Beclin 2 negatively regulates innate immune signaling and tumor development
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
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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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 4

Beclin 2 inhibits ERK1/2 signaling by targeting MEKK3 and TAK1 for autophagic degradation.

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Beclin 2 inhibits ERK1/2 signaling by targeting MEKK3 and TAK1 for autop...
(A) Schematic illustration of proinflammatory gene expression controlled by key adaptors, kinases, and transcription factors involved in NF-κB and MAPK signaling. (B) IL-6 production from WT, Becn2-KO, and sgRNA-guided Mapk3/1-KO plus Becn2-KO macrophages treated with LPS (100 ng/mL) for the indicated periods. (C) Immunoblot analysis of p-STAT3, STAT3, and ERK1/2 in WT, Becn2-KO, and sgRNA-guided Mapk3/1-KO plus Becn2-KO macrophages treated with LPS (100 ng/mL). (D) 293T cells cotransfected with the Flag–Beclin 2 plasmid alone or together with an HA-tagged plasmid were immunoprecipitated using anti-Flag beads, then immunoblotted with the indicated antibodies. (E) BMDMs were left untreated or treated by LPS (100 ng/mL) for 3 hours. Cell lysates of BMDMs were immunoprecipitated using MEKK3- or TAK1-specific antibodies against each protein, then immunoblotted with indicated antibodies. (F) Immunoblot analysis of endogenous TAK1 and MEKK3 in the lysates of 293T cells transfected with an increasing amount of pcDNA–Beclin 2 plasmid (0, 500, and 1000 ng/106 cells). (G) Immunoblot analysis of TAK1 and MEKK3 in cell lysates of WT and Becn2-KO T cells, B cells, and macrophages, neutrophils, and DCs before and after LPS (100 ng/mL, 2 hours) treatment. Samples were run on parallel gels contemporaneously. (H) 293T cells were transfected with HA-TAK1 or HA-MEKK3 alone or together with Flag–Beclin 2 plasmid, followed by treatment for 6–10 hours with 3MA (5 mM), BafA (500 nM), CQ (10 μM), MRT68921 (1 μM), or MG132 (10 μM). Cell lysates were immunoblotted with the indicated antibodies. Data are representative of at least 3 independent experiments. Statistical differences between groups were calculated using 1-way ANOVA with Tukey’s multiple comparisons test (B). *P < 0.05; **P < 0.01.