title: Phosphorylation of NLRC4 is critical for inflammasome activation.
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 pyroptosis. 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 specks during S. typhimurium infection, so phosphorylation of NLRC4 S533 probably drives conformational changes necessary for NLRC4 inflammasome activity and host innate immunity.
authors.affiliation: Department of Physiological Chemistry, Genentech Inc., 1 DNA Way, South San Francisco, California 94080, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Qu
authors.forename: Shahram
authors.initials: S
authors.lastname: Misaghi
authors.forename: Anita
authors.initials: A
authors.lastname: Izrael-Tomasevic
authors.forename: Kim
authors.initials: K
authors.lastname: Newton
authors.forename: Laurie L
authors.initials: LL
authors.lastname: Gilmour
authors.forename: Mohamed
authors.initials: M
authors.lastname: Lamkanfi
authors.forename: Salina
authors.initials: S
authors.lastname: Louie
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Kayagaki
authors.forename: Jinfeng
authors.initials: J
authors.lastname: Liu
authors.forename: László
authors.initials: L
authors.lastname: Kömüves
authors.forename: James E
authors.initials: JE
authors.lastname: Cupp
authors.forename: David
authors.initials: D
authors.lastname: Arnott
authors.forename: Denise
authors.initials: D
authors.lastname: Monack
authors.forename: Vishva M
authors.initials: VM
authors.lastname: Dixit
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D053468:CARD Signaling Adaptor Proteins / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D020170:Caspase 1 / Q000378:metabolism; D004789:Enzyme Activation; D055879:Gene Knock-In Techniques; D006801:Humans; D007113:Immunity, Innate / Q000276:immunology; D058847:Inflammasomes / Q000378:metabolism*; D053583:Interleukin-1beta / Q000276:immunology / Q000378:metabolism; D008264:Macrophages / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011487:Protein Conformation; D051745:Protein Kinase C-delta / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D012486:Salmonella typhimurium / Q000276:immunology; D016415:Sequence Alignment
chemical_list: D053468:CARD Signaling Adaptor Proteins; D002135:Calcium-Binding Proteins; D058847:Inflammasomes; D053583:Interleukin-1beta; C435692:NLRC4 protein, human; D051745:Protein Kinase C-delta; D020170:Caspase 1