Figure 7. GSK3B phosphorylates BCL2 at Ser70 to trigger the ubiquitination of BCL2. (A) IL17A eliminates the phosphorylation of BCL2 Ser70 induced by GSK3B. Cells transfected with GSK3B-HA were treated with IL17A (30 ng/ml) for 2 h. Then cell lysates were prepared and immunoprecipitated with anti-Myc antibody. The precipitates were detected by anti-p-BCL2 (Ser70) antibody. (B) Depletion of GSK3B decreased the expression of phosphorylation of BCL2 at Ser70. Cells were transfected with si-Gsk3b and BCL2-Myc for 24 h. Then cell lysates were prepared and immunoprecipitated with anti-Myc antibody. The precipitates were detected by anti-p-BCL2 (Ser70) antibody. (C) Mimicking phosphorylation at Ser70 accelerates degradation of BCL2. Cells were transfected with Myc-tagged BCL2 variants (WT, S70A, or S70D) plasmids for 24 h. Then cells were treated with CHX for the indicated times. The cells were also transfected with an identical amount of pEGFP-N1 plasmid to monitor the transfection efficiency. The expression of BCL2 or GFP was examined by western blotting with anti-Myc antibody (for BCL2) or anti-GFP antibody respectively. (D) Mimicking phosphorylation of BCL2 Ser70 promotes ubiquitination. Cells were transfected with Flag-tagged ubiquitin and Myc-tagged BCL2 variants (WT, S70A, or S70D) plasmids. Cell lysates were immunoprecipitated with anti-Myc antibody. The precipitates were blotted with anti-Flag antibody and anti-Myc antibody. (E) Schematic diagram of the mechanism of IL17A-mediated attenuation of autophagy in pulmonary fibrosis. IL17A activates PIK3CA to induce the phosphorylation of GSK3B at Ser9, which causes a decrease in the kinase activity of GSK3B. The suppressed GSK3B attenuates the phosphorylation and degradation of BCL2. Thus, enhanced expression of BCL2 promotes the association of BCL2 and BECN1 to protect against the activation of autophagy.