Introduction

The TLR family is highly conserved in vertebrates and includes 11 human homologues (1). The primary role of these receptors is to recognize molecular signa-tures associated with pathogens, termed pathogen-associated molecular patterns (PAMPs), as the first step in induction of protective innate and adaptive immune responses (2,3). Each TLR is activated by a specific PAMP ligand. For example, TLR4 is activated by lipopolysaccharide (LPS), a component of bacterial membranes (4), whereas TLR9 is activated by nucleic acids containing unmethylated CpG sequences that are frequent within bacterial DNA but largely absent from host genetic material (5). Microbial DNA can be mim-icked by short, single-stranded unmethylated CpG-containing oligodeoxynucleotides (CpGs), which serve as TLR9 ligands (6).

Toll, and subsequently, the Toll-like, receptors were initially identified on the basis of the homology of their intracellular domain to that of the interleukin-1 recep-tor (7). This shared intracellular signaling region is referred to as the Toll-IL-1 receptor (TIR) domain. On the bais of the high degree of conservation of the TIR domain among TLRs, activation of conserved patterns of signal transduction would be anticipated. Classically, TLR signaling involves an intracellular cascade involv-ing myeloid differentiation primary response gene 88 (MyD88), interleukin-1 receptor-activated kinase (IRAK), and tumor-necrosis factor receptor-associated factor 6 (TRAF6), leading to activation of Nuclear Factor kappaB (NF-κB) (8). Although this represents an accu-rate description of baseline TLR signaling, it is apparent that this system is far more complex in terms of ligand recognition and use of alternate signaling pathways (9). For example, discovery of TLR signaling that is

Journal of Receptors and Signal TransductionJournal of Receptors and Signal Transduction, 2009; 29(6): 299–311

2009

Address for Correspondence: Scott Napper, Vaccine and Infectious Disease Organization, 120 Veterinary Road, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E3, Canada. Tel: 306-966-1546; Fax 306-966-7478; E-mail: scott.napper@usask.ca

11 May 2009

07 August 2009

16 August 2009

1079-9893

1532-4281

© 2009 Informa UK Ltd

10.3109/10799890903295127

R E S E A R C H A R T I C L E

Kinome analysis of Toll-like receptor signaling in bovine monocytes

Ryan J. Arsenault1,2, Shakiba Jalal1, 2, Lorne A. Babiuk3, Andrew Potter1, Philip J. Griebel1, and Scott Napper1

1Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, 2Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, and 3University of Alberta, Edmonton, Alberta, Canada

AbstractThe Toll-like receptors (TLRs) are a family of pathogen recognition receptors that alert the host to the presence of microbial challenge. Each TLR responds to a specific microbial associated ligand. For exam-ple, TLR4 is activated by lipopolysaccharide (LPS), whereas TLR9 responds to microbial DNA (CpGs). In this report signal transduction responses of bovine monocytes to stimulation with LPS and CpG are described through a bovine-specific peptide array. In addition to confirming activation of the defined TLR pathway in bovine cells, unique phosphorylation events not previously attributed to TLR signaling are described and validated. For example, array data predicts phosphorylation of Tyr40 of Etk in response to LPS, but not CpG, stimulation as well as the activation of oxidative burst in CpG, but not LPS. This investigation confirms interspecies conservation of the TLR pathway in bovine as well as providing insight into the complexity and mechanisms of TLR signaling.

Keywords: Peptide array; bovine; kinome; Toll-like receptor; lipopolysaccharide; CpG

RST

429686

(Received 11 May 2009; revised 07 August 2009; accepted 16 August 2009)

ISSN 1079-9893 print/ISSN 1532-4281 online © 2009 Informa UK LtdDOI: 10.3109/10799890903295127 http://www.informahealthcare.com/rst

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300 Ryan J. Arsenault et al.

independent of MyD88 led to identification of alternate signaling pathways through adaptor molecules, which are specific to individual TLRs (10). This specialization enables unique, customized cellular responses that are more appropriate for the microbial challenge associated with the particular PAMP.

With the appreciation that many cellular responses occur independent of changes in transcription or translation, there is an increasing interest in defining responses at the level of protein posttranslation modifi-cation, in particular, kinase-mediated signal transduc-tion. There are two basic strategies for quantification of cellular phosphorylation activity, either through quan-tification of the phosphoprotein products resulting from the action of the kinases, the phosphoproteome, or through quantification of the activities of the kinases themselves, the kinome. Generally, phosphoproteome and kinome analysis both require highly specialized tools or information in the form of phosphorylation-specific antibodies for phosphoproteome analysis or detailed species-specific information of phosphoryla-tion events for creation of peptide arrays for kinome analysis. These prerequisites previously precluded signal transduction investigations of nontraditional animal models for which these resources were una-vailable. Recently, however, our group reported on a strategy for creation of species-specific peptide arrays representing phosphorylation events predicted in pro-teins of the target species based on phosphorylation events characterized for other species (11).

Here we compare phosphorylation-mediated sig-nal transduction in bovine monocytes in response to stimulation with LPS and CpG. These medically rel-evant PAMPs, LPS as a causative agent of sepsis and CpGs for their therapeutic applications as vaccine adjuvant and cancer treatments, provide an appropri-ate challenge for validation of the TLR pathway in a novel species as well as comparative analysis of acti-vation of distinct TLRs. These PAMPs are also of inter-est for a mechanistic comparison of TLR-mediated signal transduction initiated from an extracellular (TLR4) and intracellular (TLR9) receptor. Signaling events associated with TLR activation have been char-acterized through a variety of traditional methodolo-gies such as immunoprecipitation, knockout studies, phosphospecific antibody Western blots and kinase activity assays in a variety of human and mouse cells (10,12,13). These analyses provide a framework for comparison of signal transduction patterns induced in bovine monocytes.

Conserved and unique patterns of peptide phos-phorylation are observed in bovine monocytes in response to LPS and CpG stimulation. Relative to media-treated cells, approximately 100 peptides were differentially phosphorylated following LPS and CpG

treatments. Over half of these, many relating to known TLR-associated signaling molecules, were conserved in identity and direction of phosphorylation change for both treatment conditions. These data support the conclusion that TLR4 and TLR9 signaling is highly conserved in bovine monocytes. The remaining, differ-entially phosphorylated peptides were either specific to a particular treatment or had different (increased vs. decreased phosphorylation) trends of response for the two agonists. This presumably reflects special-ized responses initiated by each receptor. In addition, a number of phosphorylation events not previously implicated in TLR signaling were identified and vali-dated. These investigations confirm the ability of the arrays to describe complex phenotypes and provide insight into nuances of TLR signaling.

Materials and methods

Isolation of bovine blood monocytes

Blood was transferred to 50-mL polypropylene tubes and centrifuged at 1400 × g for 20 min at 20°C. White blood cells were isolated from the buffy coat and mixed with PBSA to a final volume of 35 mL. The cell suspen-sion was layered onto 15 mL of 54% isotonic PERCOLL (Amersham Biosciences, GH healthcare) and centri-fuged at 2000 × g for 20 min at 20°C. Peripheral blood mononuclear cells (PBMC) from the PERCOLL-PBSA interface were collected and washed three times with cold PBSA. Isolated PBMCs were cultured in AIM V

medium (GIBCO) supplemented with 10% heat-inac-tivated fetal bovine serum (GIBCO). Monocytes were purified from isolated PBMCs by MACS purification using CD14+ microbeads (Miltenyi Biotec Inc., Auburn, CA). Monocytes (>95% pure) were plated at 5 × 106 cells/well in 6-well plates in AIM V® medium (GIBCO) sup-plemented with 10% heat-inactivated fetal bovine serum (GIBCO). Isolated monocytes were rested overnight prior to stimulation.

Cell stimulation

Purified monocytes (25 × 106) were stimulated with 100 ng/mL LPS (Escherichia coli 0111:B4) (Sigma-

PERCOLL is a registered trademark of GE Healthcare Bio-Sciences AB in the United States of America and elsewhere; AIM V is a registered trademark of Invitrogen Corporation in the United States of America;

GIBCO is a registered trademark of Invitrogen Corporation in the United States of America and elsewhere

MACS is a registered trademark of Miltenyi Biotec GmbH in the United States of America and elsewhere;

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Comparative analysis of Toll-like receptor signaling 301

Aldrich), 5 µg/mL CpG 2007 (Merial), or media for 4 hr at 37°C. This quantity and type of LPS was previously shown to induce cellular responses in bovine mono-cytes (11,12). CpG ODNs are often species-specific in their ability to induce innate immune responses, and ODN 2007 (TCGTCGTTGTCGTTTTGTCGTT) was shown to activate bovine PBMCs (14). Cells were pel-leted and stored at −80°C before use with the peptide arrays.

Knowledge-based peptide design

A description of the rationale and process of construc-tion of the bovine-specific peptide arrays for kinome analysis have been presented elsewhere (11). Briefly, online databases such as PHOSPHOSITE (www.phosphosite.org) are compilations of experimentally reported and manually curated peptide substrates pri-marily for human and mouse with limited representa-tion of other species. In silico analysis of such online resources enables selection of phosphorylation events of interest. Peptides were selected to represent phos-phorylation events associated with a spectrum of cellu-lar events but with emphasis on responses associated with innate immunity. Bovine consensus sequences of the human and mouse peptides selected from PHOSPHOSITE were obtained by using the Blastp program from NCBI to compare collected human pep-tides against the NCBI bovine protein database. Blastp was set to retrieve short exact matches. Parsed Blastp results revealed the majority of the hit sequences had 100% identity to their query sequences, and a com-parison of the protein descriptions for the query and hit sequences confirmed they referred to the same protein.

Kinome analysis

Cell lysates were prepared and incubated with the arrays as reported previously with the exception that 25 × 106 monocytes were used for each array (11). Briefly, cell pellets were lysed with 100 µL lysis buffer (20 mM Tris-HCL pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% TRITON, 2.5 mM sodium pyrophos-phate, 1 mM Na

3VO

4, 1 mM NaF, 1 µg/mL leupeptin,

1 g/mL aprotinin, 1 mM PMSF), incubated on ice for 10 min and then spun in a microcentrifuge for 10 min at 4°C. A 70-µL aliquot of this supernatant was mixed with 10 µL of activation mix (50% glycerol, 50 uM ATP, 60 mM MgCl

2, 0.05% v/v Brij-35, 0.25 mg/mL BSA, 2

mCi/mL γ-32P-ATP) and incubated on the array for 2 hr

at 37°C. Finally, slides were washed once with Tris-buffered saline (PBS) (1 × solution; pH 7.3) containing 1% TRITON X-100, twice with 2 M NaCl containing 1% TRITON X-100 and finally in demineralized H

2O.

Following air drying, arrays were exposed to a phos-phoimager screen for one week. Images were obtained by scanning the screen with TYPHOON scanner (GE Healthcare) and then loaded on ARRAYVISION (Image Research). Intensity values for the spots and background were obtained and normalized. Statistical analyses were performed with GENESPRING (Agilent Technologies) software.

Protein kinase A (PKA) activity

OMNIA Lysate Assay kit (Biosource) was used to quantify PKA activity. Bovine monocytes (1 × 107) were stimulated with either 100 ng/mL LPS (Sigma-Alrich), 5 µg/mL CpG 2007 (Merial) or cultured in media for 4 hr at 37°C. Cells were lysed with 50 µL OMNIA Cell Extraction Buffer, and supernatant protein concentra-tion was determined by QUICK START Protein Assay (Bio-Rad). PKA assays were performed by using 50 µg of protein according to the manufacturer’s protocol.

Superoxide production

Superoxide production was measured by chemi-luminescence by established protocols with the exception that 4 × 106 bovine monocytes were used without phorbol myristate acetate activation (15). Before reading luminescence the 96-well plate was wrapped in foil and incubated at 37°C, 5% CO

2 for

20 min. Luminescence was measured at 30-sec inter-vals by using a VICTOR3V 1420 Multilabel Counter (PerkinElmer).

Western blot analysis

Human blood was collected by using 0.3% EDTA as an anticoagulant. PBMCs were isolated and monocytes were MACS purified following the same protocols

PHOSPHOSITE is a registed trademark of Cell Signaling Technology, Inc. in the United States of America and elsewhere

TRITON is a registered trademark of Union Carbide Corporation in the United States of America and elsewhere; TYPHOON is a registered trademark of GE Healthcare Bio-sciences AB in the United States of America and elsewhere.

ARRAYVISION is a registered trademark of GE Healthcare Niagara Inc. in the United States the United States of America and elsewhere; GENESPRING is a registered trademark of Agilent Technologies, Inc. in the United States of America and elsewhere; OMNIA is a registered trademark of Invitrogen Corporation in the United States of America.QUICK START is a trademark of Bio-Rad Laboratories, Inc; VICTOR3V is a trademark of PerkinElmer, Inc.

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302 Ryan J. Arsenault et al.

used to isolate bovine monocytes (11). Monocytes were cultured overnight, resuspended at 2 × 106/mL and stimulated with 100 ng/mL LPS (Sigma-Alrich), 5 µg/mL CpG 2007 (Merial), or media for 4 hr at 37°C. Stimulated cells were then pelleted and lysed with 100 µl SDS sample buffer [62.5 mM Tris-HCl (pH 6.8 at 25°C), 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.01% w/v bromophenol blue, 1 mM PMSF], denatured (5 min at 95°C), and 30 µL lysate was loaded on a 10% SDS gel and transferred to a nitrocellulose membrane. Phospho-Etk antibody (Cell Signaling Technology) and a secondary fluorescent antibody (LI-COR Biosciences) were used in accordance to the supplier’s protocol. An image of

the blot was captured by using an ODYSSEYscanner (LI-COR Biosciences).

Results

Kinome responses of bovine monocyte to LPS and CpG stimulation

Cellular extracts prepared from purified bovine mono-cytes treated with either media, LPS, or CpG ODN 2007 were subjected to kinome analysis. For each treatment condition pseudo-images representing the averaged normalized signal of each peptide were generated (Figure 1). One-sample t-tests on replicate data veri-fied individual peptides displayed high technical and biological reproducibility with high confidence levels (P < 0.05) for 219 and 220 of the 300 peptides for the CpG- and LPS-treated samples respectively (Table 1). Of these, relative to the media-treated cells, 92 and 98 pep-tides were differentially phosphorylated following LPS and CpG treatments, respectively (Figure 2). Over half of these differentially phosphorylated peptides, many relating to known TLR-associated signaling molecules, were conserved in identity and direction of phosphor-ylation change. These patterns of phosphorylation are consistent with previous investigations of TLR signal-ing in other species and cell types (16–18). These data support the conclusion that TLR4 and TLR9 signaling is highly conserved in bovine monocytes (Figure 3). This is not unanticipated as similar adaptor molecules are involved in signaling by TLR family members (9). The remaining, differentially phosphorylated peptides were either specific to a particular treatment or had differ-ent trends of response (increased vs. decreased phos-phorylation) for LPS vs. CpG stimulation presumably

0.01 3.4

A. LPS

0.01 3.1

B. CpG

0.01 3.9

C. Media

Figure 1. Phosphorylation of substrate targets on peptide arrays by cellular lysates from bovine monocytes stimulated with LPS (A), CpG ODN (B), or medium (C). Purified CD14+ bovine monocytes (25 × 106) were cultured overnight, stimulated with 100 ng/mL LPS, 5 µg/mL CpG ODN 2007, or cultured in media for 4 hr at 37°C. Monocyte lysates were prepared and incubated with peptide arrays in the pres-ence of γ-32P-ATP. After original slide images were acquired by a scan-ner, normalized, and corrected for background noise, signal strength for each spot was calculated. The average signal for each peptide was calculated from the 18 replicates, and this value was used to generate pseudoimages of peptide arrays for each condition assayed.

Phosphorylationactivity unique to CpGstimulation

Antagonisticphosphorylation activity following LPS or CpG

stimulation

Phosphorylation commonto LPS and CpG

stimulation

2822

14

56Phosphorylationactivity unique to LPSstimulation

Figure 2. Altered phosphorylation events following LPS and CpG ODN stimulation. The Venn diagram summarizes the number and pattern of significant and differentially phosphorylated peptides observed with LPS- and CpG ODN-stimulated cell lysates compared to responses from media-treated cells. Altered phosphorylation events by LPS and CpG ODN were identified with fold changes ≤ 0.7 and ≥ 1.4 when normalized signals were compared to signals from media control.

ODYSSEY is a registered trademark of Li-Cor, Inc. in the United States of America and elsewhere.

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Comparative analysis of Toll-like receptor signaling 303

LPS

TLR

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2

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.

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Table 1. Phosphorylation of substrate peptides by kinases in LPS and CpG ODN stimulated monocytes.

Phospho-protein1

Kinase Target2

Fold Change3 p-value4 Kinase Mediating Phosphorylation5 Effect of Phosphorylation6LPS CpG LPS CpG

IRAK1 T209 3.2 3.7 0.01 0.01 IRAK1 Required for self phosphorylation and activation

IRAK1 S376 1.1 7.3 0.01 0.22 IRAK4 Activation

IRAK1 S568 0.2 0.2 0.01 0.01

IRAK1 T387 1.0 1.1 0.01 0.01

IRAK1 T100 1.3 1.2 0.01 0.01 Akt1 regulates transcription

FAK Y397 2.9 2.5 0.14 0.01 FAK Regulates apoptosis, and pro-tein degradation

Etk Y40 7.2 1.6 0.01 0.01 FAK Enzymatic activation, altered intracellular location, regulates molecular association and cell motility

PKCA S657 1.1 1.0 0.01 0.01 Altered intracellular location

p40phox T154 1.7 0.4 0.01 0.01 PKCA

p47phox S370 4.6 3.1 0.01 0.01 PKCA, PKACa Enzymatic activation, and altered intracellular location

PKACa S338 3.9 2.5 0.02 0.02

PKACa T195/7 7.7 1.9 0.02 0.01

Src S74 1.0 1.0 0.01 0.01

IKKα T23 1.0 1.0 0.01 0.01 Akt2 Regulate apoptosis, and enzy-matic activation

IKKβ Y188 0.4 1.1 0.56 0.01 Src Regulates transcription, and activation

IKKγ S31 0.1 0.0 0.01 0.01 IKKβ Regulates proper NFkB activity

IKKγ S43 2.6 0.5 0.01 0.01 IKKβ

IkBα S32/6 0.8 0.5 0.06 0.62 CK2-A1(S32/6), IKK-alpha(S32/6), Nik(S32/6)

activation(S32), protein degra-dation and more(S32/6)

IkB-β T19 0.8 0.8 0.01 0.01 Protein degradation

IkB-ε S18 0.5 0.6 0.01 0.01 Protein degradation

NFkB-p100 S870/72 3.5 13.2 0.06 0.71 Nik(S870), IKK-α (S872)

Protein processing(S870), regu-lates association with cellular proteins(S870)

NFkB-p105 S337 18.4 20.5 0.02 0.01 PKACa Activation, and regulates transcription

NFkB-p65 S276 11.4 11.0 0.01 0.01 MSK1, PKACa Activation, regulates transcrip-tion, and more

NFkB-p65 S311 4.3 2.3 0.01 0.01 PKCZ Activation?

TAK1 S192 15.0 22.9 0.15 0.09 TAK1 Activation

P38-γ T185 0.4 1.1 0.16 0.32

p38-α T179/Y181 1.3 1.4 0.88 0.76 MKK3/6 (T179/Y181)

Activation, Enzymatic activa-tion, and more (T179/Y181)

ASK1 S966 0.2 0.4 0.01 0.01 PKD Inhibition

MEK1 S217 1.3 1.4 0.01 0.01 Raf1 and Cot Activation

MEK2 Y216 0.4 0.7 0.11 0.29 Inhibition

MEKK1 T1383 0.8 0.7 0.01 0.01 MEKK1 Activation

JNK2 T404/7 0.7 1.2 0.01 0.01 CK2-A1(T404/7), MKK7(T404/7)

Activation(T404/7)

Jun S63 1.9 1.6 0.01 0.01 JNK1,JNK2 Protein activation, stabilization and more

Jun S73 0.9 1.1 0.04 0.01 JNK1 Protein activation, stabilization and more

Table 1. continued on next page

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Comparative analysis of Toll-like receptor signaling 305

reflecting differential responses achieved by activation of different TLRs (Figure 3).

Interleukin-1 receptor-associated kinase 1 (IRAK1)

IRAK1 is a critical upstream kinase in the TLR signal-ing pathway (9). During activation, IRAK1 undergoes a series of phosphorylation events enabling interac-tion with TLR adaptor molecules such as TRAF6 (9). In particular, phosphorylation of Thr209 in IRAK1 is pre-requisite for TLR-mediated activation (17). Consistent with this observation, a peptide corresponding to this regulatory site of bovine IRAK1 undergoes 3.2- and 3.7-fold increases in phosphorylation following LPS and CpG stimulation (Table 2). The increased phosphoryla-tion of this peptide, under both treatment conditions, would seem to confirm IRAK1 as a conserved signal transduction kinase for both TLR4 and TLR9 in bovine monocytes.

Many proteins undergo phosphorylation at mul-tiple sites to control discrete aspects of their biology. For example, IRAK1, in addition to the activating

modification at Thr209, undergoes phosphorylation at four other sites: Thr100, Thr387, Ser376, and Ser568. The physiological significance of these modifications isn’t clearly defined. Peptides representing these sites underwent a complex pattern of modification in response to LPS and CpG stimulation. Specifically, stimulation with LPS or CpG resulted in a 5-fold decrease in phosphorylation of the peptide represent-ing Ser568 compared to media control cells, suggesting a conserved function in TLR signaling. Alternatively, phosphorylation of the peptide corresponding to Ser376 increased 7.3-fold in response to CpG treat-ment but was unchanged with LPS stimulation (Table 2), suggesting differential regulation in response to activation of different TLRs. Finally, phosphorylation of peptides representing Thr100 and Thr387 were not influenced by TLR4 or TLR9 activation.

Collectively, these patterns of peptide phosphoryla-tion are consistent with the hypothesis of prerequisite modification of IRAK1 at Thr209 for TLR-induced sig-nal transduction but also suggests phosphorylation- mediated regulation of IRAK1 is complex and

Phospho-protein1

Kinase Target2

Fold Change3 p-value4 Kinase Mediating Phosphorylation5 Effect of Phosphorylation6LPS CpG LPS CpG

ERK1 T202/4 1.0 1.1 0.01 0.01 Cot(T202/4), MEK1/2(T202/4), Lck(T204), ERK1(T204)

Activation (T202/4), enzymatic activation (T202/4)

Elk-1 S389 1.2 1.2 0.01 0.01 ERK1 Activation, and regulates transcription

Raf1 S259 1.0 1.0 0.01 0.01 PKACa Inhibition

Raf1 S499 1.0 2.3 0.01 0.01 PKACa Activation

Fos T232 0.2 0.2 0.05 0.01 ERK5, ERK2 Altered intracellular location, and activation

ERK2 Y204 1.2 1.0 0.01 0.01 MEK1

TBK1 S172 1.0 1.1 0.01 0.01 Enzymatic activation

IRF-3 S385/6 0.6 3.0 0.01 0.13 TBK1(S385/6), IKK-ε(S385)

Activation(S385), and regulates association with cellular pro-teins and more (S385/6)

IRF-3 S396/8 7.7 27.1 0.04 0.27 activation, regulates transcrip-tion, and more(S369), altered intracellular location, regulates association with cellular proteins(S368)

FADD S194 10.6 5.9 0.08 0.01

Casp8 S347 3.4 2.8 0.01 0.01 1Common name for substrate protein with a peptide for a phosphorylation site on the array.2Position and name of target amino acid on the substrate protein.3Fold changes for LPS and CpG samples were calculated by comparing the background corrected and normalized signal values of these samples to the media control.4P-values reported by Genespring software for normalized phosphorylation signals. A P-value represents confidence level on reproducibility of a signal and is evaluated by one-sample t-test that compares normalized signals (n = 18) for each peptide to a baseline value 1.5For phosphorylation sites with known upstream kinases, PHOSPHOSITE database provides names of these kinases. Available information from PHOSPHOSITE is listed here.6Curated data on PHOSPHOSITE database maintain information on effects of phosphorylation on cellular activity of a substrate protein. When available, such information was presented here.

Table 1. Continued.

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multifaceted, involving aspects of regulation through phosphorylation that are independent of TLR activa-tion (Thr100 and Thr387), conserved for various TLRs (Ser568) and specific to particular TLRs (Ser376). If these observations reflect physiological modifications of IRAK1, this would offer a mechanism by which spe-cialized responses are achieved following activation of particular TLRs. This, to our knowledge, is the first sug-gestion of differential regulation of IRAK1 in response to activation of different TLRs.

NF-κBs

The NF-κB transcription factors are key downstream effectors of TLR signaling. In addition to their well- documented regulation through programmed degrada-tion of inhibitory proteins, NF-κBs are also controlled through phosphorylation. Phosphorylation events relat-ing to regulation of key NF-κB proteins, such as p65, p100, and p105, are represented on the peptide array. As expected, the pattern of peptide phosphorylation is suggestive of increased phosphorylation of critical regu-latory sites within NF-kB and related kinases following LPS and CpG treatments.

NF-κB p105/p50

The ability for the NF-κB p105 subunit to bind DNA and function as a transcription factor depends on phos-phorylation of Ser337 (18). A peptide representing this site undergoes dramatic change in phosphorylation as a result of both LPS and CpG treatment with 20- and 18-fold increases, respectively (Table 2). This implies conserved activation of this transcription factor follow-ing TLR4 and TLR9 activation.

NF-κB p100/p52

Activation of p100 depends on phosphorylation of a series of C-terminal sites including S870 (19). Both LPS and CpG stimulation resulted in increased phosphoryla-tion of the peptide representing this site, but the effect was more pronounced with CpG (13.2-fold) vs. LPS (3.5-fold) stimulation (Table 2). The significance of the different magnitudes of phosphorylation in response

to LPS and CpG stimulation has not been functionally validated.

NF-κB p65

Investigations of the transactivation domain of the NF-κB p65 subunit indicate modification of Ser276 is essential for p65-dependent cellular responses (20). Consistent with the established role of the p65 subunit in mediating cellular responses for various Toll-like receptors, an 11-fold increase in phosphorylation of a peptide representing this site is observed upon stimula-tion with either LPS or CpG (Table 2). Phosphorylation of Ser311 has also been implicated in activation of p65 NF-κB in murine embryonic fibroblasts (21) and the corresponding bovine peptide displayed significantly increased phosphorylation following LPS and CpG stimulation (Table 2). The conserved pattern of phos-phorylation of these peptides suggests similar activa-tion of this transcription factor following TLR4 and TLR9 activation.

TAK1/MAP3K7

TAK1 is a member of the mitogen-activated kinase kinase kinase (MAP3K) family and serves as an impor-tant link between the TLR and MAPK pathways. An early intermediate of the TLR pathway, TRAF6, forms active signaling complexes with TAK1. A peptide representing Ser192 of TAK1, a known phosphorylation site for TAK1 activation (22) undergoes 22.9- and 15.0-fold increases in phosphorylation following stimulation with LPS and CpG, respectively. Conversely, a peptide correspond-ing to T184 of TAK1, which is not implicated in TLR-mediated signaling, is not differential phosphorylated in either treatment condition (Table 2).

TLR activation of the MAPK signaling pathway is further suggested by increased phosphorylation of peptides corresponding to activating events of v-raf-1 murine leukemia viral oncogene homolog 1 (Raf1), jun oncogene (Jun), and mitogen-activated protein kinase kinase 4 (MKK4) (Table 2). Raf1 is an upstream protein kinase of the MAPK pathway, MKK4 lies midpoint, and Jun is a downstream transcription factor. The increase in lysate kinase activity that phosphorylates peptides from proteins in this pathway suggests the pathway may be activated in response to these agonists.

Fas-associated death domain protein (FADD) and Caspase 8 (Casp8)

In addition to its apoptotic function, FADD also plays a role in TLR-induced proliferative responses (23). FADD is activated following TLR stimulation and induces cel-lular responses through activation of Casp8 (24,25).

CpG Media LPS

Figure 4. Etk protein phosphorylation due to LPS stimulation of CD14+ human monocytes. Phosphorylation of Etk in human mono-cytes cultured with media or stimulated with LPS (100 ng/mL) or CpG ODN 2007 (5 µg/mL) for 4 hr was detected by a phospho-Etk antibody reacted with a Western blot.

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Comparative analysis of Toll-like receptor signaling 307

FADD-mediated activation of Casp8 has been observed following LPS, and to a lesser extent, CpG treatment (25). Activation of FADD and Casp8 are associated with increased phosphorylation at Ser194 and Ser347, respec-tively (26,27). The peptide corresponding to Ser194 of FADD underwent a 10.6 -and 5.9-fold increases in phosphorylation, whereas the peptide corresponding to Ser347 of Casp8 displayed 3.4- and 2.8-fold increases in phosphorylation following LPS and CpG treatments, respectively (Table 2).

Focal adhesion kinase (FAK) and BMX non-receptor tyrosine kinase (Etk/BMX)

Activation of FAK through the TLR system has been suggested on the basis of cross-talk with MyD88 (28). It is of interest that a peptide corresponding to Y397 of FAK, a phosphorylation event associated with acti-vation of this kinase (29), increased 2.9- and 2.5-fold following LPS and CpG stimulation. FAK is known to activate members of the Bruton’s tyrosine kinase (Btk) family, which has defined roles in promoting cell migration (30) but has also been implicated in TLR signaling (31). Etk is a Btk family member, but a specific role for this kinase in TLR signaling has not been reported. Activation of Btk by FAK does, how-ever, provide a possible link with TLR signaling. The peptide arrays include a peptide corresponding to Tyr40 of Etk, the key regulatory phosphorylation site. Phosphorylation of this peptide increased 7.2-fold fol-lowing LPS stimulation but less than a 2-fold increase for the CpG treatment (Table 2).

The undefined role of Etk in TLR-mediated responses, differential phosphorylation of the Tyr40 peptide under the stimulation conditions and availa-bility of a phosphospecific antibody make this a strong candidate for detailed investigation. The phosphoryla-tion-specific monoclonal antibody available for Tyr40 of Etk is specific for human. Despite the high degree of conservation of the human and bovine Etk proteins this antibody does not cross-react with the bovine protein (data not shown) highlighting one of the tech-nical limitations of antibody-based kinome analysis for nontraditional animal models. On the basis of the hypothesis that Etk serves a conserved biological role across species the phosphorylation status of Tyr40 of Etk was evaluated in human CD14+ monocytes stimu-lated with LPS and CpG. Human monocytes express TLR4 and TLR9 and respond to LPS and CpG ligands (32,33). Consistent with the results of the array, stimu-lation of human monocytes with LPS, but not CpG, increased phosphorylation of Tyr40 of Etk (Figure 4). To our knowledge this is the first report linking Etk activation with LPS stimulation. This association was first revealed by data obtained from the peptide array highlighting the utility of this approach for analyzing cell-signaling events.

Protein kinase A (PKA)

A previous kinomic investigation of human peripheral blood mononuclear cells (PBMCs) showed that stimula-tion of these cells with LPS increased phosphorylation of numerous targets of PKA (34). PKA is a central cel-lular kinase regulated through cAMP availability as well as phosphorylation of Thr197 of the catalytic sub-unit

PKA Activity

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LPS CpG

Figure 5. PKA activity in bovine monocytes following LPS and CpG ODN stimulation. Purified CD14+ monocytes were stimulated with 100 ng/mL LPS, 5 µg/mL CpG, or culture medium for 4 hr before being lysed. Reagents were added to the lysate and incubated for 20 min at 37°C, 5% CO

2 before reading chemiluminescence every 30 sec during

a 30-min interval. Monocytes were isolated from three animals, and triplicate cultures were analyzed for each condition. The slope of the PKA activity was calculated for each replicate, and these values were averaged for each biological replicate. Student t-tests carried out on slopes of PKA (n = 9) showed significant increase for LPS (P = 0.02)- but not CpG (P = 0.2)-stimulated cells compared to medium-cultured cells. Fold changes for LPS and CpG were calculated in relation to results from medium-cultured cells.

Superoxide production

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Figure 6. Superoxide production in LPS- and CpG ODN-stimulated monocytes. Bovine monocytes were stimulated with 100 ng/mL LPS, 5 µg/mL CpG, or cultured in medium for 4 hr before the addition of luminol and HRP. The monocytes were then incubated for 20 min at 37°C, 5% CO

2 to allow the reaction to begin. Following incubation a

luminescence reading was taken every 30 sec for 30 min. Fold change calculation was based on data from the mean and one standard devi-ation of values from three biological samples that were analyzed in duplicate. Fold changes for LPS and CpG were calculated in relation to results from media-cultured cells. Student t-tests (n = 6) showed significant increase of superoxide by CpG (P = 0.04) but not LPS stim-ulation (P = 0.5) compared to medium-cultured cells.Jo

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(PKACa) (35). In bovine monocytes, LPS stimulation increased phosphorylation of a peptide representing Thr197 nearly 8-fold, whereas CpG treatment resulted in a 4-fold increase relative to media control (Table 2). The increased phosphorylation of this peptide by lysates of bovine monocytes stimulated with LPS appears consist-ent with the report of increased activity of PKA in human PBMCs under similar treatment conditions.

The different magnitude of phosphorylation of a pep-tide representing the regulatory site of PKA in response to CpG and LPS stimulation, as well as the tools to quantify the activity of this enzyme in cellular extracts, offer opportunity to determine whether the magnitude of phosphorylation of a peptide on the array correlates with the associated phenotype of the regulated protein in the context of the cell. Purified bovine monocytes, stimulated with LPS, CpG, or media, under the same conditions used for the peptide arrays were assayed for PKA activity. Relative to the media control, lysates from the LPS-treated cells demonstrated a significant (P = 0.02) increase in PKA activity, whereas CpG stimu-lation did not significantly (P = 0.2) increase PKA activity (Figure 5).

Oxidative burst

Oxidative burst, a mechanism of innate immune defense, is closely associated with activation of the Toll-like system with p47PHOX serving as a direct target for IRAK4 (36). Although differential levels of oxidative burst in response to TLR ligands have been reported in various immune cells of different species, not all TLRs use the same signaling pathway to achieve this response (37,38). P47PHOX activation involves phosphorylation at numerous residues, but Ser370 has been identified as the earliest and most significant modifications (39). The activity of NADPH oxidase is also influenced by p40PHOX, which interacts with p47PHOX in a phosphor-ylation-dependent manner to regulate oxidative burst. Phosphorylation of Thr154 of p40PHOX inhibits NADPH oxidase (40). Oxidative burst therefore depends on acti-vation of p47PHOX through phosphorylation at Ser370 as well as dephosphorylation of Thr154 of p40PHOX to remove its inhibitory action.

Phosphorylation of the peptide corresponding to S370 of p47PHOX underwent a similar increase in phos-phorylation following LPS and CpG stimulation, 4.6- and 3.1-fold, respectively. The increased phosphorylation at this site predicts activation of oxidative burst under both conditions. However, phosphorylation of the pep-tide corresponding to Thr154 of p40PHOX was decreased only in the CpG-treated monocytes (−2.5-fold) with increased phosphorylation (1.7 fold) following LPS stimulation, which predicts inhibition of oxidative burst in LPS-treated cells.

Monocytes stimulated under the same conditions as for kinome analysis were assayed for oxidative burst activity. As predicted by array data stimulation of bovine monocytes with CpGs induced significant (P = 0.04) oxi-dative burst activity. Although oxidative burst activity appears suppressed in the LPS-treated cells, this reduc-tion was not statistically significant (P = 0.5) (Figure 6). The oxidative burst assay was consistent with predicted phosphorylation changes of p40PHOX and support the conclusion that p47phox phosphorylation does not directly correlate with cellular responses. Thus kinome analysis provides direct evidence for one mechanism by which TLR signaling may result in differential activation of the oxidative burst in monocytes.

Discussion

The bovine peptide arrays used here confirm the use of this approach for characterizing complex phospho-rylation-mediated signaling events. It is important to note, however, potential limitations of this approach. For example, the efficiency of which a particular kinase modifies phosphoacceptor sites may differ when the res-idue is in either the context of an intact protein or immo-bilized peptide. This could result in either the inability for particular kinases to recognize peptide substrates or conversely recognition and modification of peptide substrates by inappropriate kinases. Furthermore, this experimental approach may offer opportunity for kinases to modify targets they wouldn’t encounter in a physi-ological setting due to patterns of expression or higher order organization within the cell. For this reason it is important that the arrays are applied as a tool to suggest patterns of phosphorylation that are validated through independent techniques. In this report arrays results are validated through a number of approaches, including phosphorylation-specific antibodies, quantification of enzyme activity, and characterization of a cellular phe-notype. Similar approaches should enable validation of virtual any event or process suggested by array data.

Despite these potential limitations the arrays were able to accurately describe signaling events within bovine monocytes following stimulation by agonists for distinct TLRs. TLR4 and TLR9 are known to share con-served signaling pathways but also appear to activate receptor-specific responses (9). This is consistent with emerging evidence that leukocytes display distinct cel-lular responses to each TLR agonist (41). Our kinome analysis confirmed that TLR4 and TLR9 activation in bovine monocytes involved a conserved pathway of signal transduction (Figure 2) consistent with previous analyses in other species and cell types (Figure 3). This also supports the conclusion of a relatively high level of accuracy when annotating orthologous genes in the

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Comparative analysis of Toll-like receptor signaling 309

bovine genome. Kinome analysis by the bovine peptide arrays also revealed a substantial component of cell signaling unique to each TLR as well as identification of phosphorylation events not previously implicated in TLR signaling.

Detection of a novel TLR signaling protein

In addition to confirmation of the known TLR pathway, modifications not previously ascribed to TLR signaling were also detected. For example, phosphorylation of Tyr40 of Etk as a result of LPS, but not CpG, stimula-tion was suggested by the array and subsequently con-firmed with phospho-specific antibody. The role of Etk in TLR signaling is not known, but it may provide a link between the TLR pathway and the downstream effects of phospholipase C (PLC), which is activated by Btk fam-ily members of which Etk is a member. PLC activation by TLR4 has been linked to an inducement of phagocy-tosis (42) and may be one of the ways that TLR4 alters immune activity during infection.

Proteins that undergo phosphorylation at multiple sites

A considerable advantage of the arrays is their ability to independently and simultaneously monitor protein number of phosphoacceptor sites of the same protein. Such an approach provides a more descriptive repre-sentation of how complex patterns of phosphorylation enable numerous phenotypically distinct isomers of a single protein. For example, the arrays confirmed prerequisite modification of IRAK1 at Thr209 for TLR-induced signal transduction but also suggest that phosphorylation- mediated regulation of IRAK1 is com-plex and multifaceted, involving aspects of regulation through phosphorylation that are independent of TLR activation (Thr100 and Thr387), conserved for various TLRs (Ser568), and specific to particular TLRs (Ser376).

Quantitative analysis of array data

In bovine monocytes, LPS stimulation increased phos-phorylation of the Thr197 peptide nearly 8-fold, whereas stimulation with CpG resulted in a 4-fold increase. The magnitude of increased phosphorylation of these pep-tides appears to correlate with the extent of activation of PKA. This indicates, at least for this example, that array data can be used for relative quantitative prediction of the associated phenotype. Such detailed interpretation of the data must be tempered with the appreciation that different proteins may have different sensitivities in terms of fold changes in phosphorylation required to initiate a particular response. Furthermore, such comparative analysis should be restricted to the extent

of phosphorylation of a given peptide under differ-ent conditions to avoid peptide-specific variables that contribute to the overall extent of phosphorylation of a particular peptide such as differences in efficiencies in recognition and modification of the peptides by the associated kinases.

Phenotypes involving phosphorylation-mediated regulation of multiple proteins

In attempting to predict cellular responses from kinomic data, it is important to consider that many phe-notypes reflect the contributions of numerous individu-ally regulated proteins. Changes in phosphorylation of a particular site may therefore contribute, but not encapsulate, the final functional output. For example, a cursory look at the kinomic data would predict com-parable activation of oxidative burst based on similar magnitudes of increased phosphorylation of the key regulatory site of p47PHOX. Understanding oxidative burst requires a broader perspective that includes regulation of p40PHOX, which in the phosphorylated state serves to inhibit oxidative burst. Following CpG, but not LPS, stimulation there is decreased phosphorylation at this corresponding peptide sequence that eliminates the suppressive effect of the protein on oxidative burst that was confirmed through functional assays. Induction of a superoxide response is consistent with the role of TLR9 activation in promoting destruction of intracel-lular pathogens.

Understanding innate immunity through cellular signaling events

While the species-specific arrays offer valuable oppor-tunity for species-comparative analysis this should be performed with the following considerations in mind. Because responses to a particular ligand can be species-specific, it is important that these natural variances in biology are considered when performing comparative analysis as well as selecting animal models of human disease. An example specific to ligands considered in this investigation of cellular responses to LPS can vary depending on both the species as well as the source of LPS (43). Furthermore, application of the arrays for species-comparative analysis should consider how spe-cies-dependent levels of expression of kinases and their targets impact the biological significance of the results. Species-specific differences in levels of expression of a particular kinase will impact the extent of phospho-rylation of the corresponding target. This would be an accurate reflection of the differing biology between two species. If, however, the array offers the opportunity for a kinase to modify a peptide target corresponding to a protein that is not expressed in a particular species then

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these results would reflect an artifact of the technology. This again emphasizes the importance of validation of array results.

Through this study we have observed the ability of the innate immune system to tailor its response based on specific host-pathogen interactions. The TLR4 stimulant LPS induces specific responses that indicate its nature as an extracellular ligand such as possible phagocytosis via PLC signaling. Likewise, the TLR9 stimulant CpG induces intracellular responses such as the production of superoxide radicals. The adaptation of the innate immune system to specific pathogens is obviously important for effective elimination of infec-tion. Understanding these specific responses as well as their regulation through kinome analysis opens the potential to alter immune response by altering kinase activity through drug therapy. This may lead to the development of immune response/pathogen-specific disease treatment with minimal adverse effects.

Acknowledgements

The authors recognize the funding support provided by Genome Canada and the resources provided by Merial Canada, Inc.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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