Fig. 3.

Validation of AMPK substrates. a Phosphoproteomic quantification of the five candidate AMPK phosphorylation sites selected for validation. ARMC10 (S45)_1 is the phosphopeptide from the shorter isoform of ARMC10 (308 residues). ARMC10 (S45)_2 is the phosphopeptide from the longer isoform of ARMC10 (343 residues). b Validation of the five selected AMPK substrates. For this experiment, constructs encoding SFB-tagged genes were transfected into WT or AMPKα1/α2 HEK293A cells. Cells were treated 16 h later with AMPK activator A769662 (100 μM) for 24 h. Cells were collected and subjected to lysis in NETN cell lysis buffer. Cell lysates were subjected to pulldown assay using S protein beads. Western blotting was conducted using indicated antibodies. c Conservation of the phosphorylation site in ARMC10 in mammals. d Association of ARMC10 with AMPK. Constructs encoding Myc-tagged ARMC10 and indicated constructs for SFB empty vector, SFB-tagged AMPKα1, SFB-tagged AMPKα2, or SFB-tagged control genes were co-transfected into HEK293T cells. The two control genes, CAMKKβ and LKB1, are two kinases that phosphorylate AMPKα and regulate its function, but they normally do not share substrates with AMPK. Cell lysates were subjected to pulldown assays with S protein beads and analyzed by Western blotting using the indicated antibodies. e Both isoforms of ARMC10 were phosphorylated by the AMPK complex at the S45 site. In vitro kinase assays were performed with γ-³²P-ATP, and PhosphorImager software was used to detect the kinases. f In vivo analysis of ARMC10 phosphorylation. Anti-phospho-S45 ARMC10 antibody was used to detect changes in the phosphorylation of ARMC10 S45 before and after treatment with the AMPK activator A796662 100 μM for 24 h in HEK293A WT or AMPKα1/α2-DKO cells