title: Activation of interferon regulatory factor 5 by site specific phosphorylation.
abstract: The cellular defense to infection depends on accurate activation of transcription factors and expression of select innate immunity genes. Interferon regulatory factor 5 (IRF5), a risk factor for systemic lupus erythematosus, is activated in response to pathogen recognition receptor engagement and downstream effector molecules. We find the nucleotide-binding oligomerization domain containing protein 2 (NOD2) receptor to be a significant activator of IRF5. Phosphorylation is key to the regulation of IRF5, but the precise phosphorylation sites in IRF5 remained to be identified. We used mass spectrometry to identify for the first time specific residues that are phosphorylated in response to TANK-binding kinase-1 (TBK-1), tumor necrosis factor receptor-associated factor 6 (TRAF6), or receptor interacting protein 2 (RIP2). RIP2, a kinase known to function downstream of NOD2, was the most effective activator of IRF5-regulated gene expression. To determine if the phosphorylated residues are required or sufficient for IRF5 activity, aspartic acid phosphomimetic substitutions or inactivating alanine substitutions were tested. 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.
authors.affiliation: Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York, United States of America.
authors.forename: Hui-Chen
authors.initials: HC
authors.lastname: Chang Foreman
authors.forename: Sarah
authors.initials: S
authors.lastname: Van Scoy
authors.forename: Tsu-Fan
authors.initials: TF
authors.lastname: Cheng
authors.forename: Nancy C
authors.initials: NC
authors.lastname: Reich
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics; D002460:Cell Line; D002467:Cell Nucleus; D006801:Humans; D050835:Interferon Regulatory Factors / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021381:Protein Transport; D053475:Receptor-Interacting Protein Serine-Threonine Kinase 2 / Q000378:metabolism; D048029:TNF Receptor-Associated Factor 6 / Q000378:metabolism; D015533:Transcriptional Activation; D054875:Ubiquitination
chemical_list: D050835:Interferon Regulatory Factors; D048029:TNF Receptor-Associated Factor 6; D017346:Protein Serine-Threonine Kinases; D053475:Receptor-Interacting Protein Serine-Threonine Kinase 2