Protein kinase IKKβ-catalyzed phosphorylation of IRF5 at Ser462 induces its dimerization and nuclear translocation in myeloid cells

Abstract

The siRNA knockdown of IFN Regulatory Factor 5 (IRF5) in the human plasmacytoid dendritic cell line Gen2.2 prevented IFNβ production induced by compound CL097, a ligand for Toll-like receptor 7 (TLR7). CL097 also stimulated the phosphorylation of IRF5 at Ser462 and stimulated the nuclear translocation of wild-type IRF5, but not the IRF5[Ser462Ala] mutant. The CL097-stimulated phosphorylation of IRF5 at Ser462 and its nuclear translocation was prevented by the pharmacological inhibition of protein kinase IKKβ or the siRNA knockdown of IKKβ or its "upstream" activator, the protein kinase TAK1. Similar results were obtained in a murine macrophage cell line stimulated with the TLR7 agonist compound R848 or the nucleotide oligomerization domain 1 (NOD1) agonist KF-1B. IKKβ phosphorylated IRF5 at Ser462 in vitro and induced the dimerization of wild-type IRF5 but not the IRF5[S462A] mutant. These findings demonstrate that IKKβ activates two "master" transcription factors of the innate immune system, IRF5 and NF-κB.

Keywords: IKKβ; IRF5; TLR7; interferon β; plasmacytoid dendritic cell.

Fig. 1.

IRF5 is required for CL097-stimulated IFNβ production but not for IFNα1 or IL-6 production in Gen2.2 cells. (A–D) Gen2.2 cells were transfected with siRNA against IRF3 (hatched bars), IRF5 (white bars), IRF7 (gray bars), or a control siRNA (black bars). After 72 h, the cells were stimulated for the times indicated with 1.0 μg/mL CL097. The mRNA encoding IFN-β (A), IFN-α1 (C), and IL-6 (D) was then measured in duplicate by qRT-PCR. Graphs show the fold increase in mRNA production relative to that measured in cells treated with control siRNA and not stimulated with CL097 and are presented as the mean + SD for one representative experiment. The mRNA levels were normalized to 18S rRNA. (B) As in A, except that IFNβ secreted into the culture medium was measured by ELISA. All experiments (A–D) were repeated three times with similar results.

Fig. 2.

IRF5 is phosphorylated by IKKβ at Ser462 in Gen2.2 cells and in vitro. (A) SILAC-labeled Gen2.2 cells were incubated without (−) or with (+) BI605906 or NG-25 stimulated for 30 min without (−) or with (+) 1.0 μg/mL CL097 and the fold increase in the abundance of the tryptic phosphopeptide LQISpNPDLK (where Sp is phosphoserine) corresponding to amino acid residues 459–467 of IRF5 was quantified as described in Materials and Methods, relative to the level in unstimulated cells (mean + SEM). The number of independent experiments performed is indicated in parentheses. (B and C) Flag–IRF5 was phosphoryated with His₆–IKKβ, GST–IKKα, or GST–TBK1 in the presence or absence of BI605906 or MRT67307 using [γ³²P]ATP (B) or unlabeled ATP (C) and the incorporation of ³²P-radioactivity into proteins or phosphorylation of Ser462 of IRF5 examined by autoradioactivity (B, Upper) or immunoblotting (C). The gel in B was also stained with Coomassie blue. (D, Lower) HEK293T cells were transfected with DNA encoding wild-type (WT) HA–IKKβ or the catalytically inactive HA–IKKβ[D166A] mutant and either Flag–IRF5[WT] or the Flag–IRF5[S462A] mutant. The cell extracts were subjected to SDS/PAGE (Upper) or native gel electrophoresis (Lower two panels) and immunoblotted with the antibodies indicated. Similar results were obtained in three independent experiments.

Fig. 3.

The mutation of Ser462 to Ala prevents the nuclear translocation of IRF5 induced by TLR7 agonists. (A) Gen2.2 cells were transfected with IRF5–GFP or IRF5[S462A]–GFP. After 48 h, the cells were stimulated for 30 min with 1.0 μg/mL CL097 or left unstimulated. The cells were fixed, centrifuged onto precoated slides, permeabilized, and stained with anti-GFP or DAPI to reveal nuclei. (B) Same as A, except that RAW264.7 cells were stimulated with R848 (1.0 μg/mL).

Fig. 4.

The nuclear translocation of IRF5–GFP is prevented by inhibitors of IKKβ (BI605906 and PS1145) or TAK1 (NG-25). (A) As in Fig. 3 except that Gen2.2 cells transfected with IRF5–GFP were incubated for 1 h without or with BI605906 (5.0 μM), PS1145 (15 μM), NG-25 (2.0 μM), or MRT67307 (2.0 μM), then stimulated without or with CL097 (1.0 μg/mL). (B) Same as A, except that RAW264.7 cells were stimulated with R848.

Fig. 5.

The nuclear translocation of the endogenous IRF5 is prevented by the inhibition or knockdown of IKKβ or TAK1 in Gen2.2 cells. (A) Gen2.2 cells were incubated for 1 h without (−) or with (+) NG-25 (2.0 μM), BI605906 (5.0 μM), PS1145 (15 μM), or MRT67307 (2.0 μM), then stimulated for 30 min with CL097 (1.0 μg/mL). Nuclei were separated from the cytosol, lysed, and the localization of IRF5 and p65/RelA analyzed by immunoblotting. GADPH and SMC-1 were used as loading controls for the cytosol and nuclear extracts, respectively. (B–D) Gen2.2 cells were transfected with control siRNA or siRNA for IKKβ (B), IKKα (C), or TAK1 (D). After 72 h, the cells were treated as in A. The cell extracts prepared before fractionation into nuclei and cytosol were immunoblotted with antibodies that recognize p38α MAP kinase (as a loading control), a phospho-specific antibody that recognizes the IKK substrate p65, and with either IKKβ (B), IKKα (C), or TAK1 (D). All of the experiments in A–D were repeated at least three times with similar results.

Fig. 6.

IKKβ activates IRF5 by phosphorylating Ser462. (A) Ser462 of IRF5 is conserved during vertebrate evolution. Identical amino acid residues are shown in white letters on a black background and conservative replacements in black letters on a gray background. (B) Ser462 of IRF5 lies in an equivalent position to Ser396 of IRF3. (C) IKKβ phosphorylates the transcription factors IRF5 and NF-κB inducing their nuclear translocation where they stimulate gene transcription. IKKβ phosphorylates the IκBα subunit of NF-κB, which leads to its ubiquitylation and degradation by the proteasome, and the p65/RelA component at two or more sites (20). The p65/RelA enters the nucleus as a complex with p50.