Regulation of IRAK-4 kinase activity via autophosphorylation within its activation loop |
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Authors: | Cheng Hong Addona Terri Keshishian Hasmik Dahlstrand Erik Lu Chafen Dorsch Marion Li Zhi Wang Anlai Ocain Timothy D Li Ping Parsons Thomas F Jaffee Bruce Xu Yajun |
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Institution: | Department of Inflammation, Millennium Pharmaceuticals, Inc., Cambridge, MA 02139, USA. hong.cheng@novartis.com |
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Abstract: | 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. |
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Keywords: | IL-1R interleukin-1 receptor TIR Toll/IL-1 plant R-gene IRAK interleukin-1 receptor-associated kinase NF-κB nuclear factor κB AP-1 activating protein-1 MyD88 a cytoplasmic adaptor protein that contains a TIR domain ip immunoprecipitation wt wild-type kd kinase dead DTT dithiothreitol |
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