Abstract
The Rip2 kinase contains a caspase recruitment domain and has been implicated in the activation of the transcriptional factor NF-kappaB downstream of Toll-like receptors, Nod-like receptors, and the T cell receptor. Although Rip2 has been linked to Nod signaling, how Nod-Rip2 proteins mediate NF-kappaB activation has remained unclear. We find Rip2 required for Nod2-mediated NF-kappaB activation and to a lesser extent mitogen-activated protein kinase activation. We demonstrate that Rip2 and IkappaB kinase-gamma become stably polyubiquitinated upon treatment of cells with the NOD2 ligand, muramyl dipeptide. We also demonstrate a requirement for the E2-conjugating enzyme Ubc13, the E3 ubiquitin ligase Traf6, and the ubiquitin-activated kinase Tak1 in Nod2-mediated NF-kappaB activation. Rip2 polyubiquitination is also stimulated when macrophages are infected with live Mycobacterium tuberculosis but not when infected with heat-killed bacteria. Consistent with our data linking Rip2 to NOD and not Toll-like receptor signaling, M. tuberculosis-induced Rip2 polyubiquitination appears MyD88-independent. Collectively, these data reveal that the NOD2 pathway is ubiquitin-regulated and that Rip2 employs a ubiquitin-dependent mechanism to achieve NF-kappaB activation.
MeSH terms
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Acetylmuramyl-Alanyl-Isoglutamine / pharmacology*
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Adjuvants, Immunologic / pharmacology*
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Animals
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Cells, Cultured
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Enzyme Activation / drug effects
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Enzyme Activation / immunology
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Extracellular Signal-Regulated MAP Kinases / genetics
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Extracellular Signal-Regulated MAP Kinases / metabolism
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I-kappa B Kinase / genetics
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I-kappa B Kinase / immunology
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I-kappa B Kinase / metabolism
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MAP Kinase Kinase Kinases / genetics
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MAP Kinase Kinase Kinases / immunology
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MAP Kinase Kinase Kinases / metabolism
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Macrophages / immunology
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Macrophages / metabolism*
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Macrophages / microbiology
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Mice
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Mice, Knockout
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Mycobacterium tuberculosis* / immunology
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Myeloid Differentiation Factor 88 / genetics
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Myeloid Differentiation Factor 88 / immunology
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Myeloid Differentiation Factor 88 / metabolism
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NF-kappa B / genetics
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NF-kappa B / immunology
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NF-kappa B / metabolism
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Nod2 Signaling Adaptor Protein / genetics
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Nod2 Signaling Adaptor Protein / immunology
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Nod2 Signaling Adaptor Protein / metabolism*
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Receptor-Interacting Protein Serine-Threonine Kinase 2
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Receptor-Interacting Protein Serine-Threonine Kinases / genetics
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Receptor-Interacting Protein Serine-Threonine Kinases / immunology
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Receptor-Interacting Protein Serine-Threonine Kinases / metabolism*
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Signal Transduction / drug effects
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Signal Transduction / immunology
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TNF Receptor-Associated Factor 6 / genetics
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TNF Receptor-Associated Factor 6 / immunology
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TNF Receptor-Associated Factor 6 / metabolism
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Toll-Like Receptors / genetics
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Toll-Like Receptors / immunology
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Toll-Like Receptors / metabolism
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Tuberculosis / genetics
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Tuberculosis / immunology
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Tuberculosis / metabolism*
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Ubiquitin / genetics
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Ubiquitin / immunology
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Ubiquitin / metabolism
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Ubiquitin-Conjugating Enzymes / genetics
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Ubiquitin-Conjugating Enzymes / immunology
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Ubiquitin-Conjugating Enzymes / metabolism
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Ubiquitin-Protein Ligases / genetics
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Ubiquitin-Protein Ligases / immunology
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Ubiquitin-Protein Ligases / metabolism
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Ubiquitination / drug effects*
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Ubiquitination / immunology
Substances
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Adjuvants, Immunologic
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Myd88 protein, mouse
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Myeloid Differentiation Factor 88
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NF-kappa B
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Nod2 Signaling Adaptor Protein
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Nod2 protein, mouse
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TNF Receptor-Associated Factor 6
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Toll-Like Receptors
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Ubiquitin
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Acetylmuramyl-Alanyl-Isoglutamine
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Ube2n protein, mouse
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Ubiquitin-Conjugating Enzymes
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Ubiquitin-Protein Ligases
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Receptor-Interacting Protein Serine-Threonine Kinase 2
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Receptor-Interacting Protein Serine-Threonine Kinases
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Ripk2 protein, mouse
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I-kappa B Kinase
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Extracellular Signal-Regulated MAP Kinases
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MAP Kinase Kinase Kinases
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MAP kinase kinase kinase 7