Smurf1 regulates its protein stability by inhibiting the polyubiquitylation of LKB1

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.