Figure 2. The Beclin 1 S90 phosphorylation site is required for autophagy induction in MCF7 and U2OS cells.
(A) Western blot results of MCF7 cells transiently transfected with empty vector, and Flag epitope-tagged wild-type Beclin 1, Beclin 1 S90A, or Beclin 1 S90E. The cells were grown in normal medium (starvation−) or HBSS (starvation+) for 3 hr in the presence or absence of 100 nM bafilomycin A1. (B) Representative images of GFP-LC3 puncta (autophagosomes) in MCF7 cells transiently co-transfected with indicated Flag-Beclin 1 constructs and a plasmid expressing GFP-LC3 and grown in normal medium or in HBSS for 3 hr (starvation) in the presence or absence of 100 nM bafilomycin A1. (C) Quantification of GFP-LC3 puncta in MCF7 cells in conditions shown in (B). Bars are mean + SEM of triplicate samples (>50 cells analyzed per sample). Similar results were observed in three independent experiments. ***p < 0.001, **p < 0.01, NS, not significant; one-way ANOVA. (D) Western blot detection of Beclin 1, p62 and LC3 in U2OS cells expressing doxycycline-inducible shRNA against beclin 1 (beclin 1 shRNA U2OS cells) following treatment with 1 μg/ml doxycycline for 4 days in cells transduced with retroviral constructs expressing indicated shRNA-resistant Flag-Beclin 1 (NTm, non-targetable mutant) plasmids. Cells were either grown in normal medium (starvation−) or in HBSS for 3 hr (starvation+) in the presence or absence of 100 nM bafilomycin A1. See Figure 2—figure supplement 1 for comparison of Beclin 1, p62, and LC3 western blots in the presence and absence of doxycycline. (E) Quantification of GFP-LC3 puncta (autophagosomes) in beclin 1 shRNA U2OS cells treated with 1 μg/ml doxycycline for 4 days and co-transfected with plasmids expressing GFP-LC3 and indicated shRNA-resistant Flag-Beclin 1 construct and grown in normal medium or in EBSS for 3 hr (starvation) in the presence or absence of 100 nM bafilomycin A1. Bars are mean + SEM of triplicate samples (>50 cells analyzed per sample). Similar results were observed in three independent experiments. **p < 0.01, *p < 0.05, NS, not significant; one-way ANOVA. (F) Beclin 1-associated VPS34 in vitro lipid kinase assay and amounts of VPS34 and ATG14 in anti-Beclin 1 immunoprecipitates of beclin 1 shRNA U2OS cells following treatment with 1 μg/ml doxycycline for 4 days and transfection with indicated shRNA-resistant Flag-Beclin 1 (NTm, non-targetable mutant) plasmids. Cells were either grown in normal medium (starvation−) or in HBSS for 2 hr (starvation+). Dots shown in upper panel represent the amount of PI3P generated in an in vitro VPS34 lipid kinase assay using anti-Flag-Beclin 1 immunoprecipitates as input. (G) Densometric quantitation of VPS34 in vitro lipid kinase activity in anti-Beclin 1 immunoprecipitates in conditions described in (F). Results shown represent mean + SEM of values in three independent experiments. Similar results were observed in each independent experiment. Shown are the relative values of VPS34 lipid kinase activity compared to those observed in cells expressing WT Beclin 1 in normal media (defined as 100%). To control for input in Beclin 1 anti-immunoprecipitates, values used to calculate VPS34 lipid kinase activity were normalized for levels of Beclin 1 determined by densitometric quantification of Beclin 1 western blot bands in anti-Beclin 1 immunoprecipitates. ***p < 0.001, **p < 0.01, NS, not significant; one-way ANOVA. See also Figure 2—figure supplement 1, Figure 1—figure supplement 2, Figure 2—figure supplement 3.
Figure 2—figure supplement 1. Doxycycline reduces Beclin 1 expression and starvation-induced autophagy in U2OS cells that express doxycycline-inducible beclin 1 shRNA.
Figure 2—figure supplement 2. Bafilomycin A1-induced Beclin 1 S90 phosphorylation is reversed by the ROS scavenger, N-acetyl-L-cysteine.
Figure 2—figure supplement 3. Effects of Beclin 1 S90 phosphorylation o on subcellular localization of the Beclin 1/VPS34/ATG14 complex.
