Figure 4. CHK2‐mediated Beclin 1 phosphorylation regulates autophagy.

• A
  The effect of Beclin 1 Ser90/93 phosphorylation on the interaction between Beclin 1 and Bcl‐2. GST‐Beclin 1 WT, AA mutant, and DD mutant (bait) were incubated with or without recombinant CHK2 in the presence of ATP and then incubated with Flag‐Bcl‐2 (preys).
• B
  Immunoprecipitation of Bcl‐2 with Flag‐Beclin 1 WT, AA mutant, and DD mutant in HCT116 cells in normal medium or after H₂O₂ (500 μM) treatment for 1 h.
• C, D
  Autophagy levels were determined in Beclin 1‐depleted H1299 cells with reconstituted expression of Beclin 1 WT, AA mutant, or DD mutant in normal medium or after H₂O₂ (500 μM) treatment for 3 h in the presence (C) or absence (D) of CHK2.
• E
  Representative electron microscopic image of an autophagosome (arrow, left panel, AP) and an autolysosome (arrow, right panel, AL) in H1299 Beclin 1 WT cells treated with H₂O₂ (500 μM) for 6 h. Scale bars, 500 nm. Electron microscopic quantification of autophagosomes (AP) and autolysosomes (AL) in H1299 cells with the indicated plasmids cultured for 6 h in normal or H₂O₂ (500 μM) treatment. All quantitative data are presented as mean ± s.e.m. from three independent experiments; *P < 0.001 compared to Beclin 1 WT; ^# P < 0.001 compared to Beclin 1 WT treated with H₂O₂; ^& P < 0.001 compared to Beclin 1 WT treated with H₂O₂ in the absence of CHK2.
• F
  Western blot detection of p‐CHK2 Thr68, CHK2, p‐Beclin 1 Ser90, p‐Beclin 1 Ser93, Beclin 1, p62, and LC3 in H1299 cells transfected with the indicated shRNA in normal medium or after H₂O₂ (500 μM) treatment for 3 h.

Source data are available online for this figure.