Fig. 3. AMPK phosphorylation of FNIP1 blocks FLCN-FNIP1 GAP activity to control RagC and thereby TFEB and TFE3 activity.

(A) WT and AMPK KO HEK293T cells were subjected to a short 991 (50 μM) time course, and lysates were immunoblotted with the indicated antibodies to probe for mTORC1 signaling. (B) WT FNIP1 and SA5 FNIP1 HEK293T cells were treated as in (A), and lysates were immunoblotted to probe for mTORC1 signaling. (C) WT parental or AMPK-null HEK29T cells were AA starved for the indicated times, and lysates were immunoblotted to examine mTORC1 signaling. (D) WT FNIP1 or SA5 FNIP1 HEK293T cells were administered with the mTOR inhibitors AZD8055 or Torin1 either individually or in combination with 991, as indicated. Lysates were subsequently immunoblotted to examine TFEB phosphorylation status. (E) WT or SA5 FNIP1 cells, stably expressing GFP-TFEB cDNA were treated with or without 50-μM 991 for 1 hour. GFP-TFEB was immunoprecipitated from the lysates, and immunoprecipitates were analyzed by Western blotting. (F) Immunoprecipitates of GFP-TFEB, stably expressed in WT FNIP1 and SA5 FNIP1 HEK293T cells, were subjected to immunoblotting to probe interactions with the Rag GTPases. (G) Quantitation of RagC colocalization with Lamp2 in (H). Data are shown as the means ± SEMs of three independent experiments. *P < 0.05; **P < 0.01; unpaired t test. (H) Representative immunofluorescence images of endogenous RagC costained with Lamp2 in WT FNIP1 and SA5 FNIP1 HEK293T cells treated with 1 hour of DMSO or 991 (50 μM). (I) WT FNIP1 or SA5 FNIP1 cells were transiently transfected with HA-RagC mutants locked in either the GTP-bound state (Q120L) or the GDP-bound state (S75N) and subsequently treated with or without 1-hour 991. Lysates were immunoblotted with the indicated antibodies. (J) HA-RagC mutants from (I) were immunoprecipitated from lysates using HA magnetic beads and immunoprecipitates analyzed by Western blotting.