Beclin 2 negatively regulates innate immune signaling and tumor development
Motao Zhu, … , Guangjun Nie, Rong-Fu Wang
Motao Zhu, … , Guangjun Nie, Rong-Fu Wang
Published August 31, 2020
Citation Information: J Clin Invest. 2020;130(10):5349-5369. https://doi.org/10.1172/JCI133283.
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Research Article Cell biology Inflammation

Beclin 2 negatively regulates innate immune signaling and tumor development

Abstract

Beclin 2 plays a critical role in metabolic regulation and obesity, but its functions in innate immune signaling and cancer development remain largely unknown. Here, we identified Beclin 2 as a critical negative regulator of inflammation and lymphoma development. Mice with homozygous ablation of BCL2-interacting protein 2 (Becn2) developed splenomegaly and lymphadenopathy and markedly increased ERK1/2 and NF-κB signaling for proinflammatory cytokine production. Beclin 2 targeted the key signaling kinases MEKK3 and TAK1 for degradation through an ATG9A-dependent, but ATG16L/Beclin 1/LC3–independent, autophagic pathway. Mechanistically, Beclin 2 recruited MEKK3 or TAK1 through ATG9A to form a complex (Beclin 2-ATG9A-MEKK3) on ATG9A+ vesicles upon ULK1 activation. Beclin 2 further interacted with STX5 and STX6 to promote the fusion of MEKK3- or TAK1-associated ATG9A+ vesicles to phagophores for subsequent degradation. Importantly, Becn2-deficient mice had a markedly increased incidence of lymphoma development, with persistent STAT3 activation. Myeloid-specific ablation of MEKK3 (Map3k3) completely rescued the phenotypes (splenomegaly, higher amounts of proinflammatory cytokines, and cancer incidence) of Becn2-deficient mice. Hence, our findings have identified an important role of Beclin 2 in the negative regulation of innate immune signaling and tumor development through an ATG9A-dependent, but ATG16L/Beclin 1/LC3–independent, autophagic pathway, thus providing a potential target for the treatment of inflammatory diseases and cancer.

Authors

Motao Zhu, Guangtong Deng, Peng Tan, Changsheng Xing, Cuiping Guan, Chongming Jiang, Yinlong Zhang, Bo Ning, Chaoran Li, Bingnan Yin, Kaifu Chen, Yuliang Zhao, Helen Y. Wang, Beth Levine, Guangjun Nie, Rong-Fu Wang

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Figure 6

Beclin 2 mediates the degradation of TAK1 and MEKK3 through an ATG9A- and ULK-dependent autophagic pathway.

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Beclin 2 mediates the degradation of TAK1 and MEKK3 through an ATG9A- an...
(A and B) WT 293T cells, sgRNA-guided ATG9A-KO 293T cells (A), and sgRNA-guided ULK1-KO 293T cells (B) were transfected with HA-TAK1 or HA-MEKK3 alone or together with Flag–Beclin 2 plasmid, followed by immunoblotting with the indicated antibodies. Blots were run contemporaneously with the same protein samples (A). Bottom panels: quantitative analysis of HA-TAK1 and HA-MEKK3 expression and degradation percentages in WT and KO cells after normalization based on band intensity of 3 independent experiments. (C and D) WT and ATG9A-KO (C) or ULK1-KO (D) 293T cells were transfected with the HA–Beclin 2 plasmid alone or together with Flag-MEKK3. Cell lysates were immunoprecipitated using anti-Flag beads, followed by immunoblotting with the indicated antibodies. Data are representative of 3 independent experiments. (E) Confocal images of WT, ATG9A-KO, and ULK1-KO 293T cells cotransfected with GFP-MEKK3 and Flag–Beclin 2, then stained with anti-Flag antibody, followed by Alexa Fluor 633–conjugated secondary antibody staining. Hoechst 33342 was applied for nucleus staining. Scale bars: 10 μm. (F) Confocal images of WT, ATG9A-KO, and ULK1-KO 293T cells transfected with GFP-MEKK3, then stained with lysotracker and Hoechst 33342. Scale bars: 10 μm. Original magnification, ×1000 (merge); ×2500 (magnified). Data are plotted as mean ± SEM. Pearson’s correlation coefficient for colocalization shown in E and F was used with Image J Coloc 2 (at least 30 cells were analyzed per condition)\. Statistical differences between EmpVec-transfected and Flag–Beclin 2–transfected cells were calculated using Student’s unpaired t test (A and B). Statistical differences between WT and KO groups were calculated using Student’s unpaired t test (A) or 1-way ANOVA with Dunnett’s multiple comparison tests (B, E, and F). *P < 0.05; **P < 0.01; ***P < 0.001.