本文關鍵詞： 蛋白磷酸激酶 GSK-3 蛋白磷酸酶-2A 磷酸化 甲基化　出處：《華中科技大學》2009年碩士論文　論文類型：學位論文

【摘要】：蛋白磷酸酯酶-2A(PP2A)是一種在真核細胞中普遍存在的絲/蘇氨酸磷酸酯酶,它參與細胞內許多信號途徑。PP2A的核心酶由一個催化亞基C和結構亞基A組成,這個核心酶可以和不同的調節(jié)B亞基結合形成具有不同催化功能的全酶。PP2A催化亞基翻譯后磷酸化和甲基化修飾可影響PP2A活性的調節(jié)。PP2A催化亞基Tyr307位點經蛋白酪氨酸激酶(如Src)磷酸化后活性下降;催化亞基Leu309位點可以被PP2A特異性甲基轉移酶(PPMT1)/甲酯酶(PME-1)甲基化/去甲基化修飾調節(jié),甲基化后可增強PP2A活性,去甲基化則使PP2A活性降低。PP2A與糖原合酶激酶-3(GSK-3)是微管相關蛋白Tau磷酸化修飾最為重要的蛋白磷酸酯酶和磷酸激酶,它們調節(jié)的失衡可造成Tau過度磷酸化,導致AD的病理學改變。在阿爾茨海默病患者腦中PP2A活性下降,其具體機制尚不明確。我們課題組已經發(fā)現(xiàn),GSK-3可上調蛋白磷酸酯酶抑制因子-2 (I2PP2A)的表達水平而下調PP2A活性,GSK-3與I2PP2A相關系數(shù)R=0.9158,GSK-3活性與PP2A活性成負相關系數(shù)R=0.9166,而I2PP2A與PP2A負相關系數(shù)R=0.7164。這些研究結果提示,GSK-3很可能還通過其他途徑調節(jié)PP2A活性。目的:為了進一步探討GSK-3對PP2Ac翻譯后的調節(jié)機制,我們在整體水平改變GSK-3的活性,在細胞水平增加GSK-3的表達,通過檢測PP2A催化亞基磷酸化和甲基化水平變化來進一步闡明GSK-3對PP2A催化亞基的翻譯后修飾調節(jié)的可能機制。材料和方法:整體水平采用Sprague Dawley (SD)大鼠,側腦室定位注射GSK-3拮抗劑SB216763(SB)和激動劑wortmannin(WT),分組如下:人工腦脊液對照組、70μM SB給藥組,100μM WT給藥組,側腦室定位注射GSK-3拮抗劑SB216763(SB)和激動劑wortmannin(WT),在注射24小時后,麻醉灌流取材,免疫組化方法檢測PP2Ac、PP2Ac磷酸化和去甲基化水平的變化;細胞水平采用HEK293/wt細胞系,用構建好的野生型GSK-3β質粒體轉染24小時后提取蛋白,用免疫印跡(western blot)檢測PP2Ac去甲基化、磷酸化及相關酶表達水平的變化。結果:給予GSK-3拮抗劑SB216763處理后,PP2Ac表達水平升高,PP2Ac去甲基化水平和磷酸化水平降低。給予PI3-Kinase抑制劑WT處理后,PP2Ac表達水平降低,PP2Ac去甲基化水平和磷酸化水平升高。GSK-3過表達后,蛋白酪氨酸激酶Src表達水平升高;蛋白磷酸酯酶甲基轉移酶(PPMT1)表達水平降低,蛋白磷酸酯酶甲酯酶(PME-1)表達水平升高。抑制Src的表達后,激活GSK-3不能使PP2Ac酪氨酸307磷酸化水平增加。結論:GSK-3可通過Src、PPMT1、PME-1調節(jié)PP2A翻譯后磷酸化和甲基化水平來改變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.
【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R341

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