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. 2013 Dec;9(12):1983-95.
doi: 10.4161/auto.26058.

Regulation of PIK3C3/VPS34 complexes by MTOR in nutrient stress-induced autophagy

Hai-Xin Yuan, Ryan C Russell, Kun-Liang Guan

Regulation of PIK3C3/VPS34 complexes by MTOR in nutrient stress-induced autophagy

Hai-Xin Yuan et al. Autophagy. 2013 Dec.

Abstract

Autophagy is a cellular defense response to stress conditions, such as nutrient starvation. The type III phosphatidylinositol (PtdIns) 3-kinase, whose catalytic subunit is PIK3C3/VPS34, plays a critical role in intracellular membrane trafficking and autophagy induction. PIK3C3 forms multiple complexes and the ATG14-containing PIK3C3 is specifically involved in autophagy induction. Mechanistic target of rapamycin (MTOR) complex 1, MTORC1, is a key cellular nutrient sensor and integrator to stimulate anabolism and inhibit catabolism. Inactivation of TORC1 by nutrient starvation plays a critical role in autophagy induction. In this report we demonstrated that MTORC1 inactivation is critical for the activation of the autophagy-specific (ATG14-containing) PIK3C3 kinase, whereas it has no effect on ATG14-free PIK3C3 complexes. MTORC1 inhibits the PtdIns 3-kinase activity of ATG14-containing PIK3C3 by phosphorylating ATG14, which is required for PIK3C3 inhibition by MTORC1 both in vitro and in vivo. Our data suggest a mechanistic link between amino acid starvation and autophagy induction via the direct activation of the autophagy-specific PIK3C3 kinase.

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Figures

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Figure 1. MTOR is required for autophagy-related PIK3C3 complex regulation. (A) ATG14 containing PIK3C3 is activated under conditions of MTOR inhibition. HEK293 cells were cultured in following conditions: nutrient-rich (NR), amino acid free (AA-, indicated time), Torin1 (100 nM, 1 h) or rapamycin (50 nM, 1 h) treatment. ATG14-containing PIK3C3 complex was immunoprecipitated with ATG14 antibody and used for lipid kinase assay. (B) Quantification of (A). (C) Induction of autophagy by MTOR inhibition. HEK293 cells were treated similarly as in (A) with or without bafilomycin A1 (Baf A1) (100 nM) treatment for 1 h before harvesting cells. LC3 and SQSTM1 level were detected using specific antibodies. (D) RHEB inhibits ATG14 containing PIK3C3 via activation of MTOR. HEK293 cells were transfected with Flag-ATG14, Myc-BECN1 and HA-PIK3C3 together with pcDNA3 or RHEB. ATG14-containing PIK3C3 complex was immunoprecipitated with Flag antibody and used for lipid kinase assay. (E) Quantification of (D). The error bars represent the standard error of the mean from independent experiments within same treatment group. Stars indicate a statistically significant difference.
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Figure 2. Rag GTPases-MTOR pathway mediates the autophagy response to amino acid starvation. (A) Rag GTPases suppress ATG14-PIK3C3 activation upon amino acid starvation. HEK293 cells were transfected with Flag-ATG14, Myc-BECN1 and HA-PIK3C3 together with pcDNA3, RRAGA-QL and RRAGC-SN, or RRAGA-TN and RRAGC-QL. ATG14-containing PIK3C3 complex was immunoprecipitated with Flag antibody and subjected to lipid kinase assay. (B) Quantification of (A). (C) Constitutively active RRAGB blocks autophagy via MTORC1 activation. HEK293A cells stably expressing control vector or RRAGBQ99L were subjected to amino acid starvation or Torin1 (100 nM) treatment with or without bafilomycin A1 (100 nM) treatment for 1 h before harvesting cells. LC3 levels were detected by immunoblot. LE: long exposure; SE: short exposure. The error bars represent the standard error of the mean from independent experiments within same treatment group. Stars indicate a statistically significant difference.
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Figure 3. MTORC1, but not MTORC2, is involved in the regulation of different PIK3C3 complexes. (A) Amino acid starvation only activates the ATG14-containing PIK3C3 lipid kinase activity. Different PIK3C3 complexes were immunoprecipitated from MEFs that were cultured in nutrient-rich or AA-free medium for 1 h, and subjected to lipid kinase assay and immunoblot. (B) Quantification of (A). (C) RPTOR knockdown activates ATG14-PIK3C3. Different PIK3C3 complexes from scrambled or Rptor KD MEFs were immunoprecipitated with corresponding antibodies and subjected to lipid kinase assay. The right panel indicates RPTOR protein levels in control or Rptor KD MEFs. (D) Rictor knockout has no effect on ATG14-PIK3C3 activity. Different PIK3C3 complexes from RICTOR WT or Rictor KO MEFs were immunoprecipitated with corresponding antibodies and subjected to lipid kinase assay. The right panel indicates RICTOR protein levels in WT or Rictor KO MEFs.
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Figure 4. MTORC1, but not MTORC2, regulates autophagy-specific PIK3C3 activity. (A) RPTOR knockdown enhances basal autophagy and autophagy-specific PIK3C3 activity. Scrambled or Rptor KD MEFs were cultured in either nutrient-rich or amino acid-free medium for 1 h. Cells were fixed and subjected to immunostaining with LC3 antibody and GST-FYVE probe. (B) Quantification of the staining in (A). (C) Rictor knockout has not effect on autophagy induction by amino acid starvation. WT and Rictor KO MEFs were treated and stained similarly as in (A). (D) Quantification of the staining in (C). The error bars represent the standard error of the mean from independent experiments within same treatment group. Stars indicate a statistically significant difference.
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Figure 5. MTORC1, but not MTORC2, regulates the ATG14-containing PIK3C3 complex in vitro. (A) MTORC1 inhibits ATG14-PIK3C3 activity in vitro. ATG14-containing complexes were immunoprecipitated with ATG14 antibody and treated with MTORC1 or MTORC2 purified from HEK293 cells in vitro. For MTORC1 treatment, reactions in the presence of Torin1 (50 nM) or without ATP were set up as controls. The immune-complexes were then subjected to lipid kinase assay. Right panels show the relative amount of MTORC1 and MTORC2 used in the assay. (B) Quantification of (A). The error bars represent the standard error of the mean from independent experiments within same treatment group. Stars indicate a significant difference. (C) Dose-dependent inhibition of ATG14-PIK3C3 by MTORC1. ATG14-containing complexes were treated with increasing amount of MTORC1 in vitro and subjected to lipid kinase assay. The presence of Torin1 is indicated (last lane). (D) The BECN1-PIK3C3 complex is not regulated by MTORC1 or MTORC2. BECN1-PIK3C3 complex were treated with MTORC1 or MTORC2 in vitro and then subjected to lipid kinase assay. (E) ATG14 increased the basal PIK3C3 activity and also confers inhibition by MTORC1. BECN1-PIK3C3 complex were immunoprecipitated with BECN1 antibody and incubated with Flag-ATG14 protein (50 ng) for 10 min. After washing, the immune-complexes were further treated with MTORC1 and finally subjected to lipid kinase assay. Immunoblot shows Flag-ATG14 bound to the BECN1-PIK3C3 complex. The numbers under top panels in (C–E) represent the relative density of each dot normalized to PIK3C3 levels.
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Figure 6. MTORC1 inhibits ATG14-PIK3C3 complex activity by phosphorylating ATG14. (A) Phosphorylation of ATG14 by MTOR. Recombinant GST-tagged ATG14 or RPS6KB were expressed and purified from E. coli, and used as substrates for in vitro MTOR kinase assay. Phosphorylation was determined by 32P-autoradiograph and the protein levels were determined by immunoblot. GST-RPS6KB was included as a positive control for MTOR kinase assay. (B) Mutation of the five phosphorylation residues abolishes ATG14 phosphorylation by MTOR in vitro. HEK293 cells were transfected with Flag-ATG14-WT or 5S/TA mutant as well as Myc-BECN1 and HA-PIK3C3. Flag-ATG14 proteins were purified by immunoprecipitation with Flag antibody and subjected to in vitro MTOR kinase assay. (C) HEK293 cells stably expressing HA-ATG14-WT or HA-ATG14-5S/TA. The HA-tagged ATG14 run slightly slower than the endogenous ATG14 (top panel). (D) Amino acid starvation activates ATG14-WT but not the ATG14-5S/TA associated PIK3C3 activity. ATG14-containing PIK3C3 complexes were immunoprecipitated from HEK293 stable cells under either nutrient-rich or -starvation conditions using HA antibody. The immune-complexes were treated with MTORC1 and then subjected to lipid kinase assay. (E) Quantification of (D). The error bars represent the standard error of the mean from independent experiments within same treatment group. Stars indicate a statistically significant difference.
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Figure 7. ATG14-5S/TA mutant promotes autophagy level. (A) Expression of ATG14-5S/TA enhances LC3-II. HEK293 cells stably expressing control vector, ATG14-WT or ATG14-5S/TA were treated with either nutrient-rich or AA-free medium for 1 h with or without presence of bafilomycin A1 (100 nM). LC3 levels were detected by immunoblot. The histogram under the blots represents quantification of each LC3-II band normalized by tubulin. (B) ATG14-5S/TA enhances SQSTM1 degradation. The histogram under the blots represents quantification of each SQSTM1 band normalized by actin. (C) ATG14-5S/TA promotes autophagy. HEK293A stable cells stably expressing control vector, ATG14-WT or ATG14-5S/TA were cultured in nutrient-rich medium for 1 h. Cells were fixed and subjected to immunostaining with GST-FYVE probe (PtdIns3P) and antibodies against HA and LC3. Scale bar: 10 μm. (D) Quantification of the staining in (C). The error bars represent the standard error of the mean from independent experiments within same treatment group. Stars indicate a statistically significant difference.
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