Smurf1通过抑制LKB1的多聚泛素化而调控其蛋白稳定性

Smurf1 regulates its protein stability by inhibiting the polyubiquitylation of LKB1

目的:为了探寻肝脏激酶B1(liver kinase B1,LKB1)的上游调控分子,并研究其对LKB1的影响。方法:首先,利用UbiBrowser数据库预测LKB1可能的上游调控分子,并根据评分筛选出可能对LKB1具有调控作用的Smurf1基因。然后,构建Smurf1和LKB1相关过表达质粒,并转染HEK293T细胞,通过Western blot检测LKB1蛋白质表达的变化。最后,运用荧光定量PCR(qPCR)、免疫共沉淀(Co-Immunoprecipitation)探索Smurf1调控LKB1可能的分子机制。结果:①成功构建LKB1和Smurf1相关过表达质粒。②在HEK293T细胞中过表达0、0.5、1、2μg的野生型Smurf1时其对应的LKB1蛋白相对表达量分别为0.0262±0.0007、0.0727±0.0003、0.1301±0.0013、0.4315±0.0010。经单因素方差分析,其结果为F=79636.433,P=0.000。与0μg对照组相比,进一步采用LSD-t法分析,0.5、1、2μg各组P值分别为0.000、0.000和0.000。③在HEK293T细胞中过表达0、0.5、1、2μg的突变型Smurf1时其对应的LKB1的蛋白相对表达量分别为0.0180±0.0001、0.0530±0.0004、0.1250±0.0010、0.2004±0.0017。经单因素方差分析,其结果为F=12887.993,P=0.000。与0μg对照组相比,进一步采用LSD-t法分析,0.5、1、2μg各组P值分别为0.000、0.000和0.000。④以GAPDH为内参,未过表达Smurf1组和过表达Smurf1组中,LKB1的相对mRNA表达水平分别为1.0851±0.4125、2.3982±1.7669。经独立样本t检验统计分析,P=0.257。⑤以β-actin为内参,未经Smurf1处理组、野生型Smurf1(WT)处理组、突变型Smurf1(C699A)处理组中LKB1的相对泛素化水平分别为1.8238±0.0278、0.0881±0.0051、0.2127±0.0062。经单因素方差分析,其结果为F=6721.953,P=0.000。与未经Smurf1处理组相比,进一步采用LSD-t法分析,野生型Smurf1组和突变型Smurf1组各组P值分别为0.000和0.000。结论:这些结果表明Smurf1通过降低LKB1的多聚泛素化从而参与其蛋白稳定性的调节。

Objective:To explore the upstream regulatory molecules of liver kinase B1(LKB1)and to investigate their effect on LKB1.Methods:First,the UbiBrowser database was used to predict the possible upstream regulatory molecules of LKB1,and the Smurf1 gene that may regulate LKB1 was screened out according to the score.Then,the Smurf1-and LKB1-related overexpression plasmids were constructed and transfected into HEK293 T cells,and the changes in the expression of LKB1 protein were detected by Western blot.Finally,the possible molecular mechanism of Smurf1 regulating LKB1 was explored by quantitative polymerase chain reaction and co-immunoprecipitation.Results:①The LKB1-and Smurf1-related overexpression plasmids were successfully constructed.②The overexpression levels of 0,0.5,1,and 2μg of wild-type Smurf1 in HEK293 T cells corresponded to LKB1 protein expression levels of0.0262±0.0007,0.0727±0.0003,0.1301±0.0013,and 0.4315±0.0010,respectively.One-way analysis of variance(ANOVA)showed a result of F=79636.433 and P=0.000.Further analysis by the least significant difference t(LSD-t)test yielded P values of 0.000,0.000,and 0.000 for the 0.5,1,and 2μg groups,respectively,compared with the 0μg control group.③The overexpression levels of 0,0.5,1,and 2μg of mutant Smurf1 in HEK293 T cells corresponded to LKB1 protein expression levels of 0.0180±0.0001,0.0530±0.0004,0.1250±0.0010,and 0.2004±0.0017,respectively.One-way ANOVA showed a result of F=12887.993 and P=0.000.Further analysis by the LSD-t test yielded P values of 0.000,0.000,and 0.000 for the 0.5,1,and 2μg groups,respectively,compared with the 0μg control group.④With GAPDH as the internal reference substance,the relative mRNA expression levels of LKB1 in the Smurf1 non-overexpression group and Smurf1 overexpression group were 1.0851±0.4125 and 2.3982±1.7669,respectively.The P value was 0.257 by independent samples t-test analysis.⑤Withβ-actin as the internal reference substance,the relative ubiquitination levels of LKB1 in the Smurf1-untreated group,wild-type Smurf1-treated group,and mutant Smurf1(C699 A)-treated group were 1.8238±0.0278,0.0881±0.0051,and 0.2127±0.0062,respectively.One-way ANOVA showed a result of F=6721.953 and P=0.000.Further analysis by the LSD-t test yielded P values of 0.000 and 0.000 for the wild-type Smurf1-treated group and mutant Smurf1-treated group,respectively,compared with the Smurf1-untreated group.Conclusion:These results indicate that Smurf1 is involved in the regulation of the stability of LKB1 protein by reducing its polyubiquitination.