Interleukin 1/Toll-like receptor-induced autophosphorylation activates interleukin 1 receptor-associated kinase 4 and controls cytokine induction in a cell type-specific manner

Abstract

IRAK4 is a central kinase in innate immunity, but the role of its kinase activity is controversial. The mechanism of activation for IRAK4 is currently unknown, and little is known about the role of IRAK4 kinase in cytokine production, particularly in different human cell types. We show IRAK4 autophosphorylation occurs by an intermolecular reaction and that autophosphorylation is required for full catalytic activity of the kinase. Phosphorylation of any two of the residues Thr-342, Thr-345, and Ser-346 is required for full activity, and the death domain regulates the activation of IRAK4. Using antibodies against activated IRAK4, we demonstrate that IRAK4 becomes phosphorylated in human cells following stimulation by IL-1R and Toll-like receptor agonists, which can be blocked pharmacologically by a dual inhibitor of IRAK4 and IRAK1. Interestingly, in dermal fibroblasts, although complete inhibition of IRAK4 kinase activity does not inhibit IL-1-induced IL-6 production, NF-κB, or MAPK activation, there is complete ablation of these processes in IRAK4-deficient cells. In contrast, the inhibition of IRAK kinase activity in primary human monocytes reduces R848-induced IL-6 production with minimal effect on NF-κB or MAPK activation. Taken together, these studies define the mechanism of IRAK4 activation and highlight the differential role of IRAK4 kinase activity in different human cell types as well as the distinct roles IRAK4 scaffolding and kinase functions play.

Keywords: Innate Immunity; Interleukin 1 (IL-1); Interleukin Receptor-associated Kinase (IRAK); Interleukin Receptor-associated Kinase 4 (IRAK4); Protein Kinases; Protein Phosphorylation; Toll-like Receptors (TLR).

FIGURE 1.

A, results of ESI-MS/MS analysis of phosphorylated IRAK4 kinase domain. Positively identified peptides are highlighted in red with underscored arrows delineating subfragments of these peptides. Designation of the peptides (supplemental Fig. S2) is denoted in red below the corresponding arrow. Positively identified phosphorylated residues are highlighted in blue with the residue number designated above. The activation loop is denoted by a green bracket. B, IRAK4 structure derived from modified Protein Data Bank structure file 2NRU. Residues Thr-342, Thr-345, Ser-346, and Thr-352 are modified with phosphates, and the C-helix and ATP binding pocket are indicated for clarity.

FIGURE 2.

A, kinetic analysis of phosphorylated and unphosphorylated forms of the kinase domain and full-length IRAK4. The kinetics were determined as described under “Experimental Procedures.” Initial rates from the progress curve show that the unphosphorylated forms of IRAK4 show an initial lag compared with the phosphorylated forms. B, enzymatic activity of activation loop mutations in the kinase domain of IRAK4. The mutant FLAG-tagged kinase domain construct of IRAK4 was purified from 293T cells and assayed by DELFIA in vitro kinase assay as described under “Experimental Procedures.” Inset, Coomassie Blue gel of purified mutant IRAK4 proteins. The 1st lane is the molecular weight marker, with the 49-kDa marker indicated, followed sequentially by IRAK4 mutants in the same order as depicted in the bar graph.

FIGURE 3.

Full-length wild-type IRAK4 phosphorylates D329A kinase domain in vitro. Wild-type and kinase-inactive forms of IRAK4 were purified from HEK 293T cells and co-incubated in the presence of 2 mm ATP, 5 mm MgCl₂ for 1 h. Reactions were analyzed by Western blotting with antibodies to pIRAK4 (Thr(P)-345/Ser(P)-346) or anti-IRAK4. Arrows indicate full-length IRAK4 (FL) and D329A kinase domain (KD).

FIGURE 4.

A, phosphorylation of wild-type and kinase-inactive D329A IRAK4 at Thr-345/Ser-346 is inducible by IL-1β. Wild-type human dermal fibroblasts were transfected with FLAG-tagged constructs of IRAK4 and immunoprecipitated (IP) with anti-FLAG antibody and Western-blotted with antibodies to IRAK4 or anti-pIRAK4 (Thr(P)-345/Ser-346). B, phosphorylation of kinase-dead IRAK4 is ablated in IRAK4^−/− human dermal fibroblasts. Wild-type and IRAK4-deficient human dermal fibroblasts were transfected with full-length FLAG-tagged D329A stimulated for 30 min with 10 ng/ml IL-1β. Samples were FLAG-immunoprecipitated and Western-blotted with antibodies to pIRAK4 or total IRAK4. Whole cell lysate (WCL) blotted with anti-IRAK4 antibody is shown for comparison of endogenous IRAK4 levels.

FIGURE 5.

Wild-type kinase domain of IRAK4 is constitutively autophosphorylated, but D329A-containing kinase domain fails to be phosphorylated in human dermal fibroblasts. A, wild-type human dermal fibroblasts were transfected with wild-type and D329A mutations of FLAG-tagged kinase domain constructs. Cells were stimulated for 30 min with 10 ng/ml IL-1β, and the lysates were blotted with pIRAK4 (Thr(P)-345/Ser(P)-346) and a polyclonal antibody to the C terminus of IRAK4. Arrows indicate position of full-length endogenous IRAK4 (FL), a nonspecific band (NS), endogenous cleaved IRAK4, and transfected kinase domain (KD). B, lysates were immunoprecipitated with anti-FLAG antibody and Western-blotted with anti-pIRAK4 and anti-FLAG antibodies. Molecular weight markers are indicated on the left margin of each blot.

FIGURE 6.

A, pharmacological inhibition of IRAK4 kinase blocks IRAK4 phosphorylation in human dermal fibroblasts. Human wild-type and IRAK4-deficient (P15) dermal fibroblasts were treated with 10 μm compound 1 for 30 min followed by stimulation with 10 ng/ml IL-1β for 30 min. Cell lysates were collected and analyzed by Western blot for pIRAK4 (Thr(P)-345/Ser(P)-346), IRAK4, and IRAK1. B, pharmacological inhibition of IRAK4 kinase blocks IRAK4 phosphorylation in human monocytes. Human monocytes were treated with 10 μm compound 1 for 30 min followed by stimulation with 1 μg/ml R848 for 15 min. Cell lysates were collected and analyzed by Western blot for pIRAK4 (Thr(P)-345/Ser(P)-346) and IRAK4.

FIGURE 7.

A, IRAK4 kinase activity is dispensable for cytokine production in human dermal fibroblasts. Human wild-type and IRAK4-deficient dermal fibroblasts were treated with 10 μm compound 1 for 30 min followed by stimulation with 10 ng/ml IL-1β. B, IRAK4 kinase activity is required for cytokine production in human monocytes. Human monocytes were treated with 10 μm compound 1 for 30 min followed by stimulation with 1 μg/ml R848. Cell supernatants were collected and analyzed by ELISA (MSD) for levels of IL-6 and TNF-α. Significance of inhibition was determined by a Student's t test in both experiments. NS, not significant; *, p < 0.03.

FIGURE 8.

A, minimal effects on NF-κB and MAPK are observed with inhibition of IRAK4 kinase in human dermal fibroblasts. Human wild-type and IRAK4-deficient dermal fibroblasts were treated with 10 μm compound 1 for 30 min followed by stimulation with 10 ng/ml IL-1β. B, minimal effects on NF-κB and MAPK are observed with inhibition of IRAK4 kinase in human monocytes. Human monocytes were treated with 10 μm compound 1 for 30 min followed by stimulation with 1 μg/ml R848 for 15 min. Cell lysates were collected and analyzed by Western blot for phospho-p65, phospho-ERK, phospho-JNK, and phospho-p38 (p-p65, p-ERK, p-JNK, and p-p38, respectively).