Sequential autophosphorylation steps in the interleukin-1 receptor-associated kinase-1 regulate its availability as an adapter in interleukin-1 signaling.
The interleukin-1 receptor-associated kinase 1 (IRAK-1) is an important adapter in the signaling complex of the Toll/interleukin-1 (IL-1) receptor family. Formation of the signaling IL-1 receptor complex results in the activation and hyperphosphorylation of IRAK-1, which leads to a pronounced shift of its apparent molecular mass in gel electrophoresis. Presently, the individual residues phosphorylated in IRAK-1 and the consequences for IRAK-1 function are unknown. We define sequential phosphorylation steps in IRAK-1, which are, in vitro, autophosphorylation. First, IRAK-1 is phosphorylated at Thr209. By fluorescence energy transfer experiments, we demonstrate that Thr209 phosphorylation results in a conformational change of the kinase domain, permitting further phosphorylations to take place. Substitution of Thr209 by alanine results in a kinase-inactive IRAK-1. Second, Thr387 in the activation loop is phosphorylated, leading to full enzymatic activity. Third, IRAK-1 autophosphorylates several times in the proline-, serine-, and threonine-rich ProST region between the N-terminal death domain and kinase domain. Hyperphosphorylation of this region leads to dissociation of IRAK-1 from the upstream adapters MyD88 and Tollip but leaves its interaction with the downstream adapter TRAF6 unaffected. This identifies IRAK-1 as a novel type of adapter protein, which employs its own kinase activity to introduce negative charges adjacent to the protein interaction domain, which anchors IRAK-1 at the active receptor complex. Thus, IRAK-1 regulates its own availability as an adapter molecule by sequential autophosphorylation.
Kollewe
Christian
C
Department of Pharmacology, Hannover Medical School, D-30623 Hannover, Germany. Christian.Kollewe@bio.uni-giessen.de
Mackensen
Anne-Christin
AC
Neumann
Detlef
D
Knop
Johannes
J
Cao
Ping
P
Li
Shyun
S
Wesche
Holger
H
Martin
Michael U
MU
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000943:Antigens, Differentiation / Q000737:chemistry; D002352:Carrier Proteins / Q000737:chemistry; D002460:Cell Line; D003001:Cloning, Molecular; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D005822:Genetic Vectors; D006801:Humans; D015151:Immunoblotting; D007375:Interleukin-1 / Q000378:metabolism*; D053592:Interleukin-1 Receptor-Associated Kinases; D047908:Intracellular Signaling Peptides and Proteins; D013058:Mass Spectrometry; D008954:Models, Biological; D008969:Molecular Sequence Data; D053594:Myeloid Differentiation Factor 88; D010766:Phosphorylation; D011233:Precipitin Tests; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011971:Receptors, Immunologic / Q000737:chemistry; D017472:Receptors, Interleukin-1 / Q000378:metabolism; D015398:Signal Transduction; D021241:Spectrometry, Mass, Electrospray Ionization; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; D000943:Antigens, Differentiation; D002352:Carrier Proteins; D007375:Interleukin-1; D047908:Intracellular Signaling Peptides and Proteins; C507779:MYD88 protein, human; D053594:Myeloid Differentiation Factor 88; D011971:Receptors, Immunologic; D017472:Receptors, Interleukin-1; C410602:TOLLIP protein, human; D013912:Threonine; D011494:Protein Kinases; D053592:Interleukin-1 Receptor-Associated Kinases