Korean J Physiol Pharmacol.  2014 Apr;18(2):155-161. 10.4196/kjpp.2014.18.2.155.

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

• Yu UY
• Yoo BC
• Ahn JH

• KMID: 2285505
• DOI: http://doi.org/10.4196/kjpp.2014.18.2.155

Abstract

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 (GSK3beta) by PP2A with PPP2R5D subunit. The level of activating phosphorylation of the GSK3beta 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.

Keyword

Alzheimer's disease; Phosphorylation; Protein phosphatase 2A; Tau protein

MeSH Terms

Alzheimer Disease
Amyloid
Catalytic Domain
Down-Regulation
Glycogen Synthase Kinase 3
Negotiating
Neurofibrillary Tangles
Phosphorylation*
Phosphotransferases
Protein Isoforms
Protein Phosphatase 2*
Spectrum Analysis
tau Proteins*
Amyloid
Glycogen Synthase Kinase 3
Phosphotransferases
Protein Isoforms
Protein Phosphatase 2
tau Proteins

Figure

• 

  Fig. 1 Hyperphosphorylation of tau protein in the AD model cell line. A FLAG-tagged tau 441 expression construct was transfected into H4 wild-type cells (H4) or H4-swe cells, which express the Swedish mutant form of APP. The phosphorylation status was examined on (A) Ser-199, (B) Ser-202/Thr-205, (C) Thr-231, (D) Ser-262, (E) Ser-404, and (F) Ser-422 with the respective phospho-specific antibodies. Total tau protein was visualized with anti-FLAG antibody. Bar graphs show the phosphorylation levels of each phosphorylation site. 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).

• 

  Fig. 2 PP2A-mediated regulation of the tau protein phosphorylation level. FLAG-tagged tau 441-transfected H4-swe cells were treated with 10 nM okadaic acid for 12 hours. The phosphorylation status was examined on (A) Ser-199, (B) Ser-202/Thr-205, (C) Thr-231, (D) Ser-262, (E) Ser-404, and (F) Ser-422 with a phospho-specific antibody corresponding to each site. Total tau protein was visualized with anti-FLAG antibody. Bar graphs show the phosphorylation levels of each phosphorylation site. Signals from H4-swe cells were normalized to values obtained for DMSO vehicle-treated 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).

• 

  Fig. 3 Regulatory B subunits are involved in site-specific regulation of tau protein phosphorylation. B regulatory subunits were knocked down with siRNA specific to each B subunit; PPP2R2A siRNA (si2A), PPP2R3A siRNA (si3A), or PPP2R5D siRNA (si5D) was transfected into H4-swe cells. (A) The expression level of each B subunit was measured with antibodies corresponding to each B subunit. (B) Phosphorylation status analysis was performed with immunoblotting for each site as indicated. Bar graphs show the phosphorylation level for each site normalized to control siRNA-treated 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).

• 

  Fig. 4 PP2A with the PPP2R5D subunit regulates the AKT-GSK3β signaling pathway. H4 cells were transfected with control siRNA or PPP2R5D siRNA (left panel), or a control vector or a PPP2R5D expression construct (right panel) as indicated. The status of inhibitory phosphorylation at Ser-9 of GSK3β was visualized with a phospho-specific antibody. H4 cells were transfected with siRNA specific for the PPP2R5D subunit, and then the status of activating phosphorylation at Thr-308 and Ser-473 was visualized with a phospho-specific antibody corresponding to each site. 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).

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