FIGURE 6.

MEK/ERK up-regulates Beclin 1 and autophagy by down-regulating mTOR activity. A, MEK/ERK failed to up-regulate Beclin 1 and autophagy when mTOR was activated. H4IIE cells were transfected with constitutively active Akt with or without 1 mm AICAR, and MEK/ERK or mTOR activation, Beclin 1 expression, and LC3 processing were determined. B, fluorescence microscopy of the GFP-LC3 cells transfected with constitutively active Akt (caAkt) with or without autophagy stimuli. C, overexpressing active Akt enhanced mTORC1 activity using GST-4EBP1 as a substrate. The ³²P incorporated into GST-4EBP1 (GST-4EBP1-P) is shown in a bar graph. D, MEK/ERK activation inhibits mTORC1 activity. H4IIE cells were stimulated with the designated autophagy stimuli or transfected with constitutively active MEK (caMEK) or constitutively active ERK (caERK) with or without 2 μm PD98059 (PD), and mTORC1 activity was determined as described in C. E, inhibiting MEK/ERK disassociated both mTORC1 and mTORC2. H4IIE cells were starved for amino acids (AA) with or without 2 μm PD98059, and Beclin 1 expression and LC3 processing as well as mTOR binding to Raptor, Rictor, or GβL were determined. F, inhibiting mTORC1 or mTORC2 individually caused a mildly increased Beclin 1 and autophagic response, but inhibiting both mTORC1 and mTORC2 caused strongly pronounced Beclin 1 levels and autophagic responses. H4IIE cells were transfected with siRNA against mTOR or against Raptor or Rictor individually or in combination, and activation of mTOR effectors, Beclin 1 expression, and LC3 processing was determined by autophagic flux assay. The Beclin 1 levels were quantified by density scanning. G, fluorescence microscopy of GFP-LC3 transfectant H4IIE cells with Raptor or Rictor knockdown individually or in combination. Shown in this figure from A to G are the representative Western blots (WB) or co-immunoprecipitate (IP) blots or quantified data (means ± S.D.) of each of three independent experiments. Ctrl, control; Kd, kinase-dead; p-, phospho-.