Glycogen Synthase Kinase-3 Regulates Phosphorylation And Methylation Of Protein Phosphatase 2A And The Underlying Mechanisms

Protein phosphatase 2A(PP2A) is a major serine/threonine phosphatase involved in many essential aspects of cellular function. PP2A is composed of structural (A), catalytic (C), and regulatory (B) subunits. The catalytic subunit (PP2Ac) undergoes post-translational modifications, including phosphorylation at tyrosine 307 catalyzed by tyrosine kinases (e.g., src) and reversible carboxyl-methylation and demethylation at its C-terminal leucine residue (Leu309), catalyzed by PP2A-methyltransferase (PPMT1) and PP2A methylesterase (PME-1), respectively. The phosphorylation level at tyrosine 307 increase is behalf of PP2A phosphatase activity decrease and methylation level at Leu309 increase is behalf of PP2A phosphatase activity increase.It has been well recognized that an imbalanced regulation in protein kinases and protein phosphatases is the direct cause for the AD-like tau hyperphosphorylation. Among various kinases and phosphatases, glycogen synthase kinase-3 (GSK-3) and protein phosphatase (PP)-2A are, respectively, the most implicated. Decreases of PP2A activity have been reported in the Alzheimer's disease (AD) brain, but the mechanism is unclear. Previous studies showed that a negative correlation between GSK-3 and PP-2A was detected. GSK-3 inhibits PP2A activity via up-regulation of inhibitor-2 of PP2A (I2PP2A). In addition, GSK-3 may affect PP2A activity via regulation of PP2A posttranslational modifications. Objective: In this study, we investigated the post-translational phosphorylation and methylation modifications of PP2A affected by GSK-3 in vitro and in vivo. Materials and methods: SB216763 (SB, inhibitor of GSK-3) or wortmannin (WT, inhibitor of PI3-K) was injected into the lateral ventricle of Sprague Dawley rats. The rats were grouped control group (Artificial Cerebrospinal Fluid), SB group (70μM), WT group (100μM) and WT (100μM) associated with SB (70μM) group. All rats were killed and separated hippocampi after 24 hours of injection. In vitro, we treated N2a and HEK293 with SB or WT one hour, including control group, SB 5μM group, SB 7μM group, WT 1μM group, WT 2μM group, SB 7μM ssociated with WT 1μM group, SB 7μM ssociated with WT 2μM group. Then, we detected the levels of proteins via western blot. Results: The level of PP2Ac increased and the levels of phosphorylated PP2Ac and demethylated PP2Ac decreased in SB groups compared with control groups. In contrast, in WT groups PP2Ac level decreased and phosphorylated PP2Ac and demethylated PP2Ac levels increased. Meanwhile, we found that the levels of tyrosine kinase src and PP2A methylesterase (PME-1) decreased and the levels of protein tyrosine phosphatase PTPαand PP2A-methyltransferase (PPMT1) inceased. In WT groups the levels of these proteins changed contrarily. Conclusion: GSK-3 regulates 1) PP2A post-translational modification of phosphorylation and dephosphorylation at tyrosine 307 by regulating tyrosine kinase src and protein tyrosine phosphatase PTPα, respectively; 2) PP2A post-translational modification of reversible carboxyl-methylation and demethylation at its C-terminal leucine residue (Leu309) by regulating PP2A-methyltransferase (PPMT1) and PP2A methylesterase (PME-1), respectively. GSK-3 inhibits PP2A activity partially by regulation post-translational modifications of phosphorylation and methylation.