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The kinase MST4 limits inflammatory responses through direct phosphorylation of the adaptor TRAF6

Nature Immunology volume 16, pages 246–257 (2015)Cite this article

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Abstract

Immune responses need to be tightly controlled to avoid excessive inflammation and prevent unwanted host damage. Here we report that germinal center kinase MST4 responded dynamically to bacterial infection and acted as a negative regulator of inflammation. We found that MST4 directly interacted with and phosphorylated the adaptor TRAF6 to prevent its oligomerization and autoubiquitination. Accordingly, MST4 did not inhibit lipopolysaccharide-induced cytokine production in Traf6−/− embryonic fibroblasts transfected to express a mutant form of TRAF6 that cannot be phosphorylated at positions 463 and 486 (with substitution of alanine for threonine at those positions). Upon developing septic shock, mice in which MST4 was knocked down showed exacerbated inflammation and reduced survival, whereas heterozygous deletion of Traf6 (Traf6+/−) alleviated such deleterious effects. Our findings reveal a mechanism by which TRAF6 is regulated and highlight a role for MST4 in limiting inflammatory responses.

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Figure 1: Fluctuation in MST4 expression in response to stimulation with LPS.
Figure 2: MST4 negatively regulates TLR signaling.
Figure 3: Suppression of TLR signaling by MST4 is dependent on its kinase activity.
Figure 4: MST4 regulates TLR signaling through direct interaction with TRAF6.
Figure 5: MST4 inhibits the autoubiquitination of TRAF6.
Figure 6: MST4 directly phosphorylates TRAF6.
Figure 7: Phosphorylation of TRAF6 by MST4 impairs its homo-oligomerization and complex assembly.
Figure 8: MST4 protects mice from inflammatory damage upon septic shock.

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Acknowledgements

We thank H. Ji (Shanghai Institute of Biochemistry and Cell Biology) for the pLKO.1-puro backbone; and D. Li, J. Zhou, G. Pei, B. Sun, L. Li, A. Lin and M. Lei for support. Supported by the 973 program of the Ministry of Science and Technology of China (2012CB910204 and 2012CB910200), the National Natural Science Foundation of China (31270808, 31300734, 31470736, 31470868, 31030021 and 81161120542), the Science and Technology Commission of Shanghai Municipality (11JC14140000 and 13ZR1446400), and the “Cross and cooperation in science and technology innovation team” project of the Chinese Academy of Sciences.

Author information

Author notes

  1. Shi Jiao, Zhen Zhang and Chuanchuan Li: These authors contributed equally to this work.

  2. zczhou@sibcb.ac.cn

Authors and Affiliations

  1. National Center for Protein Science Shanghai, State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

    Shi Jiao, Zhen Zhang, Chuanchuan Li, Min Huang, Zhubing Shi, Yanyan Wang, Xiaomin Song, Heng Liu, Chunyang Li, Min Chen, Wenjia Wang, Yun Zhao, Hongyan Wang, Catherine C L Wong, Chen Wang & Zhaocai Zhou

  2. State Key Laboratory of Protein and Plant Gene Research, Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education, School of Life Sciences, Peking University, Beijing, China

    Zhengfan Jiang

  3. Peking University-Tsinghua University Joint Center for Life Sciences, Beijing, China

    Zhengfan Jiang

  4. School of Life Science and Technology, ShanghaiTech University, Shanghai, China

    Zhaocai Zhou

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Contributions

S.J., Zhe.Z. and Chuan.L. designed and carried out most of the experiments and analyzed the data; M.H. and C.C.L.W. performed mass spectrometry analysis; Z.S. did the structural modeling; Y.W. and M.C. provided purified proteins. X.S., H.L., Chun.L. and W.W. contributed to data analysis; Zha.Z., Y.Z., Z.J., H.W. and C.W. contributed to experimental design; S.J., X.S. and Zha.Z. wrote the manuscript; and Zha.Z. supervised the project.

Corresponding authors

Correspondence to Chen Wang or Zhaocai Zhou.

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Competing interests

Zha.Z. and S.J. have filed a patent (201410705068.X) related to this work, which covers the potential application of MST4 as a biomarker for certain infectious diseases, and possible therapeutics relevant to the MST4 function in macrophages.

Integrated supplementary information

Supplementary Figure 1 Fluctuation of MST4 expression in response to LPS stimulation or bacterial infection.

(a,b) Scatter plot for MST4 mRNA levels in sepsis data sets GSE54514 (a) and GSE26378 (b). (c) Relative mRNA levels of MST4, TNF and IL6 upon LPS challenge at different time points. (d) Transcription of Mst4 in mouse AVECs, AECs, LFs, and AMs upon SL1344 infection. (e) Immunoblot (IB throughout) analysis for MST4 (arrowhead along right margin throughout) in cells transfected with Flag-MST4 and MST4-specific shRNA. (f) Immunoblot of mouse tissues with MST4 antibody. Mouse tissues were prepared and quantified using the bicinchoninic acid assay (50 mg protein loaded per sample), analyzed with polyclonal anti-MST4 antibody. Data are representative of at least two experiments.

Supplementary Figure 2 MST4 negatively regulates TLR signaling.

(a) Relative mRNA levels of TNF and I L6 in THP-1 cells expressing MST4 upon LPS challenge. (b) Production of TNF and IL-6 in THP-1 cells expressing MST4 upon LPS challenge. (c) Secretion of cytokines in shMST4 THP-1 cells in response to LPS challenge. (d) Il1b mRNA levels in PEMs expressing MST4 upon SL1344 infection. (e) Il1b mRNA levels in shMST4 PEMs challenged with different concentrations of LPS. (f) Transcription of cytokines in shMST4 THP-1 cells challenged with different concentrations of LPS. (g) Production of TNF and IL-6 in shMST4 THP-1 after LPS tolerance. (h, i) Il6 mRNA levels in control and MST4-knockdown MEFs (h) and PEMs (i) upon acute LPS challenge. *, P<0.05; **, P<0.01; ***, P<0.001 compared with control group (unpaired t-test). Data are representative of at least two experiments (mean and s.d.). EV, empty vector (same below); Scr, control shRNA.

Supplementary Figure 3 MST4 regulates TLR signaling dependent on its kinase activity.

(a) Immunoblot analysis of total IκBα in THP-1 cells after transfection with MST4 and its mutants upon LPS treatment. (b) Immunoblot of MST4 in shMST4 MEFs after transfection with Flag-MST4 and its mutants. (c) The interaction between MST4 and MO25 in the lung and bone marrow of mice treated with LPS for the indicated time period. (d) LPS-induced IL-6 production after transfection with MST4 and MO25 in THP-1 cells. **, P<0.01; ***, P<0.001 compared with control group (unpaired t-test). Data are representative of at least two experiments (means and s.d. in d).

Supplementary Figure 4 MST4 directly interacts with TRAF6.

(a) Luciferase activity detected in HEK 293T cells transfected with a NF-κB luciferase reporter and indicated plasmids. (b) NF-κB activation induced by TRAF6 after transfection with MST4 and MO25. (c) Exogenous IP for MST4 and TRAF6. (d) Co-localization of MST4 and TRAF6 in THP-1 cells upon LPS stimulation. (e) Schematic illustration of the domain organization of MST4 and TRAF6. (f, g) Co-immunoprecipitation (IP throughout) mapping of specific domains responsible for MST4 interaction with TRAF6. (h) Pulldown assay for testing a direct interaction between MST4 and TRAF6-C. *, P<0.05; **, P<0.01; ***, P<0.001 (unpaired t-test). Data are representative of at least two experiments. TRAF6-C, TRAF-C domain of TRAF6.

Supplementary Figure 5 MST4 inhibits TRAF6 autoubiquitination in a manner dependent on its kinase activity.

(a) Immunoblot of TRAF6 ubiquitination in THP-1 cells upon LPS challenge. (b) Ubiquitination of TRAF6 after transfection with MST4 or its constitutively active mutant (TE) or kinase dead mutant (KR). (c) Ubiquitination of TRAF6 in HEK 293T cells overexpressing MST4 and MO25. (d) Ubiquitination of NEMO in cells overexpressing MST4 and its mutants. Data are representative of at least two experiments.

Supplementary Figure 6 MST4 directly phosphorylates the TRAF-C domain of TRAF6.

(a) Ubiquitination and Ser/Thr phosphorylation of TRAF6 in THP-1 cells transfected with shMST4 upon LPS challenge. (b) Domain mapping of TRAF6 phosphorylation by MST4. (c) Mass spectrometric analysis of phosphorylation sites of TRAF6 by MST4. (d) In vitro kinase assay of wild-type MST4 using wild-type TRAF-C domain of TRAF6 (TRAF6-C (WT)) or its mutant with threonines 463 and 486 substituted with alanines (TRAF6-C (2A)) as substrate.

Supplementary Figure 7 MST4 interferes with the assembly of TRAF6-related signaling complex.

Gel-filtration assays for signaling complexes containing endogenous TRAF6 in TNFα-stimulated 293T cells. Fractions of gel-filtration were immunoblotted with antibody against TRAF6. Data are representative of at least two experiments.

Supplementary Figure 8 In vivo knockdown of MST4 in mice upon septic shock.

(a,b) Transcription of Mst4 (a) and Il6 (b) in the different organs from MST4-KD mice upon intraperitoneal injection with LPS. (c) Relative mRNA change of Mst4 and Il6 in MST4-KD mice compared with mock mice. (d) Plasma cytokine expression in mice treated with LPS (n=5). (e) Flow cytometry of F4/80+ macrophages isolated from spleens 2 days after clodronate liposome treatment. *, P<0.05 (unpaired t-test). Data are representative of at least two experiments (means and s.d. in d).

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Jiao, S., Zhang, Z., Li, C. et al. The kinase MST4 limits inflammatory responses through direct phosphorylation of the adaptor TRAF6. Nat Immunol 16, 246–257 (2015). https://doi.org/10.1038/ni.3097

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