Quantitative Phosphoproteomic Analysis of Signaling Downstream

Article pubs.acs.org/jpr

Quantitative Phosphoproteomic Analysis of Signaling Downstream of the Prostaglandin E2/G-Protein Coupled Receptor in Human Synovial Fibroblasts: Potential Antifibrotic Networks Casimiro Gerarduzzi,*,†,‡ QingWen He,‡ John Antoniou,§ and John A. Di Battista†,‡ †

Department of Experimental Medicine, McGill University, 687 Pine Avenue West, Montreal, Quebec H3A 1A1, Canada Department of Medicine, McGill University and the Division of Rheumatology, Royal Victoria Hospital, McGill University Health Centre, 687 Pine Avenue West, Montréal, Quebec H3A 1A1, Canada § Department of Orthopaedic Surgery, Jewish General Hospital, 3755 Cote Ste. Catherine Road, Montréal, Quebec H3T 1E2, Canada ‡

S Supporting Information *

ABSTRACT: The Prostaglandin E2 (PGE2) signaling mechanism within fibroblasts is of growing interest as it has been shown to prevent numerous fibrotic features of fibroblast activation with limited evidence of downstream pathways. To understand the mechanisms of fibroblasts producing tremendous amounts of PGE2 with autocrine effects, we apply a strategy of combining a wide-screening of PGE2-induced kinases with quantitative phosphoproteomics. Our large-scale proteomic approach identified a PKA signal transmitted through phosphorylation of its substrates harboring the R(R/X)X(S*/T*) motif. We documented 115 substrates, of which 72 had 89 sites with a 2.5-fold phosphorylation difference in PGE2-treated cells than in untreated cells, where approximately half of such sites were defined as being novel. They were compiled by networking software to focus on highlighted activities and to associate them with a functional readout of fibroblasts. The substrates were associated with a variety of cellular functions including cytoskeletal structures (migration/ motility), regulators of G-protein coupled receptor function, protein kinases, and transcriptional/translational regulators. For the first time, we extended the PGE2 pathway into an elaborate network of interconnecting phosphoproteins, providing vital information to a once restricted signalosome. These data provide new insights into eicosanoid-initiated cell signaling with regards to the regulation of fibroblast activation and the identification of new targets for evidenced-based pharmacotherapy against fibrosis. KEYWORDS: Prostaglandin E2, synovial fibroblasts, phosphosubstrate, cell signaling

■

INTRODUCTION Fibrosis is a complex chronic disease characterized by a persistent repair response. All tissues and organ systems are susceptible to fibrosis, and nearly 45% of all deaths in the developed world have an element of chronic fibroproliferative disease.1 Its pathogenesis is poorly understood but it is known to require the activity of TGFß and extracellular matrix (ECM) tension, which drive the activation of fibroblasts to transition into the effector cells of repair known as myofibroblasts (fibroblast to myofibroblast transition, FMT). The regulation of myofibroblast differentiation is a critical control point between a normal and pathological tissue repair response. In fibrotic diseases, this delicate signaling network becomes deregulated by causes that remain ill-defined, leading to the accumulation of myofibroblasts and their propagating influences on fibrosis. The presently known molecular events of myofibroblast differentiation have provided us with an understanding of numerous factors that promote and maintain this tissue repair phenotype, such as major cytoskeletal rearrangements, formation of alpha-smooth muscle actin (α-sma)-rich stress fibers, © 2014 American Chemical Society

and mature focal adhesions (FAs), all of which contribute to high levels of cytoskeletal contraction for wound closure. Prominent signaling pathways that support myofibroblastic differentiation include TGFβ-dependent signaling, Wnt/ β-catenin, Ras/Raf/MAP kinase and Rho-Associated Kinase (ROCK)-dependent pathways.2 Resolution of the myofibroblast phenotype is vital to prevent continual wound repair; otherwise, its continual contraction and deposition of ECM proteins substantiate tissue fibrosis. However, few factors have been identified to inhibit myofibroblast differentiation and even less is known regarding the signaling pathways that they undergo. One antifibrotic mediator that has recently been receiving attention is Prostaglandin E2 (PGE2), an autocrine and paracrine Special Issue: Proteomics of Human Diseases: Pathogenesis, Diagnosis, Prognosis, and Treatment Received: May 22, 2014 Published: September 16, 2014 5262

dx.doi.org/10.1021/pr500495s | J. Proteome Res. 2014, 13, 5262−5280

Journal of Proteome Research

Article

L-161982, a specific EP4 antagonist, and sulprostone were products of Cayman Chemical (Ann Arbor Michigan), while L-902688, a specific EP4 agonist, was a gift from Merck Frosst Canada (Pointe-Claire, Quebec). Sodium dodecyl sulfate (SDS), acrylamide, bis-acrylamide, ammonium persulfate, and Bio-Rad protein reagent originated from Bio-Rad Laboratories Canada (Mississauga, Ontario, Canada). Tris-base, EDTA, MgCl2, NaCl, CaCl2, chloroform, dimethyl sulfoxide (DMSO), anhydrous ethanol (95%), methanol (99%), formaldehyde, and formamide were obtained from Fisher Scientific (Nepean, Ontario, Canada). Dulbecco’s Modified Eagle Medium (DMEM, Gibco), phosphate-free DMEM, Trizol reagent, heat-inactivated fetal bovine serum (FBS), an antibiotic mixture [10 000 units of penicillin (base), 10 000 μg of streptomycin (base)], phosphate-buffered saline (PBS), and tetramethylethylenediamine (TEMED) were products of Invitrogen (Burlington, Ontario, Canada).

bioactive lipid derived from arachidonic acid (AA) by its key regulatory enzymes COX-1 and -2. PGE2 can also regulate many cellular functions partly because it signals through four distinct G-protein-coupled receptors (GPCRs) with different signaling pathways and diverse expression patterns on various cell types.3 These E prostaglandin (EP) receptors are designated EP1, EP2, EP3, and EP4, and each one’s downstream function is a reflection of its coupled intracellular signaling machinery: EP2 and EP4 receptors increase cAMP levels to activate protein kinase A (PKA); EP1 activates phospholipase C (PLC) to increase intracellular Ca2+ levels; and EP3 is predominantly known to decrease cAMP levels.3 PGE2-activated EP receptors influence a broad range of physiological and pathophysiological (e.g., cancer, arthritis) functions in essentially all organ systems through actions at the cellular/molecular level that regulate cell-cycle, cell structure/morphology, cell proliferation, and migration. The wide assortment of PGE2 functions are central to normal tissue homeostasis,4 particularly believed to have a primary role in tissue repair. Specifically, PGE2 is known to potently inhibit the FMT. However, this signaling mechanism remains elusive granted that our knowledge is limited to the proximity of its receptors. For this reason, we have decided to expand our current knowledge on the antifibrotic agent. In fact, the importance of this PGE2 counter-effect for the prevention and treatment of fibrotic diseases is emphasized by the impaired PGE2 synthesis and COX2 expression in fibroblasts isolated from human and animal fibrotic lung models5−9 and by the protective effect that PGE2 overproduction has against fibrosis.10 Furthermore, PGE2 has been shown to downregulate myofibroblast activation.11−13 Indeed, there is substantial evidence that reports reduced levels of PGE2 in fibrotic patients,5,6,14 which may be pertinent to the propagation of fibrosis. Therefore, understanding the PGE2 signaling network that is responsible for inhibiting myofibroblast differentiation will expand our understanding of endogenous antifibrotic events as well as enlighten new targets for prevention and treatment of fibrotic diseases. Our laboratory has designed a strategy of combining kinasemodified motif antibodies and mass spectrometry analysis to decipher the downstream signaling pathways through which the antifibrotic mediator PGE2 prevents a myofibroblast phenotype outcome. By quantitatively detecting the changes of molecules in various pathways, we can monitor the functional patterns that these pathways converge for an outcome. This paper describes a draft of the complete PGE2-dependent phosphoproteome/Gαs signalosome in fibroblast-like cell phenotypes (by PhosphoScan, Cell Signaling) that includes proteins associated with cellular differentiation and cytoskeletal/ adhesion structures. Furthermore, we identified the specific amino acid sequence of novel phosphorylation sites, vital information toward the control process of a protein’s activity, location, and stability. With this in mind, our current mapping of the intricate pathways of PGE2 that stem from their receptors will help us understand its antifibrotic effects so as to extract them for future studies in therapeutic development.

■

Specimen Selection and Cell Culture

Human (synovial) fibroblasts (HSFs) were chosen as our cellular model because they synthesize large amounts of PGE2 when activated, express all four subtypes of EP receptors, and are clinically relevant to the extent that they are effectors/modulators of inflammatory/immune responses in chronic diseases/ conditions with a strong inflammatory component.15−18 Synovial lining cells (HSFs) were isolated from synovial membranes (synovia) obtained at necropsy from donors with no history of arthritic disease (mean age 30 ± 27). HSFs were released by sequential enzymatic digestion with 1 mg/mL Pronase (Boehringer Mannheim, Laval, Quebec, Canada) for 1 h, followed by 6 h with 2 mg/mL collagenase (type IA, SigmaAldrich) at 37 °C in DMEM supplemented with 10% heatinactivated FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin. Released HSFs were incubated for 1 h at 37 °C in tissue culture flasks (Primaria #3824, Falcon, Lincoln Park, NJ), allowing the adherence of fibroblastic cells possibly present in the synovial preparation. In addition, flow cytometric analysis (Epic II, Coulter, Miami, FL) using the anti-CD14 (fluorescein isothiocyanate, FITC) antibody was conducted to confirm that no monocytes/macrophages were present in the synovial fibroblast preparation.15,16 The HSFs were CD45-negative and expressed no epithelial (e.g., EpiCAM) or endothelial markers but produced large amounts of hyaluronan and expressed high levels of hyaluronan synthase 2, VCAM-1, fibroblast-specific protein (FSP-1, S-100), prolyl-4-hydroylase, and collagen type I α1 chain.19,20 Fibroblast-specific proteins were detected by Western blotting, FACS analysis for surface markers, or immunocytochemistry. The cells were seeded in tissue culture flasks and cultured until confluence in DMEM supplemented with 10% FBS and antibiotics at 37 °C in a humidified atmosphere of 5% CO2/95% air. Additionally, HSFs were incubated in fresh medium containing 0.5 to 1% FBS for 24 h before the experiments; only second or third passaged HSFs were used. HEK293T cells were purchased from American Type Culture Collection (ATCC, Rockville, MD) and were grown in DMEM supplemented with 10% FBS, penicillin (100 units/mL), and streptomycin (100 μg/mL) at 37 °C in a humidified atmosphere with 5% CO2/95% air.

MATERIALS AND METHODS

Chemicals

Plasmids and Transfection Experiments

Sodium fluoride, leupeptin, aprotinin, pepstatin, phenylmethylsulfonylflouride (PMSF), actinomycin D, dithiothreitol (DTT), sodium orthovanadate, DAPI, phalloidin, Y27632, and bovine serum albumin (BSA) were products of Sigma-Aldrich Canada (Oakville, Ontario, Canada). Prostaglandin E2, butaprost,

Transient transfection experiments for reporter analysis were conducted in 6−12 well cluster plates using HEK cells and FuGENE6TM (Roche Applied Science, Indianapolis, IN) or Lipofectamine 2000 (Invitrogen) according to the 5263



Article

manufacturers’ protocols with cells at 30−40% confluence. Cells were re-exposed to a culture medium with 1% FBS for 2 h prior to the addition of the biological effectors. Transfection efficiencies were controlled in all experiments by cotransfection with a pHSV-TK-driven Renilla luciferase construct (Promega, Madison, WI). Luciferase values, expressed as enhanced relative light units, were measured in a Lumat LB 9507 luminometer (EG&G, Stuttgart, Germany) and normalized to the levels Renilla luciferase activity and cellular protein (bicinchoninic acid procedure; Pierce). In signal transduction pathway reporting systems (Stratagene/Agilent Technologies, Mississauga, ON), a reporter plasmid, containing the 17 bp (5×) Gal4 DNA binding element (UAS) fused to a TATA box upstream from the luciferase gene (pFR-LUC) was cotransfected with a construct containing the trans-activation domains of β-catenin (e.g., Gal4-β-Cat or Gal4-β-Ca (S552A) mutant) fused to GAL4 DNA binding domain and driven by a CMV promoter. The full-length Ras/Rab interactor/inhibitor 1 (RIN1) (GenBank Accession No. Q13671) expression construct was purchased from OriGene Technologies (Rockville, MD) and was originally inserted into Not1 sites of the pcmv6-XL5 vector. The S291A (Ser → Ala), S292A, and the double-mutant 291/ 292 were constructed from the wildtype (wt) RIN1 expression vector using the QuikChange kit (Stratagene, La Jolla, CA). The following primer set was used for developing the double mutant: sense, 5′-GCC AGC TGC TAC GGC GGG AGG CCG CAG TGG GGT ACC GCG TGC C-3′; antisense, 5′-GGC ACG CGG TAC CCC ACT GCG GCC TCC CTC CGT AGC AGC TGG C-3′. Base-pair substitutions were verified by double DNA sequencing.

Tandem mass spectra were collected with an LTQ-Orbitrap XL hybrid mass spectrometer using a top-ten method, dynamic exclusion repeat count of 1, and a repeat duration of 30 s. Realtime recalibration of mass error was performed using lock mass, which is used to calibrate the mass measurements on a scan-byscan basis and help maintain the performance to within ±3 ppm precision. MS spectra were collected in the Orbitrap component of the mass spectrometer and MS/MS spectra were collected in the LTQ. The raw data associated with this manuscript may be viewed from Peptide Atlas online repository under the identifier code: PASS00232. All of the MS2 spectrum numbers are available in Suppl. Table 2 in the Supporting Information. MS/MS spectra were evaluated using TurboSequest in the Proteomics Browser Package (v. 27, rev. 13) and the following parameters: peptide ion mass tolerance, 50 ppm; fragment ion mass tolerance, 1.0 Da; maximum number of differential amino acids per modification, 4; parent ion mass type, monoisotopic; fragment ion mass type, monoisotopic; maximum number of internal cleavage sites, 4; neutral losses of water and ammonia from b and y ions were considered in the correlation analysis, and the proteolytic enzyme was specified. Datadependent acquisition parameters were as follows: the mass resolution for MS1 scans was 60 000 resolution (at 400 m/z) and the scan range was 365−1800 m/z; the relative collision energy for tandem-MS was 27.0 eV. Samples were run in duplicate on the instrument. Searches were performed against the NCBI human database, released on February 13, 2014 and containing 55 565 protein sequences, in both forward and reverse sequence directions. Such searches were validated using the UniProt human database, released on July 7, 2014 and containing 157 107 sequences. Using the PhosphoSite database (www.phosphosite. org), phosphosites published in the literature were labeled as reported, while unpublished phosphosites were labeled as novel. Enzyme specificity was limited to trypsin, with at least one tryptic (Lys-or Arg-containing) terminus required per peptide and up to four missed cleavages allowed. Cysteine carboxamidomethylation was specified as a static modification, oxidation of methionine residues was allowed, and phosphorylation was allowed on serine and threonine residues only. The justification for the peptide identifications is based on the XCorr values and the DeltaCN values provided by the SEQUEST search engine. The false-positive assignment rate was approximated by taking the ratio of the reversed database assignments to the forward database assignments after filtering the initial SEQUEST search results for valid assignments based on XCorr (>1.5), mass accuracy (±5 ppm) and on the presence of the expected phospho (Ser/Thr) PKA sequence motif R(R/X)X(S*/T*). The mass accuracy range was narrowed further based on the XCorr-mass error plot for each experiment. The average false-positive assignment obtained from the SEQUEST search of the four LC−MS/MS experiments in this study was determined to be 3% for those phosphopeptides containing the basophilic motif.24 Mass spectrometric data from the supplemental table in the Supporting Information was manually screened for R(R/X)X(S*/T*) authenticity, and a minimum 2.5-fold difference between treatments was used to produce the data in Table 1, providing information regarding the relative quantification of phosphorylation. Although the A-Scores are provided in Suppl. Table 2 in the Supporting Information, the additional criterion of the sequence motif, as defined by the motif antibody, is also an important determinant for the confidence of the identification. A cutoff of 2.5 fold was set to indicate a significantly increased abundance of phosphorylated

Sample Preparation and Phosphoscan Proteomic Analysis

Samples were processed using the PhosphoScan proteomics platform as previously described.21−25 Cellular protein was extracted into Urea Lysis Buffer (20 mM HEPES, pH 8.0, 9.0 M urea, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, and 1 mM beta-glycerol-phosphate, http:// www.cellsignal.com/services/pdfs/PTMScan_Proteomics_ System_Protocol.pdf) from control and 10 min PGE2-treated HSF, sonicated at 15W output for 25 s, and centrifuged 15 min at 20 000g to remove insoluble material. Samples were sent on dry ice to Cell Signaling Technology for PhosphoScan analysis using the phospho-PKA substrate motif antibody, RRX(S*/T*) (#9624, * denotes phosphorylation). Equal amounts (16.0 ± 0.2 mg) of total cleared proteins from treated and untreated samples were reduced, carboxamidomethylated, and digested with endoproteinase GluC. Peptides were separated from nonpeptide material by solid-phase extraction with Sep-Pak C18 cartridges. Lyophilized peptides were redissolved, and phospho-peptides were isolated using a slurry of immobilized phosphorylated PKA substrate motif rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA, #9624). Peptides were eluted from the antibody-coupled resin into a total volume of 100 μL in 0.15% TFA. Eluted peptides were concentrated with PerfectPure C18 tips and digested using ProteoGenBioDigest tips (10 μL) immediately prior to LC−MS analysis. The immunoprecipitation is performed with the GluC peptides to preserve the basophilic motif for antigen recognition. The post-IP digestion with trypsin produces peptides that are more favorable for LC−MS analysis. Peptides were loaded onto a 10 cm × 75 μM PicoFrit capillary column packed with Magic C18 AQ reversed-phase resin. The column was developed with a 45 min gradient of acetonitrile in 0.125% formic acid delivered at 280 nL/min. 5264


EI24

ANKRD27 ARF GAP1

ARHGEF17

ARHGEF17

DOCK7 iso2 DOCK7 iso3 RGL2

RGL2

ANKRD27 ARFGAP1

ARHGEF17

ARHGEF17

DOCK7 DOCK7 RGL2

RGL2

navl iso2

NAV1

EI24

nav1 iso2

NAV1

FLNA FLNA lamin A/C iso2 RIL WNK1 ROCK2

nav1 iso2

NAV1

FLNA FLNA LMNA PDLIM4 WNK1 ROCK2

RC3 Mena iso2 GAB1 Nck1 AKAP2 Tks5 tensin 3 CTNNB1

DMXL2 ENAH GAB1 NCK1 PALM2-AKAP2 SH3PXD2A TNS3 CTNNB1

HSP20

AP3D1 iso5

AP3D1

HSPB6

AHNAK GIT2

Protein Name

AHNAK GIT2

Gene Name

5265

ankyrin repeat domain 27 (VPS9 domain) ADP-ribosylation factor GTPase activating protein 1 isoform a Rho guanine nucleotide exchange factor (GEF) 17 Rho guanine nucleotide exchange factor (GEF) 17 dedicator of cytokinesis 7 dedicator of cytokinesis 7 ral guanine nucleotide dissociation stimulator-like 2 ral guanine nucleotide dissociation stimulator-like 2

§1023 §361

738

439 440 736

1725

1717

etoposide-induced 2.4 isoform 2

heat shock protein, alpha-crystallin-related, B6 filamin A, alpha isoform 2 filamin A, alpha isoform 2 lamin A/C isoform 2 PDZ and LIM domain 4 isoform 2 WNK lysine deficient protein kinase 1 Rho-associated, coiled-coil containing protein kinase 2

neuron navigator 1

neuron navigator 1

AHNAK nucleoprotein isoform 1 G protein-coupled receptor kinase interacting ArfGAP 2 adaptor-related protein complex 3, delta 1 subunit isoform 1 Dmx-like 2 enabled homologue isoform a GRB2-associated-binding protein 1 NCK adaptor protein 1 PALM2-AKAP2 protein isoform 1 SH3 multiple domains 1 tensin 3 catenin (cadherin-associated protein), beta 1, 88 kDa neuron navigator 1

Description

65

§2152 §2336 10 §120 §60 1379

§16

964

453

§452

326 §265 246 §85 1083 §1016 944 §552

§158 579, 586 §758

Site

Table 1. PGE2-Induced Phospho-R(R/X)X(S*/T*) Motif Proteinsa

RAPS*VANVGSHCDLSLKIPE RLT*VSSLQE RAT*RSGAQASSTPLSPTR IQDGS*PTTSRRPS*GTGTGPE RHT*MDKDSRGAAATTTTTE RPS*RQLAPNKPS

RAS*APLPGLSAPGR

RHS*HTIGGLPE

RNST*IVLRTDSE

RNS*TIVLRTDSE

RSS*VLVTHAE RIS*SAAAPASVE RIS*SAAPSSDSSLYNAPLPE RKPS*VPDSASPADDSFVDPGER RKS*ALALRWE RNS*SFSTAR ADASSTPSFQQAFASSCTISSNGPGQRRESSSS*AE RTS*M#GGTQQQFVE

RRHS*SLPTES*DEDIAPAQQVDIVTE

Cytoskeletal/Adhesion RVT*AYTVDVTGRE RAS*RLEKQNS*TPE

Peptide

O15211

Q96N67-2 Q96N67-3 O15211

Q96PE2

Q96PE2

O14681

RRSST*ATPGVTSGPSASGTPPSE

RNS*S*IVGRRSLE RRNSS*IVGR RRS*STATPGVTSGPSASGTPPSEGGGGSFPR

RSSHGSFTRGS*LE

RSS*HGSFTRGSLE

Apoptosis RA5*SVLAQR G Protein or Regulator Q96NW4 RHT*VEDAVVSQGPE Q8N6T3 KSPSSDSWTCADTSTERRSS*DSWE

P21333 P21333 P02545-2 P50479 Q9H4A3 O75116

Q8NEY12 Q8NEY12 Q8NEY12 O14558

Q8TDJ6 Q8N8S7 Q13480 P16333 Q9Y2D5-4 Q5TCZ1 Q68CZ2 P35222

O14617-5

Q09666 Q14161

Accession

756.3485

767.3506 562.7881 994.7953

790.8166

790.8166

802.3595 948.0472

534.2718

744.0281 556.7713 641.9851 720.9705 710.6479 477.5807

715.3695

642.2985

490.9035

735.8513

589.7822 619.7927 1071.4914 803.6958 437.2311 553.2432 1224.8554 782.8270

985.1044

773.8670 838.3639

Calc. m/z

221 920

365 818 59 321 151 566

159 251

159 251

767 066 1 430 647

951 388

22 704 187 24 345 786 12 511 709 4 061 306 98 622 1 217 349

46 886 030

804 932

2 275 971

9 078 134

313 959 832 110 1 102 051 343 873 1 657 370 55 443 259 031 174 269

697 399

17 612 019 63 316

Ave. Intensities +PGE2

20 000

20 000 20 000 20 000

20 000

20 000

141 746 392 586

21 317

7 855 288 556 217 1 995 174 245 505 39 801 39 312

1 467 758

99 847

88 951

276 308

22,392 37 742 414 211 41 881 165 515 20 000 31 832 58 229

44 491

473 966 20 000

Ave. Intensities Untreated

0.039 0.039

8.0 8.0

0.005 5 × 10−4

18.3 3.0 7.6 11.1

2 × 10−6

0.003 0.014

2 × 10−4

0.01 2 × 10−5 0.01 8 × 10−5 0.053 0.014

0.001

0.007

0.018

0.003

0.006 0.003 0.263 0.027 0.005 0.107 0.006 0.003

0.015

0.032 0.105

P Value

5.4 3.6

44.6

2.9 43.8 6.3 16.5 2.5 31.0

31.9

8.1

25.6

32.9

14.0 22.0 2.7 8.2 10.0 2.8 8.1 3.0

15.7

37.2 3.2

+PGE2: Untreated

Journal of Proteome Research Article


5266

Cdc27 CLASP1 iso3 NUDE1 iso2

NuMA-1 OFD1 cytospin-A TNKS1BP1 TNKS1BP1

BIRC6 RUFY1

S6 S6

DAPK2 QSK iso4 HGK iso7

KHS1

PKD1 PANK2 PANK2 Kidins220

PDE3B PDE3B PLCL1 PLCL1

MTMR5 CUTL1 NDRG1 YAP1;YAP1 iso2 YAP1; YAP1 iso2 NFAT3

CDC27 CLASP1 NDE1

NUMA1 OFD1 SPECC1L TNKS1BP1 TNKS1BP1

BIRC6 RUFY1

RPS6 RPS6

DAPK2 KIAA0999 MAP4K4

MAP4K5

PRKD1 PANK2 PANK2 KIDINS220

PDE3B PDE3B PLCL1 PLCL1

SBF1 CUX1 NDRG1 YAP1

NFATC4

YAP1

TBC1D15 iso2

Protein Name

TBC1D15

Gene Name

Table 1. continued

death-associated protein kinase 2 KIAA0999 protein mitogen-activated protein kinase kinase kinase kinase 4 isoform 1 mitogen-activated protein kinase kinase kinase kinase 5 protein kinase D1 pantothenate kinase 2 isoform 2 pantothenate kinase 2 isoform 2 kinase D-interacting substrate of 220 kDa

§299 §411 §921

SET binding factor 1 isoform a cut-like homeobox 1 isoform b N-myc downstream regulated 1 yes-associated protein 1, 65 kDa isoform 1 yes-associated protein 1, 65 kDa isoform 1 nuclear factor of activated T-cells

1747 §1215 §330 §127

§109

§272

§295 §296 77 §93, 98

§205 §168 §169 1315 phosphodiesterase 3B, cGMP-inhibited phosphodiesterase 3B, cGMP-inhibited phospholipase C-like 1 isoform a phospholipase C-like 1 isoform a

ribosomal protein S6 ribosomal protein S6

§235 §236

433

baculoviral IAP repeat-containing 6 RUN and FYVE domain-containing 1 isoform b

nuclear mitotic apparatus protein 1 oral-facial-digital syndrome 1 SPECC1-like tankyrase 1-binding protein 1 tankyrase 1-binding protein 1

§1969 735 §832 §1545 §1554

425 74

cell division cycle protein 27 isoform 1 CLIP-associating protein 1 nuclear distribution gene E homolog 1

TBC1 domain family, member 15

Description

379 §646 §307

§70

Site G Protein or Regulator VIVDWRPLDDALDSSSILYARKDS*SSVVE

Peptide

Q14934

Q7Z574

O95248 P39880 Q92597 Q7Z574

Q13370 Q13370 Q15111 Q15111

Q15139 Q9BZ23 Q9BZ23 Q9ULH0

Q9Y4K4

Q9UIK4 A1A5A8 Q53TX8

P62753 P62753

RYS*SSGTPSSASPALSR

SHSRQAS*TDAGTAGALTPQHVR

Dif ferentiation QAARRST*STLYSQFQTAE RRHS*SVSDSQPCEPPSVGTE TAS*GSSVTSLDGTR AHS*SPASLQLGAVSPGTLTPTGVVSGPAATPT

RLS*NVSLTGVSTIR RAS*SASVPAVGASAE RRASS*ASVPAVGASAE RAS*HNELPHTE Phosphodiesterase RRS*SCVSLGE RRSS*CVSLGE RSS*IIKDPSNQK KKT*VSFSS*MPSEK

RQS*SPSCGPVAE

Translation RLS*S*LRASTSKSE RLS*S*LRAS*TSKSE Kinase ALRHPWITPVDNQQAM#VRRES*VVNLE RHT*VGVADPRTE KGS*VVNVNPTNTRPQSDTPE

Cell Cycle Regulation P30260 RSS*RLFTSDSSTTKE Q7Z460-3 TRRQS*SGSATNVASTPDNR Q9NXR1DGGERRPSS*TSVPLGDKGLDTSCR 2 Q14980 RAS*MQPIQIAE O75665 RLS*STPLPK Q69YQ0 RSS*TSSEPTPTVK Q9C0C2 WSDQGPAQTSRRPS*QGPPARSPSQDFSFIE Q9C0C2 WSDQGPAQTSRRPSQGPPARSPS*QDFSFIE Ubiquitin Conjugating System Q9NR09 INAYDPAIVQQLILSGDPSS*GVDSRRPT*LAWLE Q96T51 RAT*GNLSASCGSALR

Q8TC072

Accession

895.9073

602.7685

1102.9725 764.6622 709.8118 904.4640

655.7519 655.7519 726.8642 808.3458

791.9195 720.3302 838.3639 685.7963

677.7768

789.3963 473.2247 740.6816

527.9063 554.5617

1247.9360 800.8670

662.3102 539.7867 728.8379 1132.8517 1159.5071

891.4072 695.6542 876.4012

1105.8767

Calc. m/z

194 853

282 874

573 419 105 781 127 118 946 453

192 236 192 236 199 268 211 648

392 017 6 950 673 350 076 135 267

833 464

471 648 4 790 274 755 081

4 323 716 1 769 746

252 580 451 827

1 075 609 562 541 1 449 686 892 772 4 022 377

206 634 77 377 198 324

628 131


45 810

1 008 462

20 000 27 926 405 541 2 470 564

20 000 20 000 20 000 20 000

137 740 2 294 219 37 144 20 000

88 398

67 402 1 870 061 28 404

861 964 1 502 350

37 929 74 817

73 307 31 329 118 218 265 804 582 225

20 000 20 000 17 912

20 000


4.3

−3.6

28.7 3.8 −3.2 −2.6

9.6 9.6 10.0 10.6

2.8 3.0 9.4 6.8

9.4

7.0 2.6 26.6

5.0 1.2

6.7 6.0

14.7 18.0 12.3 3.4 6.9

10.3 3.9 11.1

31.4

+PGE2: Untreated

0.003

0.013

0.002 0.004 0.015 0.021

0.048 0.048 0.034 0.009

0.024 0.005 0.013 0.075

0.061

0.023 0.009 0.009

8 × 10−4 0.39

0.003 0.024

0.014 0.039 0.005 0.011 0.005

0.019 0.003 1 × 10−5

0.028

P Value



5267

TMCC1

TNFRSF19

TMCC1

TNFRSF19

241

64

63

975 §411

654

352

14 §196 433 39 40 398 10 §62 §602 72 4262 188

103 §412 70 §365 §366 572

291 §292

§552

Site

breast carcinoma amplified sequence 3 hypothetical protein LOC54785 hypothetical protein LOC151963 choline/ethanolamine kinase isoform a choline/ethanolamine kinase isoform a CNKSR family member 3 CUE domain-containing 1 hypothetical protein LOC116224 niban protein isoform 2 hypothetical protein LOC9764 hypothetical protein LOC84162 PREDICTED: similar to dual specificity phosphatase 22 leucine-rich repeats and calponin homology (CH) domain containing 3 pleckstrin homology-like domain, family B, member 1 fibrocystin L TBC1 domain family, member 9B (with GRAM domain) isoform b transmembrane and coiled-coil domain family 1 isoform a transmembrane and coiled-coi1 domain family 1 isoform a tumor necrosis factor receptor superfamily, member 19

cyclic AMP phosphoprotein, 19 kDa general transcription factor II, i isoform 4 nuclear receptor corepressor 1 polymerase I and transcript release factor polymerase I and transcript release factor hypothetical protein LOC91748

catenin (cadherin-associated protein), beta 1, 88 kDa ras inhibitor RIN1 ras inhibitor RIN1

Description

Q9NS68

O94876

Q94876

QS6WI1 Q66K14-2

QS6UU1

Q9H6U6 Q96GS4 Q8IYB1 Q9Y259 Q9Y259 Q6P9H4 Q9NWM3 Q96E09 Q9BZQ8 O60268 Q2LD37 Q9NRW42 Q96II8

P56211 P78347 O75376 Q6NZI2 Q6NZI2 Q6PJG2

Q13671 Q13671

P35222

Accession

RDS*VQTCGPVR

RRSS*VSPHDVQQIQADPEPE

RRS*SVSPHDVQQIQADPEPE

RISVT*R KAS*VVDPSTE

ATAALALAGRRPS*RGLAGASGR

LRRES*QYQE

RES*SVGYRVPAGSGPSLPPMPSLQE RESS*VGYRVPAGSGPSLPPMPSLQE Gene Regulation VTGDHIPTPQDLPQRKPS*LVASKLAG GIPFRRPS*TYGIPRLE RRPS*LLSE RGS*S*PDVHALLE RGS*S*PDVHALLE RRS*TRIPGTDAQAQAE Other Functions RPS*RCTGGVVVRPQAVTE RAT*ISSPLELE RGS*TTSIPSPQSDGGDPNQPDDRLAK RAS*SLSRDAE RASS*LSRDAE RFT*IADSDQLPGYSVE RSSS*GSGGGGTAGARGGGGGTAAPQE RNS*TTFPSR RAS*AILPGVLGSETLSNE SADSENDMGESPSHPSWDQDRRS*SSNE YDMRRLS*E RWS*SFPALAPLTYDNYTTET

Dif ferentiation RTS*MGGTQQQFVE

Peptide

677.8006

1218.0233

785.6960

406.2132 556.7475

540.7936

644.7880

683.6739 648.3160 926.0897 626.2422 626.2422 939.4198 769.6434 573.2589 947.4698 1029.7270 575.2418 1207.0413

702.1223 647.0035 519.2609 720.7999 720.7999 612.9623

893.4267 898.7583

782.8270

Calc. m/z

274 244

214 544

40 204

19 173 970 363 845

3 966 419

109 448

1 671 462 24 633 025 160 426 57 234 57 234 393 515 55 354 253 763 16 773 508 119 188 801 106 528 677

10 205 870 1 562 535 9 762 581 1 642 591 1 642 591 875 485

3 120 803 1 420 687

174 269


32 956

20 000

106 194

686 874 20 468

335 454

20 000

57 870 1 884 911 48 533 22 204 22 204 103 779 20 000 65 765 4 618 797 20 000 309 324 40 755

190 395 319 725 2 138 068 4 114 442 4 114 442 341 891

578 163 295 459

58 229


8.3

0.006

0.001

0.008

−2.6 10.7

0.002 0.059

0.018

0.006

0.002 8 × 10−4 0.061 0.156 0.156 0,006 0.171 0.012 0.002 0.058 0.593 0.007

0.021 0.006 0.014 2 × 10−4 2 × 10−4 0.004

0.002 0.005

0.003

P Value

27.9 17.8

11.8

5.5

28.9 13.1 3.3 2.6 2.6 3.8 2.8 3.9 3.6 6.0 2.6 13.0

53.6 4.9 4.6 −2.5 −2.5 2.6

5.4 4.8

3.0

+PGE2: Untreated

Cultured HSFs were synchronized in DMEM 1% FBS and incubated in the presence or absence of 100 nmol/L PGE2 for 10 min and processed as described in Materials and Methods. Mass spectroscopy results of peptides distinguished by different protein classes (section heads), site of phosphorylation, gene/protein name, accession number, peptide sequence, and MS/MS statistics between PGE2 and untreated cells. Listed are peptide ions with fold-changes above 2.5 or below −2.5. “§” denotes previously reported phosphorylation sites. “*” denotes phosphorylation.

a

TMCC1

TMCC1

LRCH3

LRCH3

PKHD1L1 KIAA0676 iso2

BCAS3 FLJ20014 MB21D2 CHKB CHKB CNKSR3 CUEDC1 C9orf42 C1orf24 KIAA0513 KIAA1109 DUSP22

BCAS3 C17orf59 C3orf59 CHKB CHKB CNKSR3 CUEDC1 FAM122A FAM129A KIAA0513 KIAA1109 DUSP22

PKHD1L1 TBC1D9B

ARPP-19 TFII-I iso2 N-CoR1 PTRF PTRF C14orf43

ARPP-19 GTF2I NCOR1 PTRF PTRF C140rf43

PHLDB1

Rin1 Rinl

RIN1 RIN1

PHLDB1

CTNNB1

Protein Name

CTNNB1

Gene Name

Table 1. continued




Article

protein in PGE2 treated cells.25,29 This data were utilized throughout the rest of the manuscript for functional proteomic analysis and interpretation by means of the Ingenuity Pathway Analysis (IPA) system tools 3.0., which converts large data sets into significant networks containing direct and indirect relationships among proteins based on known interactions in the literature (http://www.ingenuity.com). The association of a set of molecules with a specific IPA functional annotation is based on literature evidence. The inclusion of a particular functional annotation in the analysis results is based on a p value, calculated internally by IPA using Fisher’s Exact Test.

against actin, ROCK2, and S100A4 were products of AbCAM (Cambridge, MA). Immunocytochemistry and Confocal Microscopy

For all fluorescence imaging, a LSM 510 META confocal microscope (Carl Zeiss MicroImaging, Jena, Germany) was employed. Cells were seeded onto sterile coverslips and allowed to grow for 48 h prior to fixation. Following fixation with 4% formaldehyde in PBS, coverslips were incubated in 50 mM NH4Cl in PBS to quench remaining aldehyde groups. After washing three times with PBS, cells were permeabilized using 0.1% saponin in PBS. Nonspecific antibody binding sites were blocked using 0.2% fish skin gelatin in PBS. Cells were incubated with antiphosphopaxillin (Tyr118), or antiphospho PKA substrate motif (RRX(S*/T*)) (Cell Signaling Technologies). Primary antibodies were revealed using Alexa Fluor 488 goat antirabbit and Alexa Fluor 594 chicken antimouse secondary antibodies. Cells were mounted on glass slides using Mowiol mounting medium with antifade agent (1,4diazabicyclo[2.2.2]octane) prior to microscopy. Alexa Fluor 488 was excited by the 488 nm argon ion laser, and the fluorescence was collected using a BP505-530 emission filter, whereas Alexa Fluor 594 was excited by the 543 nm HeNe laser line and the fluorescence was collected using an LP560 emission filter. All images were acquired using a Plan-Apochromat ×63/1.40 oil differential interference contrast objective in sequential scanning (multitrack) mode with the pinholes set to obtain an optical section of ∼0.8 μm in both channels (1 Airy unit).30

Label-Free Quantitation

Using an internal proprietary software from Cell Signaling, relative quantitation was produced from the apex peak height of the corresponding peptide assignments according to previously published protocols.25−29 The basis of the software involves integrating a chromatographic alignment algorithm that performs numerous binary comparisons to generate an overall clustering strategy for the complete data set of all identified peptides by virtue of its accurate mass measurement and its retention time. MS1 peak intensities for identified motif peptides identified in at least one sample were retrieved from the ion chromatogram files of all samples using a mass precision of ∓5 ppm and retention time window of 5 min. Retention time warping (or chromatographic alignment) was performed across binary comparisons to allow retrieval of the correct peak intensity. Peak intensities for all peptides that changed in abundance between treatments were manually reviewed in the ion chromatogram files to ensure accuracy, and where necessary peak height measurements were replaced with peak areas using XCalibur software (Thermo). Manually validated intensities are reported in Suppl. Table 2 in the Supporting Information.

Cell Migration Assay

Cell migration assays were conducted using the Millipore QCMTM 24-well colorimetric cell migration assay according to the manufacturer’s instructions. Briefly, the technique is based on the Boyden chamber principle using 8 μm pore size polycarbonate membranes, which are optimal for epithelial/ fibroblast cells lineages. (0.5 to 1.0) × 106 cells were added to the upper chamber of the membrane insert in serum-free medium, and the latter was lowered into the chemoattractant medium of the outer bottom chamber; cells were allowed to migrate for 24 h at 37 °C in a CO2 incubator (5%/95% air). Nonmigrated cells were removed, membranes were rinsed, and migrated cells were recovered in extraction buffer/dye mixture.

Preparation of Cell Extracts and Western Blotting

50−100 μg of cellular protein extracted into RIPA buffer or hot SDS-PAGE loading buffer, from control and treated cells, was subjected to SDS-PAGE through 10% gels (16 × 20 cm, final concentration of acrylamide) under reducing conditions and transferred onto nitrocellulose membranes (GE Healthcare Amersham Pharmacia Biotech, Piscataway, NJ). Following blocking with 5% BLOTTO for 2 h at room temperature and washing, the membranes were incubated overnight at 4 °C with the primary antibody in Tween-TBS containing 0.25% BLOTTO. The second antirabbit antibody-HRP conjugate (Cell Signaling Technologies, Danvers, MA; 1:2000 dilution) was subsequently incubated with membranes for 1 h at room temperature, then washed extensively for 30−40 min with Tween-TBS at room temperature. Following incubation with an ECL chemiluminescence reagent (Amersham Pharmacia Biotech), membranes were prepared for autoradiography, exposed to Kodak (Rochester, NY) X-Omat film, and subjected to digital imaging system (Alpha G-Imager 2000; Canberra Packard Canada, Mississauga, Ontario, Canada) for semiquantitative measurements. The following rabbit primary antibodies were used: phospho-(Ser) PKC substrate (motif R/K-X-S-Hyd-R/K); phospho-(Ser) MAPK/CDK substrate (motif K/R-S-P-X-K/R); phospho-(Ser/Thr) ATM/ATR substrate (motif Hyd-S/T-Q); phospho (Ser/Thr) PKA substrate (motif R-R-X-S/T); phospho (Ser/Thr) AKT substrate (motif R/K-X-R/K-X-X-S/T); phospho-paxillin (Tyr118); (Cell Signaling Technologies). The rabbit antihuman antibodies

Flow Cytometry

After antibody staining (1 × 105 cells) for 30 min at 4 °C in labeling buffer, the cells were washed and analyzed by flow cytometry using FACS Calibur (Becton Dickinson) and FlowJo version 6.3.4 software. The following fluorochrome-conjugated antibodies were used for surface staining: FITC-conjugated anti-Annexin V, FITC-conjugated anticyclin B, and isotype controls (BioLegend, San Diego, CA). Propidium iodide (PI) was purchased from Sigma (St. Louis, MI). The appropriately labeled isotype control antibodies were used for each cell surface marker.

■

RESULTS

PGE2 Induces Serine/Threonine Phosphorylation of a PKA-Associated Motif

To determine the signaling cascade(s) activated by PGE2 in HSFs, we screened various kinases for their activity by Western Blot analysis using the following kinase specific phospho-motif 5268



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Figure 1. Analysis of PGE2-dependent substrate phosphorylation. (A) Antibody used for Western blot analysis and immunoprecipitation/ immunoaffinity columns that recognizes the PKA-preferred (RRX(S*/T*)) motif, whereby the serine or threonine must be phosphorylated for antibody binding. Cultured human synovial fibroblasts (HSFs) were synchronized in DMEM 1% FBS and then treated at various time points as indicated with 100 nmol/L PGE2 (B,C) or 100 nmol/L EP2 agonist (butaprost), 100 nmol/L EP4 agonist (L-902688), or 10 μM forskolin for 60 min (D). Total cell lysates were subjected to 1-D Western analysis using the PKA-preferred RRX(S*/T*) substrate antibody; β-actin antibody as a loading control as described in the Materials and Methods.

while the EP4 agonist L-902688-induced response differed in terms of magnitude (Figure 1D).

antibodies: MAPK/CDK, Akt, PKA, ATM/ATR, PKC, and CDK. We determined that PGE2 predominantly induced PKAdependent pathways (Figure 1), with no noticeable changes by the other screened kinases (Suppl. Figure 1 in the Supporting Information). The PKA phosphorylated substrate antibody that was used recognized the PKA motif RRX(S*/T*), where phosphorylated serine (S*) or threonine (T*) is fixed at position 0, arginine (R) is fixed at position −3 or −2, and any amino acid (X) is found at position −1 (Figure 1A). We previously observed that the PKA-substrate phosphorylation profile induced by PGE2 was characterized by a zenith at 10−20 min and was stable for up to 60 min (Figure 1B, C). In addition, response kinetics revealed an EC50 of 30 nM (CV = 6%), reaching a Vmax at 100 nmol/L; we used the latter concentration in all experiments.15 Forskolin, an activator of adenylate cyclase, induced essentially the same phosphosubstrate profile as PGE2, supporting the predominant role of PKA activity (Figure 1D). EP2 and EP4 receptors are known to couple PGE2 to the activation of the adenyl cyclase/cAMP/ PKA axis, and we attempted to substantiate this concept using specific/preferential EP receptor agonists as a first approach. Indeed, the EP2 agonist butapost elicited an essentially identical phosphorylation band profile and response magnitude as PGE2,

Large-Scale Identification of PGE2-Induced RRX(S*/T*) Substrates

To analyze differences in serine/threonine phosphorylation in HSFs treated with or without PGE2 at the zenith time point of 10 min, we enriched phosphorylated peptides from proteasedigested protein extracts by an immunoaffinity purification method using agarose-conjugated PKA phospho-motif antibody and analyzed them by LC−MS/MS methods as described in Figure 2A. Performed in duplicate, each sample yielded various chromatographic peak apex intensities of peptide ions derived from ion chromatogram files. In comparison, peak intensities for the same peptide ion were measured between samples to generate their corresponding fold-change values, following a label-free quantitative approach. The qualitative and quantitative data from the LC−MS/MS and SEQUEST results generated a redundant list of phosphopeptide assignments along with their associated experimental data (Suppl. Tables 1−5 in the Supporting Information). Overall, quantitative data were gathered for 146 nonredundant peptide assignments to 115 proteins, where 89 of the 146 peptide ions identified were at least 2.5 times more abundant in PGE2-treated cells than 5269



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Figure 2. Targeting PGE2-induced phosphopeptides/phosphoproteins. Cultured HSFs were synchronized in DMEM 1% FBS and incubated in the presence or absence of 100 nmol/L PGE2 for 10 min and processed as described in the Materials and Methods. (A) PGE2-induced phosphopeptides were analyzed using the PhosphoScan method as schematically depicted. Antibody used for immunoprecipitation/immunoaffinity columns preferably recognizes the RRX(S/T) motif, whereby the serine or threonine must be phosphorylated for antibody binding. (B) HEK cells were cotransfected with 1 μg of 5X GAL4−TATAA-luciferase reporter and 50 ng of Gal4-β-Cat or Gal4-β-Ca (S552A) mutant construct for 16 h in complete medium after which time the cells received a change of medium and treated with or without PGE2 (100 ng/mL) and/or Wnt (10 ng/mL) for 6 h. Cells were then lysed, and luciferase activity was measured as described in the Materials and Methods section.

Table 2. Selected PKA Substrates from PGE2-Induced Phospho-R(R/X)X(S*/T*) Motif Proteinsa

Peptide ions with fold-changes above 2.5 (green) or below −2.5 (red) known to be PKA substrates.31−40 “§” denotes previously reported phosphorylation sites. “*” denotes phosphorylation. a

PGE2-dependent pathways. Nonetheless, our data pool contained 13 phosphosites from 9 peptides previously published to be PKA-targeted, which represents 9% of total phosphorylated sites and 8% of total phosphoproteins (Table 2).31−40 To validate that the phosphorylation events are effective by PGE2, we extended a PGE2-induced phosphoprotein β-catenin at Ser552 for further analysis. We transfected HEK cells to separately express a wildtype β-catenin, a transcription factor activated by Wnt, and a mutated β-catenin with serine

in untreated cells, while 7 were at least 2.5 times less abundant. These 2.5-fold cutoff phosphotargets belong to 72 of the proteins identified (Table 1). We acknowledge that our strategy of using an antibody that targets a PKA motif does not directly prove that they are phosphorylated by PKA; however, we rely on the high stringency of the motif obtained from our results to associate such proteins as PKA substrates. Even if not bona fide PKA substrates, any differences detected with the antibody are relevant to 5270



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Figure 3. Functional distribution of phosphopeptides/phosphoproteins in PGE2-induced fibroblasts. (A) Distribution of protein classes for the total PGE2-induced phosphoproteome. Phosphosites with a 2.5-fold difference between 10 min PGE2-treated and untreated fibroblasts were used for a significant analysis (n = 72). (B) Percentile distribution of protein classes for previously reported and novel phosphosites from the total phosphoproteome (89 phosphosites from 72 phosphoproteins). (C) Distribution of protein classes for phospho-serine and phospho-threonine of the total phosphoproteome (n = 72). (D) Conservation of the PKA motif R(R/X)X(S*/T*) motif detected by antibody purification and LC−MS/ MS of our 89 phosphosites and its comparison with the amino acid frequency of known PKA substrates from Cell Signaling Technology.

552 exchanged for an alanine. These cells were treated either with or without Wnt or PGE2 and analyzed by a β-catenin reporter assay (Figure 2B). PGE2 inhibited the transcriptional activity of β-catenin with or without Wnt cotreatment. Furthermore, this inhibition occurred through the phosphorylation on Ser552 of β-catenin, as its mutation to alanine protected against the suppressive effect by PGE2. In our previous work, we validated and extensively analyzed other phosphosites for their influence by PGE2, which include ROCK2 Ser1379,41 Lamin A/C Ser10, and DOCK-7 Ser390 (data not shown).

can recognize which proteins of a particular function have been studied extensively in regards to Ser/Thr phosphorylation (Figure 3B). Basically, the PKA substrate-motif antibodybased method provided a rich source of randomly sampled peptides from different functional sources due to the dynamic range of our proteomic approach involving a mass spectrometer. The functional distribution of identified proteins with reported phospho-Ser/Thr sites places particular attention on cytoskeletal/focal adhesion, differentiation, cell cycle regulation, and kinases (Figure 3B). Proteins with cytoskeletal/adhesion functions are even more remarkable by undoubtedly containing the most phosphosites in comparison to other functionally grouped peptides (Figure 3A), notably 28% of the reported and 18% of the novel phosphoproteome (Figure 3B). The emphasis that these structural proteins have phosphorylated motifs with high PKA stringency is consistent with prior knowledge that cAMP/PKA is very influential on actin cytoskeletal dynamics and migration.42 Other common functions of identified proteins with novel sites were G proteins/regulators, correlating with the PGE2’s receptor type of GPCR. Those identified proteins with specific functions other than the ones listed were grouped as “Other functions” (Figure 3). As our purification method was capable of selecting either a phosphorylated serine or threonine, we analyzed the frequency

Mass Functional Classification of PGE2-Induced RRX(S*/T*) Phosphopeptides

Analysis of our PGE2-influenced phosphoproteome revealed peptides of several functional classes (Figure 3A) with varying amounts of previously reported and novel sites (Figure 3B). Of the identified phosphosites, 51% were previously reported to be Ser/Thr-phosphorylated, substantiating the efficiency of the RRX(S*/T*) antibody as a purification strategy (Figure 3B). The remaining 49% of phosphosites have not been documented, which makes the RRX(S*/T*) enrichment technique a vital tool in phosphoproteome wide screening of novel sites. By comparing the number of reported and novel phosphosites between the identified protein classes in our model, we 5271



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Figure 4. Canonical pathways based on the PGE2-induced RRX(S*/T*) antibody pull-down. (A) Top six significant canonical pathways predicted by Ingenuity Pathway Analysis. Values on the right y axis indicate the number of proteins from the phosphoproteome data set (n = 72) involved in each pathway while the negative of the log(p value) is plotted on the top x axis. (B) “Protein Kinase A signaling” and “Netrin Signaling” pathway from panel A is an example of a graphical representation of the top two IPA predicted canonical pathways. Enriched RRX(S*/T*) motif proteins are color-coded for their phosphorylation folds. Red, upregulated; green, downregulated; white, not part of the phosphodata set; darker intensities imply higher amounts of phosphorylation. Solid arrows represent direct interactions.

Categorizing PGE2-Induced RRX(S*/T*) Phosphoproteins into Canonical Pathways, Grouped Downstream Effects, and Networks

of each amino acid sequence and compared the distribution of each phosphorylated amino acid identified based on their protein function classification (Figure 3C). We observed the distribution of protein classes to be somewhat proportional between those phosphorylated on serine and those on threonine. As a whole, our frequency of phosphorylation being higher on serine than threonine was reflective of what is commonly observed between both amino acids (Figure 3C,D), where serine is nine times more likely to be phosphorylated than threonine.43 Furthermore, the amino acid frequency of motifs harboring a phosphorylated serine versus those with threonine bears a striking resemblance to that of published phosphomotifs belonging to PKA substrates (Figure 3D).

The complexity behind our proteomics data was analyzed and interpreted at multiple levels using IPA software to associate them into the most relevant pathways, downstream effects, and networks. Commencing with the simplest form of association, we analyzed at the molecular level the most relevant canonical upstream pathways across the entire data set to determine their key molecular roles and potential biological downstream effects (Figure 4A). Ranked statistically according to their −log(p value), the most significant pathway from our data set was the PKA pathway, correlating with the antibody immunopurification of substrates containing a PKA phosphomotif. 5272



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Figure 5. Common category effects and compiled signaling networks of the PGE2-induced RRX(S*/T*) phosphoproteome. (A) Top five effects of the PGE2-induced proteome of a RRX(S*/T*) antibody pull-down predicted by Ingenuity Pathway Analysis. Values on the right y axis indicate the number of proteins from our phosphoproteome data set (n = 72) with a 2.5-fold cutoff involved in each pathway, while the left y axis indicates the top category effects. PGE2-induced RRX(S*/T*) phosphoproteome predicted 5 networks to produce Suppl. Figure 3A in the Supporting Information which was compiled into top coherent functions (B). Protein names in bold and italic are those from our phosphodata while those in normal font are non-specified proteins.

The top five effects generated from our phosphoproteome were based on those proteins containing both the highest −log(p value), whereby the −log(0.05) of 1.3 is the minimal value of significance (Figure 5A). Each effect was further broken down into the various functions that composed it, including the list of proteins that comprised a particular function (Suppl. Figure 2 in the Supporting Information). The effect with the most associated proteins from our data set, “Cellular Assembly and Organization” (Figure 5A), has functions involving the organization of the cytoplasm and cytoskeleton, microtubule dynamics, and the formation of actin filaments and focal adhesions (Suppl. Figure 2 in the Supporting Information). The other major effects of “Cellular Movement”, “Cellular Function and Maintenance”, “Cellular Development”, and “Cellular Growth and Proliferation” also involve functions requiring the modification of the cellular architecture. Therefore, the different functional classes of our phospho-targeted proteins are known to come together from various grouped downstream effects involving the same biological trend of structural rearrangements. Furthermore, this biological trend correlates with our finding that cytoskeletal proteins make up the majority of proteins from our phosphodata (Figure 3) as well as with the two most statistically relevant signaling pathways generated from our data, PKA and

Furthermore, the PKA pathway also had more proteins from our data set than any other pathway. The second most significant pathway associated with our proteomic data was the Netrin pathway. These top two pathways are both well known for their influences on cytoskeletal/adhesion functions,42,44 correlating with our findings that the cytoskeletal/adhesion classification is the major functional group of all phosphoproteins (Figure 3). Proteins from both pathways were mapped together with their fold change in phosphorylation (Figure 4B). Therefore, from a signaling perspective, our RRX(S*/T*) phosphoproteome showed a meaningful relationship with our PKA-targeted pathway. Identifying pathways from a data set provides support that proteins are associated with one another. Although pathways have numerous potential functions, those functions complementary between them narrow down the possible downstream effects. We used IPA to analyze such downstream effects, which is established on expected casual effects between proteins and their functions. Proteins were grouped into an “Effect Category” based on their combined specific function, which is significantly calculated from the data set with a p value less than 0.05. By assessing the various functional outcomes of each phosphoprotein and their phosphorylation fold change, we were able to visualize biological trends influenced by PGE2. 5273



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for top functions and effects associated with our microscopy results: organization of cytoskeleton/cytoplasm, formation of cellular protrusions, and microtubule dynamics. Immunocytochemistry with the RRX(S*/T*) Ab confirmed the phosphorylation signal induced by PGE2 and localized its substrates (Figure 6E). Consistent with our Western Blot data, PGE2 stimulated Ser/Thr phosphorylation in fibroblasts, primarily localized in the cytoplasm at 10 min and extending into pseudopodial extensions at 40 min. The localization of such phosphosubstrates relates to their site of function (Figure 5B). As the formation of the actin cytoskeleton and adhesion structures was a common function from our grouped networks in Figure 5B and Supplementary Figure 2, it urged us to visualize their formation. Phalloidin staining of actin revealed the integrity of the cytoskeleton (Figure 6E). Nonstimulated fibroblasts had actin organized in the form of filaments, while PGE2 stimulated the disassembly of actin throughout the cytoplasm, where there was a loss of uniform cell shape. Staining for phospho-Tyr-118-paxillin, which is an indicator for focal adhesion maturation, revealed a clear distribution around the cytoplasm of untreated cells, while PGE2 treatments of 10 and 40 min removed such staining. Therefore, the kinetics of PGE2 phosphorylation of our functionally grouped cytoskeletal proteins draw a parallel with that of the disassembly of actin filaments and adhesion molecules, which are the most probable cause of the observed cytoplasm collapse and pseudopodial extensions. Our predicted networks had most of their listed top functions involving cellular architectural changes, possibly culminating in one of our listed top effects, “Cellular Movement” (Figure 5A). Of our PGE2-induced phosphoproteins, RIN1 Ser291 and Ser292 were selected to serve as a source for interpreting the effect of PGE2 on migration because RIN1 was grouped into the network involving cellular movement (Suppl, Figure 3A in the Supporting Information, Network ID 1), it was found in the top 5 “combined network” functions implicated in migration (Figure 5B), it inhibits Ras/Raf, a pathway known for its migration, wound healing, and symbiosis with TGFβ,45−49 and phosphorylation of identified RIN1 Ser292 has been published to inhibit migration.50 In this regard, HEK293T were transfected with either pCMV, RIN1 WT, RIN1 Ser291Ala, or RIN1 Ser292Ala, then seeded into a serum-free upper Boyden chamber. The transfected cells were treated with/out PGE2 and then examined for their efficiency of migration to the lower chamber containing TGFβ (migratory stimulant). The potency effect of PGE2 was made clear, as it inhibited the inducible nature of TGFβ on the migration of control pCMV cells (Figure 6F). In comparison, RIN1 WT cells were drastically inhibited to migrate with/out PGE2-treatment compared with the control. The separate RIN1 mutation of both identified phosphosites Ser291 and Ser292 released the PGE2 inhibitor effect on migration. Thus, our PGE2 phosphosites Ser291 and Ser292 of RIN1 are required for the inhibiting nature of the biolipid in cellular movement, functionally validating the correlation of our phosphodata with the prediction analysis previously described.

Netrin (Figure 4), which are both known for signaling changes in cellular structures. Once we grouped our various proteins together for expected effects, we wanted to reveal the interaction that these proteins had with one another by incorporating them into networks most pertinent to the largest set of our phospho-proteins along with other associating nonspecified proteins to complete a full interacting signaling circuit. In doing so, we would have a better idea of the protein functions and downstream effects most affected by PGE2-induced phosphorylation because phosphorylation of an interacting string of proteins associated with a particular function and effect would most likely affect their particular downstream outcome. Using IPA for its network assessment, we further grouped the phosphoproteins into their most relevant networks by interconnecting them with predicted proteins outside our data set and found that five network IDs were obtainable with our phosphodata set (Suppl. Figure 3 in the Supporting Information). As expected, the top functions and their downstream effects for each established network all involved some sort of cellular structural rearrangement. However, the diverse networks have distinct molecular interactions among themselves, where some are conducting several of the same downstream effects such as differentiation, movement, proliferation, cytoskeletal organization, and formation of cellular petrusions. To further increase the probability that a particular function/ effect was affected by PGE2-induced phosphorylation, we overlapped the five individual networks into a “shared network” for the most common functions/effects between the different interacting strings found in each network (Figure 5B). We subsequently observed a more conserved effect of “Cellular Assembly and Organization” among the networks, where the focus was around cytoskeletal organization and membrane projections. The likelihood of affecting a particular function/ effect is even higher when affecting various signaling networks that share the same outcome. Therefore, PGE2 has targeted several proteins interconnected within a shared network and, as a result, had a higher probability of affecting the functions and effects associated with that particular network. Functional Assay of PGE2 Phosphorylation Regarding Cellular Morphology, Cytoskeletal/Adhesion Structure, And Migration

The high proportion of our PGE2 phosphoproteins identified within the functional class of cytoskeletal proteins as well as being grouped together into similar cytoskeletal “pathways/ functions/effects/networks”, prompted us to re-examine the effect that PGE2 phosphorylation had on cellular morphology. In contrast with the spread-out morphology of the control fibroblasts, we observed by light microscopy that PGE2 treatment reorganized the cytoplasm into a collapsed form with pseudopodial extensions (Figure 6A). The concern of the cell status after such morphological changes extended our morphology study well beyond our time frame of analysis to show that the cells were bound and viable after 72 h of PGE2 treatment, all the while maintaining the dramatic cell structural changes (Figure 6B). In addition, cell viability and cell cycle status were evaluated using flow cytometry before and after 12 and 24 h PGE2 treatment (Figure 6C,D). PGE2 was able to arrest the cell cycle without the induction of apoptosis, suggesting that our presented signaling circuitry is appropriately presented. From our combined network data of PGE2-induced phosphoproteins (Figure 5B), we saw proteins working together

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DISCUSSION The predominant method of studying signaling pathways focuses on either a few central proteins essential to a function or generalized pulldowns that provide a rich proteome. Focusing on a few proteins gives details on known pathways but cannot incorporate adjacent influential pathways, while proteome 5274



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Figure 6. Kinetics of PGE2-induced phosphorylation and changes in fibroblast structure and migration. Human synovial fibroblasts were seeded in six-well plates and cultured for 24 h in complete medium and then incubated for 4 h in DMEM + 1% FBS prior to any treatments. Light phasecontrast microscopy was used for capturing morphological changes after the addition of 100 nmol/L of PGE2 to HSF for 60 and 90 min (A) as well as 24, 48, and 72 h (B). (C) Confluent, quiescent, and synchronized cultured HSFs were incubated for 2−4 h in complete medium containing 1% serum prior to treatment with vehicle (control), 100 nM PGE2 for 12 or 24 h, or with 10 μM of staurosporine for 24 h. Cells were then stained with PI and an Alexa-fluor FITC conjugated anti-Annex-V antibody and processed for flow cytometry, as described in the Materials and Methods. (D) (i) Confluent, quiescent, and synchronized cultured HSFs were stained with PI (1 μg/mL) and processed for flow cytometry. In (ii) and (iii), HSFs were incubated at 30−40% confluency, allowed to adhere in culture plates for 2 h, and then incubated with either 10% serum (ii) or with a 30 min pretreatment of 100 nM PGE2 (iii) and 10% serum in complete medium for a further 20 h. Cells were stained with PI and processed for flow cytometry. In (iv) control and (v) PGE2, cells treated as in (ii) and (iii) were stained with PI and an Alexa-fluor conjugated anticyclin B antibody and processed for flow cytometry. (E) HSFs were seeded in six-well glass microscope slides for 24 h in complete medium and then incubated for 4 h in DMEM + 1% FBS prior to the addition of 100 nmol/L of PGE2 for 10 and 40 min. Cells were then fixed, and confocal microscopy was used to stain for nuclei using DAPI (blue) as well as actin filaments stained with phalloidin (red), while immunocytochemistry used RRX(S*/T*) and PhosphoPaxillin (Tyr118) antibodies (green). Images are representative of >90% of the total population of cells in three separate preparations. (F) HEK cells transfected with 1 μg each pCMV empty vector, wt RIN, RINmut291, or RINmut292 were added to the upper Boyden chamber of the 8 μm pore size polycarbonate membrane insert in serum-free medium,and the latter was lowered into the TGFβ (10 ng/mL) chemoattractant medium of the outer bottom chamber. Cells were treated with/out 100 nM PGE2 and allowed to migrate for 24h at 37 °C in a CO2 incubator (5%/95% air). Nonmigrated cells were removed, membranes were rinsed, and migrated cells were recovered in extraction buffer/dye mixture. Images are representative of >90% of the total population of cells in three separate preparations. 5275



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proteins, providing some upstream insight on the downstream morphological changes that we and others induced by PGE2. There are several pieces of evidence that provide a representation of our PGE2 phosphodata for having a RRX(S*/T*) motif that is highly PKA stringent. First, a positive PKA phosphorylation Western Blot profile of PGE2 is comparable to that of PKA-associated EP2/4 agonists and forskolin (Figure 1D). Second, we identified 13 sites from 9 phosphoproteins known to be phosphorylated by PKA (Table 2). Third, the amino acid frequency within the RXXS/T motif found between our phosphodata and known PKA substrates is similar (Figure 3D). Finally, our phosphodata has a primary disposition for the PKA pathway compared with other predicted pathways (Figure 4A). Although RRX(S*/T*) is a high stringency site for PKA, the kinase can also phosphorylate a RX-X-S*/T* motif to a lesser degree, explaining the minimal amount of varying amino acids at position −2 (Figure 3D).58 In a particular case, we provide proof that the elicited PGE2 phosphosite Ser1379 on ROCK2 was mediated by PKA.41 The likelihood that a certain net effect may arise from our data set would be dependent on the frequency of proteins with the same function along with their occurrence of being grouped with different functions into the same downstream effect. Using IPA for its filtering process of sorting our data from their respective general functions to overlapping shared effects derived the best understanding of the functional role of our PGE2-mediated phospho-Ser/Thr signalosome in fibroblasts (Figure 5A). Hereafter, the grouping of multiple networks into one (Suppl. Figure 3A in the Supporting Information) deduced a composition of various cytoskeletal functions (Figure 5B) that was in agreement with our physiological model (Figure 6). PGE2 treatment created the distinct morphological features of cytoplasm collapse and cellular protrusions, which in essence requires processes that are dependent on cytoskeletal reorganization and assembly. These experiments were designed to verify the same observations of PGE2 on cellular morphology as in previous publications12,59 and to observe the overlapping rates of phosphorylation and structural changes for a certain degree of associating such changes with our phosphodata. Immunocytochemistry imaging revealed part of the collapse to involve the disassembly of actin filaments, whereby an intact cytoskeleton does not have the plasticity to undergo such changes (Figure 6E). Furthermore, shrinkage of the cytosol would require the under area of the cytoplasm to move. Such a case would lose the anchored structure of focal adhesions, which we observed through the loss of its maturation signal of phosphopaxillin. It is safe to assume that the presence of the phosphosignals does not coexist with the integrity of actin filaments and mature adhesion signals, and for this reason such signals may be responsible for their loss. Therefore, by relating these observed functions to the predicted grouped functions of our enriched PGE2-related protein networks, we were able to identify those phosphoproteins possibly involved with each particular function. Cytoskeletal features of activated fibroblasts (myofibroblasts) facilitate repair by providing the contractile forces necessary to bring together the ECM for wound closure.60 The cytoskeletal structure of myofibroblasts has the form of a contractile apparatus because it is composed of actin filament bundles that terminate at the surface in mature focal adhesions, which link the cytoskeleton to the ECM.61−64 Such structures are reinforced to support the mounting tension necessary for wound closure and repair. Therefore, the disassembly of these

pulldowns can describe proteins involved in a system, but its rich data dilutes post-translational modification signals. However, using antibodies that immunoprecipitate proteins sharing motifs recognized and modified by a particular kinase allows for a more defined analysis of proteins related in a specific signaling event. Therefore, we applied the combination of kinase-targeted phospho-serine/threonine enrichment techniques, mass spectrophotometry, as well as a recent compilation of phosphopeptide and phosphoprotein databases to identify and quantify a rich data set of random phosphorylation events at one time within a PGE2 signaling network. Overall, our procedure revealed that PGE2 induced primarily a PKA-preferred phosphorylation of substrates containing the RRX(S/T) motif. This result was coherent with the EP agonist blot, which indicated that only the PKA-associated receptors EP2/4 were able to induce a similar profile as PGE2. The PKA phosphorylation RRX(S/T) motif Ab was used to immunoprecipitate these substrates for identification by MS/MS. As a result, 89 phosphosites from 72 proteins were identified with at least 2.5 times more abundance in PGE2-treated cells than in untreated cells, while 7 phosphosites from 4 proteins were at least 2.5 times less phosphorylated. Further analysis of the 2.5-fold difference cutoff identified 49% of phosphosites to be absent from the literature, permitting them to be labeled as novel. Various phosphosites (ROCK2 Ser1379,41 DOCK-7 Ser439 and Ser440, and LaminA/C Ser10 (unpublished data)), were mutated that prevented the influence by PGE2, including our current analysis of β-Catenin phospho-Ser552 as well as RIN1 phospho-Ser291 and Ser292, to evaluate the validity of the phosphodata. Furthermore, our method of investigating PGE2 signaling networks is validated by a similar approach by Oberprieler et al. 2010,51 who applied quantitative phosphoproteomic mass spectrometry to identify a PGE2-induced PKA signaling node specific to T-cells. By characterizing our PGE2-induced phosphoproteins into various levels of signalization, starting from the smallest process (ie. phosphorylation) that would eventually propagate into a common network effect, we intended to derive those that source the antifibrotic events of the bioactive lipid. Purification of proteins containing the RRX(S*/T*) phosphomotif revealed that PGE2 primarily focused on the functional class of cytoskeletal/adhesion proteins (Figure 3A). Although proteins were grouped into other smaller distinct classes, associations exist among these classes with that of the cytoskeletal/adhesion class. For instance, the “G protein/regulator” class is known for their involvement in regulating actin dynamics.52 Also, the classes of “Cell cycle regulation” and “Differentiation” to a certain degree require an association with the cytoskeletal/ adhesion proteins in order to achieve their own processes. PGE2 as well as other agonists of the cAMP/PKA pathway have been extensively studied for their drastic effects on the cellular architecture, such as stress fiber breakdown, formation of stellate morphology, and loss of cytoplasmic volume.12,53−57 For these reasons, we consider those PGE2-targeted phosphosubstrates that were characterized within the upstream PKA pathway, our most significant predicted pathway (Figure 4B), to be the initial signals behind the morphological/cytoskeletal changes that we induced by PGE2 treatment (Figure 6A,B,E). Also known for its influences on cytoskeletal functions, the Netrin pathway was predicted as the second most significant pathway from our results. Therefore, our phosphodata provides information on signaling pathways that correlate with their most common functional class of cytoskeletal/adhesion 5276



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phosphosignal on AKAP2 would be responsible for bringing PKA to its actin-associating substrates and signaling them for the induction of our cytoskeletal breakdown. An example of a phosphotarget that could describe another of our functional outcomes is Nck1, which was predicted for its involvement in cellular movement (Suppl. Figures 2C and 3A in the Supporting Information ID 1). Our migratory results may be partly explained by Nck1 because this adaptor protein is primarily involved with the inhibition of migration.76 This speculates that the PGE2 induction of Nck1 phosphorylation is activating so as to obtain our observed prevention of fibroblast migration (Figure 6F). Tensin 3 is another interesting phosphotarget highlighted by our predicted analysis that most probably explains the loss of our focal adhesions (Suppl. Figure 2A,B in the Supporting Information). Localized within focal adhesions, tensin serves as a structural platform necessary in regulating the assembly of focal adhesion components such as paxillin.77−79 Hence, our phosphosignal by PGE2 may inhibit tensin from such an assembly to establish our loss in the focal adhesion maturation signal observed through paxillin (Figure 6E). The relevance that such stated proteins have for their respective functions provides strong speculative hypotheses for future studies regarding PGE2-targeted phosphosubstrates in antifibrotic pathways. Overall, our predicted functions allow us to narrow in on those candidate phosphoproteins so as to determine the influence of each PGE2-induced phosphosite. Understanding these PGE2 inhibitory signaling mechanisms against myofibroblasts could be of potential use for terminating a chronic deregulated repair necessary for treating fibrosis. By studying the contribution of each PGE2-induced phosphosite on those relevant substrates of cytoskeletal rearrangement and migration found in our data set, we may be able to mimic those signals that are antifibrotic for the development of several therapeutics of fibrosis. Interestingly, a method of fibrosis treatment involves ROCK2 inhibition by Y26732 or fasudil to suppress actin filament contraction, validating the potential of our phospho-Ser1379 as a target of ROCK2 inhibition for fibrotic treatment.80,81 Our method of wide-screening analysis using kinase targeted substrate antibody purification and MS/MS allowed us to expand the limited signaling knowledge of PGE2, through both statistical and experimental methods. Recognizing the underpinning of cellular responses through phosphorylation events induced by homeostatic regulators like PGE2 provides fundamental targets for pharmaceutical intervention in rehabilitating stability.

structures in our model implies that PGE2 is involved in terminating repair, where our phosphosubstrates appear to be the targets for such an event. In fact, several lines of evidence indicate that PGE2 has opposite effects to fibrogenesis. PGE2 seems to remove the effector functions of repair through its ability to inhibit fibroblast proliferation,65,66,67 decrease fibroblast chemotaxis and migration,11,68 increase collagen degradation,69 inhibit actin bundling,12,59,69 and inhibit/reverse myofibroblast differentiation.12,59 Most of these PGE2-suppressive effects on fibroblasts are mediated by the activation of EP2 and its coupled cAMP/PKA pathway.12,13,64 This evidence that PGE2 can impede the repair activities of fibroblasts implies that PGE2 signaling is beneficial in limiting the pathological features of fibrosis. However, such observations go as far as the EP2 receptor level to define a mechanism of action. Our work is in line with most of these findings through our EP2 predominance of the PKA signal but also provides a map of downstream targets by which PGE2 can disrupt actin formation and FA maturation. More importantly, we identified those PGE2-induced phosphoproteins associated with essential signaling pathways of myofibroblast activities, which include β-catenin (differentiation) (Figure 2B), RIN1 (migration) (Figure 6F), and ROCK2 (contraction).41 ROCK2 was found in numerous predicted functions from our combined 5 networks (Figure 5B), such as “Organization of Cytoplasm”, “Organization of Cytoskeleton”, and “Formation of Cytoskeleton/Actin Filaments”. Granted that these functions are well-known to be associated with ROCK2,70,71 we decided to determine the effect that our PGE2-induced phosphosite Ser1379 had on ROCK2 (Table 1) and relate it to our observed morphological outcomes. We previously showed that such a phosphorylation event inhibited ROCK2 from inducing its downstream signaling components necessary for bundling actin into stress fiber filaments,41 as a result contributing to the collapse of the cytoplasm. In addition to targeting stress fibers, PGE2 inhibition of migration would be another antirepair process because cellular movement is a vital process in localizing myofibroblasts to the wound site. Indeed, we presented the inhibitory effect of PGE2 on migration in the presence of the pro-fibrotic agent TGFβ (Figure 6F). In targeting both Rin1 phosphosites Ser291 and Ser292, we identified such sites to be necessary for PGE2 inhibition of migration. Phosphorylation of RIN1 Ser292 has been published to inhibit migration, hence further validating our results from a functional perspective.72 However, our phosphosite RIN1 Ser291 was newly discovered to be also implicated in preventing migration. Finally, we have shown that the PGE2-induced phosphorylation on Ser552 of β-catenin is inhibitory to its transcriptional activity. The implication of this would prevent a transition of fibroblasts to myofibroblasts given the positive relationship that the Wnt/ β-catenin pathway has on myofibroblast differentiation.73 According to our function prediction analysis, we speculate that several other described phosphotargets may also be explanatory to our functional outcomes, such as the involvement of Filamin A and AKAP2 in the “Organization of Cytoplasm/Cytoskeleton” (Figure 5B). Filamins bind actin proteins to organize them into network filaments,74 meaning that our PGE2-induced phosphorylated sites on Filamin A would appear to be inhibitory so as to reflect the cytoskeletal breakdown PGE2 imposed on fibroblasts. As for AKAP2, it is specifically known to colocalize PKA with actin.75 Given that the typical cytoskeletal effects of PKA are similar to our microscopy results, there is an assumption that our observed

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary Figure 1: Analysis of PGE2-dependent total substrate phosphorylation. Supplementary Figure 2: Category effects and their associated functions based on the PGE2-induced RRX(S*/T*) antibody pull-down. Supplementary Figure 3: Signaling networks of the PGE2-induced RRx(S*/T*) phosphoproteome. Supplementary Table 1: Column definitions. Supplementary Table 2: Details of MS data. Supplementary Table 3: Summary of MS data. Supplementary Table 4: Foldchange descending order. Supplementary Table 5: Fold-change by protein type. This material is available free of charge via the Internet at http://pubs.acs.org. 5277



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AUTHOR INFORMATION

Corresponding Author

*Tel: 514-934-1934, ext.36276. Fax: 514-289-8542. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Donald Halstead for his critical reading of the manuscript.

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ABBREVIATIONS

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REFERENCES

AC, adenylate cyclase; DMEM, Dulbecco’s modified Eagles medium; ECM, extracellular matrix; EP, prostaglandin E receptor; FBS, fetal bovine serum; GPCR, G-protein coupled receptor; HSF, human synovial fibroblast; IPA, Ingenuity Pathway Analysis; MAPK, mitogen-activated protein kinase; PI3K, phoshotidylinositol 3-kinase; PGE2, prostaglandin E2; PKA, protein kinase A; ROCK2, rho-associated coiled coilcontaining kinase-2; RSK, ribosomal S-6-kinase; TGF-ß, transforming factor-ß

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Quantitative Phosphoproteomic Analysis of Signaling Downstream

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