The class IA phosphatidylinositol 3-kinase p110-beta subunit is a positive regulator of autophagy

Abstract

Autophagy is an evolutionarily conserved cell renewal process that depends on phosphatidylinositol 3-phosphate (PtdIns(3)P). In metazoans, autophagy is inhibited by PtdIns(3,4,5)P(3), the product of class IA PI3Ks, which mediates the activation of the Akt-TOR kinase cascade. However, the precise function of class IA PI3Ks in autophagy remains undetermined. Class IA PI3Ks are heterodimeric proteins consisting of an 85-kD regulatory subunit and a 110-kD catalytic subunit. Here we show that the class IA p110-β catalytic subunit is a positive regulator of autophagy. Genetic deletion of p110-β results in impaired autophagy in mouse embryonic fibroblasts, liver, and heart. p110-β does not promote autophagy by affecting the Akt-TOR pathway. Rather, it associates with the autophagy-promoting Vps34-Vps15-Beclin 1-Atg14L complex and facilitates the generation of cellular PtdIns(3)P. Our results unveil a previously unknown function for p110-β as a positive regulator of autophagy in multicellular organisms.

Figure 1.

Autophagy is impaired in β^−/− MEFs. (A) β^+/+ and β^−/− MEFs were cultured in complete (untreated) or serum-free medium for 6 h, then observed under an electron microscope. Note the appearance of autophagosomes in serum-deprived β^+/+ MEFs, indicated by arrowheads. A higher magnification view of an autophagosome is shown in the inset. In β^−/− MEFs, deformed mitochondria are indicated by arrows. In serum-deprived β^−/− MEFs, aggregated ribosomes are indicated by asterisks. Nu, nucleus. (Right) Quantification of autophagic vacuoles per cell. Error bars = SEM; n = 10; *, P < 0.05; ***, P < 0.0001. (B) MEFs were labeled with ¹⁴C-valine for 24 h and cultured in complete or serum-free medium for 6 h. Degradation of long-lived proteins was measured and normalized. Data presented are averages of three independent experiments ± SEM; *, P < 0.05; ***, P < 0.0001. (C) MEFs were cultured in complete or serum-free medium for 6 h. Cells were stained with LysoTracker red and subjected to flow cytometry analysis. Data shown are representative of three independent experiments. (D) MEFs were cultured in glucose-free medium for 12 h, serum-free medium for 6 h, or treated with 25 µM etoposide for 6 h in the presence or absence of lysosomal inhibitors E64D and PepA. Cell lysates were probed for LC3 and β-tubulin. Quantification of LC3-II/β-tubulin from three independent immunoblots is shown. Data presented are the mean values normalized to β^+/+ untreated condition. *, P < 0.05; **, P < 0.005. (E) β^−/− and p110-β–reconstituted β^−/− MEFs were left untreated or serum starved for 6 h in the presence of E64D and PepA. Cell lysates were subjected to LC3 and β-tubulin immunoblotting. Quantification of LC3-II/β-tubulin from three independent experiments is shown. *, P < 0.05; **, P < 0.005. (F) MEFs with indicated genotypes expressing GFP-LC3 were treated as indicated. Cells were observed under a deconvolution fluorescence microscope. Representative images are shown. Quantification of autophagic cells was determined as described in Materials and methods. Data shown are averages of at least five blind countings ± SD. *, P < 0.05; **, P < 0.005. Bar, 20 µm.

Figure 2.

Overexpression of p110-β stimulates autophagy. (A) HEK293 cells were transfected with vector or Flag–p110-β expression construct. 48 h after transfection, cells were left untreated or serum starved for 12 h. Cell lysates were immunoblotted with indicated antibodies. Quantification of the relative LC3-II/β-tubulin ratio is shown. (B) HeLa cells were transfected with GFP-LC3 construct, together with empty vector or Flag–p110-β expression construct. 48 h after transfection, cells were cultured in complete medium or in Hank’s buffer for 4.5 h. Cells were fixed and observed under a fluorescence microscope. Representative images were taken, and quantification of autophagic cells is shown. Data presented are average of four blind countings ± SD. **, P < 0.005. Bar, 20 µm.

Figure 3.

p110-β promotes autophagy independently of the Akt–TOR pathway. (A) β^+/+ and β^−/− MEFs were left untreated or treated with 5 µM Akt inhibitor (Akti), 20 nM rapamycin (Ra), or 500 nM TGX-221 (TGX, pretreated overnight) in complete medium or in combination with glucose deprivation (12 h), or serum deprivation (6 h), all in the presence of lysosomal inhibitors E64D and PepA. Cell lysates were immunoblotted for LC3 and β-tubulin. (B) β^+/+ and β^−/− MEFs stably expressing GFP-LC3 were treated with 5 µM Akti or 20 nM rapamycin for 12 h. Cells were observed under a deconvolution fluorescence microscope. Representative images are shown. Bar, 20 µm. (C) Quantification of autophagic cells in MEFs stably expressing GFP-LC3 in response to indicated treatments. Data shown are averages of at least three blind countings ± SD. *, P < 0.05; **, P < 0.005.

Figure 4.

p110-β positively regulates PtdIns(3)P level and Vps34 catalytic activity. (A) β^+/+ and β^−/− MEFs stably expressing GFP-FYVE were observed under a deconvolution fluorescence microscope. Representative images (left) and quantification of GFP-FYVE puncta per cell (right) are shown. Bar, 20 µm. ***, P < 0.0001; n > 20; error bars = SEM. (B) Total cellular lipids were extracted from β^+/+, β^−/−, and β^+/+ MEFs treated with 1 mM 3-MA for 12 h. The lipid extracts were subjected to protein–lipid overlay analysis for PtdIns(3)P content. PtdIns(3)P standard and other PtdIns species were used as controls. Data shown are representative of three independent experiments. Quantification of total cellular PtdIns(3)P normalized against total protein is shown on the right (n = 3 for β^+/+ and β^−/−; n = 2 for β^+/+ +3-MA; **, P < 0.005; error bars = SEM). (C) β^+/+ and β^−/− MEFs were stained with antibodies against PtdIns(3)P or PtdIns(3,4,5)P₃, and subjected to flow cytometry analysis. (D and E) HEK293T cells were transfected with indicated plasmids. 48 h after transfection, cell lysates were subjected to immunoprecipitation using Myc antibody-conjugated agarose (D) or GFP antibody and protein A–conjugated agarose (E). 75% of the precipitates were assayed for Vps34 catalytic activity, and 25% of the precipitates were immunoblotted with indicated antibodies. Vps34 activity is calculated as the amount of PtdIns(3)P generated normalized against the amount of Vps34 present in the precipitates. IP reaction using IgG isotype control showed no activity over the background scintillation readings. Representative autoradiographs of the in vitro Vps34 assay results and the corresponding immunoblots are shown on the left. Quantification of Vps34 activity and Beclin 1–associated Vps34 activity are shown on the right. Data shown are average of three independent experiments ± SEM. *, P < 0.05. (F) Purified p110-β/p85-α was assayed under p110-β or Vps34 assay conditions. Results shown are the average of duplicate experiments.

Figure 5.

Early stage of autophagosome formation is suppressed in β^−/− MEFs. (A) β^+/+ and β^−/− MEFs were transfected with GFP-DFCP1 expression construct. 24 h after transfection, cells were cultured in complete medium, serum-free medium (6 h), or Hank’s buffer (2 h). Cells were fixed, observed, and imaged under a deconvolution fluorescence microscope. Only cells with moderate levels of GFP signal were quantified and imaged. Representative images are shown. Bar, 20 µm. Quantification of GFP puncta per cell is shown on the right. Error bars = SEM; n = 10; *, P < 0.05; ***, P < 0.001; N.S., nonsignificant. (B) β^+/+, β^−/−, and p110-β–reconstituted β^−/− MEFs lysates were probed for Atg5 and β-tubulin antibodies. The quantification of relative Atg5–Atg12 conjugation is shown on right. Data shown are the mean values from three independent experiments (*, P < 0.05; N.S., nonsignificant; error bars = SEM).

Figure 6.

p110-β is a component of the Vps34–Vps15–Beclin 1–Atg14L complex. (A) HEK293T cells were transfected with indicated plasmids. Cell lysates were subjected to immunoprecipitation using IgG control or GFP antibody. The lysates and precipitates were analyzed with indicated antibodies. (B) GFP-Rab5–transfected HEK293T cells were subjected to immunoprecipitation, and the precipitates were immunoblotted with indicated antibodies. (C–E) HEK293T cells were transfected with indicated plasmids. Cells were either cultured in complete medium or in serum-free medium for 12 h. Cell extracts were made and subjected to immunoprecipitation using Myc, Flag, or V5 antibodies. The precipitates together with lysates were subjected to immunoblotting with indicated antibodies. (F and G) 293T (F) or MCF10A (G) cell lysates were subjected to immunoprecipitation using IgG, Beclin 1, or p110-β antibodies. The precipitates together with cell lysates were analyzed with indicated antibodies. (H and I) β^−/− and p110-β–reconstituted β^−/− MEFs were cultured in complete medium or in serum-free medium for 6 h. Cell extracts were subjected to immunoprecipitation using IgG control, p110-β antibody (H), or Atg14L and Rubicon antibodies (I). The precipitates and cell lysates were analyzed with indicated antibodies. (J) HeLa cells were transfected with Flag–p110-β together with GFP-Atg14L constructs. Cells were left untreated or starved in Hank’s buffer for 4 h. The cells were fixed and stained with Flag and Alexa 594–conjugated antibodies, and observed under a confocal fluorescence microscope. Representative images with green, red, or merged channels are shown.

Figure 7.

p110-β promotes autophagy independently of its kinase activity. (A) β^−/− MEFs expressing GFP-LC3 were stably reconstituted with construct expressing p110-βWT or p110-βK805R mutant. Cells were left untreated or serum starved for 6 h. Cells were fixed and imaged under a deconvolution fluorescence microscope. Representative images are shown. Bar, 20 µm. (B) Quantification of autophagic cell percentage of GFP-LC3–expressing MEFs upon indicated treatments. Data shown are averages of five blind countings ± SD. *, P < 0.05; **, P < 0.005. (C) The three cell lines were cultured in complete medium, glucose-free medium (12 h), or serum-free medium (6 h) in the presence of E64D and PepA. Cell lysates were immunoblotted with indicated antibodies. Quantification of LC3-II/β-tubulin is shown at bottom. (D, top) HEK293 cells were transfected with empty vector, or constructs expressing Flag-tagged p110-βWT or p110-βN924K. p110-β PI3K activity was assayed in Flag-immunoprecipitates. Results show an autoradiograph from duplicate assays. (Bottom) β^+/+, β^−/−, and β^−/− MEFs reconstituted with p110-βN924K mutant were cultured in serum-free medium for 6 h in the presence of E64D and PepA. Cell extracts were probed for LC3 and β-tubulin, and the ratio of LC3-II/β-tubulin is shown. (E) MEFs with indicated genotypes were subject to protein–lipid overlay analysis for PtdIns(3)P content. Quantification of total cellular PtdIns(3)P normalized against total protein is shown on the right. Data shown are the mean values from three independent experiments (*, P < 0.05; N.S., nonsignificant; error bars = SEM). (F) HeLa cells were transfected with GFP-FYVE, together with vector control, Flag–p110-βWT, or Flag–p110-βK805R construct. 48 h after transfection, cells were fixed and observed under a deconvolution fluorescence microscope. Bar, 20 µm. Cells with over 100 GFP puncta were quantified and presented. Data shown within each group are the average of at least five independent countings with over 200 cells. Error bars = SD. *, P < 0.05; N.S., nonsignificant. (G and H) HEK293T cells overexpressing Myc–Vps34–V5–Vps15, Beclin 1–GFP, together with vector control or Flag–p110-βK805R were subjected to immunoprecipitation using an anti-Myc antibody (G) or a GFP antibody (H). Vps34 activity was determined. Data presented are the average of three independent experiments; error bars = SEM. *, P < 0.05. (I) HEK293T cells were transfected with the Myc–Vps34–V5–Vps15 bicistronic plasmid, together with empty vector or constructs expressing wild-type or kinase-dead p110-β mutants. Cell lysates were subject to immunoprecipitation using an anti-Myc antibody. The precipitates were immunoblotted for Myc and p110-β. (J) HEK293T cells were transfected with constructs expressing wild-type or kinase-dead p110-β mutants. Cell lysates were subject to immunoprecipitation using IgG or Beclin 1 antibody. The precipitates were probed for Beclin 1 and p110-β.

Figure 8.

Autophagy is impaired in p110-β-deficient liver and heart. (A) Gender- and age-paired 8- to 10-wk-old mice with indicated genotypes were fed or fasted for 24 h. Liver weight, body weight, and liver protein concentrations were measured. Data presented are the normalized average values ± SEM; n = 5 within each group; *, P < 0.05; N.S., nonsignificant. (B) Electron micrographic images of livers from 8-wk-old fed or 24-h fasted mice. Deformed mitochondria are indicated by arrow in the β^−/− liver. In fasted livers, autophagosomes are labeled with arrowheads. Higher magnification view of a representative autophagosome is shown in the inset. Note the swollen mitochondria in fasted β^−/− liver. Gly, glycogen area; Nu, nucleus. (C) Total lysates were made from β^+/+ and β^−/− livers, and probed for ubiquitin and β-tubulin. Data shown are representative of three independent pairs of animals. (D) Total liver lysates from fed or 24-h fasted mice with indicated genotypes were generated, and immunoblotted for p62. Ponceau S staining is shown for equal loading. (E) 8-wk-old GFP-LC3 transgenic mice with indicated liver genotypes were fed or fasted for 24 h. Cryosections of the livers were observed under a deconvolution fluorescence microscope. Representative images are shown. Bar, 20 µm. (F) GFP-LC3 transgenic mice with indicated heart genotypes were fed or fasted for 48 h. Heart cryosections were observed and representative images are shown. Bar, 20 µm. Note the increase in GFP-LC3 puncta in fasted β^+/+ liver and heart, which are impaired in fasted β^−/− mice.

Figure 9.

p110-β is required for autophagy induced by pressure overload in the heart. (A) At 8 wk of age, MerCreMer;p110-β^flox/flox mice and their littermate p110-β^flox/flox controls were injected with 1 mg tamoxifen intraperitoneally daily for 28 d. 4 wks later, transverse aortic constriction (TAC) or sham operation was performed on these tamoxifen-injected mice. 33 d later, hearts were harvested and sections were prepared for electron microscopy. Representative images are shown for each group of mice. Note the dramatic induction of autophagosomes in the β^+/+ heart, which is absent in the β^−/− heart. (B) TAC or sham operation was performed on 10–12-wk-old MCK-Cre;GFP-LC3;p110-β^flox/flox mice and their littermate GFP-LC3;p110-β^flox/flox controls. 7 d later, hearts were harvested and frozen sections prepared. GFP-LC3 puncta in the heart sections were visualized under a deconvolution fluorescence microscope. Representative images with the same magnification are shown for each group of mice. Bar, 20 µm.

Figure 10.

Schematic model for p110-β in the regulation of autophagy. Growth factors activate membrane receptors (such as RTK and GPCR), which recruit p85-p110 and stimulate the production of PtdIns(3,4,5)P₃, which in turn activates the Akt–TOR pathway and inhibits autophagy. On the other hand, p110-β (but not p110-α) localizes in the autophagy-promoting complex that contains Rab5, Vps34, Vps15, Beclin 1, and Atg14L. The complex produces PtdIns(3)P and facilitates the formation of autophagosomes.