadate, and 2 µM okadaic acid.

Crude PHF-tau for immunoblotting was prepared according to the method of Greenberg and Davies (14) with minor modifications. The supernatant of brain homogenates after centrifugation at 27,200 times g was brought to 1% Sarkosyl and incubated at 37 °C for 1 h. Sarkosyl-insoluble pellet was collected by centrifugation at 100,000 times g for 35 min and suspended in 0.7% formic acid, and the suspension was heated to 100 °C for 5 min. After centrifugation, aliquots of the supernatant were subjected to SDS-PAGE.

Purification of Fetal tau from Neonatal Rat Brain

Fetal (rat) tau was purified from 1-day-old rat brains according to the protocol described previously(7) . The supernatants prepared from neonatal brain homogenates were fractionated with ammonium sulfate (33-50%) and heated to 90 °C for 5 min. After centrifugation, cleared supernatants were applied onto a phosphocellulose (P11, Whatman) column, and after being washed with 0.1 M NaCl, crude fetal tau was eluted with 0.3 M NaCl. Crude fetal tau was further purified as described above.

Proteolytic Digestion of tau 1 Portion and Carboxyl-terminal Portion of PHF-tau

Appropriate amounts of highly purified PHF-tau were digested with Achromobacter lyticus protease I at an enzyme-to-substrate ratio of 1:100 (w/w) at 37 °C overnight. A. lyticus protease I peptides were fractionated on a Superspher RP-Select B column (2 times 119 mm, Merck) with a gradient of 0-48% acetonitrile in 0.1% trifluoroacetic acid in 24 min at a flow rate of 0.2 ml/min. Those fractions containing tau 1 and carboxyl-terminal portions (21) were pooled and subjected to further digestion with other proteases. The tau 1 portion was further digested with immobilized trypsin (Pierce) at an enzyme:substrate ratio of 1:2 to 1:5. The digest was fractionated on the same column with a gradient of 0-32% acetonitrile in 0.1% trifluoroacetic acid in 16 min. Each fraction was subjected to amino acid sequencing after ethanethiol modification and ion spray mass spectrometry (ISMS). The carboxyl-terminal portion was cleaved with Pseudomonas fragi endoproteinase AspN (1:10; Boehringer Mannheim) and then with immobilized trypsin (1:1 to 1:10). The digest was fractionated as above with the same gradient in 32 min and subjected to further analysis.

Amino Acid Sequence and Mass Spectrometric Analyses of Proteolytic Peptides

Fractionated peptides were sequenced on an Applied Biosystems 477A protein sequencer equipped with an on-line 120A PTH Analyzer or on an Applied Biosystems 473A protein sequencer. Mass spectral analysis was performed by ISMS on a PE-SCIEX API III biomolecular mass analyzer (triple stage quadrupole mass spectrometer) equipped with a standard atmospheric pressure ion source, as described previously(21) . For unknown reasons, highly phosphorylated peptides that were confirmed by sequencing often gave no significant ISMS signals. For certain phosphopeptides, collision mass spectra were obtained. In this manner Ser-396, -400, -409, and -422 were determined as the phosphorylation sites (see Table 1).



Determination of the Phosphorylation Sites

Phosphopeptides were subjected to sequence analysis after conversion to S-ethylcysteine peptides(33, 34) . In general, no peak corresponding to S-ethylcysteine was observed either by the modification of nonphosphorylated counterparts or in the cycles other than in the position of phosphoserine. Related to this, it should be noted that an amino-terminally located phosphoserine cannot be identified as S-ethylcysteine(33) . Phosphothreonine was indirectly assigned when (i) a particular phosphopeptide (determined by ISMS) contained the Ser or Thr residue; (ii) in the cycle at Thr, PTH-threonine yield was unusually poor as compared with that from the nonphosphorylated counterpart; and (iii) the assignment was compatible with ISMS data if available(34) .

Antibodies to Synthetic Phosphopeptides and Immunochemical Methods

The following phosphopeptides were synthesized and conjugated with KLH through amino- or carboxyl-terminal cysteine (Fujiya Bioscience Lab, Kanagawa): SPRHLS(P)NVSSTC, CGGHLSNVSS(P)TGSID, CGGRSRTPS(P)LPTPP, and CGGIDMVDS(P)PQLAT, where S(P) represents phosphoserine. One mg (as peptide) of each immunogen was injected into a rabbit, which received every 2 weeks 0.5 mg of immunogen as booster starting from 4 weeks after the first immunization. The titers of antisera were assessed by enzyme-linked immunosorbent assay, and rabbits were bled; the obtained antisera were designated AP409, AP413, AP214, and AP422, corresponding to the above four peptides, respectively. AP413, AP214, and AP422 were phosphopeptide-specific, whereas AP409 was reactive with both phospho- and nonphosphopeptides. AP409 became phosphopeptide-specific by passing through a nonphosphopeptide-immobilized column. Each antiserum was reactive with a phosphopeptide used as immunogen but not with three other phosphopeptides.

Other antibodies used were M4 and C5, which most likely recognize the phosphorylation on Thr-231 and Ser-396, respectively(35) . Immunoblotting was performed as described(13) .


RESULTS

Peptide Maps Suggest That tau 1 and Carboxyl-terminal Portions of PHF-tau Are More Phosphorylated Than Those of Fetal tau

We previously showed that two PHF polyclonals and two PHF monoclonals, both of which are phosphorylation-dependent and have distinct specificities, exclusively or preferentially label fetal tau but hardly act on adult tau(35, 36) . Because fetal tau is much easier to analyze, we first determined the in vivo phosphorylation sites of fetal tau(34) . The results indicate that (i) the tau 1 portion of fetal tau is phosphorylated on four Ser/Thr sites at most, and its carboxyl-terminal portion on five sites; and (ii) of 12 phosphorylation sites 8 are proline-directed and 4 are non-proline-directed (see Fig. 4; (34) ). (^2)


Figure 4: Reported phosphorylation sites of tau in vivo and by various protein kinases. a, (34) ; Ser-409 and -413 have been newly identified as the phosphorylation sites^2; b, tau protein kinase I/glycogen synthese kinase 3beta, ((45) ); c, glycogen synthase kinase 3alpha, ( (66) and (67) ); d, ((58) ); tau protein kinase II; e, (68) ; f, mitogen-activated protein kinase ((69) ); g, (59) ; h, (43) and (59) ; i, (70) ; j, protein kinase C ((71) ); k, Ca/calmodulin-dependent protein kinase ((22) ). *, a phosphorylated site.



We investigated and established the appropriate conditions under which the two portions from PHF-tau are effectively and, most importantly, reproducibly cleaved off. The tau 1 portion from PHF-tau was almost completely refractory to chymotrypsin, prolylendopeptidase, and pepsin even at high enzyme-substrate ratios. The carboxyl-terminal portion was also unusually resistant to trypsin, pepsin, AspN, and V8 protease. After trial and error, we found that (i) the tau 1 portion can be cleaved with immobilized trypsin; and (ii) the carboxyl-terminal portion generates phosphopeptides upon digestion with a high ratio of AspN followed by immobilized trypsin. Under these cleavage conditions, these two portions have been successfully digested and recovered. The choice of proteases for these portions differs from that for fetal tau (see (34) ); the two portions from PHF-tau are far more resistant to proteases (data not shown).

The peptide maps clearly show great differences between PHF-tau and fetal (rat) tau (Fig. 1, A-D), whereas the primary structures in the tau 1 and carboxyl-terminal portions are identical except for 1 residue, Asp-193 in PHF-tau for Glu in fetal rat tau(37) . The tau 1 map from PHF-tau and, to a lesser extent, the carboxyl-terminal map are characterized by the presence of poorly separated, broad peaks and very poor recovery from the HPLC column (Fig. 1, A and B). Since by dephosphorylation those portions can be easily digested with proteases, and generated peptides elute in distinct peaks (data not shown), the HPLC profiles strongly suggest that both tau 1 and carboxyl-terminal portions of PHF-tau are more phosphorylated than those in fetal tau; such excess phosphorylation most likely prevents specific cleavage, which provides an apparently distinct HPLC profile. Poor separation of generated peptides presumably reflects incomplete digestion and heterogeneous phosphorylation of those portions (see below).


Figure 1: Peptide maps of the tau 1 portion (A, C) and carboxyl-terminal portion (B, D) from PHF-tau (A, B) or fetal (rat) tau (C, D). The tau 1 and carboxyl-terminal portions generated with Achromobacter lyticus protease I were further digested. The tau 1 portion was digested with immobilized trypsin, whereas the carboxyl-terminal portion was digested with AspN followed by immobilized trypsin. The same amount of each digest was fractionated by reverse phase HPLC. Note that there are great differences between PHF-tau and fetal tau in the HPLC profiles. Fractions corresponding to numbered peaks were subjected to sequence and ISMS analyses (see Table 1). Outside the figure in A, one peak eluted at 6.5 min and was determined to be EPK (residues 222-224).



Isolation of Phosphopeptides from tau 1 and Carboxyl-terminal Portions and Determination of Phosphorylation Sites

The HPLC fractions containing phosphopeptides were identified by the extra mass number of n times 80 by ISMS (see Table 1), and the phosphorylation sites were determined by the method of S-ethylcysteine modification(33) . PHF-tau purified from three different AD brains provided very similar HPLC profiles for tau 1 and carboxyl-terminal portions. We isolated 15 phosphopeptides from each of the tau 1 and carboxyl-terminal portions (Table 1). Peak 8 from the tau 1 portion of PHF-tau contained two phosphopeptides, peptides 195-221 and 210-221 (Table 1). The former peptide specific for PHF-tau was generated because of the failure of cleavage at the carboxyl sides of the two arginyl residues. The peaks for PTH-S-ethylcysteine were present in the 4th, 5th, 8th, 14th, 16th, and 20th Edman degradation cycles (data not shown; see Table 1). This indicated that Ser-198, -202, -208, -210, and -214 in peptide 195-221 are phosphorylated. PTH-S-ethylcysteine in the fifth cycle does not distinguish between Ser-199 in peptide 195-221 and Ser-214 in peptide 210-221. In this regard, the sequencing of peak 5 was informative, showing that Ser-199 is also phosphorylated (Table 1). In the third and eighth cycles of peak 8, PTH-threonine yield was unusually poor (data not shown; Table 1), suggesting that Thr-212 and -217 in peptide 210-221 are phosphorylated. This is also supported by ISMS; peptide 210-221 should carry four phosphates, which should be assigned to Ser-210, Ser-214, Thr-212, Thr-217, or Thr-220.

Regarding the carboxyl-terminal portion, ISMS (and sequencing) of peaks 1 and 3 indicated that Ser-396, Ser-400, Thr-403, and Ser-404 are phosphorylated (Table 1). By sequencing of peak 7 (not detected by ISMS), PTH-S-ethylcysteine was identifiable in the third, sixth, and seventh degradation cycles, indicating that Ser-409, -412, and -413 are phosphorylated. Sequencing of peak 19 provided PTH-S-ethylcysteine in the second cycle, and thus Ser-422 is also phosphorylated.

Overall, Ser-198, -199, -202, -208, -210, and -214 in the tau 1 portion were assigned to probable phosphorylation sites and Thr-212 and -217 to possible sites (Table 1; Fig. 2). Likewise, in the carboxyl-terminal portion, Ser-396, Ser-400, Thr-403, Ser-404, Ser-409, Ser-412, Ser-413, and Ser-422 were assigned to probable sites. Since Thr-231, Ser-235, and Ser-262 are also the sites for abnormal phosphorylation(21) , PHF-tau is phosphorylated on 19 Ser/Thr sites at most; 10 sites are located in the amino-terminal flanking region of the microtubule-binding domain and eight sites in the carboxyl-terminal flanking region. Nine sites are proline-directed, whereas the others are non-proline-directed. Eleven phosphorylation sites including Ser-198, Ser-199, Ser-202, Thr-217, Thr-231, Ser-235, Ser-396, Ser-400, Ser-404, Ser-409, and Ser-413 are indeed shared by fetal tau (see Fig. 4; (34) ),^2 which provides a rationale for the immunochemical similarities between PHF-tau and fetal tau(35, 36) . A minor fraction of Thr-181, that is phosphorylated in fetal tau (34) appeared to be phosphorylated, but its extent was almost the same as that in normal tau (data not shown). PHF-tau-specific phosphorylation sites that are not found in fetal tau include Ser-208, Ser-210, Thr-212, Ser-214, Ser-262, Thr-403, Ser-412, and Ser-422, with two sites, Thr-212 and Ser-422, being proline-directed and the remaining six being non-proline-directed.


Figure 2: Schematic illustration of phosphorylation sites in PHF-tau. The shaded bar represents a 441-residue tau isoform(28) . Open boxes indicate homologous 18-amino acid repeats. Boldface letters and P denote phosphorylation sites and covalently bound phosphate, respectively.



To confirm the above, we raised several phosphorylation-site-specific antibodies and examined them immunochemically (Fig. 3). AP214, AP409, AP413, and AP422 were raised against synthetic peptides carrying phosphoserine 214, 409, 413, and 422, respectively. AP214 stained many neurofibrillary tangles and senile plaque neurites but few curly fibers. The others intensely stained all three PHF-containing structures as well (data not shown). On the immunoblot, AP409 and AP413 labeled both fetal tau and PHF-tau (and smear), as expected, whereas AP422 labeled only PHF-tau but not fetal tau (Fig. 3). AP214 reacted only faintly with fetal tau or PHF-tau (data not shown). This may suggest that Ser-214 is a very minor site for phosphorylation. Also it is possible that the extent of phosphorylation at Ser-214 varies greatly from case to case or is profoundly affected by postmortem dephosphorylation (see below).


Figure 3: Immunoblot of fetal tau and PHF-tau with C5 and two phosphopeptide-specific antibodies. Fetal tau and crude PHF-tau were prepared as described under ``Experimental Procedures.'' One-half µg of fetal tau (a) and 3.5 µg of PHF-tau fraction (b) were electrophoresed on a 10% polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Immobilon; Millipore Corp.), followed by immunostaining with C5(A), AP413(B), and AP422(C).



It should be noted that the above Ser/Thr sites are not necessarily uniformly phosphorylated among PHF-tau molecules (Table 1). Major phosphorylation sites are Ser-198, Ser-199, Ser-202, Thr-231, Ser-235, Ser-396, Ser-404, Ser-409, Ser-413, and Ser-422; a trace or only a small amount of nonphosphorylated counterparts for those sites were detected. Other sites appear not to be completely phosphorylated because their nonphosphorylated counterparts were always detected among other HPLC fractions (Table 1). Peptide 195-209 bears one to four or more covalently bound phosphates (Table 1). The monophosphorylated peptide 195-209 could be a mixture of three different peptides phosphorylated on Ser-198, Ser-199, or Ser-202 (peak 7 in Table 1; see also (34) ). The heterogeneity in the phosphorylation varies to some extent from case to case (data not shown). However, regarding the extent of phosphorylation on a given site, even on fetal type sites, PHF-tau is much more phosphorylated than fetal tau; for instance, Thr-231 and Ser-396 in fetal tau are phosphorylated 30% (see also (34) ).

The above observation raises the possibility that these partially phosphorylated Ser/Thr sites were originally completely phosphorylated but dephosphorylated by protein phosphatases during the post-mortem period and/or the preparation of PHF-tau. We have examined the latter possibility using four monoclonals, tau 1, AT8, M4, and C5; tau 1 is reported to recognize the nonphosphorylated state on Ser-199/-202 (38) or Ser-199(39) , whereas AT8 recognizes phosphorylation on Ser-202 (32) . The Sarkosyl pellets were prepared from AD brain in the presence or absence of various protein phosphatase inhibitors (see ``Experimental Procedures''), and the effects of the inhibitors on those phosphorylation sites were assessed. tau 1, M4, and C5 immunoreactivities showed no consistent alterations with or without those inhibitors. One area from one case suffered a considerable loss of AT8 immunoreactivities when prepared without inhibitors, but this never occurred in other areas in the same brain and other AD brains even after long preincubation. Although we cannot rule out the possibility that some dephosphorylation occurs during the preparation of PHF-tau, the above result suggests that it may not be to a large extent (see also (40) ). Related to this, it is of note that possibly Thr-217 in the tau 1 portion (data not shown), definitely Thr-231(21) , and Thr-403 and Ser-404 in the carboxyl-terminal portion (data not shown) are difficult to dephosphorylate by alkaline phosphatase treatment at 37 °C. This suggests that some of the phosphorylation sites are inaccessible by phosphatases and thus may faithfully reflect the in vivo phosphorylation state.


DISCUSSION

In this work we have rigorously determined the abnormal phosphorylation sites of PHF-tau by means of ISMS and sequencing of S-ethylcysteine peptides. Some important characteristics of PHF-tau phosphorylation emerge ( Fig. 2and Fig. 4): (i) a large number of Ser/Thr sites are phosphorylated, and this number is much larger than that of fetal tau (19 versus 12); (ii) all of the phosphorylation sites except Ser-262 are clustered in the two portions common to all six isoforms outside the microtubule-binding domain, especially in its amino-terminal flanking region (tau 1 portion); (iii) 11 of the sites are shared by fetal tau but are phosphorylated to a much larger extent; and most unexpectedly (iv) 10 sites are non-proline-directed, most of which are not phosphorylated in fetal tau and are therefore specific for PHF-tau.

Here we have focused on the phosphorylation sites in the tau 1 and carboxyl-terminal portions, because these two portions along the PHF-tau molecule were known to be the most highly phosphorylated(21) . This does not exclude the possibility that a small fraction of certain Ser/Thr residues outside those portions are phosphorylated, which can be detected by a sensitive immunochemical procedure. Ser-46 in the amino-terminal short insert (41) and Thr-123 in the adjacent portion (42) may be among such residues. Ser-137 was suggested by sequencing as one of the major phosphorylation sites in PHF-tau(15) , but we are uncertain about this because a particular peptide containing the residue was not recovered from an HPLC column(21) . Within the tau 1 portion, Thr-220 may be a phosphorylation site (Footnote i to Table 1). This residue is located penultimate to the carboxyl-terminal end of the peptide, and it is difficult to confirm the phosphorylation.

Although proline-directed phosphorylation of PHF-tau is currently highlighted, the present work has clearly shown that a number of non-proline-directed sites are also phosphorylated in PHF-tau. This unexpected result would never have been obtained using an indirect immunochemical approach; almost all well characterized PHF monoclonals are directed toward proline-directed phosphorylation(30, 31, 32, 35, 43) . Overall, the phosphorylation of PHF-tau consists of fetal type phosphorylation, additional proline-directed phosphorylation, and other non-proline-directed phosphorylation ( Fig. 2and Fig. 4). Thus, the latter two characterize PHF-tau.

Recent data (34) strongly suggest that fetal tau is phosphorylated by both tau protein kinase I/glycogen synthase kinase 3beta (44, 45, 46) and tau protein kinase II(47) . tau protein kinase II is a heterodimer, and the 30-kDa subunit (catalytic subunit) is homologous to cyclin-dependent kinase 5(48, 49, 50, 51) . It may be claimed that mitogen-activated protein kinase is also involved in the fetal type phosphorylation, because this and the former two kinases share some characteristics: abundance in neurons (52, 53) and association with microtubules(54, 55) . However, it should be noted that the three non-proline-directed sites, Ser-198, -400, and -409 become the motif for glycogen synthase kinase 3 when Ser-202, -404, and -413 are phosphorylated as in fetal tau(34, 56, 57) . In addition, Ser-413, a non-proline-directed site, which is found to be only partly phosphorylated in fetal tau, is phosphorylated by tau protein kinase I/glycogen synthase kinase 3beta in vitro(58) ^2; the glycogen synthase kinase 3 motif is not necessarily proline-directed(57) . Furthermore, the tau phosphorylation by tau protein kinase I/glycogen synthase kinase 3beta is enhanced by prior phosphorylation by tau protein kinase II(47) .

Besides fetal type sites, there are eight phosphorylation sites in PHF-tau. Among them, Ser-208 and -210 become the glycogen synthase kinase 3 recognition motif (S*XXXS(P)) when Thr-212 and Ser-214 are phosphorylated(56, 57) . Ser-214 in turn is the site phosphorylated by cAMP-dependent kinase (Fig. 4). Thus, it is tempting to speculate that PHF-tau is phosphorylated to a high degree because of synergistic action of multiple kinases. Regarding Ser-262, it has only recently been shown that a 35/41-kDa kinase, which is found in the ``brain kinase'' fraction, specifically phosphorylates the residue (59) . The remaining non-proline-directed sites, Thr-403 and Ser-412, do not conform to any known kinase motif(56, 57) . On the other hand, two protein kinases are reported to be associated with tangles; casein kinase II (60) and hemin-sensitive kinase (61) are loosely and tightly bound to tangles, respectively. Although Ser-199 and -409 become a motif for casein kinase II when Ser-202 and -412 are phosphorylated (56) , thus far, no in vitro experiment consistent with this has been reported; casein kinase II preferentially phosphorylates Thr-39 in vitro in isoforms containing a 58-amino acid amino-terminal insert(62) . There has also been no detailed description of characteristics of the hemin-sensitive kinase. Considering the above, we reach the conclusion that (i) the phosphorylation of PHF-tau cannot be achieved by a single protein kinase; and (ii) involved protein kinases would be tau protein kinase I/glycogen synthase kinase 3beta and tau protein kinase II for fetal type phosphorylation and possibly tau protein kinase I/glycogen synthase kinase 3beta, MAP kinase, and 35/41-kDa kinase for the non-fetal type phosphorylation specific for PHF-tau.

At present we do not know which kinase would work in the initial stages. Our recent observations indicate that at least the tau 1 site appears to be gradually dephosphorylated during PHF evolution. (^3)In contrast, phosphothreonine 153, which is undetectable in PHF-tau, has been found in the PHF smear (see also (13) ). (^4)Thus, it is possible that responsible kinases would work at different stages or for varying durations. In this context, it is important to determine which of the candidate kinases initiates the phosphorylation of tau in AD brain.

The consequence of PHF-tau phosphorylation is assembly incompetence (19, 20) . This contrasts with fetal tau, which despite a high degree of phosphorylation retains a lower but still significant activity(20) . Although phosphorylation on Ser-262, located in the microtubule-binding domain, is shown to have a deleterious effect on the affinity for microtubules(59) , it is only partially phosphorylated in PHF-tau(21) . Thus, its partial phosphorylation (30%) (^5)alone cannot explain the assembly incompetence of PHF-tau. In addition, even if phosphorylated on all proline-directed residues by MAP kinase, tau appears not to show such an extent of assembly incompetence as seen in PHF-tau(59) . In view of this, we may speculate that phosphorylation of non-proline-directed sites is required for almost total elimination of the assembly-promoting activity.

Currently we cannot offer a complete explanation for the similarities in the phosphorylation of PHF-tau and fetal tau. Presumably, the high degree of phosphorylation of fetal tau is explained mainly by up-regulation of tau protein kinase I/glycogen synthase kinase 3beta and tau protein kinase II in the neonatal period(46, 51) . The abnormal phosphorylation of tau in AD brain may be induced as a regenerative response in dystrophic neurites; tau protein kinase I/glycogen synthase kinase 3beta is transiently up-regulated during neuronal degeneration (63) . This may explain why PHF-tau is phosphorylated in a fetal manner in AD brain.

Very recently, Lee and colleagues(40) , using freshly prepared biopsied samples, have made an unusual observation that normal tau in control human brains is phosphorylated to an extent comparable with that found in fetal tau or PHF-tau. This phosphorylation is rapidly lost after tissue resection. The dephosphorylating activities are blocked at low temperature or with several protein phosphatase inhibitors and appear to be developmentally regulated; fetal brain has low activities. This remarkable finding strongly suggests that (i) fetal type phosphorylation is the prototype for tau phosphorylation; (ii) the extent of phosphorylation is regulated by protein phosphatases; and (iii) fetal type phosphorylation in PHF-tau may simply represent ``freezing'' of the in vivo phosphorylation state.

Thus, the most important question is how and why non-fetal type phosphorylation occurs on PHF-tau. Investigations addressing the question should provide a clue to PHF formation and an upstream event. Recent morphological observations showed that neuropil threads scarcely coexist with normal cytoskeletons including microtubules and neurofilaments(64, 65) . This strongly suggests that when PHF are being formed, microtubules have already been lost. If so, the non-fetal type phosphorylation specific for PHF-tau may be related to the loss of microtubules. In any case, the non-fetal type phosphorylation that distinguishes PHF-tau from fetal tau should be investigated in depth. This should lead us to a better understanding of the mechanism of PHF formation and neuritic dystrophy in AD brain.


FOOTNOTES

*
This work was supported by Grant-in-Aid for Specially Promoted Research 03102008 and Grant-in-Aid for Scientific Research on Priority Areas 06254101 from the Ministry of Education, Science, and Culture and by a Grant-in-Aid for Scientific Research from the Ministry of Health and Welfare, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel. 81-3-3812-2111 (ext. 3541, 3543); Fax: 81-3-5800-6852.

(^1)
The abbreviations used are: AD, Alzheimer's disease; PHF, paired helical filaments; ISMS, ion spray mass spectrometry; MAP kinase, mitogen-activated protein kinase; PTH, phenylthiohydantoin; SEC, PTH-S-ethylcysteine; HPLC, high performance liquid chromatography.

(^2)
M. Morishima-Kawashima, M. Hasegawa, K. Takio, M. Suzuki, H. Yoshida, K. Titani, and Y. Ihara, unpublished observation.

(^3)
H. Yoshida and Y. Ihara, unpublished observation.

(^4)
M. Morishima-Kawashima, M. Hasegawa, K. Takio, M. Suzuki, K. Titani, and Y. Ihara, unpublished observation.

(^5)
M. Hasegawa, M. Morishima-Kawashima, K. Takio, M. Suzuki, K. Titani, and Y. Ihara, unpublished observation.


ACKNOWLEDGEMENTS

We are grateful to C.L. Masters for providing some AD brains used in the present study, E. Vanmechelen for supplying AT8, and M. Anzai for typing the manuscript.


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