Fig. 2: AMP potentiates the ability of AK2 to inhibit BRAF activity via enhanced affinity between AK2 and BRAF.

A AK2 NMP domain is crucial to modulate BRAF kinase activity. HEK293T cells were transfected with Flag-BRAF and HA-AK2 deletion mutants for 24 h and subjected to kinase assay after immunoprecipitation (IP) assay using anti-FLAG agarose-beads. B AK2 activity-dead mutants suppresses BRAF activity in vitro. BRAF protein (10 nM) was incubated with GST-AK2 WT (1 μM) or mutant proteins (GST-AK2 K28E, R150A, and Core, 1 μM). Values are presented as mean ± SD (n = 3; *p < 0.05, **p < 0.01, ***p < 0.001). C AMP enhances the AK2–BRAF binding. After transfection of HEK293T cells with Flag-BRAF and AK2-HA, cell extracts were incubated for 12 h with 100 μM AMP or ATP and subjected to immunoprecipitation (IP) assay. D AMP binding-defective AK2 mutants lose AMP-stimulation of AK2-binding to BRAF. After transfection of HEK293T cells with BRAF and either AK2 WT or mutants (T46S or R51K), cell lysates were incubated with 100 μM AMP and subjected to immunoprecipitation assay. E Energy deprivation conditions enhance AK2-HA–Flag-BRAF binding. After transfection of HEK293T cells with Flag-BRAF and AK2-HA, cells were incubated under the following condition; glucose-free DMEM (4 h), oligomycin (1 μg/ml, 2 h), or phenformin (40 μM, 2 h). Cell lysates were subjected to immunoprecipitation (IP) assay. F Energy deprivation promotes the interaction between endogenous AK2 and BRAF. SK-Hep1 cells were treated with 2-DG (25 mM) or AICAR (1 mM) for 12 h and then subjected to immunoprecipitation (IP) assay. G AMP or ADP potentiates the AK2–BRAF binding in vitro. Purified GST-BRAF and His-AK2 proteins (each 1 μg ml⁻¹) were combined together for 1 h in the presence or absence of AMP, ADP, or ATP and then subjected to GST pull-down assay. The pulled-samples and inputs were analyzed by western blotting.