Effects Of Glycogen Synthase Kinase-3 On Protein Phosphatase 2A Catalytic Subunit Translation And Post-translational Modifications And The Underlying Mechanisms

BackgroundProtein phosphatase 2A (PP2A) is one of major brain serine/threonine protein phosphatases. In vivo, PP2A predominantly exist as heterotrimers containing a scaffolding subunit (A), a regulatory subunit (B) and a catalytic subunit (C). The highly conserved carboxy-terminal sequence of the C subunit (PP2AC) can undergo post-translational modifications, including phosphorylation and carboxyl methylation. Phosphorylation level at tyrosine-307 (pY307) was regulated by tyrosine kinases and protein tyrosine phosphataselB (PTP1B), phosphorylation transiently inactivates PP2A. Methylation of PP2AC at leucine-309 (Leu309) was catalyzed by specific PP2A methyltransferase (PPMT1), and its demethylation was catalyzed by specific PP2A methylesterase (PME-1). Demethylation of PP2A at Leu309 inhibits PP2A. Phosphorylation and methylation of PP2Ac affects PP2A substrate pecificity, targeting and cellular functions.Among the kinases and phosphatases, glycogen synthase kinase-3β(GSK-3β) and PP2A are respectively the most implicated. It has been recognized that the imbalanced regulation of protein phosphatases and protein kinases is the direct cause of tau hyperphosphorylation. In the Alzheimer's disease (AD) brain, decrease of PP2A activity has been reported; but the molecular mechanisms are unknown. Our studies have shown that a negative correlation between GSK-3βand PP2A was observed. Activation of GSK-3βinhibits PP2A with upregulation of an endogenous PP2A inhibitor, namely inhibitor-2 of PP2A (I2PP2A). In the study, we noticed that the correlation values between GSK-3βand I2PP2A (R=0.9158) and between GSK-3βand PP2A (R=-0.9166) were much higher than the correlation value between PP2A and I2PP2A (R=-0.7564), implying that GSK-3βmay affect PP2A through multiple pathways in addition to regulating I2PP2A. ObjectiveIt was aimed to investigate the effects of GSK-3βon PP2Ac expression and posttranslational modifications, including phosphorylation at Tyr307 and demethylation at Leu309 (dmL309) in vitro and in vivo and the underlying mechanisms.MethodsThe human embryonic kidney 293 (HEK293) and neuroblastoma 2a (N2a) cells were used for the study. The cells were treated with wortmannin (WO, an inhibitor of PI3-K, 1μM and 2μM), SB216763 (SB, an inhibitor of GSK-3,5μM and 7μM), WO (2μM) associated with SB (7μM), or transfected with plasmids expressing HA-tagged wild type GSK-3β(wtGSK-3β), or pSilencer plasmids silencing gene of PTPIB or tyrosine kinase (Src) or PME-1 or PPMT1. WO (100μM) or SB (70μM) in a total volume of 10μl was injected into 3-month-old Sprague Dawley (SD) rats'left lateral ventricle or 2μl lenti-wtGSK-3βor lenti-eGFP were injected into the 3-month-old C57BL/6J (C57) mice' hippocampus. GSK-3βheterozygous knockout mice (GSK-3β+/- mice), which on the C57BL/6J background are viable and express reduced levels of protein and enzymatic activity, were used. Then proteins including PP2AC, pY307, dmL309, PTPIB, Src, PME-1, PPMT1, PP2AB and cAMP response element binding protein (CREB) were detected by western blotting. And the protein levels of pY307, dmL309 and PP2Ac in mice' hippocampus injected lenti-wtGSK-3βor lenti-eGFP were detected by immunohistochemistry. The association of GSK-3βwith PTPIB, Src, PME-1 and PPMT1 were measured by co-immunoprecipitation. Activity of PTPIB and Src was analysed by ELISA. The mRNA levels of PP2AC, PTPIB, PME-1 and PPMT1 were detected via RT-PCR.ResultsWO treatment or transfection with wtGSK-3(3 plasmid decreased the levels of PP2AC and PP2AB, and increased pY307 and dmL309, while SB treatment increased PP2AC and PP2AB and decreased pY307 and dmL309 levels. PP2AC increased and pY307 and dmL309 level decreased in the hippocampus of GSK-3(3β+/- mice compared with C57 mice, and simultaneous inhibition of GSK-3 by SB prevented the WO-induced PP2A inactivation. We also found that the level of Src and PME-1 increased, and level of PTPIB and PPMT1 deceased in WO treated groups, and the levels of these proteins changed contrarily in SB treated groups. We found that GSK-3βcan modulate both PTPIB and Src in protein levels, but it only inhibits PTPIB activity with no effect on Src activity. Furthermore, only knockdown of PTPIB but not Src by siRNA eliminates the effects of GSK-3βon PP2A; GSK-3βcan modulate both PME-1 and PPMT1, knockdown of PME-1 or PPMT1 by siRNA eliminates the effects of GSK-3βon PP2A. GSK-3βphosphorylates PTPIB and PME-1 at serine residuals and activation of GSK-3βreduces the mRNA levels of PTPIB and PPMT1 and increases PME-1 mRNA level. Additionally, we also observed that GSK-3 negatively regulates the protein and mRNA levels of PP2AC, and knockdown of CREB abolishes the increase of PP2Ac induced by GSK-3 inhibition.ConclusionGSK-3βregulates the expression of PP2Ac via CREB, and it also regulates the Tyr-307 phosphorylation by PTPIB and Leu309 demethylation via PME-1 and PPMT1, respectively. Therefore, GSK-3βmay inhibit PP2A activity through decreasing the catalytic subunit expression and increasing the phosphorylation and demethylation. GSK-3 also regulates the expression level of PP2AB. As GSK-3βand PP2A are respectively the most implicated kinase and phosphatase in AD-like tau hyperphosphorylation, our data suggest that targeting GSK-3βmay simultaneously correcting the abnormality of PP2A.