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

Protein phosphatase 2A (PP2A) is a ubiquitous phosphatase found in many eukaryotic cell types and is involved in regulating a number of intracellular signalling pathways. The core enzyme is composed of two subunits: regulatory A subunit and catalytic C subunit. This core enzyme can also associate with different regulatory B subunit. Formation of multiple variants of heterotrimeric holoenzymes provides the basis for PP2A regulation. 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 lead to the pathological change of AD : 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 in our lab 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 order to further explore the mechanism of GSK-3 regulation of PP2Ac post-translationalation. We change the level of GSK-3 activity in vivo and increase transfection wild type GSK-3 plasmid in vitro. By detecting phosphorylation and methylation level of PP2Ac and further clarify the possible mechanisms of GSK-3 regulation of PP2A catalytic subunit post-translational modification. Materials and Methods: The rats were grouped control group (Artificial Cerebrospinal Fluid), SB group (70μM), WT group (100μM). SB216763 (SB, inhibitor of GSK-3) or wortmannin (WT, inhibitor of PI3-Kinase) was injected into the lateral ventricle of Sprague Dawley rats. After 24 hours of injection, we perfused and fixed brain tissue. Then, we detected the levels of total PP2Ac, P-PP2Ac(Y307) and Deme-PP2Ac(L309) by Immunohistochemistry technology. In vitro, wild-type GSK-3βand Src antisense Oligo-DNA were transfected into cell lines HEK293/wt. After 24 hours, 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. Overexpression of GSK-3 in vitro, we found that the levels of tyrosine kinase Src and PP2A methylesterase (PME-1) inceased and PP2A-methyltransferase (PPMT1) decreased.The expression of P-PP2Ac(Y307) is decreased after inhibition of Src expression. Conclusion: GSK-3 may regulate PP2A activity partially via regulation post-translational modifications of phosphorylation and methylation.