The IRAK-catalysed activation of the E3 ligase function of Pellino isoforms induces the Lys63-linked polyubiquitination of IRAK1.
The protein kinases IRAK [IL-1 (interleukin 1) receptor-associated kinase] 1 and 4 play key roles in a signalling pathway by which bacterial infection or IL-1 trigger the production of inflammatory mediators. In the present study, we demonstrate that IRAK1 and IRAK4 phosphorylate Pellino isoforms in vitro and that phosphorylation greatly enhances Pellino's E3 ubiquitin ligase activity. We show that, in vitro, Pellino 1 can combine with the E2 conjugating complex Ubc13 (ubiquitin-conjugating enzyme 13)-Uev1a (ubiquitin E2 variant 1a) to catalyse the formation of K63-pUb (Lys63-linked polyubiquitin) chains, with UbcH3 to catalyse the formation of K48-pUb chains and with UbcH4, UbcH5a or UbcH5b to catalyse the formation of pUb-chains linked mainly via Lys11 and Lys48 of ubiquitin. In IRAK1-/- cells, the co-transfection of DNA encoding wild-type IRAK1 and Pellino 2, but not inactive mutants of these proteins, induces the formation of K63-pUb-IRAK1 and its interaction with the NEMO [NF-kappaB (nuclear factor kappaB) essential modifier] regulatory subunit of the IKK (inhibitor of NF-kappaB kinase) complex, a K63-pUb-binding protein. These studies suggest that Pellino isoforms may be the E3 ubiquitin ligases that mediate the IL-1-stimulated formation of K63-pUb-IRAK1 in cells, which may contribute to the activation of IKKbeta and the transcription factor NF-kappaB, as well as other signalling pathways dependent on IRAK1/4.
Ordureau
Alban
A
MRC Protein Phosphorylation Unit, The Sir James Black Centre, College of Life Sciences, University of Dundee, Dow Street, Dundee DD15 5EH, Scotland, UK.
Smith
Hilary
H
Windheim
Mark
M
Peggie
Mark
M
Carrick
Emma
E
Morrice
Nick
N
Cohen
Philip
P
D002384:Catalysis; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D007249:Inflammation; D007375:Interleukin-1 / Q000378:metabolism; D053592:Interleukin-1 Receptor-Associated Kinases / Q000737:chemistry* / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit / Q000378:metabolism; D010766:Phosphorylation; D020033:Protein Isoforms; D014162:Transfection; D054875:Ubiquitination
C491731:IKBKG protein, human; D007375:Interleukin-1; D052002:NF-kappa B p50 Subunit; C489548:NFKB1 protein, human; D020033:Protein Isoforms; C507768:IRAK1 protein, human; C507773:IRAK4 protein, human; D053592:Interleukin-1 Receptor-Associated Kinases; D051550:I-kappa B Kinase