IRAK-1-mediated negative regulation of Toll-like receptor signaling through proteasome-dependent downregulation of TRAF6.
TRAF6 plays a crucial role in signal transduction of the Toll-like receptor (TLR). It has been reported that TRAF6 catalyzes the formation of unique Lys63-linked polyubiquitin chains, which do not lead to proteasome-mediated degradation. Here we found that stimulation of J774.1 cells with various TLR ligands led to decreases in TRAF6 protein levels that occurred at a slower rate than IκBα degradation. The decrease in TRAF6 was inhibited by proteasome inhibitors MG-132, lactacystin and N-acetyl-leucyl-leucyl-norleucinal. Among intracellular TLR signaling molecules MyD88, IRAK-4, IRAK-1, TRAF6, and IKKβ, only IRAK-1 expression downregulated TRAF6 in HEK293 cells. The amount of TRAF6 expressed either transiently or stably was also reduced by co-expression of IRAK-1 and no TRAF6 cleavage products were detected. The levels of either a TRAF6 N-terminal deletion mutant or a ubiquitin ligase-defective mutant were not affected by IRAK-1 expression. Downregulation of TRAF6 required the TRAF6-binding site (Glu544, Glu587, Glu706) of IRAK-1 but not its catalytic site (Asp340). Upon IRAK-1 transfection, no significant TRAF6 ubiquitination was detected. Instead, TRAF6-associated IRAK-1 was ubiquitinated with both Lys48- and Lys63-linked polyubiquitin chains. TRAF6 downregulation was inhibited by co-expression of the E3 ubiquitin ligase Pellino 3, whose Lys63-linked polyubiquitination on IRAK-1 is reported to compete with Lys48-linked IRAK-1 polyubiquitination. Expression of IRAK-1 inhibited IκBα phosphorylation in response to TLR2 stimulation. These results indicate that stimulation of TLRs induces proteasome-dependent downregulation of TRAF6. We conclude that TRAF6 associated with ubiquitinated IRAK-1 is degraded together by the proteasome and that IRAK-1 possesses a negative regulatory role on TLR signaling.
Research Institute of Pharmaceutical Sciences, Musashino University, Tokyo, Japan.
Masashi
M
Muroi
Ken-ichi
K
Tanamoto
D015536:Down-Regulation; D057809:HEK293 Cells; D006801:Humans; D053592:Interleukin-1 Receptor-Associated Kinases / Q000235:genetics / Q000378:metabolism*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors; D015398:Signal Transduction / Q000502:physiology*; D048029:TNF Receptor-Associated Factor 6 / Q000235:genetics / Q000378:metabolism*; D051193:Toll-Like Receptors / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism
D016328:NF-kappa B; D009838:Oligodeoxyribonucleotides; D061988:Proteasome Inhibitors; D048029:TNF Receptor-Associated Factor 6; D051193:Toll-Like Receptors; D025801:Ubiquitin; C507768:IRAK1 protein, human; D053592:Interleukin-1 Receptor-Associated Kinases; D046988:Proteasome Endopeptidase Complex