P021 Activation of interferon regulatory factor 5 in innate immune signaling by site specific phosphorylation

Abstract

Introduction A protective cellular response to infection depends on accurate activation of transcription factors and induction of select innate immunity genes. Interferon regulatory factor 5 (IRF5), a risk factor for systemic lupus erythematosus, is activated by pathogen recognition receptors and downstream signaling molecules TANK-binding kinase-1 (TBK-1), tumor necrosis factor receptor-associated factor 6 (TRAF6), or receptor interacting protein 2 (RIP2). Phosphorylation is key to the regulation of IRF5, but the precise phosphorylation sites in IRF5 remained to be identified. Methods We used mass spectrometry to identify for the first time authentic phosphorylated residues of IRF5 in response to TBK-1, TRAF6, and RIP2 that stimulate its ability to induce gene expression, accumulate in the nucleus, and promote apoptosis. To determine if the phosphorylated residues are required or sufficient for IRF5 activity, aspartic acid phosphomimetic substitutions or inactivating alanine substitutions were tested. Results RIP2, a kinase downstream of nucleotide-binding oligomerization domain containing 2 (NOD2), was the most effective activator of IRF5. Phosphorylation of carboxyl serines 451 and 462 appear the primary trigger of IRF5 function in nuclear accumulation, transcription, and apoptosis. Results indicate polyubiquitination of IRF5 does not play a major role in its transcriptional activity, and that ubiquitination and phosphorylation are independent modifications. Conclusion Ligands that stimulate the NOD2 receptor are significant activators of IRF5. RIP2 kinase that is downstream of NOD2 can phosphorylate IRF5 on carboxyl serine residues to stimulate its ability to induce transcription of cytokines such as interferons and interleukin-12 p40.