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.

TLR8 Senses Staphylococcus aureus RNA in Human Primary Monocytes and Macrophages and Induces IFN-β Production via a TAK1–IKKβ–IRF5 Signaling Pathway

“…Mycobacterium tuberculosis induces IFN-b in mouse macrophages through a nucleotide-binding oligomerization domain-containing protein 2 (NOD2)-receptor interacting protein 2 (RIP2)-TBK1-IRF5 pathway (45). Also, a NOD2-IRF5 mechanism of S. aureus-mediated IFN-b induction in mouse BMDCs was recently reported (46), and IRF5 was activated by TBK1/IKKi and RIP2 in overexpression studies (47,48). However, we found no induction of IFN-b in RNA-depleted S. aureus lysates, which most likely contain significant amounts of peptidoglycan and bacterial cell wall fragments.…”

Interferon Regulatory Factor Signalings in Cardiometabolic Diseases

“…Within the NLR family, which consists of 3 subfamilies (NODs [NOD1-5 and CIITA], NLRPs [NLRP1-14], and IPAF), NOD2 activates IRF3 via the adaptor protein IFN-β-promoter stimulator 1 and induces IFN-β expression on single-stranded RNA stimulation, 71 primarily via the downstream kinase receptor-interacting protein 2, and functions as an effective activator of IRF5. 72 In addition to these cytosolic RNA-sensing mechanisms, the cytosolic DNA-sensing system is involved in IRF signaling activation for the recognition of and response to microbial and host DNA stimuli. 73 In response to cytosolic dsDNA, direct DNA sensors, such as IFN-γ-inducible 16 and DEADbox-polypeptide 41, bind to specific DNA motifs, leading to the activation of STING, a candidate DNA sensor that is localized to the endoplasmic reticulum.…”

Corrected and Republished from: The COP9 Signalosome Interacts with and Regulates Interferon Regulatory Factor 5 Protein Stability

“…Recent support has indicated that IRF5 can be ubiquitinated via K63 linkage in response to TLR signaling and overexpression of MyD88, TRAF6, or RIP2 (18,20); however, current data are controversial with regard to the exact functional consequence of this ubiquitination and whether it alters IRF5 cellular localization and/or transactivation function. Our data indicate that in the absence of stimulation or interaction with the CSN complex, IRF5 undergoes K48-linked polyubiquitination that targets the protein for degradation.…”

“…It is well known that many proteins that become phosphorylated in response to a given stimuli are subsequently ubiquitinated, followed by proteasome-dependent degradation (32,44). IRF5 is thought to be activated by phosphorylation in response to different extracellular stimuli, including virus, DNA damage, MyD88-dependent TLR ligands, and the death receptor ligand TRAIL (2,3,12,13,19,20,58), resulting in its biological activity. In order to test the functional significance of the CSN/IRF5 interaction in a cellular system known to activate endogenous IRF5 via phosphorylation of serine (Ser), threonine (Thr), and tyrosine (Tyr) residues (12), we treated THP-1 cells with TRAIL and examined whether IRF5 activation had any effect on its degradation.…”

“…IRF5 resides in the cytoplasm of most unstimulated cells and becomes activated by posttranslational modifications that include phosphorylation, acetylation, and/or ubiquitination, resulting in translocation to the nucleus (2,13,(18)(19)(20)(21)(22). While all IRF family members share significant homology in their amino-terminal DNA binding domain (DBD), the carboxyl terminus of individual IRFs is not well conserved and thus is thought to dictate specific interactions with other proteins and IRF family members that control/mediate their distinct functions (1,21).…”