【作者】 姚秀卿；

【导师】 王建枝；

【作者基本信息】 华中科技大学 ， 病理学与病理生理学， 2011， 博士

【摘要】 [背景]蛋白磷酸酯酶2A （protein phosphatase-2A, PP2A）是一种具有多功能的酸磷酸酯,对蛋白质的丝氨酸/苏氨酸位点具有去磷酸作用。PP2A由核心酶（结构亚基A和具催化活性的亚基C组成）和调节亚基B组成全酶。活性亚基翻译后可以进行磷酸化和甲基化修饰。PP2A催化亚基（PP2AC）的羧基端307位点酪氨酸（Tyr）磷酸化/去磷酸化由蛋白酪氨酸激酶/酪氨酸磷酸酯酶调节,Tyr307位点磷酸化后PP2A活性下降。PP2Ac羧基端309位点的亮氨酸（Leu309）的甲基化/去甲基化修饰由PP2A特异性的甲基转移酶/甲酯酶调节,活性亚基的Leu309去甲基化后,PP2A的活性下降。在阿尔茨海默病（Alzheimer disease, AD）患者,PP2A和糖原合酶激酶3 （glycogen synthase kinase-3, GSK-3）分别是对微管相关蛋白Tau磷酸化修饰调节最为重要的蛋白磷酸酯酶和蛋白激酶,其活性调节失衡可造成Tau蛋白的异常过度磷酸化,从而导致AD样的病理改变。PP2A活性在AD患者脑组织中明显下降,但其具体的分子机制仍不清楚。我们最近研究发现,GSK-3可通过上调PP2A抑制因子-2（I2PP2A）的表达水平而抑制PP2A的活性。统计学分析资料显示,GSK-3活性与PP2A活性呈负相关,相关系数为R=0.9166; GSK-3与I2PP2A的相关性系数R=0.9158,而I-PP2A与PP2A活性的相关系数为R=-0.7164。这些研究结果提示,GSK-3可能还通过其它的途径调节PP2A的活性。[目的]基于上述背景,本研究拟通过在整体和细胞水平调节GSK-3活性,检测PP2Ac蛋白、Tyr307磷酸化（pY307-PP2AC）、Leu309去甲基化水平（dmL309-PP2AC）以及调节PP2A磷酸化和甲基化相关分子的变化,进一步阐明GSK-3调节PP2A活性的分子机制。[方法]将选用小鼠神经瘤细胞株N2a和人胚肾细胞株HEK293,给予磷脂酰肌醇3-激酶抑制剂wortmannin （WO,1μM和2gM,间接激活GSK-3）或GSK-3的特异性拮抗剂SB216763（SB,5μM和7μM）或WO和SB联合给药；或转染野生型GSK-3β质粒、蛋白磷酸酯酶1B （PTP1B）或蛋白酪氨酸激酶Src RNA干扰质粒、PP2A甲酯酶（PME-1）或PP2A甲基转移酶（PPMT1） RNA干扰质粒；或在整体水平,通过在Sprague Dawley大鼠脑立体定位注射10μl的100μM WO或70μM的SB或100μM WO与70μM SB联合给药；或C57BL/6小鼠海马脑立体定位注射2gl表达野生型GSK-3β的慢病毒；还选用了GSK-3β基因敲除的杂合子小鼠（GSK-3β+/- mice）为研究对象。采用免疫印迹方法（Western blotting）检测以上各样品中PP2AC、pY307-PP2AC水平,dmL309-PP2AC水平,PTP1B、Src、CREB、PME-1、PPMT1及PP2A调节亚基（PP2AB）的蛋白水平。免疫组织化学的方法检测了表达GSK-3β慢病毒后小鼠海马pY307-PP2AC、dmL309-PP2Ac和总PP2Ac的变化。免疫共沉淀的方法检测了PTP1B、Src、PME-1、PPMT1与GSK-3p的相互作用。ELISA方法检测总的蛋白酪氨酸磷酸酯酶的酶活性以及PTP1B和Src的酶活性。RT-PCR方法检测了PP2AC、PTP1B、PME-1和PPMT1的mRNA水平。[结果]当给予PI3-K抑制剂WO处理后,pS9-GSK-3p水平下降；PP2Ac蛋白水平降低；pY307、Src蛋白水平增加、PTP1B蛋白水平下降,PTP1B的酶活性降低；dmL309、PME-1蛋白水平增加,而PPMT1蛋白水平下降；PP2AB蛋白水平降低。而当给予GSK-3的特异性拮抗剂SB处理后,pS9-GSK-3p水平上升；PP2Ac蛋白表达水平增加；pY307、Src蛋白水平降低、PTP1B蛋白水平增加,PTP1B的酶活性增加；dmL309、PME-1蛋白水平降低,PPMT1蛋白水平增加；PP2AB蛋白水平升高。且SB能逆转WO诱导的上述各指标的变化。而WO或SB处理对Src的活性没有明显的改变。过表达GSK-3β野生型质粒后,PP2Ac水平下降；pY307、Src水平升高,PTP1B蛋白水平下降；dmL309、PME-1蛋白水平增加,PPMT1蛋白水平下降。GSK-3β^+/-。小鼠海马样品中检测到PP2Ac水平升高；pY307、Src蛋白水平降低,PTP1B蛋白水平升高；dmL309、PME-1蛋白水平降低,PPMT1蛋白水平升高。下调PTP1B的表达水平后能逆转SB诱导的pY307下降,而下调Src的表达后不能消除WO诱导的pY307的改变；下调PME-1的表达后逆转了WO诱导的dmL309增加,而下调PPMT1的表达逆转了SB诱导的dmL309的减少。GSK-3β与PTP1B、PME-1存在相互作用并能磷酸化PTP1B、PME-1的丝氨酸位点。给予WO处理后,可使PP2AC、PTP1B、PPMT1的nRNA水平下降,PME-1的mRNA水平增加；而给予SB处理,PP2Ac、PTP1B、PPMT1的nRNA增加,PME-1的nRNA水平下降；同时,SB能逆转WO诱导引起的PTP1B、PPMT1的mRNA水平下降和PME-1的mRNA水平升高。另外,给予SB处理后,还增加CREB的蛋白表达水平；下调CREB蛋白表达水平后,可逆转SB诱导的PP2Ac蛋白水平的增加。[结论]GSK-3β可通过分别调节CREB蛋白水平和PTP1B的活性,影响PP2Ac的mRNA和蛋白表达及其Tyr307磷酸化水平。通过调节PME-1和PPME1转录和蛋白表达,GSK-3p改变PP2Ac的Leu309去甲基化水平。此外,GSK-3β还可调节PP2AB的蛋白表达水平。由于GSK-3p和PP2A在AD患者的tau蛋白磷酸化中起关键作用,我们的研究结果提示,调节GSK-3β可达到同时控制PP2A的含量和活性,对AD药物研发有重要价值。更多还原

【Abstract】 BackgroundProtein phosphatase 2A (PP2A) is one of major brain serine/threonine protein phosphatases. In vivo, PP2A predominantly exist as heterotrimers containing a scaffolding subunit (A), a regulatory subunit (B) and a catalytic subunit (C). The highly conserved carboxy-terminal sequence of the C subunit (PP2AC) can undergo post-translational modifications, including phosphorylation and carboxyl methylation. Phosphorylation level at tyrosine-307 (pY307) was regulated by tyrosine kinases and protein tyrosine phosphataselB (PTP1B), phosphorylation transiently inactivates PP2A. Methylation of PP2AC at leucine-309 (Leu309) was catalyzed by specific PP2A methyltransferase (PPMT1), and its demethylation was catalyzed by specific PP2A methylesterase (PME-1). Demethylation of PP2A at Leu309 inhibits PP2A. Phosphorylation and methylation of PP2Ac affects PP2A substrate pecificity, targeting and cellular functions.Among the kinases and phosphatases, glycogen synthase kinase-3β(GSK-3β) and PP2A are respectively the most implicated. It has been recognized that the imbalanced regulation of protein phosphatases and protein kinases is the direct cause of tau hyperphosphorylation. In the Alzheimer’s disease (AD) brain, decrease of PP2A activity has been reported; but the molecular mechanisms are unknown. Our studies have shown that a negative correlation between GSK-3βand PP2A was observed. Activation of GSK-3βinhibits PP2A with upregulation of an endogenous PP2A inhibitor, namely inhibitor-2 of PP2A (I2PP2A). In the study, we noticed that the correlation values between GSK-3βand I2PP2A (R=0.9158) and between GSK-3βand PP2A (R=-0.9166) were much higher than the correlation value between PP2A and I2PP2A (R=-0.7564), implying that GSK-3βmay affect PP2A through multiple pathways in addition to regulating I2PP2A. ObjectiveIt was aimed to investigate the effects of GSK-3βon PP2Ac expression and posttranslational modifications, including phosphorylation at Tyr307 and demethylation at Leu309 (dmL309) in vitro and in vivo and the underlying mechanisms.MethodsThe human embryonic kidney 293 (HEK293) and neuroblastoma 2a (N2a) cells were used for the study. The cells were treated with wortmannin (WO, an inhibitor of PI3-K, 1μM and 2μM), SB216763 (SB, an inhibitor of GSK-3,5μM and 7μM), WO (2μM) associated with SB (7μM), or transfected with plasmids expressing HA-tagged wild type GSK-3β(wtGSK-3β), or pSilencer plasmids silencing gene of PTPIB or tyrosine kinase (Src) or PME-1 or PPMT1. WO (100μM) or SB (70μM) in a total volume of 10μl was injected into 3-month-old Sprague Dawley (SD) rats’left lateral ventricle or 2μl lenti-wtGSK-3βor lenti-eGFP were injected into the 3-month-old C57BL/6J (C57) mice’ hippocampus. GSK-3βheterozygous knockout mice (GSK-3β+/- mice), which on the C57BL/6J background are viable and express reduced levels of protein and enzymatic activity, were used. Then proteins including PP2AC, pY307, dmL309, PTPIB, Src, PME-1, PPMT1, PP2AB and cAMP response element binding protein (CREB) were detected by western blotting. And the protein levels of pY307, dmL309 and PP2Ac in mice’ hippocampus injected lenti-wtGSK-3βor lenti-eGFP were detected by immunohistochemistry. The association of GSK-3βwith PTPIB, Src, PME-1 and PPMT1 were measured by co-immunoprecipitation. Activity of PTPIB and Src was analysed by ELISA. The mRNA levels of PP2AC, PTPIB, PME-1 and PPMT1 were detected via RT-PCR.ResultsWO treatment or transfection with wtGSK-3(3 plasmid decreased the levels of PP2AC and PP2AB, and increased pY307 and dmL309, while SB treatment increased PP2AC and PP2AB and decreased pY307 and dmL309 levels. PP2AC increased and pY307 and dmL309 level decreased in the hippocampus of GSK-3(3β+/- mice compared with C57 mice, and simultaneous inhibition of GSK-3 by SB prevented the WO-induced PP2A inactivation. We also found that the level of Src and PME-1 increased, and level of PTPIB and PPMT1 deceased in WO treated groups, and the levels of these proteins changed contrarily in SB treated groups. We found that GSK-3βcan modulate both PTPIB and Src in protein levels, but it only inhibits PTPIB activity with no effect on Src activity. Furthermore, only knockdown of PTPIB but not Src by siRNA eliminates the effects of GSK-3βon PP2A; GSK-3βcan modulate both PME-1 and PPMT1, knockdown of PME-1 or PPMT1 by siRNA eliminates the effects of GSK-3βon PP2A. GSK-3βphosphorylates PTPIB and PME-1 at serine residuals and activation of GSK-3βreduces the mRNA levels of PTPIB and PPMT1 and increases PME-1 mRNA level. Additionally, we also observed that GSK-3 negatively regulates the protein and mRNA levels of PP2AC, and knockdown of CREB abolishes the increase of PP2Ac induced by GSK-3 inhibition.ConclusionGSK-3βregulates the expression of PP2Ac via CREB, and it also regulates the Tyr-307 phosphorylation by PTPIB and Leu309 demethylation via PME-1 and PPMT1, respectively. Therefore, GSK-3βmay inhibit PP2A activity through decreasing the catalytic subunit expression and increasing the phosphorylation and demethylation. GSK-3 also regulates the expression level of PP2AB. As GSK-3βand PP2A are respectively the most implicated kinase and phosphatase in AD-like tau hyperphosphorylation, our data suggest that targeting GSK-3βmay simultaneously correcting the abnormality of PP2A.更多还原