Figure 5.
PRKDC regulates PRKAG1 localization and activity. (A) Representative immunoblots of indicated proteins from COS7 cells transfected with GST- PRKAA2, PRKAB1-Flag and either WT HA-PRKAG1 or its phosphorylation-deficient Ser192 and Thr284 mutants (S192A or T284A). (B) In vitro kinase activity assay of AMPK complexes immunopurified with anti-Flag agarose gel from COS7 cells transfected as in (a) and left untreated or treated with 100 µM AMP (left). Error bars, SEM of 4 independent experiments. Right, representative immunoblots of indicated proteins from total cell lysates and anti-Flag IPs. (C) Representative images of MCF7-shPRKAG1 cells transiently transfected with WT or PRKAG1S192A,T284A-mCherry-Flag (left), and quantification of the subcellular localization of the transfected proteins (right). Values represent means +SD of three independent experiments with ≥ 50 randomly chosen cells analyzed in each sample. Crosses mark cells with nuclear/diffuse (white) and mainly cytoplasmic (green) localization of PRKAG1-mCherry-Flag. Scale bars: 10 µM. (D) IP of AMPK complexes with anti-Flag antibodies from lysates of MCF7 cells transiently transfected with either WT or PRKAG1S192A,T284A-mCherry-Flag. Left, representative immunoblots of indicated proteins in IPs and lysates. Right, quantification of co-precipitating PRKDC from three independent experiments +SD. (E) IP of AMPK complexes with anti-Flag antibodies from lysates of MCF7-shPRKAG1 and -shNTC cells transiently transfected with WT or PRKAG1S192A,T284A-mCherry-Flag. (F) LC3 puncta in MCF7-shPRKAG1 cells transiently transfected with WT or PRKAG1S192A,T284A-mCherry-Flag. Error bars, SEM of ≥ 7 samples with 10–30 cells analyzed in each. P-values were calculated by 2-tailed, homoscedastic student’s t-test.