I1PP2A affects tau phosphorylation via association with the catalytic subunit of protein phosphatase 2A

In Alzheimer disease (AD) brain, the level of I (1)(PP2A), a 249-amino acid long endogenous inhibitor of protein phosphatase 2A (PP2A), is increased, the activity of the phosphatase is decreased, and the microtubule-associated protein Tau is abnormally hyperphosphorylated. However, little is known about the detailed regulatory mechanism by which PP2A activity is inhibited by I (1)(PP2A) and the consequent events in mammalian cells. In this study, we found that both I (1)(PP2A) and its N-terminal half I (1)(PP2A(1-120)), but neither I (1)(PP2A(1-163)) nor I (1)(PP2A(164-249)), inhibited PP2A activity in vitro, suggesting an autoinhibition by amino acid residues 121-163 and its neutralization by the C-terminal region. Furthermore, transfection of NIH3T3 cells produced a dose-dependent inhibition of PP2A activity by I (1)(PP2A)(1). I (PP2A) and PP2A were found to colocalize in PC12 cells. I (1)(PP2A) could only interact with the catalytic subunit of PP2A (PP2Ac) and had no interaction with the regulatory subunits of PP2A (PP2A-A or PP2A-B) using a glutathione S-transferase-pulldown assay. The interaction was further confirmed by coimmunoprecipitation of I (1)(PP2A) and PP2Ac from lysates of transiently transfected NIH3T3 cells. The N-terminal isotype specific region of I (1)(PP2A) was required for its association with PP2Ac as well as PP2A inhibition. In addition, the phosphorylation of Tau was significantly increased in PC12/Tau441 cells transiently transfected with full-length I (1)(PP2A) and with PP2Ac-interacting I (1)(PP2A) deletion mutant 1-120 (I (1)(PP2A)DeltaC2). Double immunofluorescence staining showed that I (1)(PP2A) and I (1)(PP2A)DeltaC2 increased Tau phosphorylation and impaired the microtubule network and neurite outgrowth in PC12 cells treated with nerve growth factor.     

Dephosphorylation of microtubule-associated protein tau by protein phosphatase 5

Protein phosphatase 5 (PP5) is a 58-kDa novel phosphoseryl/phosphothreonyl protein phosphatase. It is ubiquitously expressed in all mammalian tissues examined, with a high level in the brain, but little is known about its physiological substrates. We found that this phosphatase dephosphorylated recombinant tau phosphorylated with cAMP-dependent protein kinase and glycogen synthase kinase-3beta, as well as abnormally hyperphosphorylated tau isolated from brains of patients with Alzheimer's disease. The specific activity of PP5 toward tau was comparable to those reported with other protein substrates examined to date. The PP5 activity toward tau was stimulated by arachidonic acid by 30- to 45-fold. Immunostaining demonstrated that PP5 was primarily cytoplasmic in PC12 cells and in neurons of postmortem human brain tissue. A small pool of PP5 associated with microtubules. Expression of active PP5 in PC12 cells resulted in reduced phosphorylation of tau, suggesting that PP5 can also dephosphorylate tau in cells. These results suggest that PP5 plays a role in the dephosphorylation of tau and might be involved in the molecular pathogenesis of Alzheimer's disease.     

Role of protein phosphatase-2A and -1 in the regulation of GSK-3, cdk5 and cdc2 and the phosphorylation of tau in rat forebrain

In Alzheimer disease brain the activities of protein phosphatase (PP)-2A and PP-1 are decreased and the microtubule-associated protein tau is abnormally hyperphosphorylated at several sites at serine/threonine. Employing rat forebrain slices kept metabolically active in oxygenated artificial CSF as a model system, we investigated the role of PP-2A/PP-1 in the regulation of some of the major abnormally hyperphosphorylated sites of tau and the protein kinases involved. Treatment of the brain slices with 1.0 microM okadaic acid inhibited approximately 65% of PP-2A and produced hyperphosphorylation of tau at Ser 198/199/202, Ser 396/404 and Ser 422. No significant changes in the activities of glycogen synthase kinase-3 (GSK-3) and cyclin dependent protein kinases cdk5 and cdc2 were observed. Calyculin A (0.1 microM) inhibited approximately 50% PP-1, approximately 20% PP-2A, 50% GSK-3 and approximately 30% cdk5 but neither inhibited the activity of cyclin AMP dependent protein kinase A (PKA) nor resulted in the hyperphosphorylation of tau at any of the above sites. Treatment of brain slices with 1 microM okadaic acid plus 0.1 microM calyculin A inhibited approximately 100% of both PP-2A and PP-1, approximately 80% of GSK-3, approximately 50% of cdk5 and approximately 30% of cdc2 but neither inhibited PKA nor resulted in the hyperphosphorylation of tau at any of the above sites. These studies suggest (i) that PP-1 upregulates the phosphorylation of tau at Ser 198/199/202 and Ser 396/404 indirectly by regulating the activities of GSK-3, cdk5 and cdc2 whereas PP-2A regulates the phosphorylation of tau directly by dephosphorylation at the above sites, and (ii) that a decrease in the PP-2A activity leads to abnormal hyperphosphorylation of tau at Ser 198/199/202, Ser 396/404 and Ser 422.     

糖尿病大鼠脑GSK-3与PP-2A失调诱导tau蛋白过度磷酸化

探讨胰岛素缺乏的糖尿病大鼠皮层糖原合酶激酶-3(GSK-3)及蛋白磷酯酶-2A(PP-2A)变化及其对tau蛋白磷酸化的作用.用链脲佐菌素(streptozotocin,STZ)建立胰岛素缺乏的糖尿病大鼠模型,用放射性配体结合实验检测了GSK-3和PP-2A的活性,蛋白质印迹检测了tau蛋白的磷酸化水平及PP-2A的表达.结果提示:在糖尿病大鼠皮层,GSK-3活性升高,PP-2A活性及表达降低,tau蛋白在Ser198/Ser199/Ser202和Ser396/Ser404位点磷酸化.应用GSK-3的选择性抑制剂Li2CO3后,GSK-3活性降低,PP-2A活性及表达恢复,tau蛋白在Ser198/Ser199/Ser202和Ser396/Ser404位点磷酸化水平降低.研究提示:糖尿病大鼠皮层GSK-3升高可能抑制PP-2A的活性,升高的GSK-3和降低的PP-2A协同促进tau蛋白的磷酸化.     

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.     

A novel monoclonal antibody DC63 reveals that inhibitor 1 of protein phosphatase 2A is preferentially nuclearly localised in human brain

Abnormal phosphorylation of tau protein represents one of the major candidate pathological mechanisms leading to Alzheimer's disease (AD) and related tauopathies. Altered phosphorylation status of neuronal tau protein may result from upregulation of tau-specific kinases or from inhibition of tau-specific phosphatases. Increased expression of the protein inhibitor 1 of protein phosphatase 2A (I1PP2A) could therefore indirectly regulate the phosphorylation status of tau. As an important step towards elucidation of the role of I1PP2A in the physiology and pathology of tau phosphorylation, we developed a novel monoclonal antibody, DC63, which recognizes I1PP2A. Specificity of the antibody was examined by mass spectrometry and Western blot. This analysis supports the conclusion that the antibody does not recognize any of the other proteins of the 9-member leucine-rich acidic nuclear phosphoprotein family to which I1PP2A belongs. Immunoblot detection revealed that the inhibitor I1PP2A is expressed throughout the brain, including the hippocampus, temporal cortex, parietal cortex, subcortical nuclei and brain stem. The cerebellum displayed significantly higher levels of expression of I1PP2A than was seen elsewhere in the brain. Imunohistochemical analysis of normal human brain showed that I1PP2A is expressed in both neurons and glial cells and that the protein is preferentially localized to the nucleus. We conclude that the novel monoclonal antibody DC63 could be successfully employed as a mass spectrometry-validated molecular probe that may be used for in vitro and in vivo qualitative and quantitative studies of physiological and pathological pathways involving I1PP2A.     

The involvement of glycogen synthase kinase-3 and protein phosphatase-2A in lactacystin-induced tau accumulation

Here, we demonstrated that lactacystin inhibited proteasome dose-dependently in HEK293 cells stably expressing tau. Simultaneously, it induces accumulation of both non-phosphorylated and hyperphosphorylated tau and decreases the binding of tau to the taxol-stabilized microtubules. Lactacystin activates glycogen synthase kinsase-3 (GSK-3) and decreases the phosphorylation of GSK-3 at serine-9. LiCl inhibits GSK-3 and thus reverses the lactacystin-induced accumulation of the phosphorylated tau. Lactacystin also inhibits protein phosphase-2A (PP-2A) and it significantly increases the level of inhibitor 1 of PP-2A. These results suggest that inhibition of proteasome by lactacystin induces tau accumulation and activation of GSK-3 and inhibition of PP-2A are involved.     

蛋白磷酸酯酶-1和-2A抑制剂对NG108-15细胞tau蛋白磷酸化的影响

Alzheimer痴呆(AD)的主要脑病理变化之一为由超磷酸化的tau蛋白组成的神经原纤维缠结(Neurofibrillary tangle,NFT)。AD的Tau蛋白异常磷酸化与蛋白磷酸酯酶(PP)-2A和-1缺陷有关。本文首先用免疫印迹法显示NG含两大组分的tau蛋白。MTT方法观察到PP-2A和PP-1抑制剂冈 田酸(Okadaic acid.OA)处理NG108-14细胞能导致细胞代谢明显下降,同时免疫印迹法显示OA能导致的NG细胞Tau蛋白磷酸化。该研究为建立AD样蛋白磷酸酯酶缺陷引起的tau蛋白磷酸化的细胞模型奠定了基础。     

Phosphothreonine-212 of Alzheimer Abnormally Hyperphosphorylated Tau is a Preferred Substrate of Protein Phosphatase-1

We isolated and characterized several phosphoseryl/phosphothreonyl phosphatase activities (P1–P11) from frontal lobe of six autopsied human brains. Of these, PP1 (P3) was a major tau phosphatase. The enzyme required metal ions and was maximally activated by Mn²⁺. Western blots with antibodies to known protein phosphatases showed PP1 and PP2B immunoreactivity. However, the removal of PP2B by immunoabsorption or its inhibition with EGTA did not result in appreciable loss of P3 activity. These observations suggest that P3 was an enriched PP1. Dephosphorylation of Alzheimer disease hyperphosphorylated tau (AD P-tau) by PP1 was site-specific. PP1 preferentially dephosphorylated pT212 (40%), pT217 (26%), pS262 (33%), pS396 (42%) and pS422 (31%) of AD P-tau. Dephosphorylation of tau at pT181, pS199, pS202, pT205, pS214, and pS404, was undetectable. Of the sites dephosphorylated, pT212 was only a substrate for PP1, as purified/enriched PP2A and PP2B from the same brains did not dephosphorylate this site.     

Acute anoxia induces tau dephosphorylation in rat brain slices and its possible underlying mechanisms

Abnormal phosphorylation of microtubule-associated protein tau plays a critical role in Alzheimer's disease (AD), together with a distinct decrease of energy metabolism in the affected brain regions. To explore the effect of acute energy crisis on tau phosphorylation and the underlying mechanisms, we incubated rat brain slices in artificial cerebrospinal fluid (aCSF) at 37 degrees C with or without an oxygen supply, or in aCSF with low glucose concentrations. Then, the levels of total, phosphorylated and unphosphorylated tau, as well as the activities and levels of protein phosphatase (PP)-1, PP-2A, glycogen synthase kinase 3 (GSK-3), extracellular signal-regulated protein kinase (ERK) and C-jun amino terminal kinase (JNK), were measured. It was found, unexpectedly, that tau was significantly dephosphorylated at Ser396/Ser404 (PHF-1), Ser422 (R145), Ser199/Ser202 (Tau-1), Thr181 (AT270), Ser202/Thr205 (AT8) and Thr231 (AT180) by acute anoxia for 30 min or 120 min. The activity of PP-2A and the level of dephosphorylated PP-2A catalytic subunit at tyrosine 307 (Tyr307) were simultaneously increased. The active forms of ERK1/2 and JNK1/2 were decreased under anoxic incubation. The PP-2A inhibitor, okadaic acid (OA, 0.75 microm), completely prevented tau from acute anoxia-induced dephosphorylation and restored the active forms of ERK1/2 and JNK1/2 to the control level. The activities and protein levels of GSK-3 and PP-1 showed no change during acute anoxia. These data suggest that acute anoxia induces tau dephosphorylation, and that PP-2A may play a key role in tau dephosphorylation induced by acute anoxia.     

Involvement of I2PP2A in the abnormal hyperphosphorylation of tau and its reversal by Memantine

The activity of protein phosphatase (PP)-2A, which regulates tau phosphorylation, is compromised in Alzheimer disease brain. Here we show that the transient transfection of PC12 cells with inhibitor-2 (I2PP2A) of PP2A causes abnormal hyperphosphorylation of tau at Ser396/Ser404 and Ser262/Ser356. This hyperphosphorylation of tau is observed only when a sub-cellular shift of I2PP2A takes place from the nucleus to the cytoplasm and is accompanied by cleavage of I2PP2A into a 20 kDa fragment. Memantine, an un-competitive inhibitor of N-methyl-D-aspartate receptors, inhibits this abnormal phosphorylation of tau and cell death and prevents the I2PP2A-induced inhibition of PP2A activity in vitro. These findings demonstrate novel mechanisms by which I2PP2A regulates the intracellular activity of PP2A and phosphorylation of tau, and by which Memantine modulates PP2A signaling and inhibits neurofibrillary degeneration.     

Alzheimer neurofibrillary degeneration: significance, etiopathogenesis, therapeutics and prevention

Alzheimer disease (AD) is multi-factorial and heterogeneous. Independent of the aetiology, this disease is characterized clinically by chronic and progressive dementia and histopathologically by neurofibrillary degeneration of abnormally hyperphosphorylated tau seen as intraneuronal neurofibrillary tangles, neuropil threads and dystrophic neurites, and by neuritic (senile) plaques of beta-amyloid. The neurofibrillary degeneration is apparently required for the clinical expression of AD, and in related tauopathies it leads to dementia in the absence of amyloid plaques. While normal tau promotes assembly and stabilizes microtubules, the abnormally hyperphosphorylated tau sequesters normal tau, MAP1 and MAP2, and disrupts microtubules. The abnormal hyperphosphorylation of tau also promotes its self-assembly into tangles of paired helical and or straight filaments. Tau is phosphorylated by a number of protein kinases. Glycogen synthase kinase-3 (GSK-3) and cyclin dependent protein kinase 5 (cdk5) are among the kinases most implicated in the abnormal hyperphosphorylation of tau. Among the phosphatases which regulate the phosphorylation of tau, protein phosphatase-2A (PP-2A), the activity of which is down-regulated in AD brain, is by far the major enzyme. The inhibition of abnormal hyperphosphorylation of tau is one of the most promising therapeutic targets for the development of disease modifying drugs. A great advantage of inhibiting neurofibrillary degeneration is that it can be monitored by evaluating the levels of total tau and tau phosphorylated at various known abnormally hyperphosphorylated sites in the cerebrospinal fluid of patients, obtained by lumbar puncture. There are at least five subgroups of AD, each is probably caused by a different etiopathogenic mechanism. The AD subgroup identification of patients can help increase the success of clinical trials and the development of specific and potent disease modifying drugs.     

Phosphorylation inhibits turnover of the tau protein by the proteasome: influence of RCAN1 and oxidative stress

Hyperphosphorylated tau proteins accumulate in the paired helical filaments of neurofibrillary tangles seen in such tauopathies as Alzheimer's disease. In the present paper we show that tau turnover is dependent on degradation by the proteasome (inhibited by MG132) in HT22 neuronal cells. Recombinant human tau was rapidly degraded by the 20 S proteasome in vitro, but tau phosphorylation by GSK3beta (glycogen synthase kinase 3beta) significantly inhibited proteolysis. Tau phosphorylation was increased in HT22 cells by OA [okadaic acid; which inhibits PP (protein phosphatase) 1 and PP2A] or CsA [cyclosporin A; which inhibits PP2B (calcineurin)], and in PC12 cells by induction of a tet-off dependent RCAN1 transgene (which also inhibits PP2B). Inhibition of PP1/PP2A by OA was the most effective of these treatments, and tau hyperphosphorylation induced by OA almost completely blocked tau degradation in HT22 cells (and in cell lysates to which purified proteasome was added) even though proteasome activity actually increased. Many tauopathies involve both tau hyperphosphorylation and the oxidative stress of chronic inflammation. We tested the effects of both cellular oxidative stress, and direct tau oxidative modification in vitro, on tau proteolysis. In HT22 cells, oxidative stress alone caused no increase in tau phosphorylation, but did subtly change the pattern of tau phosphorylation. Tau was actually less susceptible to direct oxidative modification than most cell proteins, and oxidized tau was degraded no better than untreated tau. The combination of oxidative stress plus OA treatment caused extensive tau phosphorylation and significant inhibition of tau degradation. HT22 cells transfected with tau-CFP (cyan fluorescent protein)/tau-GFP (green fluorescent protein) constructs exhibited significant toxicity following tau hyperphosphorylation and oxidative stress, with loss of fibrillar tau structure throughout the cytoplasm. We suggest that the combination of tau phosphorylation and tau oxidation, which also occurs in tauopathies, may be directly responsible for the accumulation of tau aggregates.     

Galpha12 directly interacts with PP2A: evidence FOR Galpha12-stimulated PP2A phosphatase activity and dephosphorylation of microtubule-associated protein, tau

The Galpha(12/13) family of heterotrimeric G proteins modulate multiple cellular processes including regulation of the actin cytoskeleton. Galpha(12/13) interact with several cytoskeletal/scaffolding proteins, and in a yeast two-hybrid screen with Galpha(12), we detected an interaction with the scaffolding subunit (Aalpha) of the Ser/Thr phosphatase, protein phosphatase 2A (PP2A). PP2A dephosphorylates multiple substrates including tau, a microtubule-associated protein that is hyperphosphorylated in neurofibrillary tangles. The interaction of Aalpha and Galpha(12) was confirmed by coimmunoprecipitation studies in transfected COS cells and by glutathione S-transferase (GST)-Galpha(12) pull-downs from cell lysates of primary neurons. The interaction was specific for Aalpha and Galpha(12) and was independent of Galpha(12) conformation. Endogenous Aalpha and Galpha(12) colocalized by immunofluorescent microscopy in Caco-2 cells and in neurons. In vitro reconstitution of GST-Galpha(12) or recombinant Galpha(12) with PP2A core enzyme resulted in approximately 300% stimulation of PP2A activity that was not detected with other Galpha subunits and was similar with GTPgammaS- and GDP-liganded Galpha(12). When tau and active kinase (Cdk5 and p25) were cotransfected in to COS cells, there was robust tau phosphorylation. Co-expression of wild type or QLalpha(12) with tau and the active kinase resulted in 60 +/- 15% reductions in tau phosphorylation. In primary cortical neurons stimulated with lysophosphatitic acid, a 50% decrease in tau phosphorylation was observed. The Galpha(12) effect on tau phosphorylation was inhibited by the PP2A inhibitor, okadaic acid (50 nm), in COS cells and neurons. Taken together, these findings reveal novel, direct regulation of PP2A activity by Galpha(12) and potential in vivo modulation of PP2A target proteins including tau.     

Phosphorylated PP2A (tyrosine 307) is associated with Alzheimer neurofibrillary pathology

Down-regulation of protein phosphatase 2A (PP2A) is thought to play a critical role in tau hyperphosphorylation in Alzheimer's disease (AD). In vitro phosphorylation of PP2A catalytic subunit at Y307 efficiently inactivates PP2A. A specific antibody against phosphorylated (p) PP2A (Y307) (PP2Ac-Yp307) was used to investigate possible PP2A down-regulation by known pathophysiological changes associated with AD, such as Abeta accumulation and oestrogen deficiency. Immunohistochemistry and immunofluorescence confocal microscopy showed an aberrant accumulation of PP2Ac-Yp307 in neurons that bear pretangles or tangles in the susceptible brain regions, such as the entorhinal cortical cortex and the hippocampus. Experimentally, increased PP2Ac-Yp307 was observed in mouse N2a neuroblastoma cells that stably express the human amyloid precursor protein with Swedish mutation (APPswe) compared with wild-type, and in the brains of transgenic APPswe/ presenilin (PS1, A246E) mice, which corresponded to the increased tau phosphorylation. Treating N2a cells with Abeta25-35 mimicked the changes of PP2Ac-Yp307 and tau phosphorylation in N2a APPswe cells. Knockout of oestrogen receptor (ER) alpha or ERbeta gave similar changes of PP2Ac-Yp307 level and tau phosphorylation in the mouse brain. Taken together, these findings suggest that increased PP2A phosphorylation (Y307) can be mediated by Abeta deposition or oestrogen deficiency in the AD brain, and consequently compromise dephosphorylation of abnormally hyperphosphorylated tau, and lead to neurofibrillary tangle formation.     

PTEN, a negative regulator of PI3 kinase signalling, alters tau phosphorylation in cells by mechanisms independent of GSK-3

Deregulation of PTEN/Akt signalling has been recently implicated in the pathogenesis of Alzheimer's disease (AD), but the effects on the molecular processes underlying AD pathology have not yet been fully described. Here we report that overexpression of PTEN reduces tau phosphorylation in CHO cells. This effect was abrogated by mutant PTEN constructs with either a catalytically inactive point mutation (C124S) or with only inactive lipid phosphatase activity (G129E), suggesting an indirect, lipid phosphatase-dependent process. The predominant effects of PTEN on tau appeared to be mediated by reducing ERK1/2 activity, but were independent of Akt, GSK-3, JNK and the tau phosphatases PP1 and PP2A. Our studies provide evidence for an effect of PTEN on the phosphorylation of tau in AD pathogenesis, and provide some insight into the mechanisms through which deregulation of PTEN may contribute towards the progression of tauopathy.     

蛋白磷酸酯酶抑制剂冈田酸对人神经母细胞瘤 SK-N-SH细胞系tau蛋白磷酸化水平的影响

观察蛋白磷酸酯酶-1和蛋白磷酸酯酶-2A的抑制剂冈田酸(okadaicacid,OA)对人神经母细胞瘤系SK-N-SH细胞tau蛋白磷酸化水平的变化,确定tau蛋白过度磷酸化细胞模型的合适剂量和时间。用不同剂量OA与SK-N-SH细胞共温育不同时间,用显微镜观察细胞形态变化,用Western印迹法检测磷酸化tau蛋白和非磷酸化tau蛋白在Ser202位点和Ser404位点磷酸化水平的变化。10～160nmol/LOA与SK-N-SH神经细胞温育3～24h,可引起细胞形态损伤呈剂量依赖性和时间依赖性的变化,起效剂量和时间为10nmol/L和3h。10nmol/LOA与SK-N-SH细胞温育6～24h,磷酸化tau蛋白Ser199/Ser202位点和Ser404位点的表达明显增高,非磷酸化tau蛋白Ser202位点和Ser404位点的表达明显降低,总tau蛋白含量无明显变化。OA可以作为很好的研究tau蛋白过度磷酸化的工具药,10nmol/LOA与SK-N-SH神经细胞共温育6h可以作为制备细胞模型的适宜条件。     

Inhibition of protein phosphatase 2A overrides tau protein kinase I/glycogen synthase kinase 3 beta and cyclin-dependent kinase 5 inhibition and results in tau hyperphosphorylation in the hippocampus of starved mouse.

Hyperphosphorylated tau is the major component of paired helical filaments in neurofibrillary tangles found in Alzheimer's disease (AD) brain. Starvation of adult mice induces tau hyperphosphorylation at many paired helical filaments sites and with a similar regional selectivity as those in AD, suggesting that a common mechanism may be mobilized. Here we investigated the mechanism of starvation-induced tau hyperphosphorylation in terms of tau kinases and Ser/Thr protein phosphatases (PP), and the results were compared with those reported in AD brain. During starvation, tau hyperphosphorylation at specific epitopes was accompanied by decreases in tau protein kinase I/glycogen synthase kinase 3 beta (TPKI/GSK3 beta), cyclin-dependent kinase 5 (cdk5), and PP2A activities toward tau. These results demonstrate that the activation of TPKI/GSK3 beta and cdk5 is not necessary to obtain hyperphosphorylated tau in vivo, and indicate that inhibition of PP2A is likely the dominant factor in inducing tau hyperphosphorylation in the starved mouse, overriding the inhibition of key tau kinases such as TPKI/GSK3 beta and cdk5. Furthermore, these data give strong support to the hypothesis that PP2A is important for the regulation of tau phosphorylation in the adult brain, and provide in vivo evidence in support of a central role of PP2A in tau hyperphosphorylation in AD.     

Dephosphorylation of tau by protein phosphatase 5: impairment in Alzheimer's disease

Protein phosphatase (PP) 5 is highly expressed in the mammalian brain, but few physiological substrates have yet been identified. Here, we investigated the kinetics of dephosphoryation of phospho-tau by PP5 and found that PP5 had a K(m) of 8-13 microm toward tau, which is similar to that of PP2A, the major known tau phosphatase. This K(m) value is within the range of intraneuronal tau concentration in human brain, suggesting that tau could be a physiological substrate of both PP5 and PP2A. PP5 dephosphorylated tau at all 12 Alzheimer's disease (AD)-associated abnormal phosphorylation sites studied, with different efficiency toward each site. Thr(205), Thr(212), and Ser(409) of tau were the most favorable sites; Ser(199), Ser(202), Ser(214), Ser(396), and Ser(404) were less favorable sites; and Ser(262) was the poorest site for PP5. Overexpression of PP5 in PC12 cells resulted in dephosphorylation of tau at multiple phosphorylation sites. The activity but not the protein level of PP5 was found to be decreased by approximately 20% in AD neocortex. These results suggest that tau is probably a physiological substrate of PP5 and that the abnormal hyperphosphorylation of tau in AD might result in part from the decreased PP5 activity in the diseased brains.