Article | Published:

Kinases Mst1 and Mst2 positively regulate phagocytic induction of reactive oxygen species and bactericidal activity

Nature Immunology volume 16, pages 11421152 (2015) | Download Citation

Abstract

Mitochondria need to be juxtaposed to phagosomes for the synergistic production of ample reactive oxygen species (ROS) in phagocytes to kill pathogens. However, how phagosomes transmit signals to recruit mitochondria has remained unclear. Here we found that the kinases Mst1 and Mst2 functioned to control ROS production by regulating mitochondrial trafficking and mitochondrion-phagosome juxtaposition. Mst1 and Mst2 activated the GTPase Rac to promote Toll-like receptor (TLR)-triggered assembly of the TRAF6-ECSIT complex that is required for the recruitment of mitochondria to phagosomes. Inactive forms of Rac, including the human Rac2D57N mutant, disrupted the TRAF6-ECSIT complex by sequestering TRAF6 and substantially diminished ROS production and enhanced susceptibility to bacterial infection. Our findings demonstrate that the TLR-Mst1-Mst2-Rac signaling axis is critical for effective phagosome-mitochondrion function and bactericidal activity.

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References

  1. 1.

    , & Recognition and signaling by toll-like receptors. Annu. Rev. Cell Dev. Biol. 22, 409–437 (2006).

  2. 2.

    et al. Genetic analysis of host resistance: Toll-like receptor signaling and immunity at large. Annu. Rev. Immunol. 24, 353–389 (2006).

  3. 3.

    NOX enzymes and the biology of reactive oxygen. Nat. Rev. Immunol. 4, 181–189 (2004).

  4. 4.

    & The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol. Rev. 87, 245–313 (2007).

  5. 5.

    & Current molecular models for NADPH oxidase regulation by Rac GTPase. Blood 100, 2692–2696 (2002).

  6. 6.

    , & Protein kinase C-α signals rho-guanine nucleotide dissociation inhibitor phosphorylation and rho activation and regulates the endothelial cell barrier function. J. Biol. Chem. 276, 22614–22620 (2001).

  7. 7.

    , & Phosphorylation of RhoGDI by Pak1 mediates dissociation of Rac GTPase. Mol. Cell 15, 117–127 (2004).

  8. 8.

    et al. Human neutrophil immunodeficiency syndrome is associated with an inhibitory Rac2 mutation. Proc. Natl. Acad. Sci. USA 97, 4654–4659 (2000).

  9. 9.

    et al. Dominant negative mutation of the hematopoietic-specific Rho GTPase, Rac2, is associated with a human phagocyte immunodeficiency. Blood 96, 1646–1654 (2000).

  10. 10.

    et al. Biochemical and biological characterization of a human Rac2 GTPase mutant associated with phagocytic immunodeficiency. J. Biol. Chem. 276, 15929–15938 (2001).

  11. 11.

    et al. TLR signalling augments macrophage bactericidal activity through mitochondrial ROS. Nature 472, 476–480 (2011).

  12. 12.

    & Physiological roles of mitochondrial reactive oxygen species. Mol. Cell 48, 158–167 (2012).

  13. 13.

    et al. Mitochondria contribute to LPS-induced MAPK activation via uncoupling protein UCP2 in macrophages. Biochem. J. 402, 271–278 (2007).

  14. 14.

    , , , & CARD9 facilitates microbe-elicited production of reactive oxygen species by regulating the LyGDI-Rac1 complex. Nat. Immunol. 10, 1208–1214 (2009).

  15. 15.

    et al. Disruption of the uncoupling protein-2 gene in mice reveals a role in immunity and reactive oxygen species production. Nat. Genet. 26, 435–439 (2000).

  16. 16.

    et al. Mitochondrial reactive oxygen species promote production of proinflammatory cytokines and are elevated in TNFR1-associated periodic syndrome (TRAPS). J. Exp. Med. 208, 519–533 (2011).

  17. 17.

    , & The Ste20-like protein kinase, Mst1, dimerizes and contains an inhibitory domain. J. Biol. Chem. 271, 21049–21053 (1996).

  18. 18.

    & Cloning and characterization of a human protein kinase with homology to Ste20. J. Biol. Chem. 270, 21695–21700 (1995).

  19. 19.

    & Regulation of mammalian Ste20 (Mst) kinases. Trends Biochem. Sci. 40, 149–156 (2015).

  20. 20.

    et al. Protein kinases of the Hippo pathway: regulation and substrates. Semin. Cell Dev. Biol. 23, 770–784 (2012).

  21. 21.

    et al. Mst1 and Mst2 maintain hepatocyte quiescence and suppress hepatocellular carcinoma development through inactivation of the Yap1 oncogene. Cancer Cell 16, 425–438 (2009).

  22. 22.

    , , & Role of the kinase MST2 in suppression of apoptosis by the proto-oncogene product Raf-1. Science 306, 2267–2270 (2004).

  23. 23.

    et al. Mst1 promotes cardiac myocyte apoptosis through phosphorylation and inhibition of Bcl-xL. Mol. Cell 54, 639–650 (2014).

  24. 24.

    et al. Mst1 inhibits autophagy by promoting the interaction between Beclin1 and Bcl-2. Nat. Med. 19, 1478–1488 (2013).

  25. 25.

    The hippo signaling pathway in development and cancer. Dev. Cell 19, 491–505 (2010).

  26. 26.

    , & The Hippo pathway and human cancer. Nat. Rev. Cancer 13, 246–257 (2013).

  27. 27.

    & The Hippo pathway: regulators and regulations. Genes Dev. 27, 355–371 (2013).

  28. 28.

    & The two faces of Hippo: targeting the Hippo pathway for regenerative medicine and cancer treatment. Nat. Rev. Drug Discov. 13, 63–79 (2014).

  29. 29.

    & The emerging role of the hippo pathway in cell contact inhibition, organ size control, and cancer development in mammals. Cancer Cell 13, 188–192 (2008).

  30. 30.

    et al. Mst1 and Mst2 protein kinases restrain intestinal stem cell proliferation and colonic tumorigenesis by inhibition of Yes-associated protein (Yap) overabundance. Proc. Natl. Acad. Sci. USA 108, E1312–E1320 (2011).

  31. 31.

    et al. The phenotype of human STK4 deficiency. Blood 119, 3450–3457 (2012).

  32. 32.

    et al. MST1 mutations in autosomal recessive primary immunodeficiency characterized by defective naive T-cell survival. Blood 119, 3458–3468 (2012).

  33. 33.

    et al. Mst1/Mst2 regulate development and function of regulatory T cells through modulation of Foxo1/Foxo3 stability in autoimmune disease. J. Immunol. 192, 1525–1535 (2014).

  34. 34.

    et al. A cell-intrinsic role for Mst1 in regulating thymocyte egress. J. Immunol. 183, 3865–3872 (2009).

  35. 35.

    et al. Mst1 regulates integrin-dependent thymocyte trafficking and antigen recognition in the thymus. Nat. Commun. 3, 1098 (2012).

  36. 36.

    , & Spatiotemporal regulation of the kinase Mst1 by binding protein RAPL is critical for lymphocyte polarity and adhesion. Nat. Immunol. 7, 919–928 (2006).

  37. 37.

    , , & RAPL, a Rap1-binding molecule that mediates Rap1-induced adhesion through spatial regulation of LFA-1. Nat. Immunol. 4, 741–748 (2003).

  38. 38.

    et al. Crucial functions of the Rap1 effector molecule RAPL in lymphocyte and dendritic cell trafficking. Nat. Immunol. 5, 1045–1051 (2004).

  39. 39.

    et al. The Nore1B/Mst1 complex restrains antigen receptor-induced proliferation of naive T cells. Proc. Natl. Acad. Sci. USA 105, 20321–20326 (2008).

  40. 40.

    et al. The Mst1 and Mst2 kinases control activation of rho family GTPases and thymic egress of mature thymocytes. J. Exp. Med. 209, 741–759 (2012).

  41. 41.

    et al. T cell receptor “inside-out” pathway via signaling module SKAP1-RapL regulates T cell motility and interactions in lymph nodes. Immunity 32, 541–556 (2010).

  42. 42.

    & Pathogen recognition receptors: ligands and signaling pathways by Toll-like receptors. Int. Rev. Immunol. 32, 116–133 (2013).

  43. 43.

    & Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity 34, 637–650 (2011).

  44. 44.

    & The role of ubiquitylation in immune defence and pathogen evasion. Nat. Rev. Immunol. 12, 35–48 (2012).

  45. 45.

    et al. The kinase MST4 limits inflammatory responses through direct phosphorylation of the adaptor TRAF6. Nat. Immunol. 16, 246–257 (2015).

  46. 46.

    et al. A conserved MST-FOXO signaling pathway mediates oxidative-stress responses and extends life span. Cell 125, 987–1001 (2006).

  47. 47.

    et al. Regulation of neuronal cell death by MST1-FOXO1 signaling. J. Biol. Chem. 284, 11285–11292 (2009).

  48. 48.

    et al. The c-Abl-MST1 signaling pathway mediates oxidative stress-induced neuronal cell death. J. Neurosci. 31, 9611–9619 (2011).

  49. 49.

    , & MOBKL1A/MOBKL1B phosphorylation by MST1 and MST2 inhibits cell proliferation. Curr. Biol. 18, 311–321 (2008).

  50. 50.

    et al. The E3 ubiquitin-ligase HACE1 catalyzes the ubiquitylation of active Rac1. Dev. Cell 21, 959–965 (2011).

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Acknowledgements

Supported by the National Basic Research Program (973) of China (2015CB910502 to L.C.), China's 1000 Young Talents Program (D.Z. and L.C.), the 111 Projects (B12001 and B06016), the Fundamental Research Funds for the Central Universities of China-Xiamen University (CXB2014004 to J.Z.; 20720140551 to L.C.; and 2013121034 and 20720140537 to D.Z.), the National Natural Science Foundation of China (31270918, 81222030 and J1310027 to D.Z.; 81372617, 81422018 and U1405225 to L.C.; 81472229 to L.H.; and 81302529 to X.L.), the Natural Science Foundation of Fujian (2013J06011 to D.Z. and 2014D007 to X.L.), the US National Institutes of Health (RO1 CA136567 for J.A.) and institutional funds from Massachusetts General Hospital (for J.A.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author information

Author notes

    • Jing Geng
    •  & Xiufeng Sun

    These authors contributed equally to this work.

Affiliations

  1. State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, China.

    • Jing Geng
    • , Xiufeng Sun
    • , Ping Wang
    • , Shihao Zhang
    • , Xiaozhen Wang
    • , Hongtan Wu
    • , Lixin Hong
    • , Changchuan Xie
    • , Hao Zhao
    • , Qingxu Liu
    • , Mingting Jiang
    • , Qinghua Chen
    • , Jinjia Zhang
    • , Yang Li
    • , Siyang Song
    • , Hong-Rui Wang
    • , Sheng-Cai Lin
    • , Jiahuai Han
    • , Lanfen Chen
    •  & Dawang Zhou
  2. Department of Laboratory Medicine, the First Affiliated Hospital, Medical College of Xiamen University, Xiamen, China.

    • Xun Li
  3. Institute of Immunology and the CAS Key Laboratory of Innate Immunity and Chronic Disease, Innovation Center for Cell Signaling Network, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, China.

    • Rongbin Zhou
  4. Department of Biochemistry and Molecular Biology, University of Texas, M.D. Anderson Cancer Center, Houston, Texas, USA.

    • Randy L Johnson
  5. College of Medicine, Chang Gung University, Kwei-Shan, Taiwan.

    • Kun-Yi Chien
  6. Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA.

    • Joseph Avruch
  7. Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts, USA.

    • Joseph Avruch

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Contributions

J.G., X.S., L.C. and D.Z. designed the research and helped with data analysis; J.G., X.S., P.W., S.Z., X.W., H.W., L.H., C.X., X.L., H.Z., Q.L., M.J., Q.C., J.Z., Y.L. and K.-Y.C. performed the experiments and helped with data analysis; S.S., H.-R.W., R.Z., R.L.J., S.-C.L., J.H. and J.A. contributed to discussions and provided critical reagents; and J.A., L.C. and D.Z. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Lanfen Chen or Dawang Zhou.

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DOI

https://doi.org/10.1038/ni.3268

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