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In vivo and in vitro Studies of Adaptor-clathrin Interaction
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In vivo and in vitro Studies of Adaptor-clathrin Interaction

Published on: January 26, 2011

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Clathrin complexes with the inhibitor kappa B kinase signalosome: imaging the interactome

Fabia Gamboni1, Guillermo A Escobar2, Ernest E Moore3

  • 1Department of Surgery, University of Colorado Denver, Aurora, Colorado.

Physiological Reports
|July 5, 2014
PubMed

Insights

Clathrin heavy chains (CHC) form cytoplasmic complexes with inhibitor kappa B kinase (IKK) signalosome components, likely acting as precursors to IKK-NEMO complexes in innate immunity signaling pathways.

Area of Science:

  • Cell Biology
  • Immunology
  • Molecular Biology

Background:

  • Innate immunity receptors activate the inhibitor kappa B kinase (IKK) signalosome, a complex of IKKα, IKKβ, and NEMO.
  • Previous studies showed clathrin heavy chains (CHC) influence TNFα-induced signaling and IκB phosphorylation.

Purpose of the Study:

  • To investigate the hypothesis that CHC forms cytoplasmic complexes with IKK signalosome members.
  • To elucidate the role of CHC in the assembly and function of the IKK signalosome.

Main Methods:

  • Immunoprecipitation of IKKα and NEMO interactomes followed by mass spectrometry.
  • Indirect digital immunofluorescence microscopy and Förster Resonance Energy Transfer (FRET) to visualize protein complexes.
  • Analysis of natural protein variability in A549 cells to correlate complex formation.

Main Results:

  • Mass spectrometry identified clathrin with high confidence in IKKα and NEMO interactomes.
  • CHC-IKK complexes were visualized with NEMO and HSP90 using microscopy and FRET.
  • Statistical analysis suggested CHC-IKK complexes are precursors to IKK-NEMO complexes and CHC-IKKβ complexes may derive from IKKβ-HSP90 complexes.

Conclusions:

  • Clathrin heavy chains interact with components of the IKK signalosome.
  • CHC-IKK complexes likely serve as precursors in the formation of IKK-NEMO complexes, impacting innate immunity signaling.
  • CHC-IKKβ complexes may originate from interactions with HSP90.
Keywords:
Affinity proteomicsHsp90NEMOfluorescent resonance energy transfer

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