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

This information is current as Sabine M. Ivison, Nicholas R. Graham, Cecily Q. Bernales, of September 28, 2021. Arnawaz Kifayet, Natalie Ng, Leila A. Shobab and Theodore S. Steiner J Immunol 2007; 178:5735-5743; ; doi: 10.4049/jimmunol.178.9.5735

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Protein Kinase D Interaction with TLR5 Is Required for Inflammatory Signaling in Response to Bacterial Flagellin1

Sabine M. Ivison, Nicholas R. Graham, Cecily Q. Bernales, Arnawaz Kifayet, Natalie Ng, Leila A. Shobab, and Theodore S. Steiner2

Protein kinase D (PKD), also called protein kinase C (PKC)␮, is a serine-threonine kinase that is involved in diverse areas of cellular function such as lymphocyte signaling, oxidative stress, and protein . After identifying a putative PKD phos- phorylation site in the Toll/IL-1R domain of TLR5, we explored the role of this kinase in the interaction between human TLR5 and enteroaggregative flagellin in human epithelial lines. We report several lines of evidence that implicate PKD in TLR5 signaling. First, PKD phosphorylated the TLR5-derived target 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 coimmunoprecipi- Downloaded from tation experiments, and this association was rapidly enhanced by flagellin treatment. Third, pharmacologic inhibition of PKC or PKD with Go¨6976 resulted in reduced expression and secretion of IL-8 and prevented the flagellin-induced activation of p38 MAPK, but treatment with the PKC inhibitor Go¨6983 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 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 Journal http://www.jimmunol.org/ of Immunology, 2007, 178: 5735–5743.

ince the discovery that TLR4 initiates an inflammatory naling adaptors such as MyD88 or TIR domain-containing adap- response after recognition of bacterial LPS (1), the interest tor-inducing IFN-␤. In the MyD88-dependent pathway common to S in these pattern recognition receptors and their signaling most TLRs, ligation results in phosphorylation and acti- pathways has been intense. In homodimeric or heterodimeric form, vation of IL-1R-associated kinase family members, allowing the TLRs respond to peptidoglycans, lipoproteins, LPS, dsRNA, un- recruitment of the adaptor TNFR-associated factor 6, which acti- methylated CpG-DNA, and bacterial flagellin. Ligation of these vates the TGF-␤-activated kinase 1. The latter then activates the by guest on September 28, 2021 innate immune receptors, which are poised on surface or organellar transcription factors NF-␬B (via I-␬B degradation) and AP-1 (via membranes of many cell types, including epithelial cells, macro- p38 MAPK and JNK), leading to cytokine and chemokine expres- phages and dendritic cells, initiates signaling cascades that trigger sion (reviewed in Ref. 2). secretion of inflammatory cytokines and chemokines as well as TLR5, which recognizes bacterial flagellin, is thought to trans- expression of costimulatory signals for adaptive immunity. duce signals via the MyD88-dependent pathway only (3, 4). Ac- TLRs are characterized by N-terminal leucine-rich repeats, a tivation of IL-1R-associated kinase (5), nuclear translocation of central transmembrane domain, and a cytosolic C-terminal Toll/ NF-␬B (6, 7), and activation of MEK (8), p38 MAPK, and ERK 3 IL-1R (TIR) domain, which links the receptor to proximal sig- (6) have all been demonstrated after flagellin treatment in many cell types, including professional APCs and epithelial cells. These pathways lead to the secretion of ␤-defensin (9), cytokines IL-6 Division of Infectious Diseases, University of British Columbia, Vancouver, British ␣ ␣ Columbia, Canada and TNF- (10), and/or chemokines MIP3 and IL-8 (3, 4), which Received for publication March 20, 2006. Accepted for publication February recruit and activate dendritic cells and neutrophils, respectively. 21, 2007. However, the characteristic features of TLR5 signaling that confer The costs of publication of this article were defrayed in part by the payment of page specific responses to flagellin as opposed to other TLR agonists charges. This article must therefore be hereby marked advertisement in accordance have yet to be identified. with 18 U.S.C. Section 1734 solely to indicate this fact. Using an in silico analysis to identify potential protein interac- 1 This work was supported by Grant 992840.01 from Burroughs-Wellcome Career Award in Biomedical Sciences, Operating Grant 64355 from the Canadian Institutes tion sites in the TIR domain of TLR5, we identified a putative for Health Research, by the Canada Foundation for Innovation New Opportunities protein kinase D (PKD) phosphorylation site (see Fig. 1A). PKD is Fund 4453, by New Investigator Awards from the Canadian Institutes for Health a serine-threonine kinase of the -calcium-dependent ki- Research, and by the Vancouver Coastal Health Research Institute “In It for Life” Fund (to T.S.S.). nase family, and contains diacylglycerol-binding and pleckstrin 2 Address correspondence and reprint requests to Dr. Theodore S. Steiner, Division of homology domains within its regulatory N-terminal region. The Infectious Diseases, University of British Columbia, Room D452, Heather Pavilion three known PKD subtypes PKD1 (also called protein kinase East, 2733 Heather Street, Vancouver V5Z 3J5, British Columbia, Canada. E-mail ␮ ␯ address: [email protected] C (PKC) ), PKD2, and PKD3 (PKC ) are involved in diverse aspects of cellular function including membrane trafficking, dif- 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 hair- ferentiation, proliferation, and apoptosis as well as the responses to pin RNA; siRNA, small interfering RNA; HEK, human embryonic kidney; HA, hem- BCR and TCR ligation, regulatory , and oxidative stress agglutinin; shRNA, small hairpin RNA; Ct, cycle threshold. (see reviews Refs. 11, 12). Using the interaction between the en- Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 teroaggregative Escherichia coli H18 flagellin (FliC) and human www.jimmunol.org 5736 PKD IN TLR5 SIGNALING

␮ TLR5 in epithelial cell lines as a model, we report that PKD in- at a final concentration of 0.5–1 g/ml, while Pam3CysSK4 (EMC Micro- teracts with TLR5, and that this kinase is required for transducing collections) was used at 10 ␮g/ml. Cells were pretreated with inhibitors or flagellin-stimulated inflammatory responses. DMSO vehicle for 1 h, followed by stimulation for varied amounts of time, depending on cell type and readout. For detection of PKD, cells were lysed in 50 mM Tris-HCl (pH 7.4), 1% Materials and Methods Triton X-100, 150 mM NaCl, 5 mM EDTA (pH 7.4), and Protease Inhib- Abs and vectors itor Cocktail for mammalian cells (Sigma-Aldrich). For detection of phos- phorylated , cells were lysed in 20 mM Tris (pH 7.5), 150 mM The following Abs were used: anti-PK (Serotec), anti-PKD1, anti- NaCl, 1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40, 2.5 mM sodium phospho-PKD1 (S916), anti-phospho-p38 MAPK (T180/Y182), anti- ␤ pyrophosphate, 1 mM -glycerophosphate, 2 mM Na3VO4, and Protease total p38 (Cell Signaling Technology), and anti-GAPDH (Fitzgerald Inhibitor Cocktail. Equal amounts of proteins were separated by SDS- Industries International). PAGE and blotted for Western blot analysis. TLR5 cloned into pEF6-V5His-Topo (Invitrogen Life Technologies) For analysis of secreted IL-8, in the case of Caco-2 cells, culture me- was a gift from A. Aderem (Institute for Systems Biology, Seattle, WA). dium was removed after3hofstimulus. For HEK 293T cells, longer time pEGFP was from BD Clontech. Hemagglutinin (HA)-PKD1 and points (6 or 24 h) were used due to the slower kinetics of IL-8 production. pSPKD1-1 were provided by A. Toker (Harvard University, Boston, MA). The IL-8 contents of supernatants were determined by ELISA (OptEIA; The IL-8pLuc reporter vector (13) was provided by B. Salh (University of BD Biosciences). British Columbia, Vancouver, British Columbia, Canada). The TLR5 S805A mutant was produced by the quick-change circular In vitro kinase assays mutagenesis method (Stratagene), using the following primers: 5Ј-gtac cagttgatgaaacatcaagcccatcagaggctttgtacag and 5Ј-ctgtacaaagcctctgatgg The synthetic peptides purchased from GenScript and dissolved at 3 mM in cttgatgtttcatcaactggtac. DMSO were PKD (AALVRQMSVAFFFK), TLR5 (YQLMKHQSIRG

TLR2 in pcDNA3.1 was provided by M. Smith (University of Virginia, FVQ), and S805A (YQLMKHQAIRGFVQ). Peptides were added to a final Downloaded from Charlottesville, VA); it was cloned into the KpnI and XbaI sites of pEF6/ concentration of 150 ␮M in a reaction mixture of 0.03% Triton X-100, 20 V5-HisTOPO (Invitrogen Life Technologies) using the following primers: mM HEPES (pH 7.4) with 25 ng of active recombinant PKD1 (Upstate TLR2V5F, 5Ј-aaaaggtaccatgccacatactttgtggatggtgtggg and 5Ј-aaaatctagac Biotechnology). A kinase mixture containing [␥-32P]ATP (1 ␮Ci/reaction) ␮ ␤ tctttatcgcagctctcagatttacccaaaatcc to create TLR2-V5. in 75 mM MgCl2, 500 M ATP, 20 mM MOPS (pH 7.2), 25 mM -glyc- The pSuper-based small hairpin RNA (shRNA) vector for PKD1 knock- erophosphate, 5 mM EGTA, 1 mM Na3VO4, and 1 mM DTT was added, down pSPKD1-2 as well as the scrambled version of pSPKD1-1 (pSscr1-1) and the reaction allowed to proceed for 10 min at 30°C. The reaction was was created by annealing the following oligonucleotides and cloning them stopped by the addition of 100 ␮l of 0.75% phosphoric acid, and the entire into the pSuper vector (OligoEngine): pPKD1–8 top 5Ј-gatccccgcagattca reaction spotted on squares of P81 phosphocellulose paper. Papers were http://www.jimmunol.org/ actgccataaacttcaagagagtttatggcagttgaatctgcttttta, bottom 5Ј-agcttaaaaagcag washed three times in 0.75% phosphoric acid and once in acetone, air- attcaactgccataaactctcttgaagtttatggcagttgaatctgcggg; and pscrPKD1-1A top dried, and counted for radioactivity. Samples containing no target peptide 5Ј-gatcccctgatcgggtgctgagctggttcaagagaccagctcagcacccgactatttta, bottom were used to obtain background counts. 5Ј-agcttaaaaatagtcgggtgctgagctggtctcttgaaccagctcagcacccgactaggg. All oli- gonucleotides were obtained from Operon Biotechnologies. Coimmunoprecipitation Cell culture and transfections HEK 293T cells plated at a density of 2 ϫ 105 cells/ml in 6-well plates were transiently transfected with a total of 3 ␮g of DNA consisting of Caco-2 cells were obtained from the American Type Culture Collection HA-PKD, TLR5-V5, and/or TLR2-V5. After stimulation for the indicated (ATCC) and grown in HyQ DMEM-high glucose (HyClone) supplemented times with 1 ␮g/ml flagellin, cells were washed with cold PBS and lysed with 10% FBS (Invitrogen Life Technologies), nonessential amino acids with 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM by guest on September 28, 2021 ␮ (HyClone), penicillin (100 U/ml), and streptomycin (100 g/ml) (Sigma- EGTA, 1% Octyl ␤-D-glucopyranoside, 1 mM ␤-glycerophosphate, 2.5 Aldrich). Cells were seeded at 1 ϫ 106 cells/ml in polystyrene culture mM sodium pyrophosphate, 2 mM Na3VO4, and protease inhibitor mixture dishes and used for experiments 7–14 days after becoming confluent. Hu- (Sigma-Aldrich). man embryonic kidney (HEK) 293T cells also obtained from ATCC were After preclearing 0.75–1.5 mg of total protein in 400 ␮l volume with grown in HyQ DMEM-high glucose (HyClone Laboratories) supplemented protein A-agarose (Bio-Rad) for 10 min at 4°C, 7 ␮l of anti-V5 was added with 10% heat-inactivated FBS (HyClone), 25 mM HEPES (StemCell to the supernatant and the solution was nutated overnight at 4°C. Protein Technologies), 2 mM glutamine (StemCell Technologies), and antibiotics A-agarose beads were blocked overnight with 5% BSA in lysis buffer, as earlier described. Caco-2 cells were passaged every 1–2 wk, while the washed, and added to the samples the next morning. After 1 h, the immune HEK cells were passaged two to three times per week. complexes were washed four times in modified radioimmunoprecipitation assay buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Cell transfection Nonidet P-40, 0.25% sodium desoxycholate, 2 mM Na3VO4, and protease HEK cells were seeded at 2 ϫ 105 cells/ml in growth medium without inhibitor mixture). Beads were eluted by boiling in 2ϫ SDS-PAGE loading penicillin or streptomycin in plates coated with 0.01% poly-L-lysine buffer. Proteins were then separated by SDS-PAGE, blotted onto polyvi- (Sigma-Aldrich). The following day, cells were transfected with nylidene difluoride membranes, and probed with anti-PKD1. After strip- LipofectAMINE 2000 (Invitrogen Life Technologies) according to the ping, blots were probed with anti-V5 to detect TLR2 and TLR5. manufacturer’s instructions. Medium was changed the following day, and cells were stimulated after an additional 18 h. Quantitative RT-PCR Caco cells were transfected by electroporation as follows: cells were 7 For mRNA quantitation, cells were lysed 1 h (for IL-8) or 3 h (for CCL20) trypsinized, washed in PBS, and suspended at 10 /ml in Opti-Mem (In- after flagellin stimulation. Total RNA was isolated using RNeasy mini vitrogen Life Technologies). Plasmid DNA and sheared salmon sperm ␮ ␮ columns (Qiagen), and equal amounts of RNA from each sample were DNA (Sigma-Aldrich) totaling 25–35 g was added to 400 l of cell reverse transcribed using RevertAid H Minus First-Strand cDNA Synthesis suspension. After brief incubation, cells were transferred to a 2-mm cuvette kit (Fermentas). Real-time PCR was performed using SYBR Green master and pulsed in an ECM 630 electroporator (BTX) using capacitance of 1000 ␮ mix (Applied Biosystems) and run on an Opticon thermal cycler (Bio-Rad). F, resistance 50 Ohm, and potential 250 V. Cells were allowed to rest 5 Equal volumes from each reaction were run in triplicate for 40 cycles of min at room temperature, transferred into growth medium, and seeded in 94°C for 30 s, 55°C for 30 s, and 72°C for 45 s, with fluorescent detection culture plates or dishes. after each cycle. Melting curves were performed after 40 cycles and correct Stimulation and inhibition of cells product size was confirmed by agarose electrophoresis. Primers used were: GAPDH (forward) 5Ј-gaaggtgaaggtcggagtc, (reverse) 5Ј-gagggatctcgctcct The H18 flagellin from enteroaggregative E. coli strain 042 was expressed ggaaga; CCL20 (forward) 5Ј-ccaagagtttgctcctggct, (reverse) 5Ј-tgcttgctgct in pCR-NT-T7-Topo (Invitrogen Life Technologies) in BL21 (DE3) pLysS tctgattcg; and IL-8 (forward) 5Ј-atgacttccaagctggccgtggct, (reverse) 5Ј-tctc cells and purified by metal affinity chromatography followed by polymyxin agccctcttcaaaaacttctc. B chromatography as previously described (14). Relative mRNA amounts were calculated as follows: the cycle threshold Pharmacological inhibitors (Go¨6976, Go¨6983) were obtained from Cal- (Ct) for fluorescent detection was measured for each sample, and the dif- biochem, and phorbol 12,13-dibutyrate (PDB) and IL-1␤ were from ference between the IL-8 and GAPDH cycle threshold values was calcu- Sigma-Aldrich. Go¨6976 was used at a final concentration of 3 ␮M, Go¨6983 lated (dCt). The difference in cycle threshold for untreated Caco-2 cells at 12 ␮M, PDB at 200 nM, and IL-1␤ at 10 ng/ml. FliC (flagellin) was used was subtracted from the difference in cycle threshold for each experimental The Journal of Immunology 5737

condition to yield ddCt. The fold increase in mRNA expression for each is a similar region in TLR4 that also fits the conserved PKD rec- Ϫ condition compared with the control was then calculated as 2 ddCt. ognition/phosphorylation site, none are found in other TLRs. Phos- EMSA analysis phorylation of degenerate peptide library arrays have indicated that PKD strongly prefers a leucine at Ϫ5 and an arginine or lysine at Caco-2 cells were lysed 30 min after stimulation as described above. Nu- Ϫ clear protein extracts were isolated, tested for protein concentration, and 3 relative to the target serine or threonine (19, 20). Typically, the diluted in nuclear extract buffer to 1 mg/ml. A double-stranded NF-␬B- residue at ϩ1 is hydrophobic. Surrounding residues are less im- binding probe of sequence 5Ј-agttgaggggactttcccaggc was end-labeled with portant but do affect kinase assays to varying degrees as shown in [␥-32P]ATP using polynucleotide kinase (Fermentas) and purified by Seph- detailed analyses by Doppler et al. (20). ␬ adex G-25 chromatography and ethanol precipitation. To measure NF- B Structural modeling of TLR5 suggests that the region containing activity, a reaction mixture containing 5 ␮g of nuclear extract, and 7.5 ␮l of binding buffer (20 mM HEPES, 50 mM KCl, 0.1 mM EDTA, 5% glyc- this PKD recognition site lies at the junction of the DD loop and erol, 200 ␮g/ml BSA, and 1 mM DTT (pH 7.9), and 1.5 ␮g of poly(deoxyi- the ␣D helix, in a surface-accessible location (Fig. 1B). An in nosinic-deoxycytidylic acid) (Amersham Biosciences) in 20 ␮l total vol- silico steric hindrance analysis assigned steric hindrance values of ␮ ␬ ume was incubated 20 min on ice, and 1 l of labeled NF- B probe was 1.74 and 1.88 to K802 and S805, respectively, which fall within added. Following a 15-min incubation at room temperature, reactions were separated by 4% nondenaturing PAGE, and gels were dried onto filter the lowest third of steric hindrance values, suggesting surface paper and examined by autoradiography. NF-␬B activity was measured by availability on TLR5. delayed mobility, and specificity was verified by including a 100-fold ex- cess of unlabeled NF-␬B-binding oligonucleotide in some samples. S805 is phosphorylated in flagellin-treated cells IL-8 reporter assays To measure phosphorylation of TLR5 in vitro, we immunoprecipi- Downloaded from Caco cells were transfected as described with 1 ␮g of pEGFP, 7.5 ␮gof tated TLR5-V5 from overexpressing HEK 293T cells that had IL-8pLuc, and sheared salmon sperm DNA to a total of 30 ␮g, diluted in been treated with flagellin for 10 min. The immunoprecipitates 1.2 ml of growth medium, and seeded at 100 ␮l/well in 96-well plates. were separated by SDS-PAGE, and the band corresponding to Medium was changed the following day and fluorescence verified by mi- TLR5 (absent in cells not expressing TLR5) was evident after croscopy. Transfection efficiencies of 20% were routinely achieved. After an additional 5 days, cells were pretreated with inhibitors for 1 h and then SYPRO Ruby staining. This band was excised and analyzed by stimulated with flagellin or IL-1␤ for 6 h. Supernatants were removed and mass spectrometry. As shown in Fig. 1C, the pentapeptide frag- tested for IL-8 concentration, and cells were lysed with a 1:1 mixture of ment containing S805 had a mass shift of 80 kDa, corresponding http://www.jimmunol.org/ growth medium and Bright-Glo reagent (Promega). After 2 min, cells were to the mass of a phosphate group. read in a microplate luminometer (Berthold). The lysates were recovered and GFP fluorescence measured in a Fluoroskan Ascent FL (Thermo Elec- tron). The ratio of luminescence to fluorescence in arbitrary units (to cor- PKD phosphorylates the predicted TLR5-derived consensus rect for cell number and transfection efficiency) was calculated for each peptide in vitro sample, and the fold increase in this value compared with controls within the same experiment was defined as the fold increase in expression. To gather evidence for the phosphorylation of S805 by PKD, we looked at the ability of recombinant PKD1 to phosphorylate TLR- Structural modeling of TLR5 derived peptides in direct kinase assays. A 14-mer peptide con-

The TIR domain of TLR5 was mapped with Molecular Operating Envi- taining the PKD motif in TLR5 was synthesized, as was as the by guest on September 28, 2021 ronment version 2005.10 (Chemical Computing Group) software, using the optimal PKD consensus peptide (21) as a positive control and crystal structure of the TLR2 TIR domain as a scaffold (15). To quantify TLR5-mutant S805A (target serine mutated to alanine) as a the degree of surface exposure of individual residues in the structure of the TLR5 domain of interest, we calculated the steric hindrance parameters negative control. These peptide sequences are listed in Materi-

(Rs) for all alpha-carbon atoms of the TLR5 TIR domain, as previously als and Methods. In vitro kinase assays with active, recombi- described (16, 17). nant PKD1 were used to measure the ability of this enzyme to 32 Mass spectrometric identification of TLR5 phosphorylation incorporate P from ATP. As shown in Fig. 2, the PKD con- sensus peptide displayed the highest incorporation of 32P and ␮ HEK cells transfected in 15-cm dishes were treated with flagellin (1 g/ml) the S805A-derived peptide displayed only background radioac- for 10 min, washed in PBS, and lysed by freeze-thaw in 10 mM HEPES Ϯ (pH 7.4). Lysates were spun at 15,000 ϫ g for 10 min to pellet insoluble tivity, as would be expected. The TLR5 peptide showed 26 32 material, and supernatants were then spun at 100,000 ϫ g for1htopellet 19% of the P incorporation displayed with the consensus pep- membranes. Membrane proteins were immunoprecipitated with anti-V5 as tide, confirming the identity of this TLR5 sequence as a PKD described (18) and separated by SDS-PAGE. Gels were stained with substrate. SYPRO Ruby (Invitrogen Life Technologies) and the band correspond- ing to TLR5 (verified by its absence in identically treated cells not ex- pressing TLR5) was excised, trypsinized in situ, and analyzed by liquid Treatment of epithelial cells with flagellin increases interaction chromatography-single mass spectrometry at the Nucleic Acids and Protein between TLR5 and PKD1 Synthesis Core Mass Spectrometry facility at the University of British Columbia (Vancouver, British Columbia, Canada). Predicted tryptic pep- Direct interaction between TLR5 and PKD1 was examined by tides of TLR5 with a mass shift of 80 kDa (the size of a phosphate group) coimmunoprecipitation using HEK 293T cells. Cells were tran- were specifically sought. siently transfected with HA-PKD and TLR5 containing a V5 ␮ Statistical analysis epitope tag (TLR5-V5), treated briefly (1–10 min) with 1 g/ml flagellin, and lysed with a Tris-based buffer containing 1% octyl Statistics were performed on original data using the VassarStats statistical ␤-D-glucopyranoside. One milligram of total soluble cell lysate computation website (http://vassun.vassar.edu/ϳlowry/VassarStats.htm). Except where noted, multiple groups were analyzed by ANOVA to verify was immunoprecipitated with anti-PK (which binds the V5-tag) the presence of significant differences. This analysis was followed by and immunoblotted with anti-PKD1. Although there was non- paired testing by Student’s t test or Tukey HSD post hoc test. specific pulldown of PKD as shown by the presence of PKD in cells not transfected with TLR5, interaction between TLR5 and Results PKD1 clearly increased with exposure to flagellin only in cells The putative PKD target serine in TLR5 is predicted to be overexpressing both proteins (Fig. 3). To determine the speci- surface-exposed ficity of this ligand-dependent interaction, we repeated experi- Residues 800–806 in the TIR domain of TLR5 contain a putative ments using TLR2 in place of TLR5. As seen in Fig. 3, there PKD phosphorylation motif (Fig. 1A). Interestingly, although there was background or nonspecific interaction resulting in PKD 5738 PKD IN TLR5 SIGNALING

FIGURE 1. Location of putative PKD phosphoryla- tion motif in the TIR domain of TLR5. A, Consensus PKD phosphorylation motif and corresponding motif in TLR5. B, Ribbon (left) and space-filling (right) models of the TLR5 TIR domain, with the critical amino acids of the PKD consensus motif highlighted. C, Liquid Downloaded from chromatography-mass spectrometry tracing showing the 80 kDa-shifted tryptic peptide containing S805. http://www.jimmunol.org/ by guest on September 28, 2021

pulldown that did not change after cells were treated with the phosphorylation motif. This construct was transiently expressed

TLR2 agonist Pam3CysSK4. in HEK 293T cells and IL-8 release in response to flagellin was measured. As shown in Fig. 4, transfection of cells with wild- S805 of TLR5 is required for IL-8 production type TLR5 but not the S805A mutant caused a strong increase To investigate the importance of S805 in TLR5 signaling, we in IL-8 production after treatment with flagellin. Stimulation mutated this residue to an alanine to eliminate the putative with IL-1␤ induced equal IL-8 production in both mutants, whereas Western blot analysis of V5-tagged proteins from total cell lysates confirmed comparable receptor expression in cells transfected with both constructs (data not shown).

FIGURE 3. Interaction of PKD with TLR5. Following transfection of HEK 293T cells with either TLR5-V5 or TLR2-V5 and HA-PKD, cells ␮ ␮ were stimulated with 1 g/ml FliC or 10 g/ml Pam3CysSK4, respectively, FIGURE 2. In vitro phosphorylation of TLR5-derived peptides by for 0, 5, 10, or 30 min and V5-tagged proteins were pulled down from PKD. PKD consensus peptide, TLR5 peptide, or TLR5-S805A peptide was equal amounts of cell lysate as described in Materials and Methods. combined with 25 ng of active recombinant PKD in the presence of Washed immune complexes were resolved with SDS-PAGE, blotted, and [␥-32P]ATP, as described in Materials and Methods. Results are expressed subsequently probed with anti-PKD1-specific Ab and anti-V5. Cells trans- as a ratio of maximum incorporation (ϮSD) as defined by the PKD con- fected with PKD (P) or TLR (T) alone served as negative controls. Results sensus peptide. Data are compiled from two separate experiments (n ϭ 6). are representative of at least two experiments. The Journal of Immunology 5739

Pharmacological inhibition of PKD reduces flagellin-stimulated inflammatory responses in Caco-2 cells To determine the relevance of PKD in the inflammatory TLR5 signaling of intestinal epithelial cells, we looked at the effect of pharmacological inhibition of PKD on the flagellin-induced production of IL-8 in Caco-2 cells. Although there is no known substance that specifically inhibits PKD, a combination of stau- rosporine derivatives Go¨6976 and Go¨6983 can uncover PKD in- volvement. The former selectively inhibits classical PKC isoforms as well as PKD, whereas the latter suppresses the activity of all three PKC subgroups (classic, novel, and atypical) but not PKD (22). As shown in Fig. 5, A and B, pretreatment of Caco-2 cells with Go¨6976 but not with Go¨6983 reduced both IL-8 mRNA con- centrations ( p Ͻ 0.05 by Student’s t test) and IL-8 promoter ac- tivity ( p Ͻ 0.005 by Student’s t test) after flagellin treatment com- pared with those measured after pretreatment with DMSO vehicle FIGURE 4. Mutation of S805 to alanine abrogates the TLR5 response alone. A similar response pattern was seen when mRNA concen- to flagellin in HEK 293T cells. IL-8 produced by cells transiently trans- ␣ Downloaded from fected with salmon sperm DNA (ssDNA), TLR5, or TLR5-S805A was trations of the dendritic cell chemokine CCL20 (MIP3 ), which is ,(p Ͻ also produced by Caco-2 cells in response to TLR5 ligation (23 ,ء .(measured after stimulation with 0.5 ␮g/ml FliC for6h(n ϭ 6 0.001 by Student’s t test. http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. Effect of PKC/PKD inhibitors on flagellin-stimulated chemokine production. All cells were pretreated with DMSO, 3 ␮MGo¨6976, or 12 ␮MGo¨6983 for 1 h and then stimulated with 1 ␮g/ml FliC for the indicated times (see Results for statistical significance). A, IL-8 mRNA expression was determined for Caco cells stimulated for 1 h with FliC. Total RNA was isolated, reverse transcribed and quantified by real-time PCR with primers against IL-8 and GAPDH as described in Materials and Methods (n ϭ 11). Data shown are the mean fold increase Ϯ SD vs control (for each individual experiment). B, IL-8 promoter activity in Caco-2 cells transfected with pEGFP and an IL-8 luciferase reporter plasmid was grown to confluence and stimulated with FliC for 6 h. Luciferase activity and green fluorescence were measured sequentially in cell lysates, and the ratio was calculated as a measure of promoter activity per number of transfected cells. The fold increase in luciferase activity per GFP (RLU/RFU) vs fold increase for controls was measured. Data shown are mean Ϯ SEM in each individual experiment. Results were compiled from four separate experiments. C, CCL20 expression. Caco cells were stimulated for 3 h with FliC and relative amounts of CCL20 mRNA were quantified as described in B. Data shown are the mean Ϯ SD vs control (n ϭ 2). D, IL-8 protein secretion from Caco cells stimulated with FliC or 200 nM PDB for 3 h. IL-8 concentrations in supernatants were determined by ELISA. Data were compiled from the results of at least three different experiments and represent mean Ϯ SD vs control values. 5740 PKD IN TLR5 SIGNALING

FIGURE 6. Effects of PKC/PKD inhibitors on flagellin stimulated NF-␬B translocation and p38 acti- vation in Caco cells. Cells were pretreated with DMSO, 3 ␮MGo¨6976, or 12 ␮MGo¨6983 for 1 h before addi- tion of 1 ␮g/ml FliC for 30 min. A, EMSA to measure NF-␬B nuclear migration in Caco-2 cells. Nuclear extracts Downloaded from were purified, bound to radiolabeled NF-␬B-binding oli- gonucleotide, and separated by PAGE. Densitometric measurement on autoradiographs of NF-␬B-shifted bands is from three separate experiments (top) shown as the percentage (ϮSEM). Bands corresponding to the NF- ␬B-bound probe are shown from one representative ex- http://www.jimmunol.org/ periment (bottom). B, Western blot analysis of p38 MAPK activation after FliC or IL-1␤ stimulation (top) is shown. After stimulation with either 0.5 ␮g/ml FliC or 10 ng/ml IL-1␤ for the indicated times, cells were lysed. Equal amounts of lysate proteins were separated by SDS-PAGE, blotted, and then probed sequentially with anti-phospho-p38 MAPK (T180/Y182) and with total p38. Densitometric analysis of signals from 0 and 30 min after stimulation is shown for three pooled ex-

periments (bottom). The relative intensity corresponds by guest on September 28, 2021 to the ratio of signal to maximal signal in DMSO for each experiment after correcting for differences in total .p Ͻ 0.01 by Tukey HSD ,ء .p38

were examined (Fig. 5C). Moreover, Go¨6976 but not Go¨6983 sig- NF-␬B and one leading to activation of MAPK (2). To better nificantly inhibited flagellin-induced IL-8 secretion from Caco-2 understand the downstream effects of PKD inhibition, we cells (Fig. 5D; mean reduction 75 Ϯ 9%; p Ͻ 0.0001 by Student’s looked at the effect of Go¨6976 and Go¨6983 on elements of both t test). In contrast, IL-8 release from cells stimulated with PDB pathways in Caco-2 cells. Flagellin, like other TLR agonists, esters, which activates PKD and various isoforms of PKC, was signals through I-␬B kinase-mediated I-␬B degradation and reduced by both inhibitors, demonstrating the efficacy of Go¨6983. translocation of NF-␬B to the nucleus where it activates proin- flammatory cytokines and chemokines (7). EMSA with nuclear Pharmacological PKD inhibition before flagellin treatment has extracts from inhibitor-treated, flagellin-stimulated cells ␬ no effect on NF- B activation but prevents activation of p38 showed that neither PKD nor PKC inhibition had an effect on MAPK in Caco-2 cells NF-␬B activation after flagellin stimulation (Fig. 6A), indicat- MyD88-dependent TLR signaling is thought to branch into at ing that PKD does not modulate this branch of TLR5 signaling least two separate pathways, one involving the activation of in Caco-2 cells. The Journal of Immunology 5741 Downloaded from

FIGURE 7. Effects of pharmacological and shRNA-mediated PKD inhibition on IL-8 release from flagellin-stimulated HEK 293T cells. A, Pharma- cological PKD inhibition of secreted IL-8. TLR5-transfected cells were pretreated with DMSO, 3 ␮MGo¨6976, or 12 ␮MGo¨6983 for 1 h and stimulated with 1 ␮g/ml FliC or 200 nM PDB for 6 h, and supernatants were assayed for IL-8 content by ELISA (n ϭ 4). Results are expressed as a ratio of transfected http://www.jimmunol.org/ to DMSO-treated, FliC-stimulated controls in each experiment. Data shown are mean Ϯ SD. B, Representative Western blots showing the effect of shRNA on native PKD1 expression in HEK 293T cells. Following transfection as shown, cell lysates were separated by SDS-PAGE, blotted, and probed with anti-PKD1 and either anti-GAPDH (bottom right) or anti-total p38 (bottom left) as a loading control. C, Effect of siRNA-mediated, endogenous PKD1 knockdown on IL-8 release after FliC stimulation. Cells were transfected with TLR5 and empty pSuper, pSuper expressing one of two PKD shRNA sequences (pSPKD1-1 or pSPKD1-2), or the former sequence scrambled (pSscr1-1). Transfected cells were stimulated for 6 h with 0.5 ␮g/ml FliC or 10 ng/ml IL-1␤, and IL-8 release was determined by ELISA (n ϭ 4 for IL-1; n ϭ 10 for FliC). Results were standardized to IL-8 release with empty pSuper in each experiment. See text for statistics. D, shRNA-mediated PKD1 knockdown specifically reduces FliC-induced phosphorylation of p38 MAPK. Cells were stimulated with either 0.5 ␮g/ml FliC or 10 ng/ml IL-1␤ for 0, 5, or 20 min, and equal amounts of lysate proteins were separated by SDS-PAGE. The gels were blotted and then probed with anti-phospho-p38 MAPK (T180/Y182), total p38, anti-PKD, and finally anti-V5 to detect TLR5. Blots are by guest on September 28, 2021 representative of two such experiments.

Activation of TGF␤-activated kinase 1 through TNFR-associ- caused a significant reduction in secreted IL-8 (51 Ϯ 4%, p Ͻ 0.01 ated factor 6 after TLR ligation leads not only to I-␬B kinase by ANOVA), whereas treatment with Go¨6983 had no effect, indi- activation but also to phosphorylation of MAPKs. Both ERK and cating that a PKD-dependent pathway is involved downstream of p38 MAPKs have been implicated in TLR5 signaling (6, 7). In- TLR5 ligation in these cells (Fig. 7A). Again, PDB was used as a terestingly, pretreatment of Caco cells with Go¨6976 but not positive control for the activity of Go¨6983. For shRNA experi- Go¨6983 abrogated the activation of p38 MAPK by flagellin as ments, HEK 293T cells were transiently transfected with TLR5 measured by phosphorylation of T180/Y182 (Fig. 6B). To deter- and a pSuper-based shRNA vector containing PKD1-derived se- mine whether this effect is specific for TLR5-mediated inflamma- quences. One sequence, pSPKD1-1, has been reported to specifi- tion, the experiments were repeated with cells stimulated with 10 cally silence expression of PKD1 (24). Another shRNA construct, ng/ml IL-1␤. Although densitometric analysis of three pooled ex- pSPKD1-2, was generated to ensure that effects were not due to periments showed a small reduction in phospho-p38 MAPK levels nonspecific interactions of the small interfering RNA (siRNA). As in Go¨6976-treated cells stimulated with IL-1␤ (92 Ϯ 14% de- an additional control, we generated an shRNA construct with a creased to 63 Ϯ 11%; p ϭ 0.026), the effect was far more pro- scrambled version of the PKD1-1 siRNA sequence, pSscr1-1. nounced in FliC-treated cells, in which the relative intensity of Western blot analysis shows that HEK 293T cells expressing the 96 Ϯ 4% was reduced to 15 Ϯ 3% ( p Ͻ 0.001). At both 30 and knockdown constructs had reduced PKD expression in comparison 60 min of stimulation, Go¨6976 inhibited p38 phosphorylation in with cells expressing the empty pSuper vector (Fig. 7B). FliC chal- response to FliC significantly more than it inhibited the IL-1␤ lenge of cells expressing pSPKD1-1 reduced IL-8 production in a response ( p Ͻ 0.001). consistent but modest fashion compared with cells expressing pSuper alone (61 Ϯ 11%, p Ͻ 0.02 by Student’s t test), whereas Targeted knockdown of PKD1 in TLR5-transfected HEK 293T cells expressing pSPKD1-2 were more substantially impaired in cells reduces IL-8 production and p38 MAPK activation in their IL-8 response (17 Ϯ 6%, p Ͻ 0.001 by Student’s t test). The response to flagellin negative control pSscr1-1 failed to knock down PKD expression As the specificity of pharmacological inhibitors can be a conten- (data not shown) or flagellin-induced IL-8 release (Fig. 7C). As tious issue, we took steps to verify PKD involvement in TLR5 PKD isoforms are reportedly involved in protein trafficking (25), signaling by shRNA-mediated silencing of PKD1. Because of the the above experiments were repeated with IL-1␤-induced IL-8 pro- poor transfection rate of Caco-2 cells (ϳ20%), these experiments duction to rule out the possibility of a nonspecific effect on IL-8 se- were done exclusively in HEK 293T cells. Treatment of TLR5- cretion. Although significant reduction of IL-8 in response to IL-1␤ transfected, flagellin-stimulated HEK 293T cells with Go¨6976 stimulation was observed in the supernatants of cells expressing both 5742 PKD IN TLR5 SIGNALING

pSPKD1-1 and pSPKD1-2 ( p Ͻ 0.01), the effect was much less The physiological relevance of S805 phosphorylation was es- than that exhibited after FliC stimulation ( p Ͻ 0.001, FliC vs tablished by mutation of the target serine to alanine, which elim- IL-1␤). This demonstrates that PKD inhibition has specific effects inated the IL-8 response to flagellin. Furthermore, reduction of on TLR5 signaling that are not shared by signaling pathways PKD activity, either through pharmacologic inhibition or shRNA- downstream of IL-1. More importantly, the reduction in secreted mediated gene knockdown, also reduced IL-8 release. We also IL-8 after exposure to flagellin cannot be the sole consequence of found a smaller but significant effect of PKD inhibition on IL-1␤ nonspecific effects on protein secretion. responses. This suggests that PKD is involved in more than one As previous experiments in Caco cells indicated that at least one aspect of inflammatory signaling. However, flagellin responses contribution of PKD to TLR5-linked inflammatory signaling may were consistently more dependent on PKD activity, which sug- be through the activation of p38 MAPK, we sought to verify this gests a specific role for PKD in TLR5 signaling, rather than a finding using the PKD1 knockdown vectors. Cells were transfected general role in protein secretion; the latter is of special importance with TLR5 and either pSuper or pSPKD1-2 and then stimulated with as PKD isoforms are involved in protein secretion (25). 0.5 ␮g/ml FliC. To control for specificity, 10 ng/ml IL-1␤ was added In the majority of reports concerning PKD, activation of PKD is to cells transfected with knockdown vector or control (without exog- PKC-dependent. In these cases, stimulation is followed by activa- enous TLR5). As shown in Fig. 7D, PKD shRNA significantly re- tion of phospholipase C, resulting in the production of diacylglyc- duced p38 phosphorylation in response to flagellin. This response erol and activation of novel PKC isoforms that then phosphorylate ϭ Ͻ was confirmed by densitometry (n 4, p 0.05 by Student’s t activation loop serines (S738/S742 in human PKD) within the ki- ␤ test). In contrast, IL-1 -induced p38 phosphorylation was mini- nase domain of PKD (30). However PKC-independent activation, mally inhibited (Fig. 7D). which correlates with a lack of phosphorylation of the activation Downloaded from loop serines, has been described (31, 32). We found that the ad- dition of flagellin to Caco-2 cells does not result in phosphoryla- Discussion tion of the activation loop serines, although addition of PDB ester As TLRs are positioned at the apex of the immune response, a does (data not shown). The inability of the PKC inhibitor Go¨6983 complete understanding of their signaling is required to manipulate to block flagellin-mediated IL-8 responses in Caco-2 and HEK downstream events that control both innate and acquired immu- 293T cells may indicate that PKD is constitutively active in these http://www.jimmunol.org/ nity. Consequently, much effort has gone into the elucidation of cells. In contrast, the PKC inhibitor Go¨6983 was able to block TLR signaling, especially in the case of TLR2 and TLR4. Much flagellin-stimulated IL-8 release in HeLa cells (data not shown), less is known about TLR5 signaling, although this receptor is indicating that the mechanism of PKD activation could depend on highly expressed on epithelial, endothelial, and professional im- the cell type. mune cells. In particular, TLR5 is expressed by several cell types To ascertain which downstream signaling elements are involved in the intestinal mucosa, a major site of exchange with the external in PKD modulation of TLR5 signaling, we examined the effect of environment and home to an enormous population of commensal inhibitors on two separate components of the pathway. The best . Hence, TLR5 is thought to play an important role in nor- ␬ characterized element of TLR signaling is NF- B. PKD has been by guest on September 28, 2021 mal immune homeostasis; moreover, flagellin recognition by shown to contribute to NF-␬B activation through interaction with TLR5 has recently been shown to be involved in disease states the I-␬B kinase complex, leading to degradation of I-␬B␣, in re- such as Crohn’s disease (26), systemic lupus erythematosus (27), sponse to oxidative stress in HeLa cells (24). In our experiments Salmonella gastroenteritis (28) and Legionnaire’s disease (29). Like all TLRs, TLR5 contains a TIR domain that is responsible with Caco-2 cells, PKD inhibition did not prevent activation of ␬ for interaction with intracellular signaling elements. The structure/ NF- B (as assessed by EMSA) in response to flagellin. It still ␬ function relationships within the TLR5 TIR domain have yet to be remains to be seen whether PKD plays a role in NF- B signaling elucidated, but differences in cellular responses to various TLR through alteration of either the phosphorylation status of the tran- ligands suggest that the TIR domains of TLRs mediate distinct scription factor itself or the conformation of dependent promoter activities. sites as described in other systems (33). ␬ We examined the TIR domain of TLR5 to identify potential Although NF- B activation is generally held to be the central binding sites for signaling partners, and identified a putative PKD component of TLR signaling, we have previously reported that ␬ recognition/phosphorylation site targeting S805. We therefore hy- inhibition of NF- B activation by Bay11–7082 only reduced pothesized that the phosphorylation of TLR5 by PKD is required flagellin-dependent IL-8 production by 50%, whereas inhibition of for cellular responses to flagellin. In support of this, mass spec- p38 MAPK with SB203580 reduced it by 90% (6). In this study, troscopic analysis of flagellin-treated TLR5 showed that the puta- we show that pharmacological and shRNA-mediated PKD inhibi- tive target serine was phosphorylated. Moreover, recombinant tion abrogated flagellin-mediated activation of p38. A similar PKD was able to phosphorylate a TLR5-derived peptide contain- mechanism has been described for the activation of p38 MAPK by ing the putative PKD recognition sequence. the bone morphogenic protein, BMP-2, in mouse cell lines; the More direct evidence of PKD phosphorylation of TLR5 was requirement of PKD was confirmed with siRNA-mediated PKD provided by coimmunoprecipitation, which indicated flagellin-de- silencing (31), indicating that the phenotype seen after treatment pendent interaction of PKD and TLR5. Although the results with Go¨6976 was not due to inhibition of alternative proteins up- showed some nonspecific interaction between the two proteins, the stream of p38 MAPK. It is notable that BMP-2 related PKD sig- strength of the interaction clearly increased within 5–10 min of naling seems to be independent of PKC, as may be the case in flagellin treatment. Although we also found some nonspecific in- Caco-2 and HEK 293T cells. It is still not clear how p38 MAPK teraction between PKD and TLR2, this activity did not consistently activation facilitates IL-8 production. In IL-1␤ signaling, p38 increase after receptor ligation. It is likely that ligation of TLR5 MAPK activation contributes to both AU-rich element-mediated induces conformational changes that enhance the direct or indirect IL-8 mRNA stabilization (34, 35) and to up-regulation of IL-8 interaction with PKD. Although these results provide evidence for promoter activity (13). Although loss of mRNA stabilization could phosphorylation of TLR5 by PKD, more conclusive studies are be responsible for the reduction of IL-8 mRNA after Go¨6976 treat- needed. ment, the decrease in promoter activity suggests that p38 MAPK is The Journal of Immunology 5743 involved in other aspects of flagellin-stimulated IL-8 production 16. Cherkasov, A. 2003. Inductive electronegativity scale: iterative calculation of as well. inductive partial charges. J. Chem. Inf. Comput. Sci. 43: 2039–2047. 17. Cherkasov, A. 2005. “Inductive” descriptors: 10 successful years in QSAR. Cur- This report is the first on PKD involvement in TLR-linked in- rent Computer-Aided Drug Design 1: 21–42. nate immune signaling, although PKDs are known to participate in 18. Ivison, S. M., M. A. Khan, N. R. Graham, C. Q. Bernales, A. Kaleem, C. O. Tirling, A. Cherkasov, and T. S. Steiner. 2007. 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