PI3K-AKT-GSK3β信号通路对PP2A催化亚基甲基化的调节
发布时间：2018-09-09 18:55
【摘要】：目的Tau蛋白的过度磷酸化除了使其失去生物学功能外,还容易聚集形成双螺旋丝(paired helical filaments,PHFs),后者是阿尔茨海默病(Alzheimer disease,AD)及相关tau蛋白病(tauopathies)中神经纤维缠结(neurofibrillary tangles,NFTs)的主要成分。tau蛋白的异常过度磷酸化是AD及相关神经退行性疾病的关键事件。糖原合酶激酶3b(glycogen synthase kinase-3b,GSK-3b)和磷酸酯酶2A(protein phosphotase 2A,PP2A)是最重要的催化tau蛋白磷酸化和去磷酸化的酶。GSK-3b可催化tau蛋白上多个位点的磷酸化。GSK-3b的激酶活性受PI3K信号通路的影响,即PI3K通路通过磷酸化GSK-3b第9位的丝氨酸(Ser9)而抑制GSK-3b的活性。PP2A催化亚基(PP2A catalytic subunit,PP2Ac)第309位的亮氨酸(Leu309)的甲基化(m-PP2Ac)调节其与调节亚基的亲和性,是PP2A催化tau的去磷酸化所必须的。因此,PP2Ac的甲基化水平影响其对tau的磷酸化能力。PP2A特异性的亮氨酸甲基转移酶(leucine carboxyl methyltransferase,LCMT-1)及羧基端甲基酯酶(carboxymethylesterase,PME-1)是催化PP2Ac甲基化和去甲基化的酶。我们以前的研究提示PI3K?AKT?GSK-3β信号通路与PP2A之间可能存在交互作用。本研究的目的是深入探讨PI3K-AKT-GSK3β信号通路调节PP2Ac甲基化的分子机制。方法1)在培养的HEK-293T细胞中,用生物化学与分子生物学手段上调或下调PI3K信号通路的活性,用Western blot分析这些改变对PP2Ac甲基化修饰的影响,探讨PI3K信号通路如何调节PP2Ac的甲基化。2)在HEK-293T细胞中改变GSK-3β的表达水平,用Western blot和RT-PCR分析GSK-3β对去甲基化PP2Ac、PME-1和LCMT-1表达的影响。3)通过免疫共沉淀及免疫荧光共定位的方法探讨GSK-3β与LCMT-1、PME-1的相互作用。结果我们发现胰岛素处理激活HEK-293T细胞的PI3K信号通路,促进AKT在Thr308和Ser473的磷酸化和GSK-3b在Ser9的磷酸化,使得去甲基化的PP2Ac(demethylated PP2Ac,dm-PP2Ac)的水平增加。运用si AKT下调AKT的表达后,GSK-3b的磷酸化和dm-PP2Ac水平降低,两者呈现正相关,提示PI3K信号通路可能通过GSK-3b调节PP2Ac的甲基化,进而影响其活性。过表达GSK-3b使得dm-PP2Ac水平下降,促进PP2Ac甲基化;用si RNA下调GSK-3b的表达,PP2Ac的甲基化受到抑制,但不影响AKT的磷酸化,提示GSK-3b调节PP2Ac的甲基化。进一步研究显示GSK-3b可降低PME-1蛋白水平及mRNA水平,而对LCMT-1的表达无明显影响。GSK-3β可以被LCMT-1免疫共沉淀,GSK-3b与LCMT-1在细胞浆中存在较明显的共定位,提示GSK-3b与LCMT-1可能存在相互作用。GSK-3b可以被LCMT-11-200免疫共沉淀和共定位,提示LCMT-1可能通过N-末端与GSK-3b相互作用。在转染si LCMT-1的细胞中,GSK-3β的过表达使LCMT-1表达增加和dm-PP2Ac表达减少;而在转染si PME-1的细胞中,GSK-3b使得PME-1和dm-PP2Ac的水平进一步降低,提示GSK-3β可能通过PME-1和LCMT-1调节PP2Ac甲基化。结论1)PI3K-AKT-GSK3β信号通路参与PP2Ac甲基化的调节,上调此通路,抑制PP2Ac的甲基化;2)PI3K-AKT-GSK3β信号通路主要通过GSK-3β调节PP2Ac甲基化,上调GSK-3b的活性促进PP2Ac的甲基化修饰,而下调GSK-3b表达则抑制PP2Ac的甲基化;3)GSK-3β抑制PME-1的表达;4)GSK-3b主要与LCMT-1的N-端区域存在相互作用,进而可能通过磷酸化影响其活性;5)GSK-3b可能通过PME-1和LCMT-1调节PP2Ac甲基化。
[Abstract]:Objective In addition to the loss of biological function, the overphosphorylation of Tau protein can easily agglomerate into paired helical filaments (PHFs), the main component of neurofibrillary tangles (NFTs) in Alzheimer disease (AD) and related tauopathies. Abnormal hyperphosphorylation is a key event in AD and related neurodegenerative diseases. Glycogen synthase kinase-3B (GSK-3b) and protein phosphotase 2A (PP2A) are the most important enzymes that catalyze the phosphorylation and dephosphorylation of tau. GSK-3b catalyzes the phosphorylation of multiple sites on tau. GSK-3b stimulates the activation of GSK-3b. The activity of GSK-3b was inhibited by PI3K signaling pathway, i.e. the phosphorylation of GSK-3b site 9 serine (Ser9) by PI3K pathway. The methylation of PP2A catalytic subunit (PP2Ac) at position 309 leucine (m-PP2Ac) to regulate its affinity to the regulatory subunit was necessary for PP2A catalyzed tau dephosphorylation. Therefore, the methylation level of PP2Ac affects its ability to phosphorylate tau. PP2A-specific leucine carboxyl methyltransferase (LCMT-1) and carboxymethylesterase (PME-1) are enzymes that catalyze the methylation and demethylation of PP2Ac. The purpose of this study was to investigate the molecular mechanism of PI3K-AKT-GSK3beta signaling pathway regulating PP2Ac methylation. Methods 1) In cultured HEK-293T cells, the activity of PI3K signaling pathway was up-regulated or down-regulated by biochemical and molecular biological methods, and the effect of these changes on PP2Ac A was analyzed by Western blot. To investigate how PI3K signaling pathway regulates the methylation of PP2Ac. 2) To alter the expression of GSK-3 beta in HEK-293T cells. The effects of GSK-3 beta on the expression of demethylated PP2Ac, PME-1 and LCMT-1 were analyzed by Western blot and RT-PCR. 3) To explore the relationship between GSK-3 beta and LCMT-1, PME-1 by immunoprecipitation and immunofluorescence co-localization. The results showed that insulin treatment activated PI3K signaling pathway in HEK-293T cells, promoted the phosphorylation of AKT in Thr308 and Ser473, and GSK-3b in Ser9, and increased the level of demethylated PP2Ac (dm-PP2Ac). After down-regulating the expression of AKT by si-AKT, the phosphorylation of GSK-3b and the level of dm-PP2Ac were increased. Overexpression of GSK-3b can decrease the level of dm-PP2Ac and promote the methylation of PP2Ac, and down-regulate the expression of GSK-3b with Si RNA, which can inhibit the methylation of PP2Ac, but does not affect the phosphorylation of AKT, suggesting that GSK-3b regulates the methylation of PP2Ac. Further studies showed that GSK-3b could decrease the level of PME-1 protein and mRNA, but had no significant effect on the expression of LCMT-1. GSK-3b could be co-precipitated by LCMT-1, and GSK-3b and LCMT-1 had obvious co-localization in cytoplasm, suggesting that GSK-3b might interact with LCMT-1. GSK-3b could be co-precipitated and co-precipitated by LCMT-11-200. Localization suggests that LCMT-1 may interact with GSK-3b via N-terminal. Overexpression of GSK-3beta increases the expression of LCMT-1 and decreases the expression of dm-PP2Ac in transfected cells, while GSK-3b further decreases the levels of PME-1 and dm-PP2Ac in transfected cells, suggesting that GSK-3beta may regulate PP2Ac methyl through PME-1 and LCMT-1. Conclusion 1) PI3K-AKT-GSK3 beta signaling pathway participates in the regulation of PP2Ac methylation, up-regulates this pathway and inhibits PP2Ac methylation; 2) PI3K-AKT-GSK3 beta signaling pathway regulates PP2Ac methylation mainly through GSK-3 beta, up-regulates GSK-3b activity and promotes PP2Ac methylation, while down-regulates GSK-3b expression inhibits PP2Ac methylation; 3) GSK-3) GSK-3 beta inhibits PME-1 beta signaling pathway. GSK-3b mainly interacts with the N-terminal region of LCMT-1 and may affect its activity through phosphorylation; 5) GSK-3b may regulate PP2Ac methylation through PME-1 and LCMT-1.
【学位授予单位】：南通大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741
，
本文编号：2233274
[Abstract]:Objective In addition to the loss of biological function, the overphosphorylation of Tau protein can easily agglomerate into paired helical filaments (PHFs), the main component of neurofibrillary tangles (NFTs) in Alzheimer disease (AD) and related tauopathies. Abnormal hyperphosphorylation is a key event in AD and related neurodegenerative diseases. Glycogen synthase kinase-3B (GSK-3b) and protein phosphotase 2A (PP2A) are the most important enzymes that catalyze the phosphorylation and dephosphorylation of tau. GSK-3b catalyzes the phosphorylation of multiple sites on tau. GSK-3b stimulates the activation of GSK-3b. The activity of GSK-3b was inhibited by PI3K signaling pathway, i.e. the phosphorylation of GSK-3b site 9 serine (Ser9) by PI3K pathway. The methylation of PP2A catalytic subunit (PP2Ac) at position 309 leucine (m-PP2Ac) to regulate its affinity to the regulatory subunit was necessary for PP2A catalyzed tau dephosphorylation. Therefore, the methylation level of PP2Ac affects its ability to phosphorylate tau. PP2A-specific leucine carboxyl methyltransferase (LCMT-1) and carboxymethylesterase (PME-1) are enzymes that catalyze the methylation and demethylation of PP2Ac. The purpose of this study was to investigate the molecular mechanism of PI3K-AKT-GSK3beta signaling pathway regulating PP2Ac methylation. Methods 1) In cultured HEK-293T cells, the activity of PI3K signaling pathway was up-regulated or down-regulated by biochemical and molecular biological methods, and the effect of these changes on PP2Ac A was analyzed by Western blot. To investigate how PI3K signaling pathway regulates the methylation of PP2Ac. 2) To alter the expression of GSK-3 beta in HEK-293T cells. The effects of GSK-3 beta on the expression of demethylated PP2Ac, PME-1 and LCMT-1 were analyzed by Western blot and RT-PCR. 3) To explore the relationship between GSK-3 beta and LCMT-1, PME-1 by immunoprecipitation and immunofluorescence co-localization. The results showed that insulin treatment activated PI3K signaling pathway in HEK-293T cells, promoted the phosphorylation of AKT in Thr308 and Ser473, and GSK-3b in Ser9, and increased the level of demethylated PP2Ac (dm-PP2Ac). After down-regulating the expression of AKT by si-AKT, the phosphorylation of GSK-3b and the level of dm-PP2Ac were increased. Overexpression of GSK-3b can decrease the level of dm-PP2Ac and promote the methylation of PP2Ac, and down-regulate the expression of GSK-3b with Si RNA, which can inhibit the methylation of PP2Ac, but does not affect the phosphorylation of AKT, suggesting that GSK-3b regulates the methylation of PP2Ac. Further studies showed that GSK-3b could decrease the level of PME-1 protein and mRNA, but had no significant effect on the expression of LCMT-1. GSK-3b could be co-precipitated by LCMT-1, and GSK-3b and LCMT-1 had obvious co-localization in cytoplasm, suggesting that GSK-3b might interact with LCMT-1. GSK-3b could be co-precipitated and co-precipitated by LCMT-11-200. Localization suggests that LCMT-1 may interact with GSK-3b via N-terminal. Overexpression of GSK-3beta increases the expression of LCMT-1 and decreases the expression of dm-PP2Ac in transfected cells, while GSK-3b further decreases the levels of PME-1 and dm-PP2Ac in transfected cells, suggesting that GSK-3beta may regulate PP2Ac methyl through PME-1 and LCMT-1. Conclusion 1) PI3K-AKT-GSK3 beta signaling pathway participates in the regulation of PP2Ac methylation, up-regulates this pathway and inhibits PP2Ac methylation; 2) PI3K-AKT-GSK3 beta signaling pathway regulates PP2Ac methylation mainly through GSK-3 beta, up-regulates GSK-3b activity and promotes PP2Ac methylation, while down-regulates GSK-3b expression inhibits PP2Ac methylation; 3) GSK-3) GSK-3 beta inhibits PME-1 beta signaling pathway. GSK-3b mainly interacts with the N-terminal region of LCMT-1 and may affect its activity through phosphorylation; 5) GSK-3b may regulate PP2Ac methylation through PME-1 and LCMT-1.
【学位授予单位】：南通大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741
，
本文编号：2233274
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