MEK can bypass ERK to trigger autophagy. A and B, knockdown of p90RSK or Elk did not abolish autophagy. K562 cells were transfected with siRNA against p90RSK or Elk, and LC3 processing was determined in response to amino acid (AA) starvation. C, knockdown of ERK did not abolish autophagy. K562 cells were transfected with siRNA against ERK1/2, and LC3 processing was determined in response to starvation. D, MEK can trigger autophagic response independently of ERK. K562 cells were transfected with constitutively active MEK (caMEK) or constitutively active MEK together with dominant negative ERK (dnERK), and LC3 processing was determined by autophagic flux assay. Shown in this figure from A to D are the representative Western blots of each of three independent experiments. ctrl, control; p-, phospho-.

MEK/ERK is positioned upstream of TSC in autophagic signaling. A, TSC2 was the dominant form of TSC1/2 in both H4IIE cells and K562 cells. B, AMPK activation of TSC2 was MEK/ERK-dependent. H4IIE cells were stimulated by 1 mm AICAR with or without 2 μm PD98059 (PD), and activation of AMPK or TSC2 was determined. C, activated MEK formed multiple complexes in response to autophagy stimuli. K562 and H4IIE cells were starved for amino acids (AA), and formation of the complexes including MEK were determined by non-denaturing Western blotting (WB). D, MEK and ERK associated with TSC2 in response to autophagy stimuli. H4IIE cells were starved for amino acids or treated with AICAR, and TSC2 binding to MEK or ERK was determined. E, MEK binding to TSC2 required ERK D domain. H4IIE cells were transfected with constitutively active MEK (caMEK) or constitutively active MEK mutant with the ERK D domain disrupted, and TSC2 binding to active MEK or MEK mutant without ERK D domain was determined. F, disruption of the association between MEK and TSC2 crippled TSC2 activation and the autophagic response. H4IIE cells transfected with constitutively active MEK, constitutively active MEK together with ERK siRNA, or constitutively active MEK mutant with the ERK D domain disrupted. Shown in this figure from A to F are the representative Western blots or co-immunoprecipitate (IP) blots of each of three independent experiments. p-, phospho-; Ctrl, control.

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-.

Moderately enhanced Beclin 1 causes cytoprotective autophagy, but prominently enhanced Beclin 1 causes cytodestructive autophagy. A, amino acid starvation caused a protective autophagy at early stage and a destructive autophagy at late stage. H4IIE cells were starved for amino acids, and autophagic activity represented by LC3 dots (upper panel) and cell viability (lower panel) were determined at the designated time points. B, serum starvation caused a protective autophagy at early stage and a destructive autophagy at late stage determined at the designated times as in A. C, rapamycin (10 μm) caused a protective autophagy, determined at the designated times as described in A, throughout. Data shown in A, B, and C are means ± S.D. (n = 3) (*, p < 0.05, **, p < 0.01). D, amino acid starvation caused a sustained high MEK/ERK activation and an increasingly pronounced Beclin 1 expression. Beclin 1 expression and ERK activation were quantitated by density scanning. E, serum starvation caused a sustained high MEK/ERK activation and significantly pronounced Beclin 1 expression. F, rapamycin causes a sustained moderate MEK/ERK activation and moderately enhanced Beclin 1 determined as in D. Shown in this figure from D to F are the representative Western blots and quantified data of each of three independent experiments. AVs, autophagic vacuoles; Baf, bafilomycin A1; p-, phospho-.

Autophagic activity and Beclin 1 expression is MEK/ERK-dependent. A and B, autophagic response coincided with MEK/ERK signaling. H4IIE and K562 cells were treated with various autophagy stimuli (10 μm rapamycin (Rap) for 48 h, serum depletion for 48 h, amino acid (AA) depletion for 6 h, or 50 nm vitamin D₃ (D3) for 72 h) with or without 2 μm PD98059 (PD). C, constitutive expression of active MEK or ERK triggered autophagic responses. H4IIE cells were transfected with constitutively active (ca) MEK or active ERK, and LC3 processing was determined in the presence of lysosomal protease inhibitor pepstatin A. D, fluorescence microscopy of GFP-LC3 transfectant cells undergoing the designated treatments. The percentage of cells with a punctate pattern is indicated as means ± S.D. (n = 3). E, basal MEK/ERK activity confers constitutive Beclin 1 expression. Depleting basal phospho (p)-MEK1/2 but not Raf1 by siRNA depleted basal Beclin 1. F, constitutive expression of CR3 domain of Raf1 did not trigger autophagic responses. G, autophagy stimuli-enhanced Beclin 1 was diminished by inhibition of MEK/ERK. H4IIE and K562 cells were starved for amino acids for 6 h or serum for 48 h or treated with 10 μm rapamycin (Rm) together with or without 2 μm PD98059. H, depleting Beclin 1 abolished autophagy in response to MEK/ERK activation. Beclin 1 knockdown H4IIE cells were starved for amino acids with or without 2 μm PD98059. Shown in this figure from A to G are representative Western blots or fluorescence microscopy of each of three independent experiments. ctrl, control.

MEK/ERK is positioned downstream of AMPK in autophagic signaling. A, inhibiting AMPK inhibited MEK activation in response to autophagy stimuli. H4IIE cells were starved for amino acids (AA) and treated with 10 μm compound C. B, inhibition of MEK did not inhibit AMPK activation in response to autophagy stimuli. H4IIE cells were starved for amino acids and treated with or without 2 μm PD98059 (PD), and AMPK activation was determined. C, up-regulation of Beclin 1 and induction of autophagy by AMPK activation was dependent on MEK. H4IIE cells were treated with 1 mm AICAR with or without 2 μm PD98059, and activation of AMPK and MEK, expression of Beclin 1, LC3 processing, and Beclin 1 binding to PI3KC3 were determined. D and E, MEK interacted with AMPK but not Raf1 in response to autophagy stimuli. H4IIE cells were starved for amino acids or treated with 1 mm AICAR, and physical interaction between MEK and AMPK, between Raf1 and MEK, or between AMPK and Raf1 was determined by using total (D) or phospho antibody (E). Growth hormone (GH) treatment served as a positive control for canonical Raf/MEK/ERK activation and interaction in H4IIE cells. F, specific inhibition on AMPK by kinase-dead AMPK crippled activation of MEK and autophagic activity in response to autophagy stimuli. H4IIE cells stably transfected with either rat AMPKα2 kinase-dead (KD) construct or vector control were starved for amino acids for 6 h. Expression of total AMPKα and total and phospho (p)-MEK and processing of LC3 were determined. Shown in this figure from A to F are the representative Western blots (WB) or co-immunoprecipitate (IP) blots of each of three independent experiments.

AMPK-MEK/ERK-TSC signaling functions in physiological response of autophagy in mice. A, AMPK-MEK/ERK signaling responded to autophagy stimuli in neonatal mice. Western blotting was performed to determine activation of AMPK, MEK, and ERK; expression of Beclin 1; and processing of LC3 using cell lysates prepared from the hearts and livers of mice at 5 or 120 h after birth. B, MEK/ERK interacted with AMPK or TSC1 in response to autophagy stimuli in neonatal mice. MEK or ERK co-immunoprecipitation was performed to examine their interactions with upstream regulator or downstream effector in response to autophagy or non-autophagy stimuli using the cell lysates from the hearts and livers of the mice 5 or 120 h after birth. Shown in this figure are the representative Western blots (WB) (A) or co-immunoprecipitate (IP) blots (B) of each of three independent experiments. p-, phospho-.

MEK/ERK inhibition on mTORC1 is TSC2-dependent, but its inhibition on mTORC2 is TSC2-independent. A, depleting TSC2 increased mTOR activity and crippled Beclin 1 up-regulation and LC3 processing by MEK/ERK activation. H4IIE cells stably transfected with constitutively active (ca) MEK or ERK were transiently transfected with siRNA against TSC2. B, depleting TSC2 did not affect MEK/ERK activation but crippled Beclin 1 up-regulation and LC3 processing in response to autophagy stimuli. H4IIE cells were transfected with siRNA against TSC2 and starved for amino acids (AA). C, depleting TSC2 crippled the disassembly of mTORC1 but not mTORC2 in response to autophagy stimuli. H4IIE cells were transfected with siRNA against TSC2 and starved for amino acids, and mTOR binding to Raptor, Rictor, or GβL was determined. The binding was quantified by density scanning. Shown in this figure from A to C 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; p-, phospho-.

A diagram for the non-canonical MEK/ERK signaling pathway regulating autophagy via regulating Beclin 1. Basal MEK/ERK activity maintains the activity of mTORC1 and mTORC2 by protecting them from being disassembled and thus maintains a basal Beclin 1 level incapable of triggering autophagic response. The non-canonical MEK/ERK module is activated by upstream regulator AMPK upon autophagy stimuli. Activation of MEK/ERK up-regulates the Beclin 1 level through disassembling mTORC1 and/or mTORC2 with a significantly sustained and elevated Beclin 1 level causing cytodestructive autophagy and moderately elevated Beclin 1 causing cytoprotective autophagy. Disassembling mTORC1 by MEK/ERK depends on TSC, whereas disassembling mTORC2 does not depend on TSC2. Although ERK is implicated in the regulation of autophagy, MEK can bypass ERK to trigger autophagic response.