K45A mutation of RIPK1 results in poor necroptosis and cytokine signaling in macrophages, which impacts inflammatory responses in vivo.
Receptor interacting protein kinase 1 (RIPK1) participates in several cell signaling complexes that promote cell activation and cell death. Stimulation of RIPK1 in the absence of caspase signaling induces regulated necrosis (necroptosis), which promotes an inflammatory response. Understanding of the mechanisms through which RIPK1 promotes inflammation has been unclear. Herein we have evaluated the impact of a K45A mutation of RIPK1 on necroptosis of macrophages and the activation of inflammatory response. We show that K45A mutation of RIPK1 results in attenuated necroptosis of macrophages in response to stimulation with LPS, TNFα and IFNβ in the absence of caspase signaling. Impairment in necroptosis correlated with poor phosphorylation of RIPK1, RIPK3 and reduced trimerization of MLKL. Furthermore, K45A mutation of RIPK1 resulted in poor STAT1 phosphorylation (at S727) and expression of RANTES and MIP-1α following TNF-R engagement in the absence of caspase activation. Our results further indicate that in the absence of stimulation by pathogen-associated molecular patterns (PAMPs), cellular inhibitors of apoptotic proteins (cIAPs) prevent the K45-dependent auto-phosphorylation of RIPK1, leading to resistance against necroptosis. Finally, RIPK1(K45A) mice displayed attenuated inflammatory response in vivo as they were significantly resistant against endotoxin shock, but highly susceptible against a challenge with Salmonella typhimurium. This correlated with reduced expression of IL-1β and ROS, and poor processing of caspase 8 by RIPK1(K45A) macrophages. Overall, these results indicate that K45 mediated kinase activity of RIPK1 is not only important for necroptosis but it also has a key role in promoting cytokine signaling and host response to inflammatory stimuli.
Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada.|Program in Neuroscience and Division of Neurology, The Ottawa Hospital, Ottawa, ON, Canada.
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Shutinoski
Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada.
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Alturki
Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada.
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Rijal
Pattern Recognition Receptor Discovery Performance Unit, Immuno-Inflammation Therapeutic Area, GlaxoSmithKline, Collegeville, PA 19426, USA.
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Bertin
Pattern Recognition Receptor Discovery Performance Unit, Immuno-Inflammation Therapeutic Area, GlaxoSmithKline, Collegeville, PA 19426, USA.
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Gough
Program in Neuroscience and Division of Neurology, The Ottawa Hospital, Ottawa, ON, Canada.
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Schlossmacher
Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada.
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Sad
D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D016207:Cytokines / Q000378:metabolism*; D004731:Endotoxins; D007249:Inflammation / Q000201:enzymology / Q000473:pathology*; D051034:Inhibitor of Apoptosis Proteins / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D008239:Lysine / Q000235:genetics*; D008264:Macrophages / Q000201:enzymology* / Q000473:pathology; D051379:Mice; D009154:Mutation / Q000235:genetics*; D009336:Necrosis; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D055503:Protein Multimerization; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000378:metabolism; D050794:STAT1 Transcription Factor / Q000378:metabolism; D012486:Salmonella typhimurium / Q000502:physiology; D012772:Shock, Septic / Q000473:pathology; D015398:Signal Transduction
D016207:Cytokines; D004731:Endotoxins; D051034:Inhibitor of Apoptosis Proteins; D047888:Receptors, Tumor Necrosis Factor, Type I; D050794:STAT1 Transcription Factor; C570694:MLKL protein, mouse; D011494:Protein Kinases; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; C506251:Ripk1 protein, mouse; D048031:JNK Mitogen-Activated Protein Kinases; D008239:Lysine