[논문]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

doi:10.4196/kjpp.2014.18.2.155

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

The Korean journal of physiology & pharmacology : official journal of the Korean Physiological Society and the Korean Society of Pharmacology, v.18 no.2, 2014년, pp.155 - 161

Yu, Un Young (Department of Biochemistry, Ewha Womans University College of Medicine) , Yoo, Byong Chul (Colorectal Cancer Branch, Research Institute, National Cancer Center) , Ahn, Jung-Hyuck (Department of Biochemistry, Ewha Womans University College of Medicine)

Abstract ▼ AI-Helper

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.

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가설 설정

In this study, we examined the molecular composition and substrate specificity of PP2A towards tau protein. Moreover, PPP2R5D was required for inhibition of Akt activity by dephosphorylating the activating phosphorylation sites at Thr-308 and Ser-473.

제안 방법

The final concentrations of plasmid and siRNA were 1 μg/mL and 25 nM respectively. For the experiment, pCMV6 plasmid vector containing cDNA encoding the longest human tau (tau 441) gene purchased Origene (Origen Technologies, Inc., Rockville, MD, USA) and On-Target plus smart pool siRNAs targeting human PPP2R2A, PPP2R3A and PPP2R5D and On-Target plus non-targeting siRNAs were purchased Dharmacon (Dharmacon, Thermo Fisher Scientific, Inc., Chicago, IL, USA). Expression and knockdown of proteins were confirmed by western blot analysis.

대상 데이터

After washing TBST, the membranes incubated with an anti-mouse or -rabbit HRP-conjugated secondary antibody for 2 h. The antibodies used in this study were against Flag (1：1,000) from Sigma (Sigma Aldrich, St.Louis, MO, USA), Tau (1：1,000) and phospho Tau (Ser262) (1：1,000) from Invitrogen (Invitrogen, Carlsbad, CA, USA), phospho Tau (Ser202/Thr205, Thr231) (1：500) from Pierce (Pierce Biotechnology, Inc., Rockford, IL, USA), phospho Tau (Ser199, Ser404, Ser422) (1：1,000) from Abcam (Abcam, Cambridge, UK), PP2R2A (1：1,000) from cell signalling (Cell Signalling Technology, Danvers, MA, USA), and beta-actin (1：1,000) and secondary antibody (1：2,000) from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membranes were developed by enhanced chemiluminescence detection (GE Healthcare, Little Chalfont, Buckinghamshire, UK) at LAS 3000 (Fugifilm UK Ltd.

데이터처리

Signals from H4-swe cells were normalized to values obtained for H4 wildtype cells. Statistical analyses were performed using a t-test (*p＜0.001, **p＜0.05). Data are the means± standard error of the mean (SEM, n=3).
The bar graph shows the relative signal intensity of siRNA- transfected samples compared to the control siRNA-transfected sample. Statistical analyses were performed using a t-test (*p＜0.001, **p＜0.01). Data are the means±standard error of the mean (SEM, n=3).
Bar graphs show the phosphorylation level for each site normalized to control siRNAtreated H4 cells. Statistical analyses were performed using a t-test (*p＜0.001, **p＜0.01, ***p＜0.05). Data are the means±standard error of the mean (SEM, n=3).
Signals from H4-swe cells were normalized to values obtained for H4 wildtype cells. Statistical analyses were performed using a t-test (*p＜0.001, **p＜0.05). Data are the means± standard error of the mean (SEM, n=3).
Statistical analysis and graphing were performed using Graphpad Prism 5 (Graphpad, San Diego, CA, USA). Significance was determined using a two-tailed Student’s t-test and p values＜0.

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