Protein kinase D interaction with TLR5 is required for inflammatory signaling in response to bacterial flagellin.
Protein kinase D (PKD), also called protein kinase C (PKC)mu, is a serine-threonine kinase that is involved in diverse areas of cellular function such as lymphocyte signaling, oxidative stress, and protein secretion. After identifying a putative PKD phosphorylation site in the Toll/IL-1R domain of TLR5, we explored the role of this kinase in the interaction between human TLR5 and enteroaggregative Escherichia coli flagellin in human epithelial cell lines. We report several lines of evidence that implicate PKD in TLR5 signaling. First, PKD phosphorylated the TLR5-derived target peptide in vitro, and phosphorylation of the putative target serine 805 in HEK 293T cell-derived TLR5 was identified by mass spectrometry. Furthermore, mutation of serine 805 to alanine abrogated responses of transfected HEK 293T cells to flagellin. Second, TLR5 interacted with PKD in coimmunoprecipitation experiments, and this association was rapidly enhanced by flagellin treatment. Third, pharmacologic inhibition of PKC or PKD with Gö6976 resulted in reduced expression and secretion of IL-8 and prevented the flagellin-induced activation of p38 MAPK, but treatment with the PKC inhibitor Gö6983 had no significant effects on these phenotypes. Finally, involvement of PKD in the p38-mediated IL-8 response to flagellin was confirmed by small hairpin RNA-mediated gene silencing. Together, these results suggest that phosphorylation of TLR5 by PKD may be one of the proximal elements in the cellular response to flagellin, and that this event contributes to p38 MAPK activation and production of inflammatory cytokines in epithelial cells.
Ivison
Sabine M
SM
Division of Infectious Diseases, University of British Columbia, Vancouver, British Columbia, Canada.
Graham
Nicholas R
NR
Bernales
Cecily Q
CQ
Kifayet
Arnawaz
A
Ng
Natalie
N
Shobab
Leila A
LA
Steiner
Theodore S
TS
D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D018938:Caco-2 Cells; D002227:Carbazoles / Q000494:pharmacology; D016384:Consensus Sequence; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005408:Flagellin / Q000378:metabolism*; D006801:Humans; D007211:Indoles; D008301:Maleimides; D013058:Mass Spectrometry; D009154:Mutation; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051198:Toll-Like Receptor 5 / Q000737:chemistry / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
C465664:2-(1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl)-3-(1H-indol-3-yl)maleimide; D002227:Carbazoles; D029968:Escherichia coli Proteins; D007211:Indoles; D008301:Maleimides; D010455:Peptides; D051198:Toll-Like Receptor 5; D005408:Flagellin; D012694:Serine; C089212:protein kinase D; D011493:Protein Kinase C; D048051:p38 Mitogen-Activated Protein Kinases