The Regmlating Role Of PP2A Methylation In The Cellmlar Oxidative Stress

Objective: The methylation of protein phosphatase 2A ?PP2A? is catalysed by leucine methyl transferase 1 ?LCMT1?, and the demethylation of PP2A is catalysed by protein phosphatase methyl esterase 1?PPME1?. This reversible methylation plays an important role in the reg?lation of cell growth and cell mitosis. Our previous studies have found that hydrogen peroxide ?H₂O₂?changed the methylation of PP2A. However, the underlying response relation-ship and reg?lating mechanism are not clear. In this study, we constructed the cell lines with stable high expression of PPME1 or LCMT1 to further study the role of PP2A methylation in cell?lar oxidative stress.Methods:1. Constructing the HEK293 cell lines with high expression of PPME1 or LCMT1.?1? pcDNA3.0 and pEGFP-N1 were used to construct recombinant plasmids including PPME1-pcDNA3.0, PPME1-pEGFP-N1, LCMT1-PCDNA 3.0 and LCMT1-pEGFP-N1. All these recombinant plasmids and vectors were separately transfected into HEK293 cells with Lipofectamine 3000 to construct stable cell lines including PPME1-293, PPME1-GFP-293, LCMT1-293 and LCMT1-GFP-293, as well as the control group cells pcDNA3.0-293 and pEGFP-N1-293. ?2? The expressions of fluorescent fusion protein, PPME1-GFP and LCMT1-GFP, were observed by fluorescence microscope. The mRNA expression of PPME1 or LCMT1 were analysed by Q-PCR test. The PPME1?or PPME1-GFP?, LCMT1 ?or LCMT1-GFP? and the unmethylated PP2Ac were tested by Western blot. ?3? The protein distribution of PPME1-GFP ?or LCMT1-GFP? in nucleus and cytoplasm was observated and analyzed by laser scanning confocal microscope.2. Analysising the role and mechanism of LCMT1 high expression in the cellular oxidative stress.The cell oxidative stress model in the pcDNA3.0-293 and LCMT1-293 cells was induced by gradient concentrations of H₂O₂. The cell activity changes of this two cell lines in the model were detected by resazurin reagent, and the changes of mitochondrial membrane potential were detected by using JC-1 Dye Kit; The protein expression of unmethylated PP2Ac and phosphorylated protein mTOR and its substrates P70S6K1 and 4E-BP1 were analysed by Western blot;To analyze the molec?lar reg?lating mechanism between these proteins,AMZ-30 ? PPME1 specific inhibitor ? and Rapamycin ? mTORC1 specific inhibitor ? were used to interference in the oxidative stress model.Res?lts:1. The construction results of Res?lts HEK293 cell lines with high expression of PPME1 or LCMT1.?1? The detecting results of protein expression and function of PPME1 or LCMT1 in each cell line. The protein expression levels of PPME1 ?or PPME1-GFP? and unmethylated PP2Ac in the PPME1-293 ?or PPME1-GFP-293? cells were significantly higher than that in the pcDNA3.0-293 ?or pEGFP-Nl-293? cells, p < 0.05; The protein expression level of LCMT1 ?or LCMT1-GFP? in the LCMT1-293 ?or LCMT1-GFP-293? cells was significantly higher than that in the pcDNA3.0-293 ?or pEGFP-N1-293? cells, p < 0.05, but the expression level of unmethylated PP2Ac in the LCMT1-293 ? or LCMT1-GFP-293 ? cells was lower than that in the control group cells, P < 0.05. ?2? The distribution of PPME1-GFP and LCMT1-GFP in nucleus and cytoplasm.PPME1-GFP was mainly distributed in the nucleus, and the nucleo-cytoplasmic ratio is 2.5:1, consistent with the distribution of native PPME1 in the HEK293 cells; LCMT1-GFP was equably distributed in the nucleus and the cytoplasm,the nucleo-cytoplasmic ratio is 0.95:1, consistent with the distribution of native LCMT1 in the HEK293 cells.2. The role and mechanism of LCMT1 high expression in the cellular oxidative stress.?1? H₂O₂ induced demethylation of PP2Ac. Treating with H₂O₂ induced demethylation of PP2Ac in the HEK293, both treating with AMZ-30 and LCMT1 overexpression did inhibit this demethylation. ?2? LCMT1 over-expression prevented cell survival from oxidative stress. H₂O₂ stim?lation damaged HEK293 cells, and it was not. affected by treating with AMZ-30.After treating with the same concentration of H₂O₂, the activity of LCMT1-293 cells was obviously higher than that of pcDNA3.0-293 cells, p < 0.05. It suggested that LCMT1 overexpression prevented cell survival from oxidative stress. ?3? LCMT1 overexpression protects the phosphorylation of mTORC1 pathway. Both rapamycin and H₂O₂ did inhibit the phosphorylation of mTOR and its substrates P70S6K1 and 4E-BP1 in the HEK293 cells. After treating with the same concentration of rapamycin or H₂O₂ for 1 h, the protein expression levels of phosphorylated mTOR, P70S6K1 and 4E-BP1 in the LCMT1-293 cells were higher than that in the pcDNA3.0-293 cells, p < 0.05. It suggested that LCMT1 overexpression protects the phosphorylation of mTORC1 pathway from oxidative stress. ?4? LCMT1 overexpression protected the celluar mitochondrial membrane potential ?AT?. Under the same concentration of H₂O₂, the loss degree of mitochondrial membrane potential of the LCMT1-293 cells was lower than that of the pcDNA3.0-293 cells, P < 0.05. It suggested that LCMT1 overexpression protected the celluar mitochondrial membrane potential from oxidative stress.Conclusion: ?1? In this study, we successfully constructed the cell lines with stable overepression of PPME1 or LCMT1 ?PPME1-293 and LCMT1-293?, and with fluorescence marker GFP ?PPME1-GFP-293 and LCMT1-GFP-293?. ?2? Under the condition of cell oxidative stress, LCMT1 overepression ?or PP2Ac methylation? may reduce the damage of cell viability and celluar mitochondrial function by protecting phosphorylation of mTOR and its substrates P70S6K1 and 4E-BP1.