PI3K-AKT-GSK3β Signaling Regulates Methylation Of PP2A Catalytic Subunit

Objective Hyperphosphorylation of tau makes itself easy to aggregate and form paired helical filaments(PHF), which is the major component of neurofibrillary tangles(NFTs), a hallmak of Alzheimer’s disease(AD) and other related tauopathies. Therefore, hyperphosphorylation of tau is the key event in the pathogeneses of Alzheimer’s disease and related tauopathies. Glycogen synthase kinase-3?(GSK-3?)and protein phosphotase 2A(PP2A) are two important enzymes involed in tau hyperphosphorylation.GSK-3? phosphorylates tau at multiple sites. Its activity is modulated by PI3 K pathway via phosphorylation at Ser9 negatively by PI3 K pathway. Whereas Leu309 methylation of PP2 A catalytic subunit(PP2AC) is required for its dephosphorylation activity towards tau. Therefore, methylation of PP2 Ac affects its ability to dephosphorylate tau. Specific methyltransferase 1(LCMT-1) and methylesterase 1(PME-1) are crucial enzymes that methylates and demethylates PP2 Ac at Leu309, respectively. Previously, our studies have shown the crosstalk between PI3 K and PP2 A pathways. Hence, the objective of this study is to further determine how PI3K?AKT?GSK-3β signaling regulates methylation of PP2 A by GSK-3β.Methods 1) We up-regulated or lowered the activities of PI3 K signaling pathway biochemically and molecular biologically in cultured HEK-293 T cells, analysed their effects on phosphorylation of AKT and GSK-3? and demethylation of PP2 Ac by Western blots. 2) We altered the expression of GSK-3β in HEK-293 T cells, and then investigated the effect of GSK-3β on demethylated PP2 Ac, LCMT-1 and PME-1 expression by Western blots and RT-PCR. 3) By using co-immunoprecipitation and co-localization assays, we dtermined the interactions between GSK-3β and LCMT-1 or PME-1.Results We found that activation of PI3 K pathway by insulin treatment promoted the phosphorylation of AKT at Thr308 and Ser473 and GSK-3β at Ser9 and increased demethylated PP2Ac(dmPP2Ac). Inhibition of AKT by using siAKT led to decreased phosphorylation of GSK-3β and demethylation of PP2 Ac. Phosphorylation of GSK-3? was positively correlated with demethylated PP2 Ac, suggesting that PI3 K signaling pathway may regulate methylation of PP2 Ac through GSK-3?. Overexpression of GSK-3β decreased dmPP2 Ac level. Downregulation of GSK-3β by siRNA suppressed methylation of PP2 Ac, but didn’t affect AKT phosphorylation, supporting that PI3 K signaling regulates methylation of PP2 Ac via GSK-3β. Further studies showed that GSK-3β suppressed PME-1 expression at both protein and mRNA levels, but not LCMT-1. GSK-3β could be co-immunoprecipitated by LCMT-1 and GSK-3β co-localized with LCMT-1 in cytoplasm, which suggest LCMT-1may interact with GSK-3β. GSK-3β can be co-immunoprecipitated by and co-localized with LCMT-11-200, suggesting that N-terminus of LCMT-1 may mediate the interaction with GSK-3β. GSK-3β increased LCMT-1 expression and decreased dm-PP2 Ac level in the cells co-transfected with si LCMT-1. Whearas, GSK-3β further suppressed the levels of PME-1 amd dm-PP2 Ac in the cells co-transfected with siPME-1. These results suggestthat GSK-3β may regulate methylation of PP2 Ac through PME-1 and LCMT-1.Conclusions 1) PI3K-AKT-GSK-3β signaling pathway regulates methylation of PP2 Ac. Upregulation of this pathway supresses PP2 Ac methylation. 2) PI3K-AKT-GSK-3β signaling pathway regulates methylation of PP2 Ac through GSK-3β. Upregulaion of GSK-3β activity promotes PP2 Ac methylation, while downregulation of GSK-3β suppresses the methylation of PP2 Ac. 3) GSK-3β inhibits PME-1 expression. 4) GSK3β interacts with LCMT-1 mainly through its N terminus. 5) GSK-3β may regulate PP2 Ac methylation via PME-1 and LCMT-1.