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Native Polyacrylamide Gel Electrophoresis Immunoblot Analysis of Endogenous IRF5 Dimerization
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Native Polyacrylamide Gel Electrophoresis Immunoblot Analysis of Endogenous IRF5 Dimerization

Published on: October 6, 2019

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Protein kinase IKKβ-catalyzed phosphorylation of IRF5 at Ser462 induces its dimerization and nuclear translocation in

Marta Lopez-Pelaez1, Douglas J Lamont2, Mark Peggie1

  • 1Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, and.

Proceedings of the National Academy of Sciences of the United States of America
|October 19, 2014
PubMed

Insights

Interferon regulatory factor 5 (IRF5) activation is crucial for innate immunity. Protein kinase IKKβ phosphorylates IRF5, enabling its nuclear translocation and immune response activation.

Area of Science:

  • Immunology
  • Molecular Biology
  • Cell Biology

Background:

  • Toll-like receptor (TLR) signaling is critical for innate immune responses.
  • IFN Regulatory Factor 5 (IRF5) is a key transcription factor in innate immunity.
  • The precise mechanisms regulating IRF5 activation remain under investigation.

Purpose of the Study:

  • To elucidate the role of protein kinase IKKβ in the activation of IRF5.
  • To investigate the signaling pathway leading to IRF5 phosphorylation and nuclear translocation.
  • To understand how TLR agonists trigger IRF5-mediated immune responses.

Main Methods:

  • siRNA knockdown of IRF5, IKKβ, and TAK1 in human and murine cell lines.
  • Stimulation with TLR7 agonists (CL097, R848) and NOD1 agonist (KF-1B).
  • Western blotting to detect IRF5 phosphorylation and nuclear translocation; in vitro kinase assays.

Main Results:

  • siRNA knockdown of IRF5 abrogated IFNβ production induced by TLR7 agonists.
  • TLR7 stimulation induced IRF5 phosphorylation at Ser462 and nuclear translocation, dependent on IKKβ and TAK1.
  • IKKβ directly phosphorylated IRF5 at Ser462 in vitro, promoting its dimerization.

Conclusions:

  • IKKβ is a critical upstream activator of IRF5.
  • IKKβ-mediated phosphorylation of IRF5 at Ser462 is essential for its function in innate immunity.
  • IKKβ activates both IRF5 and NF-κB, key transcription factors in the innate immune system.
Keywords:
IKKβIRF5TLR7interferon βplasmacytoid dendritic cell

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