Src家族调控Tau蛋白磷酸化作用及机制研究

Regulating effects of Src family on Tau protein phosphorylation and its mechanism

目的探讨Tau蛋白酪氨酸磷酸化对其表达的影响及Src家族在其酪氨酸磷酸化中作用及分子机制。方法采用无缝克隆方法构建Src家族(Src、Fyn、Yes、Lck、Blk、Fgr、Lyn和Hck)质粒;Western blot检测HEK-293T和SHSY-5Y细胞共转染hTau(2N4R)-V5和Src家族24h后磷酸化和总Tau蛋白表达;免疫荧光和Western blot检测HEK-293T中hTau(2N4R)酪氨酸磷酸化(Tyrosine,Y)突变质粒hTau蛋白表达;Western blot检测转染hTau的HEK-293T细胞在共转染Fyn或Src质粒后Tau、Fyn和p-Tyr蛋白表达;Western blot检测转染hTau的Fyn KO的SHSY-5Y细胞中Fyn、Tau、Y394、P62和LC3II/I表达;免疫荧光检测PS19和WT小鼠神经元Y383表达;Western blot检测PS19和WT小鼠海马神经元原代细胞中Fyn、Y383、P62和LC3II/I表达。结果Src和Fyn组HEK293T细胞的hTau-V5、Tau5、Tau5A6、Tau46和DakoTau蛋白表达显著高于Con组,差异有统计学意义(t=14.625、15.550、32.269、28.172、38.516,P<0.05);Yes、Lck、Blk、Fgr、Lyn和Hck组无明显变化(P>0.05);与Con组相比,Src家族HEK293T和SHSY-5Y细胞的pT231、pS202、pS396/404、p-S262/356和pT181蛋白表达无统计学意义(P>0.05);与WT、Y18F、Y29F、Y197F和Y310F组相比,Y394F组V5和DakoTau蛋白显著降低(t=12.762、18.964,P<0.05);共转染Fyn的Y394F组HEK293T细胞的hTau-V5、DakoTau和p-Tyr蛋白表达显著低于共转染Fyn的WT组(t=11.471、10.285、9.344,P<0.05),Fyn-GFP和Fyn蛋白表达无统计学意义(P>0.05);与共转染Src的WT相比,Y394F和5YF组的hTau-V5、DakoTau、Fyn-GFP和Fyn蛋白表达无统计学意义(P>0.05),p-Tyr蛋白表达明显降低(t=36.261、15.514,P<0.05);转染Fyn-KO的SHSY-5Y细胞中Y394、P62和hTau蛋白表达明显低于Fyn-WT组(t=15.562、39.298、22.374,P<0.05),LC3II/LC3I蛋白表达比例明显高于Fyn-WT组(t=56.261,P<0.05);PS19小鼠海马Y383(人Y394)、Fyn、P62蛋白表达明显升高(t=9.120、18.626、22.194,P<0.05),LC3II/LC3I蛋白表达显著降低(t=45.689,P<0.05)。结论Fyn蛋白调控Tau蛋白Y394位点酪氨酸磷酸化影响Tau蛋白表达,通过下调Fyn可激活自噬抑制Tau蛋白的表达。

Objective To investigate the effect of Tau protein tyrosine phosphorylation on its expression and the role and molecular mechanism of Src family in tyrosine phosphorylation.Methods The Src family(Src,Fyn,Yes,Lck,Blk,Fgr,Lyn and Hck)plasmids were constructed by seamless cloning method.The phosphorylated and total Tau protein in HEK-293 T and SHSY-5 Y cells after co-transfection of hTau(2 N4 R)-V5 and Src family plasmids for 24 h were detected by western blot.Immunofluorescence and western blot was used to detect the expression of Tau protein in HEK-293 T cells transfected with tyrosine phosphorylation mutation plasmids.Tau,Fyn and p-Tyr protein expression in HEK-293 Tcells after co-transfection of Fyn or Src plasmids were detected by western blot.Fyn,Tau,Y394,P62 and LC3 II/I expression in SHT-5 Y cells transfected with h Tau Fyn KO were detected by western blot.Immunofluorescence was used to detect the expression of Y383 in PS19 and WT mouse neurons.Western blot was used to detect the expression of Fyn,Y383,P62 and LC3 II/I in primary cells of PS19 and WT mouse hippocampal neurons.Results The expression of h Tau-V5,Tau5,Tau5 A6,Tau46 and Dako Tau protein in HEK293 T cells of Src and Fyn group was significantly higher than that of Con group(t=14.625,15.550,32.269,28.172,38.516;P<0.05).There was no significant change in Yes,Lck,Blk,Fgr,Lyn and Hck groups(P>0.05).Compared with Con group,the p T231,p S202,p S396/404,p-S262/356 and p T181 protein expression of HEK293 T and SHSY-5 Y cells of Src family was not statistically significant different(P>0.05).Compared with WT,Y18 F,Y29 F,Y197 F and Y310 F groups,the V5 and Dako Tau proteins in the Y394 F group were significantly reduced(t=12.762,18.964;P<0.05).The expressions of h Tau-V5,Dako Tau and p-Tyr proteins in HEK293 T cells of the Y394 F group co-transfected with Fyn were significantly lower than those of co-transfected Fyn WT group(t=11.471,10.285,9.344;P<0.05),Fyn-GFP and Fyn protein expression was not statistically significant different(P>0.05).Compared with WT co-transfected with Src,the expressions of h Tau-V5,Dako Tau,Fyn-GFP and Fyn protein in the Y394 F and 5 YF groups was not statistically significant different(P>0.05),and the expression of p-Tyr protein decreased significantly(t=36.261,15.514;P<0.05).The expression of Y394,P62 and h Tau protein in SHSY-5 Y cells transfected with Fyn-KO was significantly lower than those of Fyn-WT group(t=15.562,39.298,22.374;P<0.05),the expression ratio of LC3 II/LC3 I protein was significantly higher than that of Fyn-WT group(t=56.261,P<0.05);PS19 mouse hippocampal Y383(human Y394),Fyn,P62 protein expression was significantly increased(t=9.120,18.626,22.194;P<0.05),The expression of LC3 II/LC3 I protein was significantly reduced(t=45.689,P<0.05).Conclusion Fyn protein regulates the tyrosine phosphorylation at site Y394 of Tau protein,which affects the expression of Tau protein.And autophagy can be activated to inhibit Tau protein expression by down-regulation of Fyn protein.