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A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3
11:44

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Published on: January 24, 2016

Interferon regulatory factor 3 is regulated by a dual phosphorylation-dependent switch.

Daniel Panne1, Sarah M McWhirter, Tom Maniatis

  • 1Department of Biological Chemistry and Molecular Pharmacology, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.

The Journal of Biological Chemistry
|May 29, 2007
PubMed
Summary

Interferon regulatory factor 3 (IRF-3) activation involves a two-step phosphorylation process by TBK1. This process is crucial for innate immune response gene regulation.

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Published on: March 24, 2015

Area of Science:

  • Immunology
  • Molecular Biology
  • Biochemistry

Background:

  • Interferon regulatory factor 3 (IRF-3) is a key transcription factor in the innate immune response.
  • IRF-3 activation is mediated by phosphorylation by IKK epsilon and/or TBK1 kinases.
  • The exact role of distinct phosphorylation sites on IRF-3 has been debated.

Purpose of the Study:

  • To elucidate the precise mechanism of IRF-3 activation by TBK1.
  • To investigate the role of different phosphorylation sites in IRF-3 activation.
  • To clarify the sequential events leading to IRF-3 dimerization and coactivator interaction.

Main Methods:

  • In vitro phosphorylation assays using purified proteins.
  • Analysis of IRF-3 phosphorylation at specific residues (site 1 and site 2).
  • Assessment of IRF-3 dimerization and interaction with CBP/p300.

Main Results:

  • TBK1 directly phosphorylates full-length IRF-3 in vitro.
  • Phosphorylation at site 2 (Ser396-Ser405) relieves autoinhibition, enabling CBP interaction.
  • Site 2 phosphorylation facilitates subsequent phosphorylation at site 1 (Ser385/Ser386), which is essential for IRF-3 dimerization.

Conclusions:

  • A two-step phosphorylation model for IRF-3 activation by TBK1 is proposed.
  • Site 2 phosphorylation precedes and enables site 1 phosphorylation and subsequent dimerization.
  • This mechanism clarifies the sequential activation events of IRF-3 in the innate immune response.