PI3K-AKT-GSK3β信號通路對PP2A催化亞基甲基化的調節(jié)
發(fā)布時間：2018-09-09 18:55
【摘要】：目的Tau蛋白的過度磷酸化除了使其失去生物學功能外,還容易聚集形成雙螺旋絲(paired helical filaments,PHFs),后者是阿爾茨海默病(Alzheimer disease,AD)及相關tau蛋白病(tauopathies)中神經(jīng)纖維纏結(neurofibrillary tangles,NFTs)的主要成分。tau蛋白的異常過度磷酸化是AD及相關神經(jīng)退行性疾病的關鍵事件。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蛋白上多個位點的磷酸化。GSK-3b的激酶活性受PI3K信號通路的影響,即PI3K通路通過磷酸化GSK-3b第9位的絲氨酸(Ser9)而抑制GSK-3b的活性。PP2A催化亞基(PP2A catalytic subunit,PP2Ac)第309位的亮氨酸(Leu309)的甲基化(m-PP2Ac)調節(jié)其與調節(jié)亞基的親和性,是PP2A催化tau的去磷酸化所必須的。因此,PP2Ac的甲基化水平影響其對tau的磷酸化能力。PP2A特異性的亮氨酸甲基轉移酶(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。我們以前的研究提示PI3K?AKT?GSK-3β信號通路與PP2A之間可能存在交互作用。本研究的目的是深入探討PI3K-AKT-GSK3β信號通路調節(jié)PP2Ac甲基化的分子機制。方法1)在培養(yǎng)的HEK-293T細胞中,用生物化學與分子生物學手段上調或下調PI3K信號通路的活性,用Western blot分析這些改變對PP2Ac甲基化修飾的影響,探討PI3K信號通路如何調節(jié)PP2Ac的甲基化。2)在HEK-293T細胞中改變GSK-3β的表達水平,用Western blot和RT-PCR分析GSK-3β對去甲基化PP2Ac、PME-1和LCMT-1表達的影響。3)通過免疫共沉淀及免疫熒光共定位的方法探討GSK-3β與LCMT-1、PME-1的相互作用。結果我們發(fā)現(xiàn)胰島素處理激活HEK-293T細胞的PI3K信號通路,促進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。運用si AKT下調AKT的表達后,GSK-3b的磷酸化和dm-PP2Ac水平降低,兩者呈現(xiàn)正相關,提示PI3K信號通路可能通過GSK-3b調節(jié)PP2Ac的甲基化,進而影響其活性。過表達GSK-3b使得dm-PP2Ac水平下降,促進PP2Ac甲基化;用si RNA下調GSK-3b的表達,PP2Ac的甲基化受到抑制,但不影響AKT的磷酸化,提示GSK-3b調節(jié)PP2Ac的甲基化。進一步研究顯示GSK-3b可降低PME-1蛋白水平及mRNA水平,而對LCMT-1的表達無明顯影響。GSK-3β可以被LCMT-1免疫共沉淀,GSK-3b與LCMT-1在細胞漿中存在較明顯的共定位,提示GSK-3b與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可能通過N-末端與GSK-3b相互作用。在轉染si LCMT-1的細胞中,GSK-3β的過表達使LCMT-1表達增加和dm-PP2Ac表達減少;而在轉染si PME-1的細胞中,GSK-3b使得PME-1和dm-PP2Ac的水平進一步降低,提示GSK-3β可能通過PME-1和LCMT-1調節(jié)PP2Ac甲基化。結論1)PI3K-AKT-GSK3β信號通路參與PP2Ac甲基化的調節(jié),上調此通路,抑制PP2Ac的甲基化;2)PI3K-AKT-GSK3β信號通路主要通過GSK-3β調節(jié)PP2Ac甲基化,上調GSK-3b的活性促進PP2Ac的甲基化修飾,而下調GSK-3b表達則抑制PP2Ac的甲基化;3)GSK-3β抑制PME-1的表達;4)GSK-3b主要與LCMT-1的N-端區(qū)域存在相互作用,進而可能通過磷酸化影響其活性;5)GSK-3b可能通過PME-1和LCMT-1調節(jié)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.
【學位授予單位】：南通大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R741
，
本文編號：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.
【學位授予單位】：南通大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R741
，
本文編號：2233274
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