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Division of Infectious Diseases, University of British Columbia, Vancouver, British Columbia, Canada
Protein kinase D (PKD), also called protein kinase C (PKC)µ, 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.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by Grant 992840.01 from Burroughs-Wellcome Career Award in Biomedical Sciences, Operating Grant 64355 from the Canadian Institutes for Health Research, by the Canada Foundation for Innovation New Opportunities Fund 4453, by New Investigator Awards from the Canadian Institutes for Health Research, and by the Vancouver Coastal Health Research Institute "In It for Life" Fund (to T.S.S.).
2 Address correspondence and reprint requests to Dr. Theodore S. Steiner, Division of Infectious Diseases, University of British Columbia, Room D452, Heather Pavilion East, 2733 Heather Street, Vancouver V5Z 3J5, British Columbia, Canada. E-mail address: tsteiner{at}interchange.ubc.ca
3 Abbreviations used in this paper: TIR, Toll/IL-1R; PKD, protein kinase D; PKC, protein kinase C; FliC, flagellin; PDB, phorbol 12,13-dibutyrate; shRNA, small hairpin RNA; siRNA, small interfering RNA; HEK, human embryonic kidney; HA, hemagglutinin; shRNA, small hairpin RNA; Ct, cycle threshold.
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