Differential regulation of distinct Vps34 complexes by AMPK in nutrient stress and autophagy - PubMed (original) (raw)
Differential regulation of distinct Vps34 complexes by AMPK in nutrient stress and autophagy
Joungmok Kim et al. Cell. 2013.
Abstract
Autophagy is a stress response protecting cells from unfavorable conditions, such as nutrient starvation. The class III phosphatidylinositol-3 kinase, Vps34, forms multiple complexes and regulates both intracellular vesicle trafficking and autophagy induction. Here, we show that AMPK plays a key role in regulating different Vps34 complexes. AMPK inhibits the nonautophagy Vps34 complex by phosphorylating T163/S165 in Vps34 and therefore suppresses overall PI(3)P production and protects cells from starvation. In parallel, AMPK activates the proautophagy Vps34 complex by phosphorylating S91/S94 in Beclin1 to induce autophagy. Atg14L, an autophagy-essential gene present only in the proautophagy Vps34 complex, inhibits Vps34 phosphorylation but increases Beclin1 phosphorylation by AMPK. As such, Atg14L dictates the differential regulation (either inhibition or activation) of different Vps34 complexes in response to glucose starvation. Our study reveals an intricate molecular regulation of Vps34 complexes by AMPK in nutrient stress response and autophagy.
Copyright © 2013 Elsevier Inc. All rights reserved.
Figures
Fig.1
Different regulation of Vps34 complexes by glucose starvation in a manner dependent on AMPK. (A) Regulation of Vps34 complexes by glucose starvation. Wild-type MEF cells were starved for glucose (3 hrs) as indicated and the four Vps34-IP complexes were assayed for PI(3)P lipid kinase activity (n=4). (B) Subunit composition of Vps34 complexes. Vps34 protein level in each preparation (Fig.1A) was firstly examined (Top) and then normalized to determine subunit composition of each complexes (lower panels). IP efficiency of each antibody was examined by comparison of the target protein amount in the lysate before (B) and after (A) IP. (C) Relative abundance of Vps34 complexes. A quantitative immuno-depletion assay was performed with increasing amount of the indicated antibodies. Supernatants of MEF lysates after immuno-depletion (Post-IP) were examined to determine the level of Vps34 complex proteins. (D) Preparation of four different highly-enriched Vps34 complexes. Subunit composition was examined as described in Fig.1B. (E) Glucose starvation inhibits VIC1 and VIC2 but activates VIC3 and VIC4. Four different VPS34 complexes in Fig.1D were subjected to Vps34 lipid kinase assay (n=3). (F) AMPK is required for Vps34 regulation by glucose starvation. Endogenous Vps34 complexes were immuno-purified from glucose-starved MEFs as Fig.1A. The Vps34 kinase activity was normalized by the Vps34 protein levels (Fig.S1D) (n=4). Data are represented as mean ± S.D.; CON, mouse IgG-IP as a negative control. See also Figure S1.
Fig.2
AMPK directly regulates Vps34 complex through phosphorylation. (A) AMPK directly regulates Vps34 complex. Vps34 complex was immuno-purified from MEFs in glucose-rich medium and incubated with AMPK for 15 min in vitro as indicated (n=4). (B) AMPK inhibits VIC1 and VIC2 but activates VIC3 and VIC4 in vitro. Four highly-enriched Vps34 complexes obtained as Fig.1D were treated with AMPK in vitro and assayed for the lipid kinase assay (n=3). (C) AMPK regulates Vps34 complex via phosphorylation. The indicated Vps34 complexes were immuno-purified from MEFs in glucose-rich condition. The complexes were incubated with AMPK and λ PPase as indicated by arrow for order of the treatment (n=3). (D) AMPK-mediated phosphorylation is necessary and sufficient for the regulation of Vps34 complexes by glucose starvation. The Vps34 complexes were immuno-purified from glucose starved MEFs and then, treated with AMPK and λ PPase (n=3). (E) Atg14L converts Vps34-Beclin1 complex from inhibition to activation by AMPK. Vps34-Beclin1 complex was immuno-purified from U2OS cells, to which purified Atg14L-Flag/6His protein was added. Also, Atg14L containing Vps34 complex was directly prepared by Atg14L-IP from the cells as a control. The immune-complex was treated with AMPK in vitro and assayed for Vps34 lipid kinase assay (n=2). (F) Overexpression of Atg14L activates Vps34 kinase activity of Beclin1-IP in response to AMPK treatment. Vps34 complex were immuno-purified from the U2OS cell lines overexpressing Atg14L, which was induced by DOX, and then incubated with AMPK in vitro before the lipid kinase assay as indicated (n=2). Data are represented as mean ± S.D., See also Figure S2.
Fig.3
AMPK phosphorylates T163/S165 in Vps34 and S91/S94 in Beclin1. (A) AMPK directly phosphorylates Vps34 and Beclin1 in vitro. The indicated Flag-tagged Vps34 complex proteins were purified from the transfected cells and tested as substrates for AMPK in vitro by 32P-autoradiogram. (B) Sequence alignment of the AMPK phosphorylation sites in Beclin1 and other known AMPK substrates. (C) AMPK is required for glucose starvation-induced phosphorylation of Beclin1 S91/S94 and Vps34 T163/S165. (D) Activation of AMPK is sufficient to induce phosphorylation of Vps34 and Beclin1. 293A cells were incubated with compound C (C.C, 20 μM, 30 min), before glucose starvation. In parallel, 5 mM Metformin (Metf) or 25 mM 2-DG were added in glucose-rich medium for 3 hrs. See also Figure S3.
Fig.4
Phosphorylation of Beclin1 and Vps34 are required for the regulation of Vps34 complexes by glucose starvation or AMPK. (A) Beclin1 phosphorylation, but not Vps34 phosphorylation, is required for Atg14L-associated Vps34 complex activation in response to glucose starvation. Wild-type (WT), phosphorylation defective (SA), -mimetic (SD) Ha-Vps34, Myc-Beclin1 and Flag-Atg14L were co-transfected with AMPK into HEK293 cells as indicated. The cells were glucose-starved for 3 hrs (n=3). (B) Phosphorylation of Vps34 and Beclin1 is required for Vps34 regulation by AMPK. Vps34 complexes were immuno-purified by HA (Vps34), Myc (Vps34-Beclin1), or Flag (Vps34-Beclin1-Atg14L) and then incubated with AMPK for 15 min in vitro before Vps34 lipid assay (n=3). +, wild-type; A, a phosphorylation-defective mutant. (C) Atg14L determines whether Vps34-Beclin1 complex is activated or inhibited by AMPK. Vps34 complex was immuno-purified by either HA (Vps34) or Myc (Vps34-Beclin1) from the transfected HEK293 cells, to which purified Atg14L was added, and then treated with AMPK as indicated by arrow for order of the treatment. Atg14L protein level in the complexes was determined by western blot (bottom panel) (n=2). Data are represented as mean ± S.D.
Fig.5
Atg14L stimulates the phosphorylation of Beclin1, but suppresses the phosphorylation of Vps34 by AMPK. (A) Phosphorylation of Beclin1 and Vps34 is oppositely regulated by glucose starvation in Atg14L-dependent manner. Endogenous Beclin1 and Atg14L were immunoprecipitated from glucose-starved MEFs (3 hrs) as indicated. (B) Overexpression of Atg14L increases endogenous Beclin1 phosphorylation. Overexpression of Atg14L was induced in U2OS as Fig.2F. Post Atg14L-IP denotes the lysate after Atg14L immunoprecipitation, representing the Atg14L (both endogenous and overexpressed)-free fraction. (C) Atg14L directly enhances Beclin1 phosphorylation by AMPK in vitro. Beclin1 was immuno-purified from the transfected HEK293 cells, to which purified Flag-Atg14L was added. In parallel, Beclin1-Atg14L was obtained from the transfected cells for comparison (co-exp.). The complexes were treated with AMPK for the indicated time in vitro and the Beclin1 phosphorylation was examined. (D,E) Determination of the relative phosphorylation of endogenous Vps34 (D) and Beclin1 (E) proteins. The endogenous Vps34 complexes were immuno-purified from MEFs with or without glucose as indicated. Relative phosphorylation of Vps34 and Beclin1 was compared with in vitro AMPK-phosphorylated Vps34 complexes. See also Figure S4.
Fig.6
Beclin1 S91/S94 phosphorylation is required for autophagy induction. (A) Beclin1 expression level in Beclin1 knockdown (KD) and reconstituted MEFs. Scr denotes scramble shRNA control. (B) Cells expressing the Beclin1 S91/94A mutant are compromised in LC3 lipidation in response to glucose starvation (n=3). (C) Beclin1 S91/S94A mutant is defective in autophagosome formation. The indicated MEFs were starved with glucose (3 hrs) and the number of LC3 puncta was measured by endogenous LC3 staining (top). Also, PI(3)P level was determined by counting the spots of immunostaining using GST-2xFYVE protein as a probe (middle). The number of LC3 and PI(3)P double-positive puncta was counted by the overlap of LC3 and GST-2xFYVE staining and % was shown (bottom). (10-15 randomly selected images of the cells, n=3). See Fig.S5C for confocal images. (D) Beclin1 S91/84A mutant is defective in autophagy vacuole formation. The indicated MEFs were starved with glucose for 3 hrs and the autophagy vacuoles were examined on electron microscopy (EM). The numbers of autophagosome/autolysosome-like structures (AV) from 15-20 randomly selected cells were counted (See the representative images in Fig.S5D and S6C). (E) Phosphorylation of Beclin1 S91/S94 is specifically involved in glucose starvation-induced autophagy. The indicated Beclin1-MEFs were incubated with either glucose-free or 50 nM rapamycin-containing culture medium for 3 hrs. Autophagy flux was examined in the presence of 10 mM NH4Cl. Data are represented as mean ± S.D., See also Figure S5.
Fig.7
Vsp34 phosphorylation is required for cell survival but not autophagy in response to starvation (A) Vps34 expression level in Vps34-MEFs. (B,C) The Vps34 T163/S165A mutant does not compromise autophagy induction in response to glucose starvation as evidenced by LC3 lipidation (B) and by LC3-positive puncta formation (C, See Fig.S6B for confocal images). (D) Vps34 phosphorylation is required for PI(3)P reduction in response to glucose starvation. PI(3)P level in glucose-starved MEFs was determined by quantitative PI(3)P ELISA assay. The PI(3)P level was normalized by the amount of proteins used in the assay (n=6; *, p<0.05; **, p<0.01). (E) Vps34 phosphorylation is required for cell survival in response to glucose starvation. Beclin1 or Vps34-MEF cells were starved with glucose for 24 hrs and then the viability was measured by FACS analysis using AnexinV and propidium iodide (PI) double-staining (n=4; **, p<0.01). (F) A schematic model of Vps34 regulation by AMPK in response to energy starvation. Data are represented as mean ± S.D., See also Figure S6.
References
- Backer JM. The regulation and function of Class III PI3Ks: novel roles for Vps34. Biochem J. 2008;410:1–17. - PubMed
- Byfield MP, Murray JT, Backer JM. hVps34 is a nutrient-regulated lipid kinase required for activation of p70 S6 kinase. J Biol Chem. 2005;280:33076–33082. - PubMed
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