PA stimulates mTORC1 kinase activity in vitro. A, raptor was immunoprecipitated (IP) from HEK293 cells as described in Fig. 1A and subjected to in vitro kinase assays using GST-S6K1 as the substrate. PA or PC vesicles were added at 100 μm and 200 μm prior to kinase assays in the indicated samples. Vesicle buffer was added as control wherever lipid vesicle was not added. The phospho-S6K1 (pS6K1) and GST-S6K1 blots with raptor immunoprecipitates were quantified by densitometry, and the relative ratios of phospho-S6K1 versus GST-S6K1 were calculated with control (no vesicles) designated as 1. B, rictor was immunoprecipitated from HEK293 cells and subjected to in vitro kinase assays using His-Akt as the substrate. The phospho-Akt and His-Akt blots were quantified as described in A. The data shown in the graphs are mean ± S.D. of three independent experiments. Each data point is compared with the control by one-sample t test, and significantly different data points are indicated. *, p < 0.05; **, p < 0.01.

PA antagonizes FKBP38 inhibition of mTORC1 kinase activity in vitro. mTORC1 was immunoprecipitated with anti-raptor antibody from HEK293 cells as described in Fig. 1A and subjected to in vitro kinase assays using GST-S6K1 as a substrate. A, FKBP38 and FKBP12 were added at increasing amounts as indicated prior to kinase assays. B, PA or PC vesicles at 100 μm were added together with FKBP38 prior to kinase assays. Vesicle buffer was added as control wherever lipid vesicle was not added. Each experiment was performed at least three times, and the representative Western blot analyses (or Coomassie blue stain for FKBP38 in A) are shown.

PA activates mTORC1 in the absence of FKBP38. A, HEK293 cells were infected with lentivirus expressing FKBP38 shRNA, puromycin-selected, serum-starved, and then stimulated with 300 μm C8-PA followed by Western analysis of lysates. B, cells were infected with lentiviruses expressing shRNA for FKBP38, or PLD1, or both, puromycin-selected, and followed by serum starvation and then Western blot analysis. C, the Western blot analysis results represented by blots in B were quantified by densitometry, and the relative ratios of phospho-S6K1 versus S6K1 and p4EBP1 versus 4EBP1 were calculated with control (no shRNA) designated as 1. D, mTORC1 was immunoprecipitated by anti-raptor as described in Fig. 1A from cells expressing FKBP38 shRNA or a hairpin of scrambled sequence as control and subjected to in vitro kinase assays with or without PA or PC vesicles at 100 μm. Vesicle buffer was added as control wherever lipid vesicle was not added. The Western blot analysis results were quantified by densitometry, and the relative ratios of phospho-S6K1 versus GST-S6K1 were calculated with control (no vesicles) designated as 1. The data shown are mean ± S.D. or representative blots of three independent experiments. Each data point is compared with the control by one-sample t test, and significantly different data points are indicated. *, p < 0.05; **, p < 0.01.

PA activates mTORC1 autophosphorylation in cells. A, HEK293 cells were serum-starved overnight and then stimulated with 300 μm C8-PA for 30 min. Vesicle buffer was added as control wherever lipid vesicle was not added. Cells were lysed in lysis buffer, and mTORC1 and mTORC2 were immunoprecipitated (IP) with anti-raptor and anti-rictor antibodies, respectively, and washed with the same buffer, followed by Western blotting. B, cells were serum-starved overnight and then stimulated with 300 μm C8-PA or 20% serum for 30 min followed by cell lysis and Western blotting. Each experiment was performed at least three times, and the representative blots are shown.

PA disrupts FKBP38-mTOR interaction. A, GST pull-down assays were performed with purified mTOR(1967–2191) and GST-FKBP38 with GST as a negative control. Western blot analyses are shown. Note that some free GST was present in the GST-FKBP38 protein preparation. B, GST-FKBP38 was preincubated with varying concentrations of PA (+) or PC (−) vesicles prior to addition of purified mTOR-255 and subsequent pull-down assays. C, HEK293 cells were cotransfected with HA-FKBP38 and FLAG-mTOR, followed by serum-starvation and stimulation with 300 μm C8-PA for 30 min. HA-FKBP38 was immunoprecipitated (IP) followed by Western blot analysis. D, cells were infected with lentivirus expressing PLD1 shRNA, puromycin-selected, and then cotransfected with HA-FKBP38 and FLAG-mTOR, followed by immunoprecipitation of HA-FKBP38 and subsequent Western blot analysis. Each experiment was performed at least three times, and the representative blots are shown.

PA and FKBP38 antagonize each other in the regulation of mTORC1 signaling in cells. A, HEK293 cells were cotransfected with Myc-S6K1 and HA-FKBP38, serum-starved, and then stimulated with 300 μm C8-PA followed by Western blot analysis of cell lysates. B, cells were transfected and starved as in A and stimulated with 10% serum with or without C8-PA followed by Western blot analysis of cell lysates. Vesicle buffer was added as control wherever lipid vesicle was not added. Each experiment was performed at least three times, and the representative blots are shown.