Regulation of IRAK-4 kinase activity via autophosphorylation within its activation loop.
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.
Cheng
Hong
H
Department of Inflammation, Millennium Pharmaceuticals, Inc., Cambridge, MA 02139, USA. hong.cheng@novartis.com
Addona
Terri
T
Keshishian
Hasmik
H
Dahlstrand
Erik
E
Lu
Chafen
C
Dorsch
Marion
M
Li
Zhi
Z
Wang
Anlai
A
Ocain
Timothy D
TD
Li
Ping
P
Parsons
Thomas F
TF
Jaffee
Bruce
B
Xu
Yajun
Y
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
D053592:Interleukin-1 Receptor-Associated Kinases