IRAK-1-mediated negative regulation of Toll-like receptor signaling through proteasome-dependent downregulation of TRAF6

Biochim Biophys Acta. 2012 Feb;1823(2):255-63. doi: 10.1016/j.bbamcr.2011.10.003. Epub 2011 Oct 18.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Down-Regulation
  • HEK293 Cells
  • Humans
  • Interleukin-1 Receptor-Associated Kinases / genetics
  • Interleukin-1 Receptor-Associated Kinases / metabolism*
  • NF-kappa B / genetics
  • NF-kappa B / metabolism
  • Oligodeoxyribonucleotides / metabolism
  • Proteasome Endopeptidase Complex / metabolism*
  • Proteasome Inhibitors
  • Signal Transduction / physiology*
  • TNF Receptor-Associated Factor 6 / genetics
  • TNF Receptor-Associated Factor 6 / metabolism*
  • Toll-Like Receptors / genetics
  • Toll-Like Receptors / metabolism*
  • Ubiquitin / metabolism

Substances

  • NF-kappa B
  • Oligodeoxyribonucleotides
  • Proteasome Inhibitors
  • TNF Receptor-Associated Factor 6
  • Toll-Like Receptors
  • Ubiquitin
  • IRAK1 protein, human
  • Interleukin-1 Receptor-Associated Kinases
  • Proteasome Endopeptidase Complex