Abstract

NLRC4 is a cytosolic member of the NOD-like receptor family that is expressed in innate immune cells. It senses indirectly bacterial flagellin and type III secretion systems, and responds by assembling an inflammasome complex that promotes caspase-1 activation and pyroptosis1,2,3,4,5,6. Here we use knock-in mice expressing NLRC4 with a carboxy-terminal 3×Flag tag to identify phosphorylation of NLRC4 on a single, evolutionarily conserved residue, Ser 533, following infection of macrophages with Salmonella enterica serovar Typhimurium (also known as Salmonella typhimurium). Western blotting with a NLRC4 phospho-Ser 533 antibody confirmed that this post-translational modification occurs only in the presence of stimuli known to engage NLRC4 and not the related protein NLRP3 or AIM2. Nlrc4−/− macrophages reconstituted with NLRC4 mutant S533A, unlike those reconstituted with wild-type NLRC4, did not activate caspase-1 and pyroptosis in response to S. typhimurium, indicating that S533 phosphorylation is critical for NLRC4 inflammasome function. Conversely, phosphomimetic NLRC4 S533D caused rapid macrophage pyroptosis without infection. Biochemical purification of the NLRC4-phosphorylating activity and a screen of kinase inhibitors identified PRKCD (PKCδ) as a candidate NLRC4 kinase. Recombinant PKCδ phosphorylated NLRC4 S533 in vitro, immunodepletion of PKCδ from macrophage lysates blocked NLRC4 S533 phosphorylation in vitro, and Prkcd−/− macrophages exhibited greatly attenuated caspase-1 activation and IL-1β secretion specifically in response to S. typhimurium. Phosphorylation-defective NLRC4 S533A failed to recruit procaspase-1 and did not assemble inflammasome specks7 during S. typhimurium infection, so phosphorylation of NLRC4 S533 probably drives conformational changes necessary for NLRC4 inflammasome activity and host innate immunity.

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Acknowledgements

We thank P. Broz, A. Paler Martinez, M. Roose-Girma, X. Rairdan, C. Kung, V. Asghari and S. Mukund for technical support, and K. O’Rourke for editorial assistance.

Author information

Author notes

    • Yan Qu
    •  & Shahram Misaghi

    These authors contributed equally to this work.

Affiliations

  1. Department of Physiological Chemistry, Genentech Inc., 1 DNA Way, South San Francisco, California 94080, USA

    • Yan Qu
    • , Kim Newton
    • , Nobuhiko Kayagaki
    •  & Vishva M. Dixit
  2. Department of Early Stage Cell Culture, Genentech Inc., 1 DNA Way, South San Francisco, California 94080, USA

    • Shahram Misaghi
    •  & Salina Louie
  3. Department of Protein Chemistry, Genentech Inc., 1 DNA Way, South San Francisco, California 94080, USA

    • Anita Izrael-Tomasevic
    •  & David Arnott
  4. Department of Immunology, Genentech Inc., 1 DNA Way, South San Francisco, California 94080, USA

    • Laurie L. Gilmour
    •  & James E. Cupp
  5. Department of Medical Protein Research, VIB, B-9000 Ghent, Belgium

    • Mohamed Lamkanfi
  6. Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium

    • Mohamed Lamkanfi
  7. Department of Bioinformatics and Computational BiologyGenentech Inc., 1 DNA Way, South San Francisco, California 94080, USA

    • Jinfeng Liu
  8. Department of Pathology, Genentech Inc., 1 DNA Way, South San Francisco, California 94080, USA

    • László Kömüves
  9. Department of Microbiology and Immunology, Stanford School of Medicine, California, 94305, USA

    • Denise Monack

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Contributions

Y.Q., S.M., A.I.-T., D.A., L.L.G., J.E.C., L.K. and S.L. designed and conducted experiments; K.N. generated the Nlrc4 F/F mice; J.L. performed bioinformatics analyses; M.L., N.K. and D.M. discussed the study; Y.Q., K.N. and V.M.D. wrote the manuscript.

Competing interests

Y.Q., S.M., A.I.-T., K.N., L.L.G., J.E.C., S.L., N.K., J.L., L.K., D.A. and V.M.D. are employees of Genentech, Inc.

Corresponding author

Correspondence to Vishva M. Dixit.

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https://doi.org/10.1038/nature11429

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