Regulatory B Subunits of Protein Phosphatase 2A Are Involved in Site-specific Regulation of Tau Protein Phosphorylation

Un Young Yu,Byong Chul Yoo,Jung-Hyuck Ahn 대한생리학회-대한약리학회 2014 The Korean Journal of Physiology & Pharmacology Vol.18 No.2

Overexpression of amyloid precursor protein with the Swedish mutation causes abnormal hyperphos-phorylation of the microtubule-associated protein tau. Hyperphosphorylated isoforms of tau are major components of neurofibrillary tangles, which are histopathological hallmarks of Alzheimer’s disease. Protein phosphatase 2A (PP2A), a major tau protein phosphatase, consists of a structural A subunit, catalytic C subunit, and a variety of regulatory B subunits. The B subunits have been reported to modulate function of the PP2A holoenzyme by regulating substrate binding, enzyme activity, and subcellular localization. In the current study, we characterized regulatory B subunit-specific regulation of tau protein phosphorylation. We showed that the PP2A B subunit PPP2R2A mediated dephosphory-lation of tau protein at Ser-199, Ser-202/Thr-205, Thr-231, Ser-262, and Ser-422. Down-regulation of PPP2R5D expression decreased tau phosphorylation at Ser-202/Thr-205, Thr-231, and Ser-422, which indicates activation of the tau kinase glycogen synthase kinase 3 beta (GSK3<em>β</em>) by PP2A with PPP2R5D subunit. The level of activating phosphorylation of the GSK3<em>β</em> kinase Akt at Thr-308 and Ser-473 were both increased by PPP2R5D knockdown. We also characterized B subunit-specific phos-phorylation sites in tau using mass spectrometric analysis. Liquid chromatography-mass spectrometry revealed that the phosphorylation status of the tau protein may be affected by PP2A, depending on the specific B subunits. These studies further our understanding of the function of various B subunits in mediating site-specific regulation of tau protein phosphorylation.

Regulatory B Subunits of Protein Phosphatase 2A Are Involved in Site-specific Regulation of Tau Protein Phosphorylation

Yu, Un Young,Yoo, Byong Chul,Ahn, Jung-Hyuck The Korean Society of Pharmacology 2014 The Korean Journal of Physiology & Pharmacology Vol.18 No.2

Overexpression of amyloid precursor protein with the Swedish mutation causes abnormal hyperphosphorylation of the microtubule-associated protein tau. Hyperphosphorylated isoforms of tau are major components of neurofibrillary tangles, which are histopathological hallmarks of Alzheimer's disease. Protein phosphatase 2A (PP2A), a major tau protein phosphatase, consists of a structural A subunit, catalytic C subunit, and a variety of regulatory B subunits. The B subunits have been reported to modulate function of the PP2A holoenzyme by regulating substrate binding, enzyme activity, and subcellular localization. In the current study, we characterized regulatory B subunit-specific regulation of tau protein phosphorylation. We showed that the PP2A B subunit PPP2R2A mediated dephosphorylation of tau protein at Ser-199, Ser-202/Thr-205, Thr-231, Ser-262, and Ser-422. Down-regulation of PPP2R5D expression decreased tau phosphorylation at Ser-202/Thr-205, Thr-231, and Ser-422, which indicates activation of the tau kinase glycogen synthase kinase 3 beta ($GSK3{\beta}$) by PP2A with PPP2R5D subunit. The level of activating phosphorylation of the $GSK3{\beta}$ kinase Akt at Thr-308 and Ser-473 were both increased by PPP2R5D knockdown. We also characterized B subunit-specific phosphorylation sites in tau using mass spectrometric analysis. Liquid chromatography-mass spectrometry revealed that the phosphorylation status of the tau protein may be affected by PP2A, depending on the specific B subunits. These studies further our understanding of the function of various B subunits in mediating site-specific regulation of tau protein phosphorylation.

The Change in Circadian Rhythms in P301S Transgenic Mice is Linked to Variability in Hsp70-related Tau Disaggregation

한송미,장유정,김은영,박선아 한국뇌신경과학회 2022 Experimental Neurobiology Vol.31 No.3

Circadian disruption often involves a neurodegenerative disorder, such as Alzheimer’s disease or frontotemporal dementia, which are characterized by intraneuronal tau accumulations. The altered sleep pattern and diurnal rhythms in these disorders are the results of tau pathology. The circadian disturbance in reverse is thought to develop and potentially aggravate the condition. However, the underlying mechanism is not fully understood. In this study, perturbed oscillations in BMAL1 , the core clock gene, were observed in P301S tau transgenic mice. Tau fractionation analysis of the hippocampus revealed profound fluctuations in soluble and insoluble tau protein levels that were in opposite directions to each other according to zeitgeber time. Interestingly, a diurnal oscillation was detected in the heat shock 70 kDa protein 1A (Hsp70) chaperone that was in-phase with soluble tau but out-of-phase with insoluble tau. Tau protein levels decreased in the soluble and insoluble fractions when Hsp70 was overexpressed in HEK293T cells. Transfection of the BMAL1 carrying vector was continual with the increase in Hsp70 expression and diminished tau protein levels, and it was effectively attenuated by the knockdown of Hsp70, suggesting that Bmal1 could modulate tau protein by Hsp70. Our results suggest that altered circadian oscillations affect tau status and solubility by modulating Hsp70 expression in an experimental model of tau pathology. These findings suggest Hsp70 as a possible pathogenic link between circadian disruption and aggravations of tau pathology.

Regulatory B Subunits of Protein Phosphatase 2A Are Involved in Site-specific Regulation of Tau Protein Phosphorylation

유은영,유병철,안정혁 대한약리학회 2014 The Korean Journal of Physiology & Pharmacology Vol.18 No.2

Overexpression of amyloid precursor protein with the Swedish mutation causes abnormal hyperphosphorylationof the microtubule-associated protein tau. Hyperphosphorylated isoforms of tau are majorcomponents of neurofibrillary tangles, which are histopathological hallmarks of Alzheimer’s disease. Protein phosphatase 2A (PP2A), a major tau protein phosphatase, consists of a structural A subunit,catalytic C subunit, and a variety of regulatory B subunits. The B subunits have been reported tomodulate function of the PP2A holoenzyme by regulating substrate binding, enzyme activity, andsubcellular localization. In the current study, we characterized regulatory B subunit-specific regulationof tau protein phosphorylation. We showed that the PP2A B subunit PPP2R2A mediated dephosphorylationof tau protein at Ser-199, Ser-202/Thr-205, Thr-231, Ser-262, and Ser-422. Down-regulation ofPPP2R5D expression decreased tau phosphorylation at Ser-202/Thr-205, Thr-231, and Ser-422, whichindicates activation of the tau kinase glycogen synthase kinase 3 beta (GSK3β ) by PP2A withPPP2R5D subunit. The level of activating phosphorylation of the GSK3β kinase Akt at Thr-308 andSer-473 were both increased by PPP2R5D knockdown. We also characterized B subunit-specific phosphorylationsites in tau using mass spectrometric analysis. Liquid chromatography-mass spectrometryrevealed that the phosphorylation status of the tau protein may be affected by PP2A, depending onthe specific B subunits. These studies further our understanding of the function of various B subunitsin mediating site-specific regulation of tau protein phosphorylation.

14-3-3 Protein Mediates Phosphorylation of Microtubule-associated Protein Tau by Serum- and Glucocorticoid-induced Protein Kinase 1

천재순,강상순,Taegun Kwon,이은정,Chang Hyun Kim,한연수,홍순광,Sounghee Hyun 한국분자세포생물학회 2004 Molecules and cells Vol.18 No.3

The microtubule-associated protein, tau, is involved in numerous neuronal processes such as vesicle transport, microtubule-plasma membrane interaction and the intracellular localization of proteins. Tau is known to be phosphorylated by several kinases such as mitogen activated protein kinase, microtubule affinity regulating kinase, and protein kinase A. We found a putative serum- and glucocorticoid- induced protein kinase 1 (SGK1) phosphorylation site within the 207GSRSRTPSLP216 tau amino acid sequence. We report here that SGK1 phosphorylates Ser214 of Tau. Using a pull-down assay, we found that 14-3-3θ interacts with SGK1 and tau to form a ternary protein complex that leads to phosphorylation of tau. 14-3-3 and phosphorylated tau were mainly co-localized in the nucleus of COS-1 cells. These results demonstrate that 14-3-3 scaffolds tau with SGK1 to facilitate the phosphorylation of tau at Ser214 and to regulate its subcellular localization.

한국의 알츠하이머병 환자에서 아세틸콜린 에스터레이스 억제제의 뇌척수액 베타아밀로이드 1-42 분획과 인산화타우단백 농도에 미치는 효과

이은희,윤영철,박광열,민주홍,권오상,이현옥,홍현종 대한신경과학회 2008 대한신경과학회지 Vol.26 No.3

Background: Alzheimer’s disease (AD) is characterized by the pathology of amyloid plaques and tau-associated neurofibrillary tangles. Acetylcholine esterase (AChE) transforms the β-amyloid monomer into an oligomer, and increases β-amyloid aggregation in the brain. Increased β-amyloid breaks the cytoskeleton of the brain by hyperphosphorylation of the tau protein. Previous studies support that AChE inhibitor has an inhibitory effect on toxicity of the β-amyloid and phophorylated tau protein. The purpose of this study was to analyze the CSF β-amyloid 1-42 (Aβ1-42) and phosphorylated tau protein in AD and determine their difference depending on whether AChE inhibitor was taken or not. Methods: Subjects included 16 AD, 14 normal controls, and 15 disease controls. Nine of AD group had taken an AChE inhibitor while the remainder had not. The CSF Aβ1-42 and phosphorylated tau were measured by ELISA. Results: The CSF Aβ1-42 levels were lower in AD patients than in other groups (p<0.01). We also found increased CSF Aβ1-42 levels in the AChE inhibitor users, compared with non-users. Conclusions: The level of CSF Aβ1-42 may have a diagnostic value in the patients with cognitive impairments. Also, we may expect the effect of AChE inhibitor on Alzheimer’s pathology by measuring CSF Aβ1-42 levels. Therefore, the level of CSF Aβ1-42 may serve as a biological surrogate marker for AD treatment. Background: Alzheimer’s disease (AD) is characterized by the pathology of amyloid plaques and tau-associated neurofibrillary tangles. Acetylcholine esterase (AChE) transforms the β-amyloid monomer into an oligomer, and increases β-amyloid aggregation in the brain. Increased β-amyloid breaks the cytoskeleton of the brain by hyperphosphorylation of the tau protein. Previous studies support that AChE inhibitor has an inhibitory effect on toxicity of the β-amyloid and phophorylated tau protein. The purpose of this study was to analyze the CSF β-amyloid 1-42 (Aβ1-42) and phosphorylated tau protein in AD and determine their difference depending on whether AChE inhibitor was taken or not. Methods: Subjects included 16 AD, 14 normal controls, and 15 disease controls. Nine of AD group had taken an AChE inhibitor while the remainder had not. The CSF Aβ1-42 and phosphorylated tau were measured by ELISA. Results: The CSF Aβ1-42 levels were lower in AD patients than in other groups (p<0.01). We also found increased CSF Aβ1-42 levels in the AChE inhibitor users, compared with non-users. Conclusions: The level of CSF Aβ1-42 may have a diagnostic value in the patients with cognitive impairments. Also, we may expect the effect of AChE inhibitor on Alzheimer’s pathology by measuring CSF Aβ1-42 levels. Therefore, the level of CSF Aβ1-42 may serve as a biological surrogate marker for AD treatment.

Direct force measurements reveal that protein Tau confers short-range attractions and isoform-dependent steric stabilization to microtubules

Chung, Peter J.,Choi, Myung Chul,Miller, Herbert P.,Feinstein, H. Eric,Raviv, Uri,Li, Youli,Wilson, Leslie,Feinstein, Stuart C.,Safinya, Cyrus R. National Academy of Sciences 2015 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.112 No.47

<P><B>Significance</B></P><P>The microtubule-associated protein Tau is known to stabilize microtubules against depolymerization in neuronal axons, ensuring proper trafficking of organelles along microtubules in long axons. Abnormal interactions between Tau and microtubules are implicated in Alzheimer’s disease and other neurodegenerative disorders. We directly measured forces between microtubules coated with Tau isoforms by synchrotron small-angle X-ray scattering of reconstituted Tau–microtubule mixtures under osmotic pressure (mimicking molecular crowding in cells). We found that select Tau isoforms fundamentally alter forces between microtubules by undergoing a conformational change on microtubule surfaces at a coverage indicative of an unusually extended Tau state. This gain of function by longer isoforms in imparting steric stabilization to microtubules is essential in preventing microtubule aggregation and loss of function in organelle trafficking.</P><P>Microtubules (MTs) are hollow cytoskeletal filaments assembled from αβ-tubulin heterodimers. Tau, an unstructured protein found in neuronal axons, binds to MTs and regulates their dynamics. Aberrant Tau behavior is associated with neurodegenerative dementias, including Alzheimer’s. Here, we report on a direct force measurement between paclitaxel-stabilized MTs coated with distinct Tau isoforms by synchrotron small-angle X-ray scattering (SAXS) of MT-Tau mixtures under osmotic pressure (<I>P</I>). In going from bare MTs to MTs with Tau coverage near the physiological submonolayer regime (Tau/tubulin-dimer molar ratio; Φ<SUB>Tau</SUB> = 1/10), isoforms with longer N-terminal tails (NTTs) sterically stabilized MTs, preventing bundling up to <I>P</I><SUB>B</SUB> ∼ 10,000–20,000 Pa, an order of magnitude larger than bare MTs. Tau with short NTTs showed little additional effect in suppressing the bundling pressure (<I>P</I><SUB>B</SUB> ∼ 1,000–2,000 Pa) over the same range. Remarkably, the abrupt increase in <I>P</I><SUB>B</SUB> observed for longer isoforms suggests a mushroom to brush transition occurring at 1/13 < Φ<SUB>Tau</SUB> < 1/10, which corresponds to MT-bound Tau with NTTs that are considerably more extended than SAXS data for Tau in solution indicate. Modeling of Tau-mediated MT–MT interactions supports the hypothesis that longer NTTs transition to a polyelectrolyte brush at higher coverages. Higher pressures resulted in isoform-independent irreversible bundling because the polyampholytic nature of Tau leads to short-range attractions. These findings suggest an isoform-dependent biological role for regulation by Tau, with longer isoforms conferring MT steric stabilization against aggregation either with other biomacromolecules or into tight bundles, preventing loss of function in the crowded axon environment.</P>

Atomic Level Investigations of Early Aggregation of Tau43 in Water II. Tau43-Aβ42 vs. Tau43-Tau43 Dimerizations

Prathit Chatterjee,Myung Keun Cho,Huong T. D. Bui,Sihyun Ham 대한화학회 2021 Bulletin of the Korean Chemical Society Vol.42 No.8

Amyloid ? (A?) senile plaques and Tau neurofibrillary tangles (NFTs) are major hallmarks of Alzheimer's disease (AD). However, early stages of Tau aggregation are still limitedly recognized. Here, we present atomistic molecular dynamics simulations and thermodynamics characterizations of heterogeneous Tau43-A?42 and homogeneous Tau43-Tau43 dimerization processes. Two-stage approaching-accommodation mechanism after individual diffusive regime is observed. The approach step involves opposing forces driving two distant monomers to come closer to each other, which are the decrease in protein internal and water-induced energies, respectively. In the accommodation step, a decrease in protein internal energy is the main driving force for stable compact structure formation. While the charged residues differently initiate and stabilize the dimer structures, the hydrophobic residues (11VQIVYK16 in Tau43 and 39VVIA42 in A?42) facilitate the formation of compact dimers, in agreement with experiments. Our results of Tau43-A?42 and Tau43-Tau43 dimerization will illuminate early onset mechanisms of AD pathology and corresponding therapeutic initiatives.

인체 폐암조직에서 Phospholipase C-$\gamma1$의 활성화 단백, AHNAK의 발현양상

오윤정,박준성,최소연,정성철,이선민,황성철,이이형,한명호,이기범,류한영,하만준,배윤수,이서구,Oh, Yoon-Jung,Park, Chun-Seong,Choi, So-Yeon,Cheong, Seong-Cheoll,Lee, Sun-Min,Hwang, Sung-Chul,Lee, Yi-Hyeong,Hahn, Myung-Ho,Lee, Kyi-Beom,Ryu, Han 대한결핵및호흡기학회 1999 Tuberculosis and Respiratory Diseases Vol.47 No.3

배경: Phospholipase C(PLC)는 세포의 성장, 분화, 변형(transformation)과 관련된 세포내 신호 전달과정에 중추적인 역할을 하는 효소이다. 이들 중 PLC-$\gamma$는 tyrosine kinase의 인산화에 의해 주로 활성화되는 데, 최근에 phosphatidic acid(PA), phosphatidy-linositol 3, 4, 5-trisphosphate($PIP_3$), tau 단백에 의한 활성화 기전이 밝혀진 바 있다. 특히 tau 단백은 bovine brain에서 arachidonic acid와 함께 PLC-$\gamma$를 활성화시키는 것으로 알려져 PLC-$\gamma$와 $PLA_2$ 사이의 cross-talk이 이루어질 가능성이 제시되고 있다. 최근 보고에 의하면 tau 단백과 같은 기전으로 PLC-${\gamma}1$ 활성화시키는 단백이 bovine lung에서 발견되었고, 이 활성화 단백을 정제 및 클론하여 AHNAK 단백임이 확인된 바 있다. 또한 PLC-${\gamma}1$이 유방암, 대장암, 위암 등에서 증가되어 있어 발암 과정과 연관되어 있음이 보고되어 왔으나 PLC-${\gamma}1$의 활성화 단백인 AHNAK 단백에 대해서는 질병과 관련되어 연구된 것이 아직 없는 실정이며 저자 등은 폐암 조직과 정상 폐조직에서 AHNAK 단백의 발현 양상을 연구하여 폐암의 발암과정에 AHNAK 단백이 관여함을 밝히고자 하였다. 대상 및 방법: 아주대학교 병원에 내원하여 폐암으로 수술을 받은 환자의 폐암 조직과 동일 환자의 정상 폐조직에서 AHNAK 단백의 발현양상을 western blot 분석과 면역조직화학적 염색방법을 통하여 조사하였다. 결과: 14예의 편평상피암 세포조직 중 8예 (57.1 %)와 14예의 선암 세포조직 모두에서 정상 대조군에 비해 AHNAK 단백의 발현이 증가하였고, 70 kDa~200kDa의 여러가지 분자량을 가지는 띠모양으로 나타났다. 면역조직화학적 염색에서도 정상 폐조직보다 폐암 조직내에서 강한 발색반응을 보였다. 결론: PLC-${\gamma}1$의 활성화 단백인 AHNAK 단백이 폐암 조직에서 정상 조직보다 과발현된 것은, AHNAK 단백이 PLC-${\gamma}1$을 활성화시켜 폐암의 발생 기전에 관여할 수 있음을 뒷받침한다고 하겠다. Background: Phospholipase C(PLC) plays a central role in cellular signal transduction and is important in cellular growth, differentiation and transformation. There are currently ten known mammalian isozymes of PLC reported to this date. Hydrolysis of phosphatidylinositol 4,5-bisphosphate($PIP_2$) by PLC produces two important second messengers, inositol 1,4,5-trisphosphate($IP_3$) and diacylglycerol. PLC-${\gamma}1$, previously, was known to be activated mainly through growth factor receptor tyrosine kinase. Other mechanisms of activating PLC-yl have been reported such as activation through tau protein in the presence of arachidonic acid in bovine brain and activation by $IP_3$, phosphatidic acid, etc. Very recently, another PLC-${\gamma}1$ activator protein such as tau has been found in bovine lung tissue, which now is considered to be AHNAK protein. But there has been no report concerning AHNAK and its associated disease to this date. In this study, we examined the expression of the PLC-${\gamma}1$ activator, AHNAK, in lung cancer specimens and their paired normal. Methods: From surgically resected human lung cancer tissues taken from twenty-eight patients and their paired normal counterparts, we evaluated expression level of AHNAK protein using immunoblot analysis of total tissue extract Immunohistochemical stain was performed with primary antibody against AHNAK protein. Results: Twenty-two among twenty-eight lung cancer tissues showed overexpression of AHNAK protein (eight of fourteen squamous cell lung cancers, all of fourteen adenocarcinomas). The resulting bands were multiple ranging from 70 to 200 kDa in molecular weight and each band was indistinct and formed a smear, reflecting mobility shift mainly due to proteolysis during extraction process. On immunohistochemistry, lung cancer tissues showed a very heavy, dense staining with anti-AHNAK protein antibody as compared to the surrounding normal lung tissue, coresponding well with the results of the western blot Conclusion: The overexpression of PLC-${\gamma}1$ activator protein, AHNAK in lung cancer may provide evidence that the AHNAK protein and PLC-${\gamma}1$ act in concerted manner in carcinogenesis.

Tau Protein Mediated Activation of Phospholipase C-γlsozyme is Dependent on the Presence of Arachidonic Acid

Hwang, Sung Chul,Hwang, Sung Hee 아주대학교 1996 아주의학 Vol.1 No.2

We have previously reported tyrosine kinase-independent activation of phospholipase C (PLC)-γ isozymes. The proteins that activated PLC- γ 1 have been purified from bovine brain and were identified as members of the tau family of microtubule-associated proteins'. Activation of PLC- γ isozymes is dependent on the composition of various lipids. The presence of unsaturated fatty acids such as arachidonic acid (AA) markedly enhances tau protein induced activation of PLC-γ while saturated fatty acids being ineffective in stimulating the activation. Maximal (15- to 20-fold) activation was observed in the presence of 15 μM tau protein with 25 uM AA. The effect of tau and arachidonic acid induced activation was specific and most prominent for PLC-γ isozymes in the presence of submicromolar concentrations of Ca^(2+). The effect was inhibited by the presence of phosphatidylcholine. Other polyunsaturated fatty acids, such as linoleic, linolenic, oleic and palmitoleic acids in the substrate mixture, induced similar activation but the effects were less prominent. These results suggest that, in tissues where tau protein is present, the receptors and signals that are coupled to the phospholipase A; may activate PLC- γ isozymes indirectly, without tyrosine phosphorylation, through hydrolysis of phosphatidylcholine and by generating AA, in the presence of tau protein. This process may work to amplify the signals and provide the linkage between the two different phospholipase systems.