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Protein kinase D interaction with TLR5 is required for inflammatory signaling in response to bacterial flagellin

Sabine M Ivison1, Nicholas R Graham, Cecily Q Bernales

  • 1Division of Infectious Diseases, University of British Columbia, Vancouver, British Columbia, Canada.

Journal of Immunology (Baltimore, Md. : 1950)
|April 20, 2007
PubMed

Insights

Protein kinase D (PKD) phosphorylates Toll-like receptor 5 (TLR5) at serine 805, a key step in the epithelial cell response to enteroaggregative Escherichia coli flagellin, activating p38 MAPK and inflammatory cytokine production.

Area of Science:

  • Immunology
  • Cellular Biology
  • Biochemistry

Background:

  • Protein kinase D (PKD) is a serine-threonine kinase involved in various cellular processes.
  • Toll-like receptor 5 (TLR5) recognizes bacterial flagellin, initiating immune responses.
  • A potential PKD phosphorylation site was identified in the TLR5 domain.

Purpose of the Study:

  • To investigate the role of PKD in TLR5 signaling upon interaction with enteroaggregative Escherichia coli flagellin.
  • To elucidate the mechanism of flagellin-induced TLR5 activation in human epithelial cells.

Main Methods:

  • In vitro kinase assays using a TLR5-derived peptide.
  • Mass spectrometry to identify phosphorylation sites on TLR5.
  • Site-directed mutagenesis of TLR5 (Serine 805 to Alanine).
  • Co-immunoprecipitation to assess TLR5-PKD interaction.
  • Pharmacological inhibition of PKC and PKD.
  • Small hairpin RNA (shRNA) mediated gene silencing of PKD.
  • Measurement of IL-8 expression and secretion.
  • Analysis of p38 MAPK activation.

Main Results:

  • PKD directly phosphorylated TLR5 at Serine 805 in vitro and in vivo.
  • Mutation of Serine 805 abolished flagellin-induced cellular responses.
  • Flagellin treatment enhanced the interaction between TLR5 and PKD.
  • PKD inhibition reduced IL-8 production and p38 MAPK activation.
  • PKD, not other PKC isoforms, mediated the flagellin-induced p38 MAPK and IL-8 response.

Conclusions:

  • PKD-mediated phosphorylation of TLR5 at Serine 805 is a critical early event in the response to bacterial flagellin.
  • This phosphorylation event contributes to p38 MAPK activation and subsequent inflammatory cytokine production in epithelial cells.
  • PKD is a key regulator of TLR5 signaling in response to enteroaggregative E. coli flagellin.

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