Protein Kinase D Interaction with TLR5 Is Required for Inflammatory Signaling in Response to Bacterial Flagellin

Abstract

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.

ATP is released by monocytes stimulated with pathogen-sensing receptor ligands and induces IL-1β and IL-18 secretion in an autocrine way

“…This difference is conceivably caused by the fact that mouse macrophages do not express the flagellin binding site TLR5, whereas human monocytes do (40), as also supported by the strong production of IL-8 induced by flagellin (see Figs. 3C and 5C), consistent with TLR5 activation (41). Not only PAMPs but also DAMPs, such as MSU, elicit a P2X 7 R-dependent IL-1␤ secretion, in contrast with a previous observation (22) that blocking P2X 7 R did not affect the MSU-induced release of IL-1␤.…”

PKC and PKC differentially regulate Legionella pneumophila-induced GM-CSF

“…PKCe deficient mice had defects in clearance of both Gram-positive (TLR2) and Gram-negative (TLR4) bacterial infections. In an other study, activation of PKC isoforms by TLR5 through flagellin contributes to production of inflammatory cytokines in epithelial cells [45]. Besides, PKCa has been shown to control TLR2 dependent activation of NF-kB [46].…”

DICAM inhibits osteoclast differentiation through attenuation of the integrin αVβ3 pathway

“…DICAM has one putative binding site for protein kinase C (PKC)-m in the extracellular juxta-transmembrane region (19) and recent studies have demonstrated the involvement of PKC-m in p38 MAP kinase-mediated signaling. (29,30) However, rDICAM containing only the extracellular domain inhibited osteoclast differentiation, which was rescued by high-dose M-CSF, implying that DICAMmediated inhibition of osteoclastogenesis is mostly through integrin b3 signaling. In the ''outside-in'' signaling model that connects integrin b3 and p38 MAP kinase, integrin first activates c-Src and consequently phosphorylates PLC-g. (4) When we assessed c-Src phosphorylation induced by RANKL, the phosphorylation of c-Src was not attenuated, but rather was slightly upregulated by DICAM (data not shown).…”