蛋白质酪氨酸激酶2通过PI3K/AKT/Gsk3b信号调控Tau磷酸化  

Protein Tyrosine Kinase 2 Regulates Tau Phosphorylation ThroughPI3K/AKT/Gsk3β Signaling Pathway

作　　者：柳正 王朝晖[1] LIU Zheng;WANG Zhaohui(Department of Neurology,Hanyang Hospital,Wuhan University of Science and Technology(Wuhan Hanyang Hospital),Wuhan 430050,China)

机构地区：[1]武汉科技大学附属汉阳医院(武汉市汉阳医院)神经内科,湖北武汉430050

出　　处：《标记免疫分析与临床》2022年第3期464-470,共7页Labeled Immunoassays and Clinical Medicine

基　　金：湖北省卫生健康科研基金资助项目(编号:WJ2019H217)。

摘　　要：目的探究蛋白质酪氨酸激酶2(protein tyrosine kinase,Pyk2)在学习记忆障碍小鼠模型中的表达及对神经元细胞Tau蛋白磷酸化的影响和分子机制。方法免疫组化检测APPswe/PS1ΔE9(APP/PS1)双转基因AD和WT小鼠脑组织Pyk2蛋白表达;免疫印迹检测Pyk2、Tau5、Tau46、CP13、AT180、PHF-1、12E8和AT270蛋白表达;免疫印迹检测SHSY-5Y细胞共转染pcDNA6.2-Pyk2-GFP和pcDNA6.2-hTau(2N4R)-V5质粒24h后Tau5、Tau46、CP13、AT180、PHF-1、12E8和AT270蛋白表达;包装PLOK1-Pyk2 shRNA慢病毒,转染SHSY-5Y细胞构建Pyk2敲低细胞系,转染hTau(2N4R)质粒24h后,Western blot检测Tau5、Tau46、CP13、AT180、PHF-1、12E8、AT270、P110α、P110β、P85α、p-P85α、AKT、p-AKT(Ser473)、p-AKT(Thr308)、Gsk3β和p-Gsk3β(Ser9)蛋白表达。结果与WT组相比,APP/PS1双转基因AD小鼠皮质和海马组织中Pyk2蛋白表达明显增加。海马CA1和CA3功能区Pyk2蛋白表达明显增加。海马DG功能区Pyk2蛋白表达明显减少。与WT组相比,APP/PS1双转基因AD小鼠海马组织中Tau5、Tau46 CP13[Phospho-Tau(Ser202)]、AT180[Phospho-Tau(Thr231)]、PHF-1[Phospho-Tau(Ser396/404)]、12E8[Phospho-Tau(Ser262/356)]和AT270[Phospho-Tau(Thr181)]蛋白表达明显增加。与转染0.6μg Tau-V5相比,共转染0.6μg Tau-V5和0.3μg Pyk2-GFP组的V5、Tau5、Tau46 CP13[Phospho-Tau(Ser202)],AT180[Phospho-Tau(Thr231)]和AT270[Phospho-Tau(Thr181)]蛋白表达没有明显变化。共转染0.6μg Tau-V5和0.3μg Pyk2-GFP组的PHF-1[Phospho-Tau(Ser396/404)]和12E8[Phospho-Tau(Ser262/356)]蛋白表达明显增加。与转染1.0g Tau-V5的WT SHSY-5Y细胞相比,共转染1.0g Tau-V5和Pyk2 KDSHSY-5Y细胞组的V5、Tau5、Tau46、CP13[Phospho-Tau(Ser202)]、AT180[Phospho-Tau(Thr231)]和AT270[Phospho-Tau(Thr181)]蛋白表达没有明显变化。PHF-1[Phospho-Tau(Ser396/404)]和12E8[Phospho-Tau(Ser262/356)]蛋白表达明显减少。与转染1.0g Tau-V5的WT SHSY-5Y细胞相比,共转染1.0g Tau-V5和Pyk2 KD SHSY-5Y细胞组的P110β、P85α、p-AKT(Thr308Objective To explore the expression of protein tyrosine kinase 2(PYK2)in a mouse model of learning and memory impairment and its effect on neuronal cell Tau protein phosphorylation and underlying molecular mechanisms.Methods Immunohistochemical method was conducted to detect Pyk2 protein expression in APPswe/PS1ΔE9(APP/PS1)double transgenic AD and WT mouse brain tissue;Western blot was used to detect Pyk2,Tau5,Tau46,CP13,AT180,PHF-1,12E8 and AT270 protein expression levels,as well as the expressions of Tau5,Tau46,CP13,AT180,PHF-1,12E8 and AT270 levels after 24 hours of co-transfection of SHSY-5Y cells with pcDNA6.2-Pyk2-GFP and pcDNA6.2-hTau(2N4R)-V5 plasmids PLOK1-Pyk2 shRNA lentivirus.SHSY-5Y cells were transfected to construct a Pyk2 knockdown cell line.After transfection with hTau(2N4R)plasmid for 24 hours,western blotting was used to detect Tau5,Tau46,CP13,AT180,PHF-1,12E8,AT270,P110α,P110β,P85α,p-P85α,AKT,p-AKT(Ser473),p-AKT(Thr308),Gsk3βand p-Gsk3β(Ser9)protein expression levels.Results Compared with the WT group,the expression of Pyk2 protein in cortex and hippocampus of APP/PS1 double transgenic AD mice was significantly increased.The expression of Pyk2 protein in hippocampal CA1 and CA3 functional areas increased significantly.The expression of Pyk2 protein in hippocampal DG functional area was significantly reduced.Compared with the WT group,Tau5,Tau46,CP13(Phospho-Tau(Ser202)),AT180(Phospho-Tau(Thr231)),PHF-1(Phospho-Tau(Ser396/404)),12E8(Phospho-Tau(Ser262/356))and AT270(Phospho-Tau(Thr181))protein expressions increased significantly.Compared with transfection with 0.6μg Tau-V5,when co-transfected with 0.6μg Tau-V5 and 0.3μg Pyk2-GFP groups of V5,Tau5,Tau46,CP13(Phospho-Tau(Ser202)),AT180(Phospho-Tau(Thr231))and AT270(Phospho-Tau(Thr181))protein expressions did not change significantly.The protein expressions of PHF-1(Phospho-Tau(Ser396/404))and 12E8(Phospho-Tau(Ser262/356))of the group of co-transfected with 0.6μg Tau-V5 and 0.3μg Pyk2-GFP increased significantly.Compared with WT SHSY-5Y

关 键 词：蛋白质酪氨酸激酶2 微管结合蛋白 磷脂酰肌醇-3-激酶 蛋白激酶B 阿尔茨海默氏病 

分 类 号：R591.2[医药卫生—内科学]