Fig 1.
PMA activates mTOR and induces its accumulation in perinuclear lysosomes.
(A) Time dependent changes of phosphorylated and total S6K in HEK293 cells treated with 100 nM PMA 5 hours after serum starvation. (B) The levels of S6K, ERK, and GSK3β were evaluated in HEK293 cells pretreated with MEK inhibitor (U0126), RSK inhibitor (BI-D1870) or rapamycin for 1 hour at the indicated concentrations, prior to treatment with 100 nM PMA for 1 hour. (C) Confocal microscope images of immunofluorescence labeling of mTOR and LAMP1 in HEK293 cells treated with 100 nM PMA for 30 or 60 min 5 hours after serum starvation. Scale bar, 10 μm (D) PLA to evaluate the proximity of mTOR and LAMP1 in HeLa cells treated with 100 nM PMA 5 hours after serum starvation. Bar graph in (D) represents the number of dots per cell obtained from three independent experiments. *** P < 0.001; Student T-Test). Scale bar, 10 μm. (E) Confocal microscope images of immunofluorescence labeling of mTOR and LAMP1, and (F) the levels of phosphorylated and total S6K in HEK293 cells treated with 100 nM PMA or 500 nM 10-methyl-aplog-1 for 1 hour following serum starvation for 5 hours. Scale bar in (E), 20 μm. Images are representative of three fields examined from three independent experiments. Western blots are representative of at least three independent experiments.
Fig 2.
Lysosomal mTOR is adjacent to, but distinct from, the perinuclear cPKC-containing endosomes.
Confocal microscopy images of immunofluorescence labeling of (A) GFP and RAB11, (B) GFP and mTOR, (C) GFP and LAMP1, and (D) mTOR and EEA1 in HEK293 cells were transfected with PKCα-GFP and starved for 5 hours prior to treatment with 100 nM PMA for 30 or 60 min. Images are representative of three fields examined from three independent experiments. Scale bar, 20 μm in (A-B), 10 μm in enlarged images in (C and D).
Fig 3.
Formation of the perinuclear cPKC-containing endosomes, but not perinuclear mTOR-containing lysosomes, requires endocytosis, cPKC, and PLD activity.
(A) Confocal microscopy images of immunofluorescence labeling of GFP and mTOR in PKCα-GFP-overexpressing HEK293 cells pretreated with 3uM Gö 6976 for 1 hour followed by treatment with 100nM PMA for 1 hour. Confocal microscope images of immunofluorescence labeling of mTOR and lysosomal markers (LAMP1 or LAMP2) in HEK293 cells pretreated with (B) 3 uM PLD2 inhibitor, VU 0364739 for 1 hour or (C) 400 mM sucrose for 30 min followed by treatment with 100nM PMA for 1 hour. Images are representative of three fields examined from three independent experiments. Scale bar, 10 μm in (A-C). (A’-C’) Quantification of co-localization for the respective experiments.
Fig 4.
PKCη is required for mTORC1 activation by regulating mTOR localization.
(A) Confocal microscopy images of immunofluorescence labeling of GFP and mTOR in PKCα-GFP-overexpressed HEK293 starved for 5 hours and then pretreated with 3 uM Bis for 1 hour followed by 100 nM PMA for 1 hour. Scale bar, 20 μm. (B) The level of phosphorylated and total S6K in HEK293 cells pretreated with 3 uM bisindolylmaleimide I (Bis) for 1 hour prior to treatment with 100 nM PMA for 1 hour. (C) Western blot analysis of phosphorylated and total S6K in HEK cells transfected with control, PKCη, PKCδ, PKCε, or PKCtheta siRNA and then treated with 100 nM PMA 5 hours after serum starvation. Bottom panels show efficiency of siRNAs on respective types of PKCs by protein level (PKCδ, PKCε) or mRNA (PKCη, PKCtheta). (D) Confocal microscope images of immunofluorescence labeling of mTOR and LAMP2 in HEK cells transfected with control or PKCη siRNA and treated with 100 nM PMA for 1 hour. Scale bar, 20 μm and 10 μm in enlarged images. Images are representative of three fields examined from three independent experiments. Western blots are representative of at least three independent experiments.
Fig 5.
PMA-induced translocation of mTOR to the lysosomes requires RAG, Ragulator, and v-ATPase.
(A) Western blot analysis of the level of phosphorylated and total S6K and (B) Confocal microscope images of immunofluorescence labeling of mTOR, LAMP1, and GFP in PKCα-GFP stably overexpressed HEK293 cells transfected with control or RAGB siRNA and treated with 100 nM PMA for 1 hour after serum starvation. Right panel shows efficiency of siRNA by RAGB mRNA level. Scale bar, 10 μm. (C) The level of phosphorylated and total S6K in HEK293 cells transfected with control, LAMTOR 1, or LAMTOR 3 siRNA and treated with 100 nM PMA for 1 hour after serum starvation. Right panels show efficiency of each siRNA on their targets by the protein level. (D) The level of phosphorylated and total S6K in HEK293 cells pretreated with 1 uM concanamycin A (Con A) for 1 hour followed by 100 nM PMA for 1 hour. Images are representative of three fields examined from three independent experiments. Western blots are representative of at least three independent experiments.
Fig 6.
Co-ordinated activation of classical and novel PKC isoforms is required for full mTORC1 activation by PMA.
A schematic summarizing the major findings of the study. PMA activation of mTORC1 requires activation of two separate pathways: a cPKC-dependent pathway that culminates in perinuclear PLD2 accumulation, and a nPKC-dependent pathway acting through PKCη that promotes mTORC1 translocation to perinuclear lysosomes.