Fig. 4.

Tankyrase-RNF146 axis is involved in regulation of LKB1 complex integrity and LKB1 phosphorylation. a Tankyrase inhibitor treatment enhances LKB1/STRAD/MO25 complex formation. HEK293A cells were treated with DMSO or the tankyrase inhibitors JW55. WIKI4, XAV939, or G007-LK, and subjected to immunoprecipitation/western blotting. We use the ImageJ to calculate the band densitometry of western blots, and get the relative ratio by comparing the densitometry of MO25/LKB1 or STRAD/LKB1, and we get three relative ratios from three independent experiments. The relative ratio data was analyzed by the One-way ANOVA test and we used an F-test to compare variances, a P value of <0.05 was considered to indicate statistically significant differences, ** means P-value of <0.01 and *** means P-value of <0.001 (shown in Supplementary Data 4). b TNKS1/2 depletion increases LKB1 complex formation. HEK293A cells were transduced with the indicated shRNAs and cell lysates were subjected to immunoprecipitation/western blotting. c Expression of TNKS1 but not the TNKS1-PD mutant disrupts LKB1 complex formation. shRNA-resistant inducible TNKS1 or TNKS1-PD were transducted into TNKS1/2-knockdown HEK293A cells, and cell lysates were subjected to immunoprecipitation. d Loss of RNF146 promotes LKB1 complex formation. HEK293A cells were transduced with the indicated shRNAs and cell lysates were subjected to immunoprecipitation/western blotting. e G007-LK treatment promotes complex formation of WT but not E130/138 A mutant of LKB1 with STRAD/MO25. Indicated cells were treated without or with G007-LK and cell lysates were subjected to pull-down assay. f Overexpression of RNF146 inhibits complex formation of WT but not E130/138 A mutant of LKB1 with STRAD/MO25. HeLa cells stable expressed wild-type or the E130/138 A mutant of LKB1 was transfected without or with Myc-RNF146. Cell lysates were subjected to pull-down assay. g TNKS1/2 depletion induces LKB1 phosphorylation at Ser428. HEK293A cells were transduced with the indicated shRNAs, and phosphorylation of LKB1 at different sites was assessed with the corresponding antibodies. h Knockdown of RNF146 promotes LKB1 phosphorylation. HEK293A cells were transduced with the indicated shRNAs and subjected to western blotting. Statistical significance was determined by a two-tailed, unpaired Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001