|Regulation of IRAK-4 kinase activity via autophosphorylation within its activation loop. |
Hong Cheng, Terri Addona, Hasmik Keshishian, Erik Dahlstrand, Chafen Lu, Marion Dorsch, Zhi Li, Anlai Wang, Timothy D Ocain, Ping Li, Thomas F Parsons, Bruce Jaffee, Yajun Xu
Biochemical and biophysical research communications
Interleukin-1 stimulation leads to the recruitment of MyD88, interleukin-1 receptor-associated kinase 1 (IRAK-1) and interleukin-1 receptor-associated kinase 4 (IRAK-4) to the IL-1 receptor. The formation of the IL-1 receptor complex triggers a series of IRAK-1 autophosphorylations, which result in activation. IRAK-4 is upstream of IRAK-1 and may act as IRAK-1 kinase to transmit the signal. To date, there is no upstream kinase reported for IRAK-4; the activation mechanism of IRAK-4 remains poorly understood. Here, for the first time, we report three autophosphorylation sites that are responsible for IRAK-4 kinase activity. LC-MS/MS analysis has identified phosphorylations at T342, T345, and S346, which reside within the activation loop. Site-directed mutants at these positions exhibit significant reductions in the catalytic activity of IRAK-4 (T342A: 57%; T345A: 66%; S346A: 50%). The absence of phosphorylation in kinase-dead IRAK-4 indicates that phosphorylations in the activation loop result from autophosphorylation rather than from phosphorylation by an upstream kinase. Finally, we demonstrate that autophosphorylation is an intramolecular event as wild-type IRAK-4 failed to transphosphorylate kinase-inactive IRAK-4. The present data indicate that the kinase activity of IRAK-4 is dependent on the autophosphorylations at T342, T345, and S346 in the activation loop.
|Severe impairment of interleukin-1 and Toll-like receptor signalling in mice lacking IRAK-4. |
Suzuki, Nobutaka, et al.
Nature, 416: 750-6 (2002)
Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns, and members of the pro-inflammatory interleukin-1 receptor (IL-1R) family, share homologies in their cytoplasmic domains called Toll/IL-1R/plant R gene homology (TIR) domains. Intracellular signalling mechanisms mediated by TIRs are similar, with MyD88 (refs 5-8) and TRAF6 (refs 9, 10) having critical roles. Signal transduction between MyD88 and TRAF6 is known to involve the serine-threonine kinase IL-1 receptor-associated kinase 1 (IRAK-1) and two homologous proteins, IRAK-2 (ref. 12) and IRAK-M. However, the physiological functions of the IRAK molecules remain unclear, and gene-targeting studies have shown that IRAK-1 is only partially required for IL-1R and TLR signalling. Here we show by gene-targeting that IRAK-4, an IRAK molecule closely related to the Drosophila Pelle protein, is indispensable for the responses of animals and cultured cells to IL-1 and ligands that stimulate various TLRs. IRAK-4-deficient animals are completely resistant to a lethal dose of lipopolysaccharide (LPS). In addition, animals lacking IRAK-4 are severely impaired in their responses to viral and bacterial challenges. Our results indicate that IRAK-4 has an essential role in innate immunity.