Modulation of Tau Phosphorylation Within Its Microtubule-Binding Domain by Cellular Thiols

Tau is a microtubule-stabilizing protein that is functionally modulated by alterations in its phosphorylation state. Because phosphorylation regulates both normal and pathological tau functioning, it is of importance to identify the signaling pathways that regulate tau phosphorylation in vivo. The present study examined changes in tau phosphorylation and function in response to modulation of cellular thiol content. Treatment of cells with phenylarsine oxide, which reacts with vicinal thiols, selectively increased tau phosphorylation within its microtubule-binding domain, at the non-Ser/Thr-Pro sites Ser262/356, while decreasing tau phosphorylation at Ser/ Thr-Pro sites outside this region. This increase in tau phosphorylation correlated with a decrease in the amount of tau associated with the cytoskeleton and decreased microtubule stability. Phenylarsine oxide-induced tau phosphorylation was inhibited by oxidants and by the protein kinase inhibitor staurosporine. Although staurosporine completely eliminated the increase in tau phosphorylation at Ser262/356, as detected by immunostaining with 12E8, it had a comparatively minor effect on the changes in tau localization induced by phenylarsine oxide. The results suggest that regulation of cellular thiols is important for modulating tau phosphorylation and function in situ. Additionally, although phosphorylation of Ser262/356 decreases tau's interaction with the cytoskeleton, phosphorylation of these residues alone is not sufficient for the phenylarsine oxide-induced changes in tau localization.     

Microtubule affinity-regulating kinase 1 (MARK1) is activated by electroconvulsive shock in the rat hippocampus

Electroconvulsive shock (ECS) induces phosphorylation and dephosphorylation of many signaling molecules in the rat brain. While studying phosphorylated proteins in the rat brain after ECS, we observed a 100-kDa protein that cross-reacted with anti-phospho-p70 S6 kinase antibody, which was subsequently purified and identified as microtubule affinity-regulating kinase 1 (MARK1). Purified MARK1 was phosphorylated at the Ser and Thr residues. MARK1 activation and subsequent Tau phosphorylation in the hippocampus after ECS was confirmed by an in-gel kinase assay using tau protein as a substrate. MARK1 was maximally activated between 2 and 5 min after ECS, and Tau phosphorylation at Ser262 was also increased at 2 min and lasted to 1 h after ECS. Taken together, we concluded that ECS activated MARK1 and subsequently phosphorylated Tau at Ser262. Both MARK1 activity and Tau phosphorylation were increased in the rat hippocampus after chronic ECS where axonal remodeling was apparent. In order to investigate the physiologic stimuli which are involved in the activation of MARK1, SH-SY 5Y cells were treated with brain-derived neurotrophic factor or 60 mm KCl. Both stimuli were capable of inducing MARK activation.     

Luteolin Reduces Zinc-Induced Tau Phosphorylation at Ser262/356 in an ROS-Dependent Manner in SH-SY5Y Cells

In brain, excess zinc alters the metabolism of amyloid precursor protein, leading to ??-amyloid protein deposition, one of the hallmarks of Alzheimer??s disease (AD) pathology. Recently, it has been reported that zinc accelerates in vitro tau fibrillization, another hallmark of AD. In the current study, we examined the effect of high-concentration zinc on tau phosphorylation in human neuroblastoma SH-SY5Y cells. We found that incubation of cells with zinc resulted in abnormal tau phosphorylation at Ser262/356. Moreover, the current study has investigated whether luteolin (Lu), a bioflavonoid, could decrease zinc-induced tau hyperphosphorylation and its underlying mechanisms. Using Western blot and protein phosphatase activity assay, activities of tau kinases and phosphatase were investigated. Our data suggest (1) that zinc induces tau hyperphosphorylation at Ser262/356 epitope and (2) that Lu efficiently attenuates zinc-induced tau hyperphosphorylation through not only its antioxidant action but also its regulation of the phosphorylation/dephosphorylation system.     

Phosphorylation of microtubule-associated protein tau by AMPK-related kinases

Microtubule-associated protein tau is abnormally hyperphosphorylated in the intracellular filamentous inclusions seen in neurodegenerative disorders with dementia, such as Alzheimer's disease and other tauopathies. Microtubule-associated protein/microtubule-affinity regulating kinases (MARKs) have previously been identified as kinases which phosphorylate KxGS motifs in the tandem repeats of tau. They are members of the 5'-AMP-activated protein kinase (AMPK)-related kinases in the Ca(2+)/calmodulin-dependent protein kinase group. In this study, we examined the ability of AMPK-related kinases, brain-specific kinases 1 and 2, maternal embryonic leucine-zipper kinase, MARK1, and salt-inducible kinase (SIK), to phosphorylate tau. We found that they phosphorylated S262 and S356 in KxGS motifs in the repeats of tau, thus resulting in immunoreactivity with antibody 12E8. MARK1 and SIK most effectively phosphorylated tau, and their down-regulation resulted in a reduction of 12E8-labelling. BX 795, an inhibitor of MARK1 and SIK, reduced 12E8-immunolabelling of tau in rat cortical neurons. These findings reveal a significant contribution of AMPK-related kinases to the phosphorylation of tau at S262/S356.     

Dephosphorylation of tau protein by calcineurin triturated into neural living cells

Alzheimer disease and related dementia are characterized by the presence of hyperphosphorylated tau aggregated into filaments. The role of tau phosphorylation in the fibrillogenesis has not yet been unraveled. Therefore, it is important to know which phosphatases can dephosphorylate tau protein in vivo. The effect of recombinant purified calcineurin (CN(PP2B)) and several calcineurin mutants on tau phosphorylation was studied in two neuronal like cell lines PC12 and SH-SY5Y. The modulation of tau phosphorylation at Ser199/Ser202, Ser396/Ser404, Ser262/Ser356, and Thr181 sites was examined in these cell lines using the phosphorylation state-dependent antitau antibodies Tau 1, PHF1, 12E8, and AT270. The results have shown that CN directly dephosphorylates all of those sites of tau protein. Recombinant calcineurin introduced into cells that have previously been treated with okadaic acid and cyclosporin A, which are inhibitors of phosphatases (PP1/PP2A and PP2B), has a direct effect on the phosphorylation status on all phosphorylation sites studied. We conclude that calcineurin is (besides PP2A) a important modulator of tau phosphorylation in vivo.     

Phosphorylation that detaches tau protein from microtubules (Ser262, Ser214) also protects it against aggregation into Alzheimer paired helical filaments

One of the hallmarks of Alzheimer's disease is the abnormal state of the microtubule-associated protein tau in neurons. It is both highly phosphorylated and aggregated into paired helical filaments, and it is commonly assumed that the hyperphosphorylation of tau causes its detachment from microtubules and promotes its assembly into PHFs. We have studied the relationship between the phosphorylation of tau by several kinases (MARK, PKA, MAPK, GSK3) and its assembly into PHFs. The proline-directed kinases MAPK and GSK3 are known to phosphorylate most Ser-Pro or Thr-Pro motifs in the regions flanking the repeat domain of tau: they induce the reaction with several antibodies diagnostic of Alzheimer PHFs, but this type of phosphorylation has only a weak effect on tau-microtubule interactions and on PHF assembly. By contrast, MARK and PKA phosphorylate several sites within the repeats (notably the KXGS motifs including Ser262, Ser324, and Ser356, plus Ser320); in addition PKA phosphorylates some sites in the flanking domains, notably Ser214. This type of phosphorylation strongly reduces tau's affinity for microtubules, and at the same time inhibits tau's assembly into PHFs. Thus, contrary to expectations, the phosphorylation that detaches tau from microtubules does not prime it for PHF assembly, but rather inhibits it. Likewise, although the phosphorylation sites on Ser-Pro or Thr-Pro motifs are the most prominent ones on Alzheimer PHFs (by antibody labeling), they are only weakly inhibitory to PHF assembly. This implies that the hyperphosphorylation of tau in Alzheimer's disease is not directly responsible for the pathological aggregation into PHFs; on the contrary, phosphorylation protects tau against aggregation.     

Inhibition of PP-2A upregulates CaMKII in rat forebrain and induces hyperphosphorylation of tau at Ser 262/356

The regulation of the activity of CaMKII by PP-1 and PP-2A, as well as the role of this protein kinase in the phosphorylation of tau protein in forebrain were investigated. The treatment of metabolically active rat brain slices with 1.0 microM okadaic acid (OA) inhibited approximately 65% of PP-2A and had no significant effect on PP-1 in the 16000xg tissue extract. Calyculin A (CL-A), 0.1 microM under the same conditions, inhibited approximately 50% of PP-1 and approximately 20% of PP-2A activities. In contrast, a mixture of OA and CL-A practically completely inhibited both PP-2A and PP-1 activities. The inhibition of the two phosphatase activities or PP-2A alone resulted in an approximately 2-fold increase in CaMKII activity and an approximately 8-fold increase in the phosphorylation of tau at Ser 262/356 in 60 min. Treatment of the brain slices with KN-62, an inhibitor of the autophosphorylation of CaMKII at Thr 286/287, produced approximately 60% inhibition in CaMKII activity and no significant effect on tau phosphorylation at Ser 262/356. The KN-62-treated brain slices when further treated with OA and CL-A did not show any change in CaMKII activity. In vitro, both PP-2A and PP-1 dephosphorylated tau at Ser 262/356 that was phosphorylated with purified CaMKII. These studies suggest (i) that in mammalian forebrain the cytosolic CaMKII activity is regulated mainly by PP-2A, (ii) that CaMKII is the major tau Ser 262/356 kinase in brain, and (iii) that a decrease in PP-2A/PP-1 activities in the brain leads to hyperphosphorylation of tau not only by inhibition of its dephosphorylation but also by promoting the CaMKII activity.     

Phosphorylation modulates the local conformation and self-aggregation ability of a peptide from the fourth tau microtubule-binding repeat

Phosphorylation of tau protein modulates both its physiological role and its aggregation into paired helical fragments, as observed in Alzheimer's diseased neurons. It is of fundamental importance to study paired helical fragment formation and its modulation by phosphorylation. This study focused on the fourth microtubule-binding repeat of tau, encompassing an abnormal phosphorylation site, Ser356. The aggregation propensities of this repeat peptide and its corresponding phosphorylated form were investigated using turbidity, thioflavin T fluorescence and electron microscopy. There is evidence for a conformational change in the fourth microtubule-binding repeat of tau peptide upon phosphorylation, as well as changes in aggregation activity. Although both tau peptides have the ability to aggregate, this is weaker in the phosphorylated peptide. This study reveals that both tau peptides are capable of self-aggregation and that phosphorylation at Ser356 can modulate this process.     

14-3-3zeta is an effector of tau protein phosphorylation

Neurofibrillary tangles associated with Alzheimer's disease are composed mainly of paired helical filaments that are formed by the aggregation of abnormally phosphorylated microtubule-associated protein tau. 14-3-3, a highly conserved protein family that exists as seven isoforms and regulates diverse cellular processes is present in neurofibrillary tangles (Layfield, R., Fergusson, J., Aitken, A., Lowe, J., Landon, M., Mayer, R. J. (1996) Neurosci. Lett. 209, 57-60). The role of 14-3-3 in Alzheimer's disease pathogenesis is not known. In this study, we found that the 14-3-3zeta isoform is associated with tau in brain extract and profoundly stimulates cAMP-dependent protein kinase catalyzed in vitro phosphorylation on Ser(262)/Ser(356) located within the microtubule-binding region of tau. 14-3-3zeta binds to both phosphorylated and nonphosphorylated tau, and the binding site is located within the microtubule-binding region of tau. From brain extract, 14-3-3zeta co-purifies with microtubules, and tubulin blocks 14-3-3zeta-tau binding. Among four 14-3-3 isoforms tested, beta and zeta but not gamma and epsilon associate with tau. Our data suggest that 14-3-3zeta is a tau protein effector and may be involved in the abnormal tau phosphorylation occurring during Alzheimer's disease ontogeny.     

GSK-3beta directly phosphorylates and activates MARK2/PAR-1

In Alzheimer disease (AD), the microtubule-associated protein tau is found hyperphosphorylated in paired helical filaments. Among many phosphorylated sites in tau, Ser-262 is the major site for abnormal phosphorylation of tau in AD brain. The kinase known to phosphorylate this particular site is MARK2, whose activation mechanism is yet to be studied. Our first finding that treatment of cells with LiCl, a selective inhibitor of another major tau kinase, glycogen synthase kinase-3beta (GSK-3beta), inhibits phosphorylation of Ser-262 of tau led us to investigate the possible involvement of GSK-3beta in MARK2 activation. In vitro kinase reaction revealed that recombinant GSK-3beta indeed phosphorylates MARK2, whereas it failed to phosphorylate Ser-262 of tau. Our further findings led us to conclude that GSK-3beta phosphorylates MARK2 on Ser-212, one of the two reported phosphorylation sites (Thr-208 and Ser-212) found in the activation loop of MARK2. Down-regulation of either GSK-3beta or MARK2 by small interfering RNAs suppressed the level of phosphorylation on Ser-262. These results, respectively, indicated that GSK-3beta is responsible for phosphorylating Ser-262 of tau through phosphorylation and activation of MARK2 and that the phosphorylation of tau at this particular site is predominantly mediated by a GSK-3beta-MARK2 pathway. These findings are of interest in the context of the pathogenesis of AD.     

Microtubule affinity–regulating kinase 4 with an Alzheimer's disease-related mutation promotes tau accumulation and exacerbates neurodegeneration

Accumulation of the microtubule-associated protein tau is associated with Alzheimer''s disease (AD). In AD brain, tau is abnormally phosphorylated at many sites, and phosphorylation at Ser-262 and Ser-356 plays critical roles in tau accumulation and toxicity. Microtubule affinity–regulating kinase 4 (MARK4) phosphorylates tau at those sites, and a double de novo mutation in the linker region of MARK4, ΔG316E317D, is associated with an elevated risk of AD. However, it remains unclear how this mutation affects phosphorylation, aggregation, and accumulation of tau and tau-induced neurodegeneration. Here, we report that MARK4^ΔG316E317D increases the abundance of highly phosphorylated, insoluble tau species and exacerbates neurodegeneration via Ser-262/356–dependent and –independent mechanisms. Using transgenic Drosophila expressing human MARK4 (MARK4^wt) or a mutant version of MARK4 (MARK4^ΔG316E317D), we found that coexpression of MARK4^wt and MARK4^ΔG316E317D increased total tau levels and enhanced tau-induced neurodegeneration and that MARK4^ΔG316E317D had more potent effects than MARK4^wt. Interestingly, the in vitro kinase activities of MARK4^wt and MARK4^ΔG316E317D were similar. When tau phosphorylation at Ser-262 and Ser-356 was blocked by alanine substitutions, MARK4^wt did not promote tau accumulation or exacerbate neurodegeneration, whereas coexpression of MARK4^ΔG316E317D did. Both MARK4^wt and MARK4^ΔG316E317D increased the levels of oligomeric forms of tau; however, only MARK4^ΔG316E317D further increased the detergent insolubility of tau in vivo. Together, these findings suggest that MARK4^ΔG316E317D increases tau levels and exacerbates tau toxicity via a novel gain-of-function mechanism and that modification in this region of MARK4 may affect disease pathogenesis.     

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.     

MARKing tau for tangles and toxicity

In healthy neurons, tau proteins regulate microtubule function in the axon. In the brains of individuals with Alzheimer's disease, tau is hyperphosphorylated and aggregated into intraneuronal deposits called neurofibrillary tangles (NFTs). Hyperphosporylation dislodges tau from the microtubule surface, potentially resulting in compromised axonal integrity and the accumulation of toxic tau peptides. Recent biochemical and animal model studies have re-evaluated tau phosphorylation and other aspects of neurofibrillar pathology. The results indicate that phosphorylation of tau's microtubule-binding domain by the protein kinase MARK primes tau for hyperphosphorylation by the kinases GSK-3 and Cdk5, which in turn triggers the aggregation of tau into filaments and tangles. Toxic consequences for the neuron might be exacerbated by tangle formation but are already evident during the early steps of the process.     

P70 S6 kinase mediates tau phosphorylation and synthesis

Currently, we found that the 70-kDa p70 S6 kinase (p70S6K) directly phosphorylates tau at S262, S214, and T212 sites in vitro. By immunoprecipitation, p-p70S6K (T421/S424) showed a close association with p-tau (S262 and S396/404). Zinc-induced p70S6K activation could only upregulate translation of total S6 and tau but not global proteins in SH-SY5Y cells. The requirement of p70S6K activation was confirmed in the SH-SY5Y cells that overexpress wild-type htau40. Level of p-p70S6K (T421/S424) was only significantly correlated with p-tau at S262, S214, and T212, but not T212/S214, in Alzheimer's disease (AD) brains. These suggested that p70S6K might contribute to tau related pathologies in AD brains.     

MARK4 is a novel microtubule-associated proteins/microtubule affinity-regulating kinase that binds to the cellular microtubule network and to centrosomes

The MARK protein kinases were originally identified by their ability to phosphorylate a serine motif in the microtubule-binding domain of tau that is critical for microtubule binding. Here, we report the cloning and expression of a novel human paralog, MARK4, which shares 75% overall homology with MARK1-3 and is predominantly expressed in brain. Homology is most pronounced in the catalytic domain (90%), and MARK4 readily phosphorylates tau and the related microtubule-associated protein 2 (MAP2) and MAP4. In contrast to the three paralogs that all exhibit uniform cytoplasmic localization, MARK4 colocalizes with the centrosome and with microtubules in cultured cells. Overexpression of MARK4 causes thinning out of the microtubule network, concomitant with a reorganization of microtubules into bundles. In line with these findings, we show that a tandem affinity-purified MARK4 protein complex contains alpha-, beta-, and gamma-tubulin. In differentiated neuroblastoma cells, MARK4 is localized prominently at the tips of neurite-like processes. We suggest that although the four MARK/PAR-1 kinases might play multiple cellular roles in concert with different targets, MARK4 is likely to be directly involved in microtubule organization in neuronal cells and may contribute to the pathological phosphorylation of tau in Alzheimer's disease.     

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 self-assembly ability of the first microtubule-binding repeat from tau and its modulation by phosphorylation

Aggregation of abnormally phosphorylated tau in the form of tangs of paired helical filaments (PHFs) is one of the hallmarks of Alzheimer's disease (AD) and other tauopathies. It is of fundamental importance to study the mechanism of PHF formation and its modulation by phosphorylation. In this work, we have focused on the first microtubule-binding repeat of tau encompassing an abnormal phosphorylation site Ser262. The assembly propensities of this repeat and its corresponding phosphorylated form were investigated by turbidity and electron microscopy. Additionally, conformation of the two peptides is also analyzed through circular dichroism (CD) and NMR spectroscopy. Our results reveal that both of them are capable of self-assembly and phosphorylation at Ser262 could speed up the process of assembly. A possible mechanism of PHF formation is proposed and enhancing effect of phosphorylation on assembly provides an explanation to its toxicity in Alzheimer's disease.     

AMP-activated protein kinase (AMPK) is a tau kinase, activated in response to amyloid β-peptide exposure

Hyperphosphorylation of tau is a hallmark of Alzheimer's disease and other tauopathies. Although the mechanisms underlying hyperphosphorylation are not fully understood, cellular stresses such as impaired energy metabolism are thought to influence the signalling cascade. The AMPK (AMP-activated protein kinase)-related kinases MARK (microtubule-associated protein-regulating kinase/microtubule affinity-regulating kinase) and BRSK (brain-specific kinase) have been implicated in tau phosphorylation, but are insensitive to activation by cellular stress. In the present study, we show that AMPK itself phosphorylates tau on a number of sites, including Ser2?2 and Ser3??, altering microtubule binding of tau. In primary mouse cortical neurons, CaMKKβ (Ca2+/calmodulin-dependent protein kinase kinase β) activation of AMPK in response to Aβ (amyloid-β peptide)-(1-42) leads to increased phosphorylation of tau at Ser2?2/Ser3?? and Ser33??. Activation of AMPK by Aβ-(1-42) is inhibited by memantine, a partial antagonist of the NMDA (N-methyl-D-aspartate) receptor and currently licensed for the treatment of Alzheimer's disease. These findings identify a pathway in which Aβ-(1-42) activates CaMKKβ and AMPK via the NMDA receptor, suggesting the possibility that AMPK plays a role in the pathophysiological phosphorylation of tau.     

Differential effects of an O-GlcNAcase inhibitor on tau phosphorylation

Abnormal hyperphosphorylation of microtubule-associated protein tau plays a crucial role in neurodegeneration in Alzheimer's disease (AD). The aggregation of hyperphosphorylated tau into neurofibrillary tangles is also a hallmark brain lesion of AD. Tau phosphorylation is regulated by tau kinases, tau phosphatases, and O-GlcNAcylation, a posttranslational modification of proteins on the serine or threonine residues with β-N-acetylglucosamine (GlcNAc). O-GlcNAcylation is dynamically regulated by O-GlcNAc transferase, the enzyme catalyzing the transfer of GlcNAc to proteins, and N-acetylglucosaminidase (OGA), the enzyme catalyzing the removal of GlcNAc from proteins. Thiamet-G is a recently synthesized potent OGA inhibitor, and initial studies suggest it can influence O-GlcNAc levels in the brain, allowing OGA inhibition to be a potential route to altering disease progression in AD. In this study, we injected thiamet-G into the lateral ventricle of mice to increase O-GlcNAcylation of proteins and investigated the resulting effects on site-specific tau phosphorylation. We found that acute thiamet-G treatment led to a decrease in tau phosphorylation at Thr181, Thr212, Ser214, Ser262/Ser356, Ser404 and Ser409, and an increase in tau phosphorylation at Ser199, Ser202, Ser396 and Ser422 in the mouse brain. Investigation of the major tau kinases showed that acute delivery of a high dose of thiamet-G into the brain also led to a marked activation of glycogen synthase kinase-3β (GSK-3β), possibly as a consequence of down-regulation of its upstream regulating kinase, AKT. However, the elevation of tau phosphorylation at the sites above was not observed and GSK-3β was not activated in cultured adult hippocampal progenitor cells or in PC12 cells after thiamet-G treatment. These results suggest that acute high-dose thiamet-G injection can not only directly antagonize tau phosphorylation, but also stimulate GSK-3β activity, with the downstream consequence being site-specific, bi-directional regulation of tau phosphorylation in the mammalian brain.     

糖原合成酶激酶-3对τ蛋白磷酸化作用的研究

糖原合成酶激酶 ３（ Ｇ Ｓ Ｋ ３）在 ３０℃与 τ蛋白保温 ４ ｈ 可催化 １７±０４ ｍ ｏｌ磷酸参入 １ ｍ ｏｌτ蛋白 将磷酸化的 τ蛋白经胰蛋白酶消化, Ｆｅ Ｃｌ３ 亲和柱分离及 Ｃ１８反相高压液相层析纯化后,再用高压电泳,手工 Ｅｄｍ ａｎ 降解及自动氨基酸序列分析等检测技术,对其磷酸化位点进行鉴定 结果发现： Ｇ Ｓ Ｋ ３ 可使 τ蛋白 Ｔｈｒ １８１, Ｓｅｒ １８４, Ｓｅｒ ２６２, Ｓｅｒ ３５６ 和 Ｓｅｒ ４００ 发生磷酸化 其中 Ｓｅｒ ２６２ 和 Ｓｅｒ ４００ 为 Ａｌｚｈｅｉｍ ｅｒ 病（ Ａ Ｄ）τ蛋白的异常磷酸化位点根据上述磷酸化作用仅轻度抑制τ蛋白生物学活性,推测： Ａ Ｄ τ蛋白 Ｓｅｒ ２６２ 和 Ｓｅｒ ４００ 的磷酸化可能不是决定其生物功能的关键性位点,单纯 Ｇ Ｓ Ｋ ３ 不能复制 Ａ Ｄ 样 τ蛋白的病理改变 