糖原合酶激酶-3对蛋白磷酸酯酶-2A催化亚基磷酸化和甲基化修饰的调节及机制

发布时间：2018-01-24 09:09

本文关键词： 蛋白磷酸激酶 GSK-3 蛋白磷酸酶-2A 磷酸化甲基化　出处：《华中科技大学》2009年硕士论文　论文类型：学位论文

【摘要】：蛋白磷酸酯酶-2A(PP2A)是一种在真核细胞中普遍存在的丝/苏氨酸磷酸酯酶,它参与细胞内许多信号途径。PP2A的核心酶由一个催化亚基C和结构亚基A组成,这个核心酶可以和不同的调节B亚基结合形成具有不同催化功能的全酶。PP2A催化亚基翻译后磷酸化和甲基化修饰可影响PP2A活性的调节。PP2A催化亚基Tyr307位点经蛋白酪氨酸激酶(如Src)磷酸化后活性下降;催化亚基Leu309位点可以被PP2A特异性甲基转移酶(PPMT1)/甲酯酶(PME-1)甲基化/去甲基化修饰调节,甲基化后可增强PP2A活性,去甲基化则使PP2A活性降低。PP2A与糖原合酶激酶-3(GSK-3)是微管相关蛋白Tau磷酸化修饰最为重要的蛋白磷酸酯酶和磷酸激酶,它们调节的失衡可造成Tau过度磷酸化,导致AD的病理学改变。在阿尔茨海默病患者脑中PP2A活性下降,其具体机制尚不明确。我们课题组已经发现,GSK-3可上调蛋白磷酸酯酶抑制因子-2 (I2PP2A)的表达水平而下调PP2A活性,GSK-3与I2PP2A相关系数R=0.9158,GSK-3活性与PP2A活性成负相关系数R=0.9166,而I2PP2A与PP2A负相关系数R=0.7164。这些研究结果提示,GSK-3很可能还通过其他途径调节PP2A活性。目的:为了进一步探讨GSK-3对PP2Ac翻译后的调节机制,我们在整体水平改变GSK-3的活性,在细胞水平增加GSK-3的表达,通过检测PP2A催化亚基磷酸化和甲基化水平变化来进一步阐明GSK-3对PP2A催化亚基的翻译后修饰调节的可能机制。材料和方法:整体水平采用Sprague Dawley (SD)大鼠,侧脑室定位注射GSK-3拮抗剂SB216763(SB)和激动剂wortmannin(WT),分组如下:人工脑脊液对照组、70μM SB给药组,100μM WT给药组,侧脑室定位注射GSK-3拮抗剂SB216763(SB)和激动剂wortmannin(WT),在注射24小时后,麻醉灌流取材,免疫组化方法检测PP2Ac、PP2Ac磷酸化和去甲基化水平的变化;细胞水平采用HEK293/wt细胞系,用构建好的野生型GSK-3β质粒体转染24小时后提取蛋白,用免疫印迹(western blot)检测PP2Ac去甲基化、磷酸化及相关酶表达水平的变化。结果:给予GSK-3拮抗剂SB216763处理后,PP2Ac表达水平升高,PP2Ac去甲基化水平和磷酸化水平降低。给予PI3-Kinase抑制剂WT处理后,PP2Ac表达水平降低,PP2Ac去甲基化水平和磷酸化水平升高。GSK-3过表达后,蛋白酪氨酸激酶Src表达水平升高;蛋白磷酸酯酶甲基转移酶(PPMT1)表达水平降低,蛋白磷酸酯酶甲酯酶(PME-1)表达水平升高。抑制Src的表达后,激活GSK-3不能使PP2Ac酪氨酸307磷酸化水平增加。结论:GSK-3可通过Src、PPMT1、PME-1调节PP2A翻译后磷酸化和甲基化水平来改变PP2A的活性。
[Abstract]:Protein phosphatase (PP2A) is a common serine / threonine phosphatase in eukaryotic cells. The core enzyme involved in many signaling pathways. PP2A is composed of a catalytic subunit C and a structural subunit A. This core enzyme can bind with different regulatory B subunits to form a whole enzyme. PP2A catalytic subunit with different catalytic functions. PP2A catalytic subunit translation phosphorylation and methylation modification can affect the regulation of PP2A activity. PP2A catalyzes. Translocation of protein tyrosine Kinase (Tyr307) at the Tyr307 site of Acetylated Subunit. For example, the activity of SRC decreased after phosphorylation. The Leu309 site of catalytic subunit can be modified by PP2A specific methyltransferase PME-1 / methyl esterase PME-1) methylation / demethylation. Methylation can enhance the activity of PP2A. Demethylation reduces the activity of PP2A. PP2A and glycogen synthase kinase (GSK-3) are the most important protein phosphatase and phosphokinase modified by microtubule-associated protein Tau phosphorylation. The imbalance they regulate can cause excessive phosphorylation of Tau, leading to pathological changes in AD. The specific mechanism of the decrease of PP2A activity in the brain of patients with Alzheimer's disease is unclear. GSK-3 upregulated the expression of protein phosphatase inhibitor 2 2 PP2A and down-regulated the activity of GSK-3 and I 2PP2A, the correlation coefficient between GSK-3 and I 2PP2A was R0. 9158. The negative correlation coefficient between GSK-3 activity and PP2A activity was 0.9166, while the negative correlation coefficient between I2PP2A and PP2A was 0.7164. GSK-3 may also regulate the activity of PP2A in other ways. Objective: to further explore the mechanism of GSK-3 regulation of PP2Ac after translation. We changed the activity of GSK-3 at the overall level and increased the expression of GSK-3 at the cellular level. By detecting the phosphorylation and methylation level of PP2A catalytic subunits, the possible mechanism of GSK-3 post-translational modification of PP2A catalytic subunits was further elucidated. Materials and methods:. The overall level is Sprague Dawley (. SD) rats. Lateral ventricle localization injection of GSK-3 antagonist SB216763 and agonist wortmanninine WTG was divided as follows: artificial cerebrospinal fluid (ACSF) control group. In 70 渭 M SB group, 100 渭 M WT group was treated with GSK-3 antagonist SB216763 and agonist wortmanninnin WT2 respectively. The changes of phosphorylation and demethylation of PP2Acn PP2Ac were detected by immunohistochemical method after 24 hours of injection. At the cell level, HEK293/wt cell line was used and the constructed wild-type GSK-3 尾 plasmid was transfected for 24 hours to extract the protein. PP2Ac demethylation was detected by Western blot. Results: the expression of PP2Ac was increased after the treatment with GSK-3 antagonist SB216763. The levels of demethylation and phosphorylation of PP2Ac decreased, and the expression of PP2Ac decreased after treatment with PI3-Kinase inhibitor WT. When PP2Ac demethylation level and phosphorylation level increased. GSK-3 overexpression, protein tyrosine kinase Src expression level increased; The expression level of protein phosphatase methyltransferase (PPMT1) was decreased, and the expression of protein phosphatase methyl ester enzyme (PME-1) was increased after inhibiting the expression of Src. Activation of GSK-3 could not increase the phosphorylation level of PP2Ac tyrosine 307. PME-1 regulates the levels of post-translational phosphorylation and methylation of PP2A to change the activity of PP2A.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R341

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