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 7

Beclin 2 binds to STX5/6 to promote the fusion of ATG9A vesicles with phagophores for MEKK3 degradation.

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Beclin 2 binds to STX5/6 to promote the fusion of ATG9A vesicles with ph...
(A) WT, sgRNA-guided STX5-KO, STX6-KO, or STX5:STX6-DKO 293T cells were transfected with HA-MEKK3 alone or together with Flag–Beclin 2 plasmid, followed by immunoblotting with the indicated antibodies. The blots were run contemporaneously with the same protein samples. Right panel: quantitative analysis of HA-MEKK3 expression and degradation percentage. (B) Cell lysates of 293T cells were immunoprecipitated using anti-STX5 or anti-STX6 antibody, respectively, and then immunoblotted with the indicated antibodies. WCL, whole cell lysates (C) Representative TEM images of WT and STX5:STX6 DKO 293T cells cooverexpressing Flag–Beclin 2 and MEKK3-APEX2 processed in parallel under identical conditions. Black arrows indicate MEKK3-associated vesicles; white arrowheads indicate MEKK3-containing autophagosome. (D) WT, STX5-KO, and STX6-KO cells were cotransfected with Flag-ATG9A and HA–Beclin 2, while BECN2 KO 293T cells were transfected with Flag-ATG9A alone. Half of the cells were left untreated for immunoisolation of Flag-ATG9A+ vesicles; the other half were pretreated by CQ (10 μM, 4 hours) to inhibit the fusion of autophagosomes with lysosomes for the enrichment of autophagosomes. Cells were resuspended in specific fractionation buffers for isolation of Flag-ATG9A+ vesicles or autophagosome enrichment, respectively, followed by immunoblotting with indicated antibodies. (E) Confocal images of WT and STX5:STX6-DKO 293T cells transfected with GFP-MEKK3 and Flag–Beclin 2, then stained with lysotracker. Scale bars: 10 μm. Pearson’s correlation coefficient for colocalization was used with Image J Coloc 2. Graph represents mean ± SEM (at least 30 cells were analyzed per condition). Data are representative of 3 independent experiments. Statistical differences between EmpVec-transfected and Flag–Beclin 2–transfected cells were calculated using Student’s unpaired t test (A). Statistical differences of degradation percentages between WT and KO groups were calculated using 1-way ANOVA with Dunnett’s multiple comparison test (A). Statistical analyses of colocalization between groups were calculated using Student’s unpaired t test (E). *P < 0.05; **P < 0.01; ***P < 0.001.