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

摘要:

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、2 μg的野生型Smurf1时其对应的LKB1蛋白相对表达量分别为0.026 2±0.000 7、0.072 7±0.000 3、0.130 1±0.001 3、0.431 5±0.001 0。经单因素方差分析，其结果为F=79 636.433，P=0.000。与0 μg对照组相比，进一步采用LSD-t法分析，0.5、1、2 μg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、2 μg的突变型Smurf1时其对应的LKB1的蛋白相对表达量分别为0.018 0±0.000 1、0.053 0±0.000 4、0.125 0±0.001 0、0.200 4±0.001 7。经单因素方差分析，其结果为F=12 887.993，P=0.000。与0 μg对照组相比，进一步采用LSD-t法分析，0.5、1、2 μg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1.085 1±0.412 5、2.398 2±1.766 9。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1.823 8±0.027 8、0.088 1±0.005 1、0.212 7±0.006 2。经单因素方差分析，其结果为F=6 721.953，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。

Abstract:

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 HEK293T 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 2 μg of wild-type Smurf1 in HEK293T cells corresponded to LKB1 protein expression levels of 0.026 2±0.000 7，0.072 7±0.000 3，0.130 1±0.001 3，and 0.431 5±0.001 0，respectively. One-way analysis of variance（ANOVA） showed a result of F=79 636.433 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 2 μg groups，respec－tively，compared with the 0 μg control group. ③The overexpres－sion levels of 0，0.5，1，and 2 μg of mutant Smurf1 in HEK293T cells corresponded to LKB1 protein expression levels of 0.018 0 ±0.000 1，0.053 0±0.000 4，0.125 0±0.001 0，and 0.200 4±0.001 7，respectively. One-way ANOVA showed a result of F=12 887.993 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 2 μg groups，respectively，compared with the 0 μg 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 1.085 1±0.412 5 and 2.398 2±1.766 9，respectively. The P value was 0.257 by independent samples t-test analysis. ⑤With β-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（C699A）-treated group were 1.823 8±0.027 8，0.088 1±0.005 1，and 0.212 7±0.006 2，respectively. One-way ANOVA showed a result of F=6 721.953 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，com－pared 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.