title: Regulation of IRAK-4 kinase activity via autophosphorylation within its activation loop.
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.
authors.affiliation: Department of Inflammation, Millennium Pharmaceuticals, Inc., Cambridge, MA 02139, USA. hong.cheng@novartis.com
authors.forename: Hong
authors.initials: H
authors.lastname: Cheng
authors.forename: Terri
authors.initials: T
authors.lastname: Addona
authors.forename: Hasmik
authors.initials: H
authors.lastname: Keshishian
authors.forename: Erik
authors.initials: E
authors.lastname: Dahlstrand
authors.forename: Chafen
authors.initials: C
authors.lastname: Lu
authors.forename: Marion
authors.initials: M
authors.lastname: Dorsch
authors.forename: Zhi
authors.initials: Z
authors.lastname: Li
authors.forename: Anlai
authors.initials: A
authors.lastname: Wang
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Ocain
authors.forename: Ping
authors.initials: P
authors.lastname: Li
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Parsons
authors.forename: Bruce
authors.initials: B
authors.lastname: Jaffee
authors.forename: Yajun
authors.initials: Y
authors.lastname: Xu
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002460:Cell Line; D004789:Enzyme Activation; D005246:Feedback / Q000502:physiology; D006801:Humans; D053592:Interleukin-1 Receptor-Associated Kinases / Q000737:chemistry* / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology*; D008969:Molecular Sequence Data; D011485:Protein Binding; D015398:Signal Transduction / Q000502:physiology*; D013329:Structure-Activity Relationship
chemical_list: D053592:Interleukin-1 Receptor-Associated Kinases