Protein Phosphatase 2A Catalytic Subunit &alpha

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Mar 28, 2013 - located with MyD88 into the nuclei of tolerant macro- phages establishes ... to NL cells, T or TL cells showed an ET-specific phenotype of .... DN under TL (Figure 3A, right), indicating that MyD88 interacts ..... (Left) The fold change of phosphorylation caused by OA (y axis) under TL calculated in Figure S3B.
Cell Reports

Report Protein Phosphatase 2A Catalytic Subunit a Plays a MyD88-Dependent, Central Role in the Gene-Specific Regulation of Endotoxin Tolerance Ling Xie,1,5 Cui Liu,1,5 Li Wang,1,4 Harsha P. Gunawardena,1 Yanbao Yu,1 Ruyun Du,4 Debra J. Taxman,3 Penggao Dai,1 Zhen Yan,1 Jing Yu,1 Stephen P. Holly,1 Leslie V. Parise,1 Yisong Y. Wan,2,3 Jenny P. Ting,2,3 and Xian Chen1,2,4,* 1Department

of Biochemistry and Biophysics Comprehensive Cancer Center 3Department of Microbiology and Immunology University of North Carolina, Chapel Hill, NC 27599, USA 4Department of Chemistry, Fudan University, Shanghai 20032, China 5These authors contributed equally to this work *Correspondence: [email protected] http://dx.doi.org/10.1016/j.celrep.2013.01.029 2Lineberger

SUMMARY

MyD88, the intracellular adaptor of most TLRs, mediates either proinflammatory or immunosuppressive signaling that contributes to chronic inflammationassociated diseases. Although gene-specific chromatin modifications regulate inflammation, the role of MyD88 signaling in establishing such epigenetic landscapes under different inflammatory states remains elusive. Using quantitative proteomics to enumerate the inflammation-phenotypic constituents of the MyD88 interactome, we found that in endotoxintolerant macrophages, protein phosphatase 2A catalytic subunit a (PP2Ac) enhances its association with MyD88 and is constitutively activated. Knockdown of PP2Ac prevents suppression of proinflammatory genes and resistance to apoptosis. Through sitespecific dephosphorylation, constitutively active PP2Ac disrupts the signal-promoting TLR4-MyD88 complex and broadly suppresses the activities of multiple proinflammatory/proapoptotic pathways as well, shifting proinflammatory MyD88 signaling to a prosurvival mode. Constitutively active PP2Ac translocated with MyD88 into the nuclei of tolerant macrophages establishes the immunosuppressive pattern of chromatin modifications and represses chromatin remodeling to selectively silence proinflammatory genes, coordinating the MyD88-dependent inflammation control at both signaling and epigenetic levels under endotoxin-tolerant conditions. INTRODUCTION Toll-like receptors (TLRs) activate the innate immune system by mounting appropriate inflammatory responses to contain infection or repair damaged tissues (Drexler and Foxwell, 2010). To 678 Cell Reports 3, 678–688, March 28, 2013 ª2013 The Authors

avoid harmful effects of persistent signaling caused by the continual presence of stimuli, cells become transiently unresponsive by acquiring tolerance to chronic inflammation, leading to a negative consequence that tumor cells can escape immunosurveillance (Rakoff-Nahoum and Medzhitov, 2009). In transmitting inflammatory signals, by mechanisms yet to be elucidated, the intracellular adaptor protein MyD88 (myeloid differentiation primary response 88) of most TLRs acts as a double-edged sword promoting both protective and harmful inflammation (Huang et al., 2008). Recent work has revealed that inflammation control is achieved by a gene-specific mechanism in which distinct chromatin modifications contribute to selective silencing of TLR4induced proinflammatory or tolerizable (T class) genes under an endotoxin tolerance (ET)-associated chronic inflammatory state (Foster et al., 2007). Major questions remain unanswered including the following: (1) How does MyD88 mediate both acute and chronic inflammatory responses? (2) What is the driving force for ‘‘converting’’ proinflammatory MyD88 to an immunosuppressive mediator? (3) How are distinct, inflammation-specific patterns of chromatin modifications differentially established at the T class promoters? Emerging evidence suggests that an effective, unharmful inflammatory response is intrinsically regulated by subtly distinct intracellular protein interactions, i.e., ‘‘interactomes’’ (proteinprotein interaction network) involved in signal transduction (Liew et al., 2005). During TLR signaling, MyD88 serves as a scaffold that coordinates protein complex assembly through sequential recruitment of specific proteins varying from TLR molecules to downstream proteins (Dai et al., 2009; Wang et al., 2006). Based on our previous identification of multiple signal regulators that interact with MyD88 in a timely and orderly manner to tightly regulate the amplitude and duration of TLR signaling (Dai et al., 2009), we reason that MyD88 may regulate either acute or chronic inflammation via assembling different, inflammation-phenotypic interactomes.

RESULTS As a Core Component of MyD88 Interactome in ET Macrophages, PP2Ac Is Chronically Activated First, by using our amino-acid-coded mass tagging (AACT)based quantitative proteomic approach (Chen et al., 2000) with modifications for interactome screening (Figure 1A), we dissected the MyD88-interacting complexes assembled in RAW cells under different inflammatory states including (1) no stimulation (N), (2) challenging with a single high LPS dose (LPS responsive, NL), (3) priming with a low LPS dose (LPS tolerant, T), and (4) challenging T cells with a high-dose LPS (TL). Compared to NL cells, T or TL cells showed an ET-specific phenotype of immunosuppression and resistance to apoptosis (Figure S1A). Through phenotypic interactome analysis (Figure 1A), we found that MyD88 interacts with different sets of proteins in NL versus TL macrophages (unpublished data): together with many negative immune regulators (Liew et al., 2005) including a negative TLR regulator Flii (Wang et al., 2006), protein phosphatase catalytic subunit a (PP2Ac) was found recruited into the MyD88 interactome specifically in ET cells (Figures 1B and S1B). Because approximately 20% of the components in the TL-specific MyD88 interactome contain the domains interacting with PP2Ac (Figure S1C) that is generally considered as a suppressor of proinflammatory kinases (Junttila et al., 2008), this inflammation-phenotypic proteomic finding suggested that PP2Ac plays a central, MyD88-dependent, immunosuppressive role during ET. Given that neither expression nor stability of PP2Ac was affected by LPS-induced inflammation (Figure S2A), we compared the PP2Ac activity in RAW cells under different inflammatory conditions. Compared to naive (N) cells, whereas its activity was little changed under NL, PP2Ac was highly activated with a prolonged stimulation and was sustained under TL (Figure 1C), indicating the activity-based, inflammation-phenotypic function of PP2Ac. To clarify the MyD88 dependence of PP2Ac activation, we measured PP2Ac activity in paired wild-type (WT) and MyD88-depleted (MyD88/) (Figure S2B) bone marrow-derived macrophages (BMDMs) under different inflammatory states induced by a variety of TLR agonists. Similar to RAWs, PP2Ac activity showed a 2-fold increase in TL WT BMDMs but less than 10% changes in MyD88/ BMDMs under N, NL, and TL (Figure 1D). The TLR9 agonist CpG, which triggers only MyD88-dependent pathways, induced a greater increase in PP2Ac activity in TL WT BMDMs than that induced by LPS, which activates both MyD88-dependent and -independent pathways (Figure 1E), whereas no differences in PP2Ac activity were observed between WT and MyD88/ BMDMs when the TLR3 agonist Poly (I:C) stimulated only MyD88-independent pathways (Figure 1F). These agonist-specific effects indicated the MyD88-dependent activation of PP2Ac in chronically inflamed cells. Second, to clarify how PP2Ac activity is regulated in tolerant macrophages, we respectively used either LPS-conditioned medium, i.e., that separated from the LPS-pretreated RAWs, or TNF-a or ionizing radiation (IR) to trigger inflammation in BMDMs. Under chronic exposure, all non-TLR stimuli caused a similar degree of tolerance-specific activation of PP2Ac in both WT and MyD88/ BMDMs (Figure 1G). In a coordinate manner,

unlike its LPS-inducible nature in WT but not in MyD88/ BMDMs (Figure 1H, left), phospho-p65 triggered by non-TLR stimuli showed similar time-dependent induction in both types of BMDMs (Figure 1H, right), indicating that stimuli-induced, secondary, NFkB-regulated production of inflammatory cytokines such as TNF-a causes tolerance-specific PP2Ac activation. Because TNF-a is a T class gene and its release is MyD88 promoted (Figure S2B), this secondary effect on PP2Ac activation was therefore more pronounced in the WT BMDMs directly stimulated by the TLR agonists that trigger MyD88-dependent pathways for producing these T class cytokines. Only during the chronic inflammation mediated by TLR2/TLR4/TR9 is the sustained PP2Ac activation MyD88 dependent. Constitutively Active PP2Ac Regulates Immunosuppression during ET To determine the correlation of persistently or constitutively activated PP2Ac with immunosuppression, we measured cytokine release from stable RAW lines expressing either shRNA for knocking down PP2Ac (PP2Ac knockdown [KD]) or shRNA for GFP (WT control) (Figures 2A and S2C). Under NL, most cytokines showed LPS-inducible release in both PP2Ac KD and WT RAWs (Figure 2B). In contrast, compared to the LPS-tolerized WT RAWs showing reduced production of T class cytokines, the LPS-primed PP2Ac KD cells were hyperresponsive to high-dose LPS stimulation and released greater amounts of cytokines. Furthermore, we determined the effect of constitutively active PP2Ac on the expression of 84 mouse genes encoding inflammatory factors, most known to be regulated by NFkB (Figure S2D). Compared with their levels in WT RAWs, 34 out of 84 genes showed more than a 30% increase (Table S1), some of which were validated by qPCR (Figure 2B). Also, T class genes (IL6 or IL10) had dramatically enhanced expression in the PP2Ac KD cells even under TL, reversing their suppressed expression in TL WT cells. In contrast, under NL or TL, PP2Ac KD had little effect on the LPS-inducible trend of Fpr1 (Figure 2B), a nontolerizeable gene (Foster et al., 2007), suggesting that constitutively active PP2Ac is involved specifically in T class gene regulation under ET. Furthermore, to determine whether the constitutively active form of PP2Ac directly contributes to acquiring ET, we transfected constructs expressing either WT or a dominant-negative (DN) mutant of PP2Ac (Chang et al., 2011) into the PP2Ac KD RAWs. Under TL, whereas expression of WT PP2Ac restored the tolerance with reduced IL6, the PP2Ac DN mutant with similar expression failed to suppress this T class gene (Figure 2C), indicating that constitutively active PP2Ac participates in the gene-specific control of chronic inflammation by selectively suppressing T class genes. Constitutively Active PP2Ac Promotes the MyD88Dependent Survival of ET Cells through Broadly Targeting Proinflammatory Kinases and Proapoptotic Factors To identify PP2Ac-target pathways through which ET is regulated by constitutively active PP2Ac, we profiled the phosphoproteomic changes in PP2Ac KD versus WT RAWs under TL (Figure S3A). Some of the PP2Ac-target sites Cell Reports 3, 678–688, March 28, 2013 ª2013 The Authors 679

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(Figure S3B) that were identified based on their phosphorylation increases in TL PP2Ac-depleted cells were in the components of the TL-specific MyD88 interactome (Figure S1B), including Rps6 (a substrate of p70S6K), Rap1 (a MAPK component), Akt1, and a mTORC1/C2 component Raptor, revealing that through site-specific dephosphorylation, constitutively active PP2Ac represses the proinflammatory/proapoptotic activity of the PI3K-mTOR-p70S6K-Akt pathway. In addition, we found that constitutively active PP2Ac targeted AP1/c-Jun, the mediator of stimuli-induced apoptosis (Li et al., 2004) (Figure S3C): under TL, in contrast to its decreased abundance in WT RAWs, phosphorylation of c-Jun Ser63 was significantly increased when PP2Ac was depleted (Figure 2D). Thus, similar to the antiapoptotic nature of PP2A (Kong et al., 2004), PP2Ac, when constitutively activated, negatively regulates the proapoptotic activity of c-Jun by directly dephosphorylating its proapoptotic pSer63. We next determined how PP2Ac KD affects the ET-characteristic resistance to apoptosis. First, we evaluated cell survival/ proliferation in PP2Ac KD RAWs under N, NL, and TL in the presence of cycloheximide (CHX) that can amplify LPS-induced apoptosis (Suzuki et al., 2004). Compared with the ET-specific increase of the surviving WT cells, PP2Ac KD led to reduced colony formation, indicating an increase in apoptotic/dead cells even under TL (Figure 2E). Similarly, in flow cytometry analysis, consistent with what was shown for cytokine release (Figure 2B), PP2Ac KD RAWs showed some degree of LPS-inducible apoptosis/cell death even in nonstimulated cells (Figure 2F), indicating the cell sensitization due to PP2Ac depletion. The NL WT RAWs showed LPS-inducible apoptosis, whereas this induction was suppressed under TL. In contrast, a dramatically increased susceptibility to apoptosis was observed even for T or TL PP2Ac KD cells. The consistent results from both assays (Figures 2E and 2F) indicated that PP2Ac depletion reduces the resistance of ET macrophages to apoptosis. Furthermore, because MyD88 promotes tumor growth by either modulating particular kinase pathways (Lee et al., 2010) or inhibiting apoptosis (Rakoff-Nahoum and Medzhitov, 2007), we next examined whether the PP2Ac effect on apoptotic propensity is MyD88 dependent. Because PP2Ac is highly abundant and robust, and nondividing BMDMs have extremely low transfection efficiency and shRNA stability, very low percentages of endogenous PP2Ac KD could be achieved in BMDMs (data not shown). Given that the phosphatase inhibitor okadaic acid (OA) at concentrations 95% incorporations of M or H was achieved and then each was either left unstimulated (‘N’) or was subjected to a single LPS stimulation at 1 mg/ml for 15 min (‘NL’) or was first primed with 0.1 mg/ml LPS for 24 hr followed by the second LPS challenge at 1 mg/ml for 15 min (‘TL’). After LPS stimulation, the cells from each pool were harvested, washed with ice-cold phosphate-buffered saline (PBS), lysed in 50 mM Tris-HCl, 150 mM NaCL, 1%NP-40 supplemented with protease inhibitor cocktail (Sigma), and centrifuged at 13,000 rpm for 20 min. The supernatants were retained for further processing. Following the manufacturer’s protocol (Miltenyi Biotec) approximately 10 mg proteins extracted from each of the cell pools was incubated with 100 ml of mMACS Protein A Microbeads coupled with anti-MyD88 antibody at 4 C for overnight, then loaded to Miltenyi mColumn, and washed with lysis buffer five times. The immunoprecipitate originated from each cell pool was eluted with SDS sample buffer. The total amount of each immunoprecipitate obtained from the L, or M, or H cell pool was measured using BCA protein assay and validated based on the immunoblotting intensity of MyD88. The equal amount of each immunoprecipitate was mixed, and the mixture was then subjected to 1D SDS PAGE separation, in-gel digestion. The gel-extracts of peptide mixtures were desalted using PepClean C18 spin columns (Pierce, Rockford, IL) according to manufacturer’s directions, and re-suspended in an aqueous solution of 0.1% formic acid. Samples were analyzed via reversed phase LC-MS/MS using a nano-LC ultra2D system coupled to a Velos Orbitrap mass spectrometer (Thermo Scientific, San Jose, CA). LC-MS experiments were performed in a data-dependent mode with Full-MS (externally calibrated to a mass accuracy of < 5 ppm, and a resolution of 60 000 at m/z 400) followed by CIDMS/MS of the top 10 most intense ions. Mass spectra were processed and quantified using the Andromeda and MaxQuant software suite (Max Planck Institute) against a mouse uniprot database. All searches were carried out using cysteine carbamidomethylation as a fixed modification while methionine oxidation and protein N-terminal acetylation, and phosphorylation at STY residues were selected as dynamic modifications. AACT/SILAC quantitation was performed in MaxQuant [ver. 1.2.2.5] with the Andromeda search engine.(Cox et al., 2011) The searches were carried out initially at 200 ppm. Precursor ion mass tolerance followed by a main search of the m/z, and retention time corrected features using a precursor ion mass tolerance of 5 ppm. Peptides were confidently identified using a target-decoy approach with a peptide false-discovery-rate (FDR) of 1% and a protein FDR of 5%. Phosphorylation sites were localized using PTM score with phosphorylation site localization FDR of 1%. Data processing and statistics were performed using Perseus [ver. 1.2.0.17] (Cox and Mann, 2011). Protein quantitation was performed on biological replicate runs and a two sample t test statistics was used with a p-value of 5% to report statistically significant fold-changes. In this experiment, mass tagging with both Arg and Lys was performed to increase the phosphoproteomic coverage. Cells were grown in the media supplemented with either 12C6-Arg/12C6-Lys (K0R0, ‘‘L’’), or 13C6-Arg/13C6-Lys (K6R6, ‘‘M’’), or 13C615N4Arg/13C615N2-Lys (K8R10, ‘‘H’’). AACT/SILAC quantitation (KR/K+6 R+6/K+8 R+10) analysis was carried out similarly as previously described (Du et al., 2010; Xie et al., 2009). The AACT ratios of the proteins were derived from the comparison of the extracted ion chromatogram peak areas of all matched light (L) peptides with those of the medium (M) or heavy (H) peptides. The ratios of M/L or H/ L were also verified by visual inspection of the raw mass spectra. In MS spectra, for each arginine-containing peptide, a set of three

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isotope signals with the mass spacing of 6 Da and 10 Da was observed as L isotope peak was originally from the non-stimulated cells, and the 6 Da (M) or 10 Da heavier (H) isotope peaks came from either NL or TL cells, respectively. In reference to the criteria previously established by our group (Xie et al., 2009) as well as others (Zhang et al., 2006), with respect to close to equal isotope intensity of MyD88, an approximate 30% increases in isotope intensity or a M/L or H/L value over 1.3 was determined as the quantitative threshold to distinguish the LPS-induced MyD88 complex components while the H/L ratios of most of non-specific contaminants were found close to or less than unity. The relative ratio of MyD88 in the immunoprecipitate mixture was used to normalize the ratios of all identified and quantified MyD88-interacting proteins. The isotope peak intensity ratio, i.e., M/L or H/L indicates the quantitative change in binding between a protein and MyD88 under either LPS-responsive or LPS-tolerant condition relative to unstimulated cells. The relative abundance of the bait protein, MyD88, in each immunoprecipitate, was found at 1:0.94:0.83 and then calibrated to 1. The normalized ratio was also calculated for an interactor indicating its MyD88-bound population. M/L and H/L ratios for each protein were determined by averaging the ratio found for at least two unique AACT-containing peptides. Most proteins were identified in high confidence with more than one peptide. For those proteins that were identified by a single peptide, the MS/MS spectra were manually inspected to ensure correct identification. For most proteins, the relative standard deviation for quantification between peptides of the same protein was less than 20%. Functional Category Analysis of the Identified MyD88-Interacting Proteins Biological processes and molecular functions of the MyD88-interacting proteins were categorized by PANTHER (http://www. pantherdb.org/) and DAVID (http://david.abcc.ncifcrf.gov/). RNA Preparation and Real-Time PCR Total RNA was isolated using RNeasy kit (QIAGEN). First-strand cDNA was synthesized by M-MLV reverse transcriptase (promega) and diluted 10 times for quantitative PCR. Real-time PCR was performed using SYBR Green. All measurements were normalized against GAPDH as the internal controls. The sequence information of primers was included in Table S1. PCR Array Raw264.7 cells expressing shRNA against GFP or PP2Ac were treated with 0.1 mg/ml LPS for 24 hr (T) or left untreated (N). Total RNA was extracted. Relative gene expression was quantified using RT2 Profiler PCR Array Mouse Inflammatory Cytokines & Receptors (SABiosciences). Fold induction of genes at T compared to that at N was normalized with the amount of GAPDH. Primary BMDM Culture Bone marrow progenitors were rinsed out from femur and tibia bones of BL6 wild-type or MyD88 / mice and cultured for 6 days in the DMEM supplemented with 10% FBS and 20% L929-conditioned media (LCM), 100 units/ml penicillin, and 100 mg/ml streptomycin (Skinner et al., 1994). AACT-Based Quantitative Analysis of LPS-Induced, PP2Ac-Targeted Phosphorylations at the MyD88 Immunoprecipitated from AACT-Labeled HEK TLR4-MD2-CD14 Stable 293 Cells The AACT labeling of the HEK293 cells stably expressing TLR4-MD2-CD14 was carried out based on the protocol previously described (Gunawardena et al., 2011; Wang et al., 2005, 2006). The HEK293 stable TLR4-MD2-CD14 cells under N, NL, TL, OA+N, OA+NL, OA+TL conditions were labeled alternatively by either regular 12C6-arginine (L), or 13C6-arginine (M) before Flagtagged MyD88 co-transfection and LPS stimulation. The Flag-tagged MyD88 was then immunoprecipitated from lysates of each labeled or unlabeled cells before equal mixing for four different AACT-labeled immunoprecipitate sets as N-NL, N-TL, NL(OA+NL), TL-(OA+TL). SILAC/AACT-based quantitation was based on extracted ion chromatograms (XIC) of peaks corresponding to L- and M-labeled tryptic peptides of MyD88. The peak areas of both phosphopeptide and non-phosphopeptide counterparts were used to calculate phosphorylation site occupancy (%) for all four conditions by adapting a recently reported method (Olsen et al., 2010) with our modifications illustrated in Figures S4A and S4B for both the identification and quantitation of phosphorylation sites and their LPS- or OA-induced changes. Systemic Identification of the LPS-Inducible Phosphorylation Sites Targeted by Chronic-Active PP2Ac through AACT-Based Quantitative Phosphoproteomic Analysis Similar to what has been illustrated above (also by Figure S3A), the cells stably expressing shRNA for GFP in ‘‘L’’ media (N), stable isotope-tagged with both R and K ‘‘M’’ with 0.1 mg/ml LPS for 24 hr then challenged with 1.0 mg/ml LPS for 15 min (TL), or expressing shRNA for PP2Ac in double-tagged ‘‘H’’ media (TL) were harvested and lyzed in buffer (8 M Urea, 50 mM Tris pH 8.0, 75 mM NaCl, 1 mM MgCl2, 500 units Benzonase) containing with protease phosphatase inhibitor cocktail set I and II (Calbiochem). Three lysates were mixed by equal amount of total protein 5 mg each. The mixture was first reduced with DTT followed by alkylation with iodoacetamide. The proteins were digested with endoproteinase Lys-C (Wako USA). The solution was then diluted 4-fold with 25 mM Tris pH 8.0, 1 mM CaCl2 and further digested with trypsin (Promega). The digestion was stopped by addition of TFA to 0.4% final concentration. The peptide solution was desalted on Sep-Pak Light C18 cartridge (Waters) and dried. The peptides S2 Cell Reports 3, 678–688, March 28, 2013 ª2013 The Authors

were dissolved in 30% ACN, 0.1% TFA and loaded on 1 ml Resource 15S (GE Healthcare) column for strong cation exchange chromatography (SCX). Linear gradient from 5 mM to 100 mM KCl in 30% ACN, 5 mM KH2PO4, 0.1% TFA was performed followed by high salt elution with 350 mM KCl in 30% ACN, 5 mM KH2PO4, 0.1% TFA. The enrichment of phosphorylated peptides was performed directly in SCX fractions (Larsen et al., 2005). Briefly, 1-5 mg of 5 mm Titansphere beads (GL Sciences) suspended in 80% ACN/1% TFA was added to each fraction and incubated for 30 min at room temperature. The beads were collected by centrifugation and washed with 150 ml 60% ACN/1% TFA three times, then transferred on top of a C8 disc (Empore) placed in 200 ml pipette-tip. Bound phosphopeptides were eluted with 15% NH4OH/40% CAN. Elutes were dried and desalted on StageTip containing 4 3 1 mm C18 extraction disk (3M). Clonogenic Survival Assay Raw264.7 cells were induced to ET/TL condition as described above. 1 mg/ml CHX was added in after 24 hr low-dose LPS priming and the cells were harvested 6 hr after the second stimulation. Then cells were counted in hemocytometer and re-plated onto 6-well plates respectively with 150, 300, 600 viable cells in duplicated wells. After growing for 9 days, cell colonies were washed with PBS, fixed with a mixture of methanol-acetic acid (3:1 in volume ratio) for 10 min, stained with 10% Giemsa (Ricca) (Gaddameedhi et al., 2012). Colonies containing more than 20 cells were counted and percentage of the survived cells under each condition was determined compared to the number of colonies on non-stimulated plates. Flow Cytometric Analysis Annexin V-FITC/Propidium iodide (PI) staining (Annexin V/PI apoptosis kit, Invitrogen) was used to determine the OA effect on cell viability under different inflammatory conditions. Briefly, BMDMs were seeded onto 35 cm dishes and incubated with or without 20nM OA. 24 hr after the last stimulation, the cells were re-suspended in binding buffer, incubated with Annexin V and propidium iodide at room temperature for 15 min in the dark, and analyzed by flow cytometry using FACScan (Becton Dickinson). Constructs of HA-Tagged MyD88-GyrB Fusion Protein and Site-Directed Mutagenesis Because of the propensity of cytosolic MyD88 in forming aggregates that interferes the dynamic binding of MyD88 to other proteins, by following the similar strategy of expressing FLAG-MyD88-GyrB fusion protein which exists in cells as monomer or dimer (Into et al., 2010) we made HA-tagged MyD88-GyrB fusion protein. MyD88 was cloned into pcDNA4 with HA tag at its N-terminus and Gyrb at C terminus. PP2Ac was cloned into pcDNA4 with C-terminal Myc-tag. Site-specific mutants of either MyD88 or PP2Ac were generated with QuikChange Site-Directed Mutagenesis Kit. The primer sequences were listed in Table S2. Antibody Production The peptide of human MyD88 with an additional cysteine at N-terminus, C-TKFAL(p)SLSPGA was synthesized. The phosphorylated serine is the mouse S242 (NP_034981) corresponding to its human counterpart S255 (NP_002459). The peptide was conjugated with carrier protein using Imject Maleimide Activated Carrier Protein Spin Kits (Thermo Scientific). The antibody was produced in rabbits at Pocono Rabbit Farm and Lab (Canadensis, PA). Transfection, Immunoprecipitation, and Immunoblotting Analysis HEK 293 stable TLR4-MD2-CD14 cell line was transfected with indicated constructs by jetPRIME transfection reagent. The cells were harvested from each N, NL, TL condition, and lysed with lysis buffer containing 0.5% NP-40, 10 mM Tris pH 7.5, 150 mM NaCl, 0.4 mM EDTA, 2 mM Na3VO4, 1x phosphotase inhibitor cocktail (Pierce), 1x protease inhibitor cocktail (sigma-aldrich). Cell lysates were incubated with affinity beads for overnight at 4 C on rotator. Affinity beads were washed, eluted and subjected to immunoblot analysis. Nuclear and Cytoplasmic Fraction Raw264.7 cells under each different inflammatory condition were washed with cold PBS once, scraped, and collected by centrifugation. The nuclear and cytoplasmic proteins were fractionated with NE-PER Nuclear and cytoplasmic Extraction Reagents (Thermo Scientific) according to the manufacturer’s instructions. Immunofluorescence For the localization experiments, the fixed cells were incubated with mouse anti-PP2Ac and rabbit anti-MyD88 followed by incubation with Alexa-488-conjugated anti-mouse IgG and Alexa-568-conjugated anti-rabbit IgG. Nuclei were visualized with DAPI. The confocal images were obtained on LeicaSP2 AOBS Upright Laser Scanning confocal microscope. ChIP ChIP experiments were conducted by dual cross-linking with DSG and formaldehyde sequentially (Porro and Perini, 2007). Following an overnight incubation with Brg-1 antibody (Santa Cruz) the cross-linked products were reversed, and the released DNA was purified by QIAquick PCR purification Kit (QIAGEN), and processed to quantitative PCR assay. Cell Reports 3, 678–688, March 28, 2013 ª2013 The Authors S3

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Figure S1. Inflammation Phenotypes of LPS Differentially Irritated Raw264.7 Cells, and Quantitative Proteomic Identification of TL-Specific MyD88-Interacting Partners, Related to Figure 1 (A) Inflammation-specific phenotypes (i.e., N, NL, TL) were characterized by (Top) the changes of IkBa and phosphor-p65 immunoblotted by indicated antibodies, (Middle) LPS-induce DNA fragmentation, and (Bottom) TNFa release measured by ELISA. (B) (Top) Mass spectra of the AACT-containing peptides of the indicated TL-specific MyD88-interacting partners. Shown for each TL-specific interactor is one spectrum representative of at least two peptides identified by MS/MS. (bottom) Immunoblot analysis of the MyD88 immunoprecipitates from Raw264.7 under N, NL, and TL condition respectively with indicated antibodies. The abundance of MyD88 in immunoprecipitates was used as the loading control, b-actin in whole cell lysate (WCL) was as the input control. (C) PP2Ac is at the Central Node of the Protein Interaction Network in the TL-specific MyD88 Interactome. Assembled by data-dependent domain-based network analysis, 84 out of a total of 433 TL-specific MyD88 interactors were found interacting in the PP2Ac-coordinated network (detailed proteomic data will be reported elsewhere). Using GO-based String PPI database, interactors were also clustered according to specific pathways.

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Figure S2. Effects of LPS-Induced Inflammation on PP2Ac Expression, Differential LPS Response of MyD88 / BMDMs, and Differential Cytokine Production in the RAW Cells Stably Expressing shRNA-GFP or shRNA-PP2Ac, Related to Figures 1, 2, and 3 (A) (Top) From the Raw264.7 cells under each of different inflammatory conditions, N, NL, and TL, total RNA was extracted and subjected to the analysis using semiquantitative PCR, GAPDH was used as the loading control, (Middle and Bottom) Raw264.7 cells were primed with either CHX for 3 hr or MG-132 for 1 hr with indicated doses before harvest. LPS was added to induce either NL or TL. The abundance of PP2Ac protein was immunoblotted for whole cell lysate. cMyc was used as the positive control to show effects of the chemicals priming. Gamma-tubulin was used as the loading control. (B) (left) MyD88 expression was completely abolished in MyD88 / BMDMs, (right) the release of TNFa in WT and MyD88 / BMDM under N, NL and TL was quantified with qPCR. (C) different clones (#363, 364, 365, 366, and 367) showing different efficiency in PP2Ac knock-down, and all RAW cells transfected by empty vector (EV) or shGFP showed no effect on PP2Ac knock-down. (D) Raw264.7 cells under tolerant condition over that in naive cells. Cells were treated with 0.1 mg/ml LPS for 24 hr (T) or left untreated (N). Total RNA was extracted and cDNA was synthesized. Relative gene expression was quantified using RT2 Profiler PCR Array Mouse Inflammatory Cytokines & Receptors. Fold induction of gene at T compared to that at N was normalized with amount of GAPDH. The threshold of upregulated genes was set at R 1.3, while downregulated genes at % 0.7.

S6 Cell Reports 3, 678–688, March 28, 2013 ª2013 The Authors

Figure S3. AACT-Based Quantitative Phosphoproteomic Approach to Identify PP2Ac Target Sites, and Cell Viability of the Differentially Stimulated BMDMs, Related to Figure 2, 3, and 4 (A) Schematic design of identification of the LPS-inducible phosphorylation sites and those targeted by chronically active PP2Ac. (B) A heat map of the clustering the proteins and their PP2Ac target phosphorylation sites. Red color indicates the increasing level of a phosphorylated peptide. ‘Wt-TL/Wt-N’, or KD-TL/Wt-N’, or ‘KD-TL/Wt-TL’ represents the ratio change of indicated phospho-peptides in the wild-type Raw cells under TL versus nonstimulated N condition, or TL PP2Ac knock-down (KD) versus non-stimulated N wild-type cells or TL PP2Ac knock-down (KD) versus TL wild-type cells respectively. (C) Immunoblot analysis of changes in phosphorylation of c-Jun ser63 in the nucleus of the wild-type BMDMs under N, NL, TL conditions without or with OA pretreatment. (D) All irritated cells were allowed a 24 hr post-irritation recovery before flow assay. For the cells subjected to OA treatment, cells were incubated without or with 20 nM OA for 6 hr prior to either acute or prolonged LPS stimulation, stained with FITC-conjugated Annexin V and propidium iodide (PI), and analyzed by flow cytometry. The percentage of each of three cell populations was found as indicated in each box, respectively, for viable cells (lower left), early apoptotic cells (lower right), and late apoptotic/necrotic cells (upper right/left).

Cell Reports 3, 678–688, March 28, 2013 ª2013 The Authors S7

Figure S4. AACT-Based Quantitative Proteomic Identification of LPS-Induced, PP2Ac Target Phosphorylation Sites on MyD88, Related to Figures 3 and 4 (A) Extracted ion chromatograms (XIC) of the signal peaks corresponding to the L-labeled untreated cells (OA-) and M-labeled OA-treated cells (OA+) of the MyD88 peptide FALSLSPGVQQK with serine 242/244 phosphorylations (Top panel), and its non-phosphorylated counterparts (Bottom panel) under NL or TL condition. (B) Quantitative analysis of phosphorylation changes, (Top) The ratios of the peak areas of phosphopeptide (x), non-phosphopeptide (y), and total protein ratio (z) were obtained from experimental data and applied directly in the equations to obtain phosphorylation site occupancy (%) for different cellular states NL+OA, NLOA, and TL+OA, TL-OA. (Bottom) effects of PP2Ac inhibition on the LPS-induced serine phosphorylation at 244/244 (peptide239FALpSLSPGVQQK) of MyD88 isolated from the NL or TL stable TLR4-expressing 293 cells. The phosphorylation stoichiometry (%) is given on the y axis. The inset shows the fold change (%) of phosphorylation in either NL or TL cells caused by OA treatment. (C) (Left) Dot-plot of the generated antibodies against phospho-serine 242, and (right) Immunoblot analysis of specificity of anti-phospho-Ser242 antibody using MyD88 wild-type or mutants.

S8 Cell Reports 3, 678–688, March 28, 2013 ª2013 The Authors