Phosphorylation of microtubule-associated protein tau by Ca2+/calmodulin-dependent protein kinase II in its tubulin binding sites

The paired helical filaments (PHF) found in Alzheimer's 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 Ca(2+)/calmodulin-dependent 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.     

The pathology of the neuronal cytoskeleton in Alzheimer's disease.

The two characteristic neuropathological lesions of Alzheimer's disease are the neurofibrillary tangles and the senile plaques. Neurofibrillary tangles are made of abnormal filaments (PHF) accumulating in neurons and mainly composed of a modified form of the microtubule-associated protein tau (PHF-tau). Senile plaques are composed of a cluster of dystrophic neurites surrounding an extracellular deposit of amyloid fibers made of a 42 amino-acid peptide (beta-amyloid peptide). The abnormal filaments contain the complete sequences of the different tau isoforms. The PHF-tau proteins can be distinguished from the normal tau proteins by the presence of several phosphorylated sites. One of these sites is phosphorylated by a calcium-calmodulin-dependent kinase. The relationship between PHF-tau and the cytoskeletal pathology in Alzheimer's disease is further discussed.     

Two-Dimensional Characterization of Paired Helical Filament-Tau from Alzheimer's Disease: Demonstration of an Additional 74-kDa Component and Age-Related Biochemical Modifications

Abstract: PHF-tau proteins are the major components of the paired helical filament (PHF) from Alzheimer's disease (AD) neurofibrillary lesions. They differ both qualitatively and quantitatively in their degree of phosphorylation when compared with native tau proteins. However, little is known about the extent and heterogeneity of phosphorylated sites or the isoform composition and the isoelectric variants of PHF-tau. Therefore, we have characterized PHF-tau proteins from cortical brain tissue homogenates of 13 AD patients using two-dimensional gel electrophoresis. Whatever the topographical origin of brain tissue homogenates, PHF-tau proteins shared the same two-dimensional gel electrophoresis profile made of a tau triplet of 55, 64, and 69 kDa. A 74-kDa hyperphosphorylated tau component was detected particularly in the youngest and most severely affected AD patients. This additional component of hyperphosphorylated tau was shown to correspond to the longest brain tau isoform. Furthermore, the isoelectric points of PHF-tau from older AD patients were significantly more basic, indicating a lower degree of phosphorylation. These results show that the severity of neurofibrillary degeneration of AD is modulated by age.     

Epitope mapping of mAbs AT8 and Tau5 directed against hyperphosphorylated regions of the human tau protein

Post-mortem diagnosis of Alzheimer's disease relies on high numbers of senile plaques and neurofibrillary tangles (NFTs) stained in distinct brain areas. NFTs mostly consist of hyperphosphorylated versions of the microtubule attached tau protein (PHF-tau) with more than 30 serine and threonine phosphorylation sites identified so far. Characterization of hyperphosphorylated tau regions and the hope to develop robust assays for early AD diagnosis relies mostly on phosphorylation-dependent monoclonal antibodies (mAbs) recognizing only disease-specific phosphorylation patterns. Here, we report that anti-PHF-tau mAb AT8 recognizes an epitope doubly phosphorylated at serine 202 and threonine 205, which was not influenced by a third phosphate group at serine 199. But mAb AT8 was cross-reactive to two doubly phosphorylated motifs containing either serines 199 and 202 or serines 205 and 208 of the human tau sequence. The epitope of anti-tau mAb Tau5 was mapped to the human tau sequence 218-225, which is not phosphorylated in vivo.     

Novel phosphorylation sites in tau from Alzheimer brain support a role for casein kinase 1 in disease pathogenesis

Tau in Alzheimer disease brain is highly phosphorylated and aggregated into paired helical filaments comprising characteristic neurofibrillary tangles. Here we have analyzed insoluble Tau (PHF-tau) extracted from Alzheimer brain by mass spectrometry and identified 11 novel phosphorylation sites, 10 of which were assigned unambiguously to specific amino acid residues. This brings the number of directly identified sites in PHF-tau to 39, with an additional six sites indicated by reactivity with phosphospecific antibodies to Tau. We also identified five new phosphorylation sites in soluble Tau from control adult human brain, bringing the total number of reported sites to nine. To assess which kinases might be responsible for Tau phosphorylation, we used mass spectrometry to determine which sites were phosphorylated in vitro by several kinases. Casein kinase 1delta and glycogen synthase kinase-3beta were each found to phosphorylate numerous sites, and each kinase phosphorylated at least 15 sites that are also phosphorylated in PHF-tau from Alzheimer brain. A combination of casein kinase 1delta and glycogen synthase kinase-3beta activities could account for over three-quarters of the serine/threonine phosphorylation sites identified in PHF-tau, indicating that casein kinase 1delta may have a role, together with glycogen synthase kinase-3beta, in the pathogenesis of Alzheimer disease.     

Non-proline-dependent protein kinases phosphorylate several sites found in tau from Alzheimer disease brain

Of 21 phosphorylation sites identified in PHF-tau 11 are on ser/thr-X motifs and are probably phosphorylated by non-proline-dependent protein kinases (non-PDPKs). The identities of the non-PDPKs and how they interact to hyperphosphorylate PHF-tau are still unclear. In a previous study we have shown that the rate of phosphorylation of human tau 39 by a PDPK (GSK-3) was increased several fold if tau were first prephosphorylated by non-PDPKs (Singh et al., FEBS Lett 358: 267-272, 1995). In this study we have examined how the specificity of a non-PDPK for different sites on human tau 39 is modulated when tau is prephosphorylated by other non-PDPKs (A-kinase, C-kinase, CK-1, CaM kinase II) as well as a PDPK (GSK-3). We found that the rate of phosphorylation of tau 39 by a non-PDPK can be stimulated if tau were first prephosphorylated by other non-PDPKs. Of the four non-PDPKs only CK-1 can phosphorylate sites (thr 231, ser 396, ser 404) known to be present in PHF-tau. Further, these sites were phosphorylated more rapidly and to a greater extent by CK-1 if tau 39 were first prephosphorylated by A-kinase, CaM kinase II or GSK-3. These results suggest that the site specificities of the non-PDPKs that participate in PHF-tau hyperphosphorylation can be modulated at the substrate level by the phosphorylation state of tau.Abbreviations PHF paired helical filaments - A-kinase cyclic AMP-dependent protein kinase - CaM kinase II calcium/calmodulin-dependent protein kinase II - C-kinase calcium/phospholipid-dependent protein kinase - CK-1 casein kinase-1 - CK-2 casein kinase-2 - GSK-3 glycogen synthase kinase-3 - MAP kinase mitogen-activated protein kinase - PDPK proline-dependent protein kinase     

Protein sequence and mass spectrometric analyses of tau in the Alzheimer's disease brain.

Tau with unusually slow mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis was purified from the Sarkosyl-insoluble pellet of Alzheimer's disease brain homogenates. Such species of tau (PHF-tau) are considered to construct the framework of the sodium dodecyl sulfate-soluble form of paired helical filaments (PHF). Detailed comparison of peptide maps of PHF-tau and normal tau before and after dephosphorylation pointed to three anomalously eluted peaks which contained abnormally phosphorylated peptides, residues 191-225, 226-240, 260-267, and 386-438, according to the numbering of the longest tau isoform (Goedert, M., Spillantini, M. G., Jakes, R., Rutherford, D., and Crowther, R. A. (1989) Neuron 3, 519-526). Protein sequence and mass spectrometric analyses localized Thr-231 and Ser-235 as the abnormal phosphorylation sites and further indicated that each tau 1 site (residues 191-225) and the most carboxyl-terminal portion of the protein (residues 386-438) carries more than two abnormal phosphates. Ser-262 was also phosphorylated in a fraction of PHF-tau. Modifications other than phosphorylation, removal of the initiator methionine, and N alpha-acetylation at the amino terminus and deamidation at 2 asparaginyl residues were found in PHF-tau, but these modifications were also present in normal tau.     

Tau Released from Paired Helical Filaments with Formic Acid or Guanidine Is Susceptible to Calpain-Mediated Proteolysis

Abstract: Paired helical filaments (PHFs), a characteristic neuropathologic finding in Alzheimer's disease brain, are abnormal fibrillary forms of hyperphosphorylated tau (PHF-tau), which have been shown to be highly resistant to calpain digestion. Either excessive phosphorylation or fibrillary arrangement of tau proteins in PHFs may play a role in proteolytic resistance by limiting access to calpain recognition/digestion sites. To determine the contribution of the fibrillary conformation, isolated PHFs were subjected to treatment with either formic acid or guanidine. Both procedures effectively abolished the fibrillary structure of PHF but preserved PHF-tau immunoreactivity using a panel of antibodies that recognize nonphosphorylated and phosphorylated epitopes. These treatments also significantly increased the sensitivity of PHF-tau polypeptides to calpain proteolysis as shown by significant decreases in the half-life ( t _1/2) from the infinite with native PHF to 44 min and 4.4 min in formic acid- or guanidine-treated samples, respectively. In contrast, the sensitivity of normal fetal tau (3.4 min) was either decreased (5.9 min) or unaffected (3.6 min) by similar treatment. Our results indicate that after guanidine treatment, the sensitivity of PHF to calpain resembles that of fetal tau. These results strongly suggest that the fibrillary structure of PHF-tau, rather than hyperphosphorylation, is the major factor responsible for the resistance of abnormal filaments to calpain-mediated proteolysis.     

Neighbored phosphorylation sites as PHF-tau specific markers in Alzheimer's disease

Neurofibrillary tangles, which represent a major pathological hallmark in Alzheimer's disease (AD), are deposits of the hyperphosphorylated microtubule-associated tau protein (PHF-tau). However, a link between the phosphorylation pattern and the cause or the progress of AD is still missing. The work reported here focused on PHF-tau specific local phosphorylation patterns at Thr212/Ser214 and Thr231/Ser235 using monoclonal antibodies (mAb) generated against correspondingly modified peptides. The binding motifs of the obtained six mAbs were characterized with non-, mono-, and double-phosphorylated peptides as well as terminally shortened sequences. Five mAbs stained neurofibrillary tangles, neuritic plaques, and neuropil threads from autoptic brains of AD cases. Four mAbs recognized PHF-tau without significant cross-reactivity towards normal human tau, bovine tau, and dephosphorylated PHF-tau in ELISA and Western blot analysis. Thus, double phosphorylation is sufficient to distinguish PHF-tau from all other tau versions and there is no need to postulate any PHF-tau specific conformation for this region.     

Proline-directed phosphorylation of human Tau protein.

The primary sequence of the microtubule-associated protein tau contains multiple repeats of the sequence -X-Ser/Thr-Pro-X-, the consensus sequence for the proline-directed protein kinase (p34cdc2/p58cyclin A). When phosphorylated by proline-directed protein kinase in vitro, tau was found to incorporate up to 4.4 mol of phosphate/mol of protein. Isoelectric focusing of the tryptic phosphopeptides demonstrated the presence of five distinct peptides with pI values of approximately 6.9, 6.5, 5.6-5.9, 4.7, and 3.6. Mapping of the tryptic phosphopeptides by high performance liquid chromatography techniques demonstrated three distinct peaks. Data from gas phase sequencing, amino acid analysis, and phosphoamino acid analysis suggest that proline-directed protein kinase phosphorylates tau at four sites. Each site demonstrates the presence of a proline residue on the carboxyl-terminal side of the phosphorylated residue. Two phosphorylation sites are located adjacent to the three-repeat microtubule-binding domain that has been found to be required for the in vivo co-localization of tau protein to microtubules. Two other putative phosphorylation sites are located within the identified epitope of the monoclonal antibody Tau-1. Phosphorylation of these sites altered the immunoreactivity of tau to Tau-1 antibody. Since the neuronal microtubule-associated protein tau is multiply phosphorylated in Alzheimer's disease, and Tau-1 immunoreactivity is similarly reduced in neurofibrillary tangles and enhanced after dephosphorylation, phosphorylation at one or more of these sites may correlate with abnormally phosphorylated sites in tau protein in Alzheimer's disease.     

Alzheimer's disease-like phosphorylation of the microtubule-associated protein tau by glycogen synthase kinase-3 in transfected mammalian cells

Background: Paired helical filaments (PHFs) are a characteristic pathological feature of Alzheimer's disease; their principal component is the microtubule-associated protein tau. The tau in PHFs (PHF-tau) is hyperphosphorylated, but the cellular mechanisms responsible for this hyperphosphorylation have yet to be elucidated. A number of kinases, including mitogen-activated protein (MAP) kinase, glycogen synthase kinase (GSK)-3α, GSK-3β and cyclin-dependent kinase-5, phosphorylate recombinant tau in vitro so that it resembles PHF-tau as judged by its reactivity with a panel of antibodies capable of discriminating between normal tau and PHF-tau, and by a reduced electrophoretic mobility that is characteristic of PHF-tau. To determine whether MAP kinase, GSK-3α and GSK-3β can also induce Alzheimer's disease-like phosphorylation of tau in mammalian cells, we studied the phosphorylation status of tau in primary neuronal cultures and transfected COS cells following changes in the activities of MAP kinase and GSK-3.Results Activating MAP kinase in cultures of primary neurons or transfected COS cells expressing tau isoforms did not increase the level of phosphorylation for any PHF-tau epitope investigated. But elevating GSK-3 activity in the COS cells by co-transfection with GSK-3α or GSK-3β decreased the electrophoretic mobility of tau so that it resembled that of PHF-tau, and induced reactivity with eight PHF-tau-selective monoclonal antibodies.Conclusion Our data indicate that GSK-3α and/or GSK-3β, but not MAP kinase, are good candidates for generating PHF-type phosphorylation of tau in Alzheimer's disease. The involvement of other kinases in the generation of PHFs cannot, however, be eliminated. Our results suggest that aberrant regulation of GSK-3 may be a pathogenic mechanism in Alzheimer's disease.     

Abnormal tau proteins from Alzheimer's disease brains. Purification and amino acid analysis

Abnormal tau proteins (PHF-tau) were isolated from Alzheimer's disease brains by treatment of paired helical filament enriched-fractions with perchloric acid and boiling of the acid precipitable fraction with beta-mercaptoethanol. These proteins were purified further by a second perchloric acid treatment. The purified PHF-tau proteins were soluble in buffers devoid of sodium dodecyl sulfate. However, they were similar to the abnormal tau extracted from paired helical filaments with sodium dodecyl sulfate, also named A68, in molecular mass (68, 64, and 60 kDa), isoelectric point (pI 5.5-6.5), reactivity with anti-tau antibodies, and in requirement for alkaline phosphatase treatment to bind the Tau-1 antibody. Compared to normal tau, the soluble PHF-tau contained 100% more glycine and 35% less lysine residue. The results suggest that besides phosphorylation other types of modification may be involved in differentiating PHF-tau from normal tau.     

Inactivation of integrin-linked kinase induces aberrant tau phosphorylation via sustained activation of glycogen synthase kinase 3beta in N1E-115 neuroblastoma cells

Integrin-linked kinase (ILK) is a focal adhesion serine/threonine protein kinase with an important role in integrin and growth factor signaling pathways. Recently, we demonstrated that ILK is expressed in N1E-115 neuroblastoma cells and controls integrin-dependent neurite outgrowth in serum-starved cells grown on laminin (Ishii, T., Satoh, E., and Nishimura, M. (2001) J. Biol. Chem. 276, 42994-43003). Here we report that ILK controls tau phosphorylation via regulation of glycogen synthase kinase-3beta (GSK-3beta) activity in N1E-115 cells. Stable transfection of a kinase-deficient ILK mutant (DN-ILK) resulted in aberrant tau phosphorylation in N1E-115 cells at sites recognized by the Tau-1 antibody that are identical to some of the phosphorylation sites in paired helical filaments, PHF-tau, in brains of patients with Alzheimer's disease. The tau phosphorylation levels in the DN-ILK-expressing cells are constant under normal and differentiating conditions. On the other hand, aberrant tau phosphorylation was not observed in the parental control cells. ILK inactivation resulted in an increase in the active form but a decrease in the inactive form of GSK-3beta, which is a candidate kinase involved in PHF-tau formation. Moreover, inhibition of GSK-3beta with lithium prevented aberrant tau phosphorylation in the DN-ILK-expressing cells. These results suggest that ILK inactivation results in aberrant tau phosphorylation via sustained activation of GSK-3beta in N1E-115 Cells. ILK directly phosphorylates GSK-3beta and inhibits its activity. Therefore, endogenous ILK protects against GSK-3beta-induced aberrant tau phosphorylation via inhibition of GSK-3beta activity in N1E-115 cells.     

Cell cycle-dependent phosphorylation and microtubule binding of tau protein stably transfected into Chinese hamster ovary cells.

Tau protein, a neuronal microtubule-associated protein, is phosphorylated in situ and hyperphosphorylated when aggregated into the paired helical filaments of Alzheimer's disease. To study the phosphorylation of tau protein in vivo, we have stably transfected htau40, the largest human tau isoform, into Chinese hamster ovary cells. The distribution and phosphorylation of tau was monitored by gel shift, autoradiography, immunofluorescence, and immunoblotting, using the antibodies Tau-1, AT8, AT180, and PHF-1, which are sensitive to the phosphorylation of Ser202, Thr205, Thr231, Ser235, Ser396, and Ser404 and are used in the diagnosis of Alzheimer tau. In interphase cells, tau becomes phosphorylated to some extent, partly at these sites; most of the tau is associated with microtubules. In mitosis, the above Ser/Thr-Pro sites become almost completely phosphorylated, causing a pronounced shift in M(r) and an antibody reactivity similar to that of Alzheimer tau. Moreover, a substantial fraction of tau is found in the cytoplasm detached from microtubules. Autoradiographs of metabolically labeled Chinese hamster ovary cells in interphase and mitosis confirmed that tau protein is more highly phosphorylated during mitosis. The understanding of tau phosphorylation under physiological conditions might help elucidate possible mechanisms for the hyperphosphorylation in Alzheimer's disease.     

Phosphorylation sites on tau identified by nanoelectrospray mass spectrometry: differences in vitro between the mitogen-activated protein kinases ERK2, c-Jun N-terminal kinase and P38, and glycogen synthase kinase-3beta

The stress-activated kinases c-Jun N-terminal kinase (JNK) and p38 are members of the mitogen-activated protein (MAP) kinase family and take part in signalling cascades initiated by various forms of stress. Their targets include the microtubule-associated protein tau, which becomes hyperphosphorylated in Alzheimer's disease. It is necessary, as a forerunner for in vivo studies, to identify the protein kinases and phosphatases that are responsible for phosphate turnover at individual sites. Using nanoelectrospray mass spectrometry, we have undertaken an extensive comparison of phosphorylation in vitro by several candidate tau kinases, namely, JNK, p38, ERK2, and glycogen synthase kinase 3beta (GSK3beta). Between 10 and 15 sites were identified for each kinase. The three MAP kinases phosphorylated Ser202 and Thr205 but not detectably Ser199, whereas conversely GSK3beta phosphorylated Ser199 but not detectably Ser202 or Thr205. Phosphorylated Ser404 was found with all of these kinases except JNK. The MAP kinases may not be strictly proline specific: p38 phosphorylated the nonproline sites Ser185, Thr245, Ser305, and Ser356, whereas ERK2 was the most strict. All of the sites detected except Thr245 and Ser305 are known or suspected phosphorylation sites in paired helical filament-tau extracted from Alzheimer brains. Thus, the three MAP kinases and GSK3beta are importantly all strong candidates as tau kinases that may be involved in the pathogenic hyperphosphorylation of tau in Alzheimer's disease.     

Tau protein and neurodegeneration

Many of the human neurodegenerative conditions involve a reorganization of the neuronal cytoskeleton. The way in which the cytoskeleton is reorganized may provide a clue to the nature of the insult causing the neurodegeneration. The most common of these conditions is Alzheimer's disease, in which microtubules are lost from neurites that fill up with filamentous structures. One component of the filamentous structures is the microtubule-associated protein (MAP), tau. The tau protein is the product of a single gene expressed predominantly in neurons. The tau gene undergoes complex alternative splicing that is regulated both by development, and by the particular neuronal cell population in which it is expressed. Tau protein can be further modified, following its translation by phosphorylation at several sites. Much of the recent interest in the transition of tau to an abnormal state within a tangle-bearing neuron has focused on phosphorylation. A group of proteins that migrate slightly more slowly than tau, designated PHF-tau, are found in regions of the Alzheimer brain rich in dystrophic neurites, are hyperphosphorylated, fail to bind to microtubules, have distinct solubility properties, and can be derived from fractions of paired helical filaments (PHF).     

Ubiquitination and abnormal phosphorylation of paired helical filaments in Alzheimer's disease

The most characteristic cellular change in Alzheimer's disease is the accumulation of aberrant filaments, the paired helical filaments (PHF), in the affected neurons. There is growing evidence from a number of laboratories that dementia correlates better with the accumulation of PHF than of the extracellular amyloid, the second major lesion of Alzheimer's disease. PHF are both morphologically and biochemically unlike any of the normal neurofibrils. The major polypeptides in isolated PHF are microtubule-associated protein tau. Tau in PHF is phosphorylated differently from tau in microtubules. This abnormal phosphorylation of tau in PHF occurs at several sites. The accumulation of abnormally phosphorylated tau in the affected neurons in Alzheimer's disease brain precedes both the formation and the ubiquitination of the neurofibrillary tangles. In Alzheimer's disease brain, tubulin is assembly competent, but the in vitro assembly of microtubules is not observed. In vitro, the phosphate groups in PHF are less accessible than those of tau to alkaline phosphatase. The in vitro dephosphorylated PHF polypeptides stimulate microtubule assembly from bovine tubulin. It is hypothesized that a defect in the protein phosphorylation/dephosphorylation system is one of the earliest events in the cytoskeletal pathology in Alzheimer's disease. Production of nonfunctional tau by its phosphorylation and its polymerization into PHF most probably contributes to a microtubule assembly defect, and consequently, to a compromise in both axoplasmic flow and neuronal function. Index Entries: Alzheimer's disease; mechanisms of neuronal degeneration; neurofibrillary changes; paired helical filaments: biochemistry; microtubule-associated protein tau; abnormal phosphorylation; ubiquitination; microtubule assembly; axoplasmic flow; protein phosphorylation/dephosphorylation.     

Synthesis and conformational properties of phosphopeptides related to the human tau protein

In the brains of Alzheimer's disease patients, the tau protein dissociates from the axonal microtubule and abnormally aggregates to form a paired helical filament (PHF). One of the priorities in Alzheimer research is to determine the effects of abnormal phosphorylation on the local structure. A series of peptides corresponding to isolated regions of tau protein have been successfully synthesized using Fmoc-based chemistry and their conformations were determined by 1H NMR spectroscopy and circular dichroism (CD) spectroscopy. Immunodominant peptides corresponding to tau-(256-273), tau-(350-367) and two phosphorylated derivatives in which a single Ser was phosphorylated at positions 262 and 356, respectively, were the main focus of the study. A direct alteration of the local structure after phosphorylation constitutes a new strategy through which control of biological activity can be enforced. In our study on Ser262 in R1 peptide and Ser356 in R4 peptide, phosphorylation modifies both the negative charge and the local conformation nearby the phosphorylation sites. Together, these structural changes indicate that phosphorylation may act as a conformational switch in the binding domain of tau protein to alter specificity and affinity of binding to microtubule, particularly in response to the abnormal phosphorylation events associated with Alzheimer's disease.     

Characterization of In Vitro Glycation Sites of Tau

Abstract: Tau is a microtubule-associated protein that loses microtubule binding activity and aggregates into paired helical filaments (PHFs) in Alzheimer's disease. Nonenzymic glycation is one of the posttranslational modifications detected in PHF-tau, but not in normal tau. PHF-tau has reduced ability to bind to microtubules. To determine whether glycation of tau occurs in its microtubule binding domains, we have characterized in vitro glycation sites of the longest isoform of tau, which has four microtubule binding domains (Tau-4). The identified glycation sites are Lys-87, 132, 150, 163, 174, 225, 234, 259, 280, 281, 347, 353, and 369. We have also studied glycation of another isoform of tau, which has only three microtubule binding domains (Tau-3). This isoform is modified by glucose 15–20% more slowly than Tau-4. However, the glycation sites appear to be the same in both isoforms, except for Lys-280 and 281; these are located in the second microtubule binding domain, which is missing in Tau-3. Lys-150, 163, and 174 are located within or proximal to the sequence of tau that is involved in the microtubule nucleation activity, and Lys-259, 280, 281, 347, 353, and 369 are located in the microtubule binding domains. Glycation at these sites can affect the functional properties of tau, and advanced glycation at these sites might lead to the formation of insoluble aggregates similar to the ones seen in Alzheimer's disease.     

Implication of brain cdc2 and MAP2 kinases in the phosphorylation of tau protein in Alzheimer's disease.

Brain tau protein is phosphorylated in vitro by cdc2 and MAP2 kinases, obtained through immunoaffinity purification from rat brain extracts. The phosphorylation sites are located on the tau molecule both upstream and downstream of the tubulin-binding motifs. A synthetic peptide comprising residues 194-213 of the tau sequence, which contains the epitope recognized by the monoclonal antibody tau-1, is also efficiently phosphorylated in vitro by cdc2 and MAP2 kinases. Phosphorylation of this peptide markedly reduces its interaction with the antibody tau-1, as it has been described for tau protein in Alzheimer's disease. Both cdc2 and MAP2 kinases are present in brain extracts obtained from Alzheimer's disease patients. Interestingly, the level of cdc2 kinase may be increased in patient brains as compared with non-demented controls. These results suggest a role for cdc2 and MAP2 kinases in phosphorylating tau protein at the tau-1 epitope in Alzheimer's disease.