Proteomic Analysis of Palmitoylated Platelet Proteins

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PLATELETS AND THROMBOPOIESIS e-Blood Proteomic analysis of palmitoylated platelet proteins

*Louisa Dowal,1 *Wei Yang,2-4 Michael R. Freeman,2,3 Hanno Steen,4,5 and Robert Flaumenhaft1

1Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center, Boston, MA; 2Urological Diseases Research Center, Department of Urology, Children’s Hospital Boston, Boston, MA; 3Department of Surgery, Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA; 4Proteomics Center, Children’s Hospital Boston, Boston, MA; and 5Department of Pathology, Harvard Medical School and Children’s Hospital Boston, Boston, MA

Protein palmitoylation is a dynamic pro- try, followed by identification using liquid transcript-1 (TLT-1) as its expression is cess that regulates membrane targeting chromatography-tandem mass spec- restricted to platelets and megakaryo- of proteins and protein-protein interac- trometry. This global analysis identified cytes. We determined that TLT-1 is a tions. We have previously demonstrated > 1300 proteins, of which 215 met crite- palmitoylated protein using metabolic la- a critical role for protein palmitoylation in ria for significance and represent the beling with [3H]palmitate and identified platelet activation and have identified pal- platelet palmitoylome. This collection the site of TLT-1 palmitoylation as cys- mitoylation machinery in platelets. Using includes 51 known palmitoylated pro- teine 196. The discovery of new platelet a novel proteomic approach, Palmitoyl teins, 61 putative palmitoylated pro- palmitoyl protein candidates will provide Protein Identification and Site Character- teins identified in other palmitoylation- a resource for subsequent investigations ization, we have begun to characterize specific proteomic studies, and 103 new to validate the palmitoylation of these the human platelet palmitoylome. Palmi- putative palmitoylated proteins. Of these proteins and to determine the role palmitoylated proteins were enriched from candidates, we chose to validate the pal- toylation plays in their function. (Blood. membranes isolated from resting plate- mitoylation of triggering receptors ex- 2011;118(13):e62-e73) lets using acyl-biotinyl exchange chemis- pressed on myeloid cell (TREM)–like Introduction

Platelets are key mediators of hemostasis and thrombosis, and their resting platelets, we8 and others9-13 observe incorporation of acute response at sites of vascular injury requires a rapid and [3H]-palmitate into platelet proteins, which indicates that this fatty complex series of signaling events. Posttranslational modifications, acid is being cycled even in the resting state. Although palmitate such as phosphorylation,1 have been shown to influence platelet- accounts for 74% of the fatty acids linked to proteins by a thioester signaling pathways, thus regulating platelet function, and palmitoyl- bond in the platelet,10 protein palmitoylation remains a poorly ation, which is also a reversible modification, has been proposed to understood posttranslational modification in platelets. These obser- play an analogous regulatory role.2-3 Palmitoylation is the most vations prompted us to assess the global scope of palmitoylation in common form of S-acylation, which is the covalent attachment of platelets. long chain fatty acids via thioester bonds to cysteine residues.4 Proteomics offers a powerful means to study the biology and Palmitoylation involves the attachment of the 16-carbon saturated biochemistry of the anucleate platelet.14-15 Recently, palmitoy- fatty acid palmitate. Although attachment of palmitate enhances the lomes in yeasts,16 rat neurons,17 and human prostate cancer cells18 hydrophobicity of a protein, its function extends beyond that of a have been described. In our efforts to define the platelet palmitoy- simple membrane anchor as palmitoylation has been shown to lome, we have purified and identified palmitoylated proteins from regulate protein trafficking, sorting, stability, and activity.5 Thus, membranes of resting platelets using the Palmitoyl Protein Identifi- the addition of this lipid group to a protein has functional cation and Site Characterization (PalmPISC) method that we consequences. Palmitoylation is unique among the different types recently developed.18 In this study, we present a comprehensive of lipid modifications in that it is reversible and does not require a identification of palmitoylated platelet proteins. This strategy specific sequence motif. The reversible nature of palmitoylation identified 215 significantly enriched palmitoylated platelet protein functions as a regulatory mechanism or molecular switch, directing candidates. Of these, 51 are known palmitoylated proteins and protein-lipid and protein-protein interactions6-7 and plays a role in 61 were identified in proteomic studies of other cells with medium the regulation of cellular responses to external stimuli. to high confidence, indicating that they are most likely palmitoy- We have previously shown that platelets possess palmitoylation lated. The remaining 103 palmitoyl protein candidates have not machinery and require palmitoylation for activation because chemi- previously been shown to be palmitoylated. As proof of this cal inhibition of protein palmitoylation results in abrogation of concept, we validated the palmitoylation of triggering receptors platelet aggregation and decreased incorporation of platelets into expressed on myeloid cell (TREM)–like transcript-1 (TLT-1), a thrombi in a murine laser-induced model of vascular injury.8 In platelet- and megakaryocyte-specific protein, and identified its

Submitted May 9, 2011; accepted July 11, 2011. Prepublished online as Blood The publication costs of this article were defrayed in part by page charge First Edition paper, August 2, 2011; DOI 10.1182/blood-2011-05-353078. payment. Therefore, and solely to indicate this fact, this article is hereby marked ‘‘advertisement’’ in accordance with 18 USC section 1734. *L.D and W.Y. contributed equally to this study. This article contains a data supplement. © 2011 by The American Society of Hematology

e62 BLOOD, 29 SEPTEMBER 2011 ⅐ VOLUME 118, NUMBER 13 From www.bloodjournal.org by guest on September 11, 2016. For personal use only.

BLOOD, 29 SEPTEMBER 2011 ⅐ VOLUME 118, NUMBER 13 PALMITOYLATED PLATELET PROTEINS e63 palmitoylation site. These experiments further our understanding Mass spectrometry of the scope of palmitoylation in platelets and will provide a basis Tryptic peptides were analyzed by on-line nanoflow reversed-phase high for studying the dynamics and function of palmitoylation in these performance liquid chromatography (Eksigent nanoLC-2D) connected to putative palmitoyl proteins. an LTQ Orbitrap mass spectrometer (Thermo Scientific) essentially as 18 described. Samples were loaded onto an in-house packed C18 column (Magic C18,5␮m, 200 Å, Michrom Bioresources) with 15-cm length and 100-␮m inner diameter, and separated at ϳ 200 nL/min with 60-minute Methods linear gradients from 5% to 35% acetonitrile in 0.2% formic acid. Survey spectra were acquired in the Orbitrap analyzer with the resolution set to a Chemicals and reagents value of 30 000. Lock mass option was enabled in all measurements, and Unless otherwise noted, all chemicals were obtained from Sigma-Aldrich. decamethylcyclopentasiloxane background ions (at m/z 371.10123) were Complete, EDTA-free protease inhibitor cocktail (11873580001) was used for real-time internal calibration as described previously.20 Up to 5 of obtained from Roche Diagnostics. [3H]Yohimbine (NET 659250UC), guanos- the most intense multiply charged ions per cycle were fragmented and ine triphosphate (GTP)[␥-35S] (NEG 030H250UC), and [3H]palmitic acid analyzed in the linear ion trap. (NET043) were obtained from PerkinElmer Life and Analytical Sciences. Tris(2-carboxyethyl)phosphine (TCEP; 77720) and N-[6-(biotinamido)hexyl]-3Ј- Database searching, spectral counting, and statistical analysis (2Ј-pyridyldithio)propionamide (biotin-HPDP; 21341) were obtained from The Thermo raw files were deposited at Tranche (https://proteomecommons. Thermo Scientific. Coomassie Brilliant Blue and polyvinylidene fluoride (PVDF) org/dataset.jsp?i ϭ 76 216) and will be made publically accessible on membranes were obtained from Bio-Rad. Iodoacetamide (RPN6302) was publication. Raw data were analyzed using MaxQuant Version 1.0.13.13.21 purchased from GE Healthcare. MS-grade trypsin (V5280) was obtained from The parameters were set as follows. In the Quant module, Singlets was Promega. Anti–human TREML1/TLT-1 primary antibody (AF2394) was ob- selected; oxidation (M), acetyl (protein N-term), carbamidomethyl (C), tained from R&D Systems, and anti–human Cdc42 (610928) antibody was and N-ethylmaleimide (C) were set as variable modifications; no fixed obtained from BD Transduction Laboratories. FITC-conjugated secondary modifications were allowed; concatenated IPI human database Version 3.52 antibodies were obtained from Pierce Biotechnology. Anti-G␤ primary antibody 1 (74 190 forward sequences and 74 190 reverse sequences) downloaded (sc-379) and Protein A/G PLUS-agarose (sc-2003) were obtained from Santa from www.maxquant.org was used for database searching; all other Cruz Biotechnology. parameters used were default values. In the Identify module, all parameters used were default values, except that maximal peptide posterior error Platelet and platelet membrane preparation probability was set as .05. False discovery rates for protein identification and peptide identification were both set at 1%. The relative protein Platelet-rich plasma that had outdated within 24 hours before use was ϩ Ϫ obtained from the Beth Israel Deaconess Medical Center Blood Bank. abundance changes between the paired HA and HA samples were 22 Platelets were washed 3 times and assessed by flow cytometry using a determined using a label-free spectral counting approach. Statistical 18 P-selectin expression assay.8 Only platelet preparations demonstrating analysis was performed as previously described with minor modifications. resting P-selectin levels comparable with resting fresh platelets were Briefly, the spectral counts were merged over both biologic replicates, and further processed. An aliquot of the selected platelets was tested to all zeros were replaced with 1 in the merged dataset to avoid division by confirm normal P-selectin expression in response to 100␮M SFLLRN. zero. Given that spectral counting is not accurate for proteins with very Resting, washed platelets were centrifuged, and platelet pellets were low spectral counts, statistical analysis was only performed on the proteins with a spectral count of at least 3. The protein-wise log2- resuspended in ice-cold lysis buffer (5mM Tris-HCl, pH 7.5, and 5mM ϩ Ϫ ethylene glyco-bis(b-aminoethyl ester)-N,N,NЈ,NЈ-tetraacetic acid) plus transformed HA /HA ratios were calculated and then clustered using a protease inhibitor cocktail to 4 mL/g wet weight. Platelets were lysed by Bayesian information criterion-based Gaussian mixture model. The result- successive rounds of sonication; and after removal of intact cells by low ing 2 Gaussian components represent the log-ratio distributions of protein speed centrifugation, the lysates were pooled. Membranes were pelleted sets largely dominated by contaminating proteins or by palmitoyl proteins. at 50 000g and flash frozen at Ϫ80°C. To assess the quality of the P values were computed based on the distribution of the contaminating platelet membrane preparation, immunoblotting for the peripheral protein-dominant dataset. membrane protein G␤1 was performed. To further evaluate the integrity of the membrane preparation, [3H]yohimbine binding and GTP[␥-35S] Western blot analysis 19 binding were evaluated. Only preparations in which the receptors For immunoblotting, separated proteins were transferred to PVDF mem- within the membrane retained the ability to bind agonists and stimulate branes and blocked in TBS/0.1% Tween-20/5% milk for 1 hour at room GTP turnover on stimulation were used for liquid chromatography-mass temperature. Blots were probed with anti–human TREML1/TLT-1 primary spectrometry (LC-MS) studies. antibody (1:1000) or anti–human Cdc42 (1:500) overnight at 4°C followed by 1-hour incubation at room temperature with FITC-conjugated secondary Platelet palmitoyl protein purification, separation, and trypsin antibodies (1:1000) to detect TLT-1 or Cdc42. Blots were analyzed with an digestion Amersham Typhoon 9400 molecular imager.

Proteins were prepared from 100 mg of platelet membranes using our Labeling platelet proteins with [3H]palmitate and adaptation of acyl-biotinyl exchange (ABE) chemistry as previously immunoprecipitation of TLT-1 described.18 Briefly, membrane proteins were denatured with SDS, reduced with 10mM TCEP, and alkylated with 50mM N-ethylmaleimide (NEM) to Washed platelets (2 mL at 1 ϫ 109 platelets/mL) were radiolabeled with block nonpalmitoylated cysteines. After methanol/chloroform precipita- 100 ␮Ci/mL [3H]palmitic acid for 1 hour at 37°C, in PIPES/NaCl buffer tions to remove excess NEM, proteins were treated with 0.75M hydroxyl- with 3.6 mg/mL BSA. Platelets were activated as described in the text, amine (HA) and 1mM biotin-HPDP to replace palmitoyl groups with diluted 5 times with PIPES/NaCl buffer, and pelleted to remove unincorpo- biotinyl groups. The in vitro biotinylated (previously palmitoylated) rated [3H]palmitic acid. Platelets were then resuspended in RIPA buffer proteins were enriched by streptavidin affinity purification, specifically (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 158mM NaCl, eluted by TCEP, and concentrated by methanol/chloroform precipitation. 10mM Tris-HCl, pH 7.6, 1mM phenylmethylsulfonyl fluoride, and Enriched proteins were separated on a 12% SDS-PAGE gel and stained with protease inhibitor cocktail) and allowed to lyse on ice for 5 minutes. The Coomassie Brilliant Blue. Each gel lane was cut into 5 slices before lysate was centrifuged for 5 minutes at 16 000g, and the supernatant was reduction with 10mM dithiothreitol, alkylation with 55mM iodoacetamide, used as the RIPA soluble fraction. The platelet lysate was precleared by and in-gel digestion with trypsin.18 adding 20 ␮L of Protein A/G beads and incubating for 30 minutes at From www.bloodjournal.org by guest on September 11, 2016. For personal use only.

e64 DOWAL et al BLOOD, 29 SEPTEMBER 2011 ⅐ VOLUME 118, NUMBER 13

Figure 1. Analysis of ABE-purified palmitoylated proteins from platelet membranes. (A) Electrophoretic analysis. Lane 1 indicates molecular weight marker; lane 2, experimental samples (HAϩ) with hydroxylamine treatment; and lane 3, control samples (HAϪ) without hydroxylamine treatment. (B) The correlation between proteins identified in 2 separate rounds of spectral counting analysis. Data were fit with a linear function (R2 ϭ 0.8645). (C) Distribution of log(HAϩ/HAϪ) ratios. Bayesian Information Criterion-based Gaussian Mixture modeling suggested that the distribution of log-ratios (solid line) is composed of 2 Guassian components (dashed line). The left Gaussian represents the log-ratio distribution of most contaminating proteins and some palmitoyl proteins, whereas the right Guassian represents the log-ratio distribution of most palmitoyl proteins and a small number of contaminants. To distinguish palmitoyl proteins from contaminating proteins, P values were calculated based on the distribution of the left Gaussian. Proteins with P Ͻ .05 were treated as significant and accepted as palmitoyl protein candidates.

4°C with end-over-end rotation. Beads were pelleted, and TLT-1 was chromatography and eluted with TCEP. This approach enriched for immunoprecipitated from the supernatant using 2.5 mg/mL human TREML1/ a large number of proteins (Figure 1A) that were separated and TLT-1 primary antibody overnight at 4°C with end-over-end rotation. visualized by SDS-PAGE followed by in gel digestion and ␮ Protein/immune complexes were pulled out by addition of 20 L of Protein LC-MS/MS. A/G beads and incubation for 60 minutes at 4°C with end-over-end LC-MS/MS and MaxQuant analyses of 2 biologic replicates rotation. Beads were then washed 3 times with 1 mL of RIPA buffer, and immunoprecipitates were eluted from the beads by the addition of 20 ␮L of enriched palmitoyl proteins resulted in the identification of Ͼ SDS sample buffer. [3H]Palmitate-labeled platelet proteins were then 1300 proteins (supplemental Table 3, see the Supplemental separated by SDS-PAGE and transferred onto a PVDF membrane, which Materials link at the top of the article), with a false discovery rate of was exposed to a tritium detection screen for 2 weeks and then analyzed 1% for both proteins and peptides. Plotting both rounds of spectral using an Amersham Typhoon 9400 molecular imager. Tritium blot band counting against each other revealed good agreement between intensities were normalized for the amount of protein loaded. experimental datasets (R2 ϭ 0.8645; Figure 1B). Label-free spectral counting quantitation and statistical analysis showed that 215 Identification of TLT-1 palmitoylation site nonredundant proteins were significantly enriched (P Ͻ .05) in ϩ Ϫ Enriched palmitoyl proteins were separated by SDS-PAGE. Because the the HA sample over that of the HA control (Figure 1C). To apparent molecular weight of TLT-1 is ϳ 37 kDa, a gel slice containing 30- ensure the accuracy of protein identification based on a single to 50-kDa proteins was excised. Proteins were tryptically digested in gel unique peptide, the MS/MS spectra of all platelet palmitoyl without reduction and alkylation. Tryptic peptides were extracted and protein candidates were manually verified (supplemental Figure analyzed by LC tandem MS (LC-MS/MS) as described in “Mass spectrom- 1). Proteins that were considered significantly enriched had an etry.” Free cysteines in the identified peptides are candidate palmitoylation HAϩ/HAϪ ratio Ͼ 3(P Ͻ .05); and of the 215 enriched proteins, sites because nonpalmitoylated cysteines were blocked by NEM before 103 proteins are not known to be palmitoylated or have not been ABE reaction. described in other palmitoyl proteomic studies (Table 1). The remaining 112 identified proteins are known to be palmitoylated or are palmitoyl protein candidates identified in other proteomic Results studies (Table 2). Purification and identification of palmitoylated platelet proteins For graphic representation of the proteins identified by LC-MS/ MS, the summed spectral counts for each protein in the HAϩ Palmitoylated proteins were enriched from platelet membranes that sample were plotted against the summed spectral counts for each were prepared from resting platelets pooled from 6 aphaeresis protein in the HAϪ sample (Figure 2A). For the majority of proteins packs, and purification of palmitoylated proteins was carried out identified, there was substantial representation in both the HAϩ and using our adaption of ABE chemistry.18 After extraction of platelet HAϪ samples, which resulted in a HAϩ/HAϪratio Ͻ 3 (gray dots, membrane proteins, all disulfide bonds were reduced with TCEP, a Figure 2A). Clustered around the x-axis, with the known palmitoy- reducing agent that does not cleave thioester bonds, and free thiols lated proteins (green circles, Figure 2A), are the newly identified were blocked with the alkylating agent, NEM. The sample was split palmitoyl protein candidates (open black circles, Figure 2A). into 2 groups, which were treated with parallel protocols: an Well-established, known palmitoylated platelet proteins identified experimental group in which the thioester bonds were specifically in our study are also indicated (blue dots, Figure 2A) and include ϩ Ϫ 11,12 11,12 12 25 cleaved with neutral HA and a control group (HA ), which was the G␣ subunits Gq, Gi, and G13, platelet glycoprotein 4, not treated with hydroxylamine. The HAϪ group represents back- and CD6326 (Table 2). Several highly abundant platelet proteins ground binding to the steptavidin beads, which may be the result of known to be palmitoylated were identified in the list of 1300 purified endogenous biotinylation, nonspecific biotinylation, or nonspecific proteins but failed to demonstrate a HAϩ/HAϪ ratio Ն 3 (red dots, binding. After the newly formed thiols were labeled with biotin- Figure 2A). This group includes P-selectin,27 tubulin,28 platelet 11 29 11 11 HPDP, biotinylated proteins were enriched by streptavidin affinity glycoprotein Ib, PECAM, G␣z, platelet glycoprotein IX, and From www.bloodjournal.org by guest on September 11, 2016. For personal use only.

BLOOD, 29 SEPTEMBER 2011 ⅐ VOLUME 118, NUMBER 13 PALMITOYLATED PLATELET PROTEINS e65

Table 1. Novel candidates of the platelet palmitoylome Protein name Gene name Ratio P

Apoptosis Myc target protein 1 MYCT1 3.10 .039094 Placenta-derived apoptotic factor PDAF 4.00 .015866 Cell growth and/or maintanence Tetraspanin-33 TSPAN33 6.25 .002368 Claudin-5 CLDN5 3.33 .030650 Dynein heavy chain 1, axonemal DNAH1 6.00 .002867 Metalloproteinase inhibitor 1 TIMP1 3.29 .032192 Lysophosphatidylcholine acyltransferase 1 LPCAT1 4.00 .015866 Transmembrane protein 97 TMEM97 3.00 .043487 DNA repair Histone H2A H2AFX 3.00 .043487 Immune response CKLF-like MARVEL transmembrane domain-containing protein 3 CMTM3 11.00 .000116 CKLF-like MARVEL transmembrane domain-containing protein 5 CMTM5 7.43 .001013 Early activation antigen CD69 CD69 5.00 .006457 Minor histocompatibility protein HA-1 HMHA1 3.00 .043487 Ion transport Calcium release-activated calcium channel protein 1 ORAI1 5.00 .006457 Metabolism ͓3-methyl-2-oxobutanoate dehydrogenase ͓lipoamide͔͔ kinase BCKDK 4.00 .015866 ␣-amylase 1 AMY1A 3.00 .043487 ATPase family AAA domain-containing protein 3A ATAD3A 3.00 .043487 Catechol-O-methyltransferase COMT 4.50 .010000 Dihydrolipoamide branched chain transacylase E2 DBT2 8.00 .000690 Dolichyldiphosphatase 1 DOLPP1 3.00 .043487 GPI ethanolamine phosphate transferase 1 PIGN 3.00 .043487 Probable cysteinyl-tRNA synthetase CARS2 4.00 .015866 Protein disulﬁde-isomerase TMX3 TMX3 15.00 .000016 Suppressor of Lec15 MPDU1 3.00 .043487 Thioredoxin-related transmembrane protein 1 TMX1 3.45 .027084 Thioredoxin-related transmembrane protein 4 TMX4 6.00 .002867 V-type proton ATPase 116-kDa subunit ␣ isoform 2 ATP6V0A2 3.00 .043487 Protein metabolism 26S proteasome non-ATPase regulatory subunit 11 PSMD11 6.00 .002867 CAAX prenyl protease 1 homolog ZMPSTE24 4.00 .015866 Copper chaperone for superoxide dismutase CCS 4.00 .015866 Cystatin-S CST4 3.00 .043487 E3 ubiquitin-protein ligase MARCH2 MARCH 6.00 .002867 Eukaryotic translation elongation factor 1 ⑀-1 EEF1E1 3.00 .043487 Eukaryotic translation initiation factor 5A-1 EIF5A 6.00 .002867 GrpE protein homolog 1 GRPEL1 3.00 .043487 Leucyl/cystinyl aminopeptidase LNPEP 4.00 .015866 RING ﬁnger protein 11 RNF11 3.50 .025869 Signal transduction Atlastin-1 ATL1 5.00 .006457 CKLF-like MARVEL transmembrane domain-containing protein 7 CMTM7 4.00 .015866 Endothelial cells scavenger receptor SCARF1 10.00 .000202 Filaggrin FLG 3.00 .043487 GTP-binding protein Rheb RHEB 4.00 .015866 Leucine-rich repeat-containing protein 32 LRRC32 21.00 .000002 Metalloreductase STEAP3 STEAP3 6.00 .002867 Nuclear receptor subfamily 4 group A member 2 NR4A2 3.00 .043487 Platelet-derived endothelial cell growth factor ECGF1 3.00 .043487 Rho-related GTP-binding protein RhoQ RHOQ 3.00 .043487 Tescalcin TESC 18.00 .000005 Tetraspanin-15 TSPAN15 17.00 .000007 Transmembrane protein 11 TMEM11 7.00 .001366 Transmembrane protein 50A TMEM50A 3.25 .033406 Transmembrane protein 55A TMEM55A 3.83 .018614 Transcription regulator activity 39S ribosomal protein L12 MRPL12 3.00 .043487 Oxidoreductase HTATIP2 HTATIP2 3.00 .043487

To date, these 103 proteins have not been shown to be palmitoylated and represent putative palmitoylated platelet proteins. Data are the result of 2 independent experiments, and proteins are listed according to the biologic process they mediate as deﬁned by the HPRD. Also shown is the HAϩ/HAϪ ratio and corresponding P value. From www.bloodjournal.org by guest on September 11, 2016. For personal use only.

e66 DOWAL et al BLOOD, 29 SEPTEMBER 2011 ⅐ VOLUME 118, NUMBER 13

Table 1. Novel candidates of the platelet palmitoylome (continued) Protein name Gene name Ratio P

Transport BET1 homolog BET1 10.00 .000202 BET1-like protein BET1L 11.00 .000116 Cholesteryl ester transfer protein CETP 4.00 .015866 MLN64 N-terminal domain homolog STARD3NL 4.00 .015866 Nonspeciﬁc lipid-transfer protein SCP2 9.00 .000366 Proteolipid protein 2 PLP2 3.00 .043487 SID1 transmembrane family member 1 SIDT1 4.00 .015866 Sodium/hydrogen exchanger 9 SLC9A9 3.67 .021908 Solute carrier family 43 member 3 SLC43A3 5.00 .006457 Syntaxin-2 STX2 3.00 .043487 Syntaxin-10 STX10 4.00 .015866 Syntaxin-11 STX11 6.18 .002507 Tandem C2 domains nuclear protein TC2N 42.00 .000000 Vesicle transport protein SFT2C SFT2D3 6.00 .002867 Vesicle-associated membrane protein 5 VAMP5 15.00 .000016 Unknown Catechol-O-methyltransferase domain-containing protein 1 COMTD1 3.50 .025869 CD99 antigen-like protein 2 CD99L2 3.00 .043487 Chronic lymphocytic leukemia deletion region gene 6 protein CLLD6 4.50 .010000 COMM domain-containing protein 9 COMMD9 3.00 .043487 Cyclin-Y CCNY 9.00 .000366 Exocyst complex component 3-like protein 2 EXOC3L2 4.00 .015866 Leptin receptor gene-related protein LEPROT 6.00 .002867 Lipoma HMGIC fusion partner LHFP 8.00 .000690 Lipoma HMGIC fusion partner-like 2 protein LHFPL2 6.40 .002115 LITAF-like protein CDIP 6.00 .002867 Major facilitator superfamily domain-containing protein 6 MFSD6 4.00 .015866 Malectin MLEC 4.90 .007035 Metallo-␤-lactamase domain-containing protein 2 MBLAC2 12.00 .000068 Mps one binder kinase activator-like 3 MOBKL3 5.00 .006457 Oligosaccharyltransferase complex subunit OSTC OSTC 3.00 .043487 PDZK1-interacting protein 1 PDZK1IP1 8.00 .000690 Prolactin-inducible protein PIP 4.00 .015866 Protein EFR3 homolog A EFR3A 9.67 .000245 Protein FAM78A FAM78A 3.00 .043487 Protein S100-A14 S100A14 3.00 .043487 Putative uncharacterized protein C1orf150 C1orf150 10.00 .000202 Putative uncharacterized protein C1orf95 C1orf95 3.00 .043487 SEC6-like protein C14orf73 C14orf73 24.00 .000001 Small VCP/p97-interacting protein SVIP 3.00 .043487 Tetraspanin-14 TSPAN14 9.80 .000227 Transmembrane BAX inhibitor motif-containing protein 1 TMBIM1 6.00 .002867 Transmembrane protein 222 TMEM222 10.00 .000202 Transmembrane protein 50B TMEM50B 8.00 .000690 Transmembrane protein with metallophosphoesterase domain TMPPE 5.00 .006457 Uncharacterized protein C22orf25 C22orf25 7.00 .001366 Uncharacterized protein KIAA2013 KIAA2013 7.00 .001366 UPF0389 protein FAM162A FAM162A 8.50 .000500 UPF0598 protein C8orf82 C8orf82 11.00 .003588 UPF0733 protein C2orf88 C2orf88 8.00 .000116

CD926 (supplemental Table 1). Included among the known palmi- Using the Human Protein Reference Database (HPRD),30 we toylated proteins is the palmitoylated form of Cdc42, which classified all the known and putative palmitoylated proteins by previously had been described in neurons17 and identified as a biologic process (Figure 3). We find that the largest percentage of putative palmitoyl protein in human prostate cancer cells.18 To palmitoylated platelet protein candidates (31.3%) is involved in confirm that we were observing the palmitoylated form of Cdc42 in signal transduction processes. A substantial number of proteins our preparation, samples were stained using antibodies directed (18.3%) involved in transport processes, which includes the against Cdc42. We found Cdc42 only in the HA palmitoyl-protein movement of vesicles and small molecules and ions within or out and not the HAϪ sample, indicating that platelets contain the of the cell, were found to be palmitoylated as well. Although close palmitoylated form of Cdc42 (Figure 2B). to half of these transport proteins are known to be palmitoylated or From www.bloodjournal.org by guest on September 11, 2016. For personal use only.

BLOOD, 29 SEPTEMBER 2011 ⅐ VOLUME 118, NUMBER 13 PALMITOYLATED PLATELET PROTEINS e67

Table 2. Known palmitoylated proteins of the platelet palmitoylome Protein name Gene name Ratio P Source

Cell growth and/or maintenance Claudin-3 CLDN3 16.50 .000009 Proteomic studies18 Desmoplakin DSP 2.90 .048440 Proteomic studies17 Flotillin-1 FLOT1 14.80 .000018 Manual 55-kDa erythrocyte membrane protein MPP1 3.00 .043487 Uniprot Immune response Complement component 1 Q subcomponent-binding protein C1QBP 3.00 .043487 Proteomic studies17 HLA class I histocompatibility antigen, B-27 ␣-chain HLA-B 9.00 .000366 Manual Interferon-induced transmembrane protein 3 IFITM3 3.00 .043487 Uniprot Linker for activation of T-cell family member 1 LAT 7.33 .001082 Uniprot Protein folding Calnexin CANX 2.88 .049774 Proteomic studies18 Metabolism 3-mercaptopyruvate sulfur transferase MPST 3.33 .030650 Proteomic studies17 4-aminobutyrate aminotransferase ABAT 4.00 .015866 Proteomic studies17 ADP-ribosyl cyclase 1 CD38 5.00 .006457 Proteomic studies17 Acid ceramidase ASAH1 5.25 .005232 Proteomic studies18 ATP synthase subunit ␥ ATP5L 4.00 .015866 Proteomic studies17 CTD small phosphatase-like protein CTDSPL 8.25 .000586 Proteomic studies18 Cytochrome b-c1 complex subunit Rieske UQCRFS1 3.00 .043487 Proteomic studies17 Glutaminase kidney isoform GLS 3.00 .043487 Proteomic studies17 NAD(P) transhydrogenase NNT 7.92 .000726 Proteomic studies17 Probable phospholipid-transporting ATPase IF ATP11B 11.00 .000116 Proteomic studies18 Sn1-speciﬁc diacylglycerol lipase-␤ DAGLB 29.00 1.29E-07 Proteomic studies18 Protein metabolism Cation-dependent mannose-6-phosphate receptor M6PR 7.00 .001366 Manual DnaJ homolog subfamily C member 5 DNAJC5 27.00 2.29E-07 Uniprot Endothelin-converting enzyme 1 ECE1 35.00 2.72E-08 Manual F-box/LRR-repeat protein 20 FBXL20 6.00 .002867 Proteomic studies18 Signal transduction Adenylate cyclase type 6 ADCY6 3.40 .028628 Proteomic studies17 ADP-ribosylation factor 5 ARF5 3.00 .043487 Proteomic studies17 ADP-ribosylation factor-like protein 15 ARL15 5.25 .005232 Proteomic studies18 Casein kinase I ␥1 isoform CSNK1G1 3.00 .043487 Proteomic studies17 Casein kinase I isoform ␥3 CSNK1G3 8.00 .000690 Proteomic studies17 CD151 antigen CD151 3.00 .043487 Uniprot CD63 antigen CD63 3.67 .021908 Manual CD82 antigen CD82 4.00 .015866 Manual CDC42 small effector protein 1 CDC42SE1 3.00 .043487 Uniprot CDC42 small effector protein 2 CDC42SE2 9.00 .000366 Uniprot Cell division control protein 42 homolog CDC42 3.00 .043487 Manual Choline transporter-like protein 2 CTL2 27.00 2.29E-07 Proteomic studies18 Disks large-associated protein 4 DLGAP4 6.00 .002867 Proteomic studies17 Disheveled-associated activator of morphogenesis 1 DAAM1 9.00 .000366 Proteomic studies17 Dystroglycan DAG1 4.00 .015866 Proteomic studies17 Erbb2-interacting protein ERBB2IP 8.88 .000395 Manual Flotillin-2 FLOT2 10.55 .000149 Manual G protein-coupled receptor kinase 6 GRK6 8.00 .000690 Probable G(i) ␣-1 GNAI1 3.80 .019226 HPRD G(i) ␣-2 GNAI2 3.25 .033301 Manual G(i) ␣-3 GNAI3 4.86 .007300 HPRD G(q) ␣ GNAQ 5.17 .005609 HPRD G(s) ␣ GNAS 5.80 .003352 Manual G␣-11 GNA11 6.00 .002867 HPRD G␣-13 GNA13 7.45 .000998 Uniprot G␣-15 GNA15 4.00 .015866 Manual GTPase Hras HRAS 7.00 .001366 Uniprot GTPase Nras NRAS 5.25 .005232 Uniprot Junctional adhesion molecule C JAM3 5.14 .005722 Proteomic studies17 Kalirin KALRN 7.50 .000965 Proteomic studies17 Linker for activation of T-cell family member 2 LAT2 7.00 .001366 Probable Mitochondrial import inner membrane translocase subunit TIM50 TIMM50 4.00 .015866 Proteomic studies24

Known palmitoylated proteins as catalogued by the Uniprot or HPRD databases or by review of published palmitoylation-related research articles (manual). Also included in this list are proteins characterized as being palmitoylated by similarity, probably palmitoylated, or potentially palmitoylated as defined by the Uniprot database. A total of 61 of the identified proteins were discovered as palmitoyl protein candidates in other proteomic studies.17,18,24 Data are the result of 2 independent experiments, and proteins are grouped according to the biologic process they mediate as defined by the HPRD. Also shown is the HAϩ/HAϪ ratio and corresponding P value. From www.bloodjournal.org by guest on September 11, 2016. For personal use only.

e68 DOWAL et al BLOOD, 29 SEPTEMBER 2011 ⅐ VOLUME 118, NUMBER 13

Table 2. Known palmitoylated proteins of the platelet palmitoylome (continued) Protein name Gene name Ratio P Source

Phosphatidylinositol 4-kinase type 2-␣ PI4K2A 10.00 .000202 Manual Phosphatidylinositol 4-kinase type 2-␤ PI4K2B 3.00 .043487 Manual Phospholipid scramblase 1 PLSCR1 20.50 .000002 Potential Platelet glycoprotein 4 CD36 6.14 .002569 Uniprot Prostacyclin receptor PTGIR 10.00 .000202 Uniprot Proto-oncogene tyrosine-protein kinase Fyn FYN 4.91 .006980 HPRD Proto-oncogene tyrosine-protein kinase Yes YES1 10.00 .000202 Proteomic studies18 Raftlin RFTN1 5.00 .006457 Probable Ras-related protein Rap-2a RAP2A 28.00 .000000 by similarity Ras-related protein Rap-2b RAP2B 3.93 .017042 by similarity Ras-related protein Rap-2c RAP2C 11.50 .000089 by similarity Ras-related protein R-Ras RRAS 7.44 .001002 Manual Regulator of G-protein signaling 19 RGS19 15.00 .000016 Uniprot Semaphorin-4D SEMA4D 6.00 .002867 Proteomic studies17 Sortilin SORT1 3.00 .043487 Proteomic studies17 Stomatin STOM 3.29 .031966 Uniprot Tetraspanin-9 TSPAN9 6.67 .001737 Proteomic studies18 Thromboxane A2 receptor TBXA2R 4.00 .015866 Manual Transmembrane protein 55B TMEM55B 3.50 .025869 Proteomic studies17 Type I inositol-1,4,5-trisphosphate 5-phosphatase INPP5A 10.67 .000139 Potential Tyrosine-protein kinase Lyn LYN 5.00 .006457 by similarity Transcription regulator activity Histone H2B type 1-N HIST1H2BN 6.00 .002867 Proteomic studies17 Transport AFG3-like protein 2 AFG3L2 4.00 .015866 Proteomic studies17 ATP-binding cassette subfamily B member 6 ABCB6 5.00 .006457 Proteomic studies18 Choline transporter-like protein 1 CTL1 14.20 .000024 Proteomic studies18 Cytochrome b5 type B CYB5B 3.67 .021908 Proteomic studies17 Golgin subfamily A member 7 GOLGA7 17.00 .000007 Uniprot Multidrug resistance-associated protein 4 ABCC4 3.00 .043487 Proteomic studies18 Phospholipid scramblase 3 PLSCR3 13.00 .000042 Probable Phospholipid scramblase 4 PLSCR4 7.00 .001366 Probable Pituitary tumor-transforming gene 1 protein-interacting protein PTTG1IP 19.50 .000003 Proteomic studies18 PRA1 family protein 2 PRAF2 14.00 .000026 Proteomic studies17 Protein tweety homolog 3 TTYH3 11.00 .000116 Proteomic studies17 Secretory carrier-associated membrane protein 1 SCAMP1 10.25 .000175 Proteomic studies17 Secretory carrier-associated membrane protein 2 SCAMP2 3.95 .016681 Proteomic studies17 Secretory carrier-associated membrane protein 3 SCAMP3 16.00 .000011 Proteomic studies17 Secretory carrier-associated membrane protein 4 SCAMP4 3.00 .043487 Proteomic studies17 Sodium/potassium-transporting ATPase subunit ␣-1 ATP1A1 3.00 .043487 Proteomic studies17 SNAP-23 SNAP23 3.74 .020385 HPRD Syntaxin-8 STX8 11.00 .000116 Manual Syntaxin-12 STX12 4.00 .015866 Proteomic studies17 Trafﬁcking protein particle complex subunit 3 TRAPPC3 8.67 .000450 by similarity Transferrin receptor protein 1 TFRC 3.00 .043487 Uniprot Vesicle-associated membrane protein 3 VAMP3 8.43 .000523 Proteomic studies18 Vesicle-associated membrane protein 4 VAMP4 10.00 .000202 Proteomic studies17 Vesicle-associated membrane protein 7 VAMP7 3.69 .021361 Proteomic studies18 Unknown 3-oxoacyl-͓acyl-carrier-protein͔ synthase OXSM 6.00 .002867 Proteomic studies17 Abhydrolase domain-containing protein FAM108B1 FAM108B1 8.33 .000556 Proteomic studies18 Coiled-coil domain-containing protein 109A CCDC109A 4.50 .010000 Proteomic studies17 Endoplasmic reticulum-Golgi intermediate compartment protein 3 ERGIC3 3.00 .043487 Proteomic studies17 Abhydrolase domain-containing protein FAM108A1 FAM108A1 7.50 .000965 Proteomic studies23 Protein FAM49B FAM49B 21.00 .000002 Proteomic studies18 Protein LYRIC gene-1 protein LYRIC 18.00 .000005 Proteomic studies17 Transmembrane protein 63A TMEM63A 7.29 .001118 Proteomic studies18 Transmembrane protein 63B TMEM63B 3.00 .043487 Proteomic studies18 UPF0404 protein C11orf59 C11orf59 5.71 .003588 Proteomic studies18

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Figure 2. Global analysis of the platelet palmitoylome. (A) Graphical depiction of the 1300 proteins identified in resting platelet membranes. Gray dots represent identified proteins not meeting the criteria for significance. Candidate palmitoyl proteins (E) cluster around the x-axis with known palmitoylated proteins and putative palmitoyl proteins identified in other palmitoylation- specific proteomic studies (green dots). Also shown are well-established palmitoylated platelet proteins identified (blue dots) and not identified (red dots) as being palmitoylated in this study. Inset: Expanded view of the graph for proteins with Ͻ 50 spectral counts. Diagonal line in each graph indicates the HAϩ/HAϪ cut-off. (B) Western blotting analysis of ABE-purified proteins from platelet membranes, as prepared for proteomic analysis, in the pres- ence and absence of HA using antibodies directed against Cdc42. Also shown is the total protein input for the HAϩ and HAϪ samples.

have been described in other proteomic studies, the rest are newly TLT-1 is a palmitoyl protein candidate identiﬁed palmitoyl candidates. This group includes syntaxins-2, -10, and -11 and VAMP-5. These results indicate that signaling and Because the expression of TLT-1 is restricted to megakaryocytes transport proteins represent approximately half of all palmitoylated and platelets, we chose it for further analysis. TLT-1 demonstrated ϩ Ϫ ϭ platelet proteins identiﬁed in this study. an HA /HA ratio of 2.5 (P .076). The full-length form of

Figure 3. The biologic processes mediated by proteins of the platelet palmitoylome. Analysis of the platelet palmitoylome indicates that many of the identiﬁed platelet proteins are involved in signal transduction pathways and transport processes. Each protein was assigned a biologic process as deﬁned by the HPRD, which is Gene Ontology compliant. From www.bloodjournal.org by guest on September 11, 2016. For personal use only. e70 DOWAL et al BLOOD, 29 SEPTEMBER 2011 ⅐ VOLUME 118, NUMBER 13

Figure 4. TLT-1 is enriched in HA؉ samples in resting and activated platelets. (A) Domain organization of full-length TLT-1 and the TLT-1 splice variant depicting an Ig-like V-type domain (blue) with 4 cysteine residues followed by a single-pass transmembrane (TM) domain (green). The TLT-1 splice variant has a truncated cytoplasmic tail and does not contain the 3 intracellular cysteines or ITIM region.32 (B) Western blot analysis of TLT-1 using ABE-puriﬁed palmitoylated proteins from resting and thrombin-activated platelets. Arrow indicates full-length TLT-1; and arrowhead, a 25 kDa TLT-1 splice variant. Bands seen directly below full-length TLT-1 and bands seen below the TLT-1 splice variant are degradation products.33 Also shown are 20 ␮g of the sample inputs that represent the protein sample added to the streptavidin agarose beads.

TLT-1 has a 126-amino acid cytosolic C-terminal tail, which Full-length TLT-1 and its degradation product were palmitoylated includes an immunoreceptor tyrosine-based inhibitory motif (ITIM) because the HAϩ samples were enriched for full-length TLT-1 in region and a proline-rich region that may mediate protein-protein resting and activated platelets compared with the HAϪ samples interactions.31 The cytosolic tail also contains 3 cysteine residues (Figure 4B). In contrast, the TLT-1 splice variant was not enriched. (Figure 4A), and analysis of the sequence using the palmitoylation Bands seen for TLT-1 in the HAϪ sample represent background site predicting software, CSS-Palm 2.0, indicated that TLT-1 was binding to the streptavidin beads, which may be the result of probably palmitoylated only on cysteine 196 (Cys196).32 TLT-1 improper biotinylation or nonspecific binding. Western blot analy- exists as 2 species: a full-length form with a molecular mass of sis of the ABE-purified samples in the resting and activated HAϩ 35 kDa and a splice variant with a molecular mass of 25 kDa. The lanes did not demonstrate a change in the palmitoylation state of splice variant differs from full-length TLT-1 in that it has a TLT-1 on platelet activation under the conditions in which this truncated cytosolic region, which encodes a short, 14-amino acid assay was performed (Figure 4B). These results suggest that 31 tail (Figure 4A). In addition, amino acids 190 to 199 of the splice full-length TLT-1, but not the TLT-1 splice variant, is palmitoylated variant differ from the full-length sequence. Instead of GNRL- in platelets. GVCGRF, the splice variant encodes for ESLLSGPPRQ, which does not contain the predicted palmitoylation site at Cys196 TLT-1 is a novel palmitoylated platelet protein (Uniprot database). To confirm that TLT-1 is palmitoylated and to determine which To determine whether TLT-1 is a bona fide palmitoylated protein, 3 TLT-1 species is palmitoylated, we directly assessed samples we metabolically labeled platelets with [ H]palmitic acid and 3 purified by ABE chemistry for TLT-1. Platelets from outdated assayed for incorporation of [ H]palmitate. Resting platelets were 3 aphaeresis packs were washed and divided into 2 equal aliquots. labeled with [ H]palmitic acid for 1 hour at 37°C and then divided One was left resting and the other was stimulated with 1 U/mL into 2 equal portions. One portion remained resting, and the other was activated with 1 U/mL ␣-thrombin. Samples were lysed and ␣-thrombin for 5 minutes at room temperature. Prostaglandin E1 was added to both samples, and the platelets were immediately TLT-1 was immunoprecipitated (Figure 5A). The minor band seen pelleted and dissolved in lysis buffer. Palmitoylated proteins were at 33 kDa represents a TLT-1 degradation product.33 Subsequent subsequently purified using the ABE method from membranes audioradiography revealed that full-length TLT-1 incorporated prepared from resting and thrombin-activated platelets, and were [3H]palmitate in both resting and activated platelets (Figure 5B). separated by gel electrophoresis and transferred to a PVDF The 25-kDa splice variant, which was immunoprecipitated with membrane (Figure 4B). Immunoblotting of ABE-purified TLT-1 full-length TLT-1 (Figure 5A lanes 3 and 4), did not incorporate demonstrated 3 major bands corresponding to full-length TLT-1, a [3H]palmitate (Figure 5B), as predicted by the absence of the previously described degradation product,33 and the splice variant. cytosolic cysteine residues and studies of TLT-1 using ABE From www.bloodjournal.org by guest on September 11, 2016. For personal use only.

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source of error is the copurification of contaminating proteins, a common issue for affinity purification-based methods. Nonspecific copurification of nonpalmitoylated proteins in the HAϪ sample will lower the HAϩ/HAϪ ratio and is a limitation of the PalmPISC method. In addition, the widely used spectral counting quantitation approach is only semiquantitative. Development of better methods to reduce the background contribution of contaminating proteins is currently ongoing and will increase the sensitivity and specificity for identification of palmitoyl proteins in platelets. Yield of palmitoyl proteins could also be improved with better solubiliza- tion of the membranes to release more integral membrane proteins. Studies are currently underway to test an alternative method using pressure cycling technology34 to extract more proteins from platelet

Figure 5. [3H]Palmitate incorporation confirms that TLT-1 is a palmitoylated membranes. protein. Platelets were metabolically labeled with [3H]palmitic acid and separated Approximately 50% of identified palmitoyl proteins mediate into resting (lane 1) and thrombin-activated (lane 2) samples and lysed. TLT-1 was signal transduction and transport processes. Because palmitoyl- then immunoprecipitated from resting (lane 3) and thrombin-activated platelet lysates ation is a dynamic lipid modification, the palmitoylation state of (lane 4). Samples were subjected to Western blot analysis (A) or audioradiography to assess [3H] palmitate incorporation (B). Full-length TLT-1 is the band corresponding platelet proteins may present another layer of regulation governing to 37 kDa. This blot is representative of 3 independent experiments. the localization and protein-protein interactions necessary to carry out these processes. Included in our list of signaling proteins is the chemistry (Figure 4). These studies confirm that TLT-1 is a platelet palmitoylated form of Cdc42. This result was intriguing because palmitoyl protein. Cdc42 exists as 2 isoforms: a prenylated form that has ubiquitous To further characterize the palmitoylation of TLT-1, we used expression and a palmitoylated form that has been described 17 PalmPISC to identify the candidate palmitoylation site. In this previously in the rat neuronal palmitoyl proteome. Immunoblot- approach, palmitoylated cysteines can be readily distinguished ting of the ABE-purified palmitoylated proteins demonstrated the from nonpalmitoylated cysteines because nonpalmitoylated cys- HA dependence of Cdc42 (Figure 2C) in our preparations and teines are irreversibly blocked by NEM, whereas palmitoylated indicates that platelets possess this palmitoylated isoform. Unlike 3 cysteines become free cysteines after the ABE reaction and TCEP metabolic labeling with [ H]palmitate, the ABE method provides a elution. The resulting spectrum of a peptide derived from TLT-1, snapshot of all the palmitoylated proteins in a cell at a particular LGVCGR, shows a free cysteine residue based on the m/z moment in time. Although ABE methodology cannot give informa- difference of 103 between the y2 and y3 fragment ion (Figure 6). tion about the rate of palmitate turnover, when combined with a The fact that this cysteine (Cys196) is free and not NEM-modified global proteomic approach, it can provide the identity of proteins indicates that Cys196 is a putative palmitoylation site. This result is for further targeted study. in agreement with the CSS-Palm prediction that Cys196 is a We chose TLT-1 as a palmitoyl protein candidate for further candidate palmitoylation site of TLT-1. study because its palmitoylation has not previously been described and its expression is restricted to megakaryocytes and platelets.35 The limited expression pattern of TLT-1 suggests that it may play a specific role in platelet function and makes it a potential target for Discussion the modulation of hemostasis and thrombosis because antibodies Our analysis of the platelet palmitoylome resulted in the identifica- directed against TLT-1 are able to block thrombin-mediated platelet 33 tion of Ͼ 1300 proteins. We found that 215 of these proteins were aggregation. Furthermore, TLT-1 knockout mice demonstrate significantly enriched in the HAϩ sample, suggesting that they are palmitoyl proteins. However, there are several reasons to think that even more palmitoylated proteins exist in platelets. First, we did not observe some of the known palmitoylated proteins as expected in the platelet palmitoylome. These proteins include members of the acyl-transferase family, known as DHHC proteins, which have been observed in other proteomic studies.18 The reason for this could lie in the low abundance of these proteins. Second, to avoid falsely identifying palmitoyl protein candidates, we used strict cut-off values to define the HAϩ enriched dataset and excluded actual palmitoyl proteins. For example, TLT-1, which was not significantly enriched in the HAϩ sample, was confirmed to be a palmitoylated platelet protein (Figure 5). Similarly, we did not identify all of the platelet proteins that have previously been found to be subject to palmitoylation. P-selectin, tubulin, platelet glyco- proteins IX and Ib, PECAM, G␣z, and CD9 were identified (Figure 1B red dots; supplemental Table 2), but their HAϩ/HAϪ ratios did not meet the stringent threshold (ratio Ն 3, P Ͻ .05) that was used Figure 6. Representative tandem mass spectrum of a candidate palmitoyl to identify candidate palmitoyl proteins. It is possible that, although peptide derived from TLT-1 protein. Enriched palmitoyl proteins were separated by SDS-PAGE and a gel slice containing 30- to 50-kDa proteins excised. Proteins were these proteins are highly abundant in platelets, their palmitate- digested in gel, followed by the extraction of tryptic peptides, which were analyzed by modified, membrane-associated forms are not. Another potential LC-MS/MS. Free cysteines in the purified peptides are candidate palmitoylation sites. From www.bloodjournal.org by guest on September 11, 2016. For personal use only. e72 DOWAL et al BLOOD, 29 SEPTEMBER 2011 ⅐ VOLUME 118, NUMBER 13

Figure 7. Venn diagrams representing the overlap between the platelet and rat neuronal palmitoylomes. Number of overlapping proteins identiﬁed in the platelet (solid line) and rat neuronal (dashed line) palmitoylomes (A). Number of overlapping known palmitoylated proteins identiﬁed in the platelet and rat neuronal palmitoylomes (B).

extended tail bleeding times and are predisposed to hemorrhage in neuronal palmitoylome.17 We chose the neuronal palmitoylome for an inflammatory model. Platelets from these mice are defective in comparison because megakaryocytes/platelets and neurons share aggregation in response to adenosine diphosphate and U46619 structural and functional characteristics, and there are numerous stimulation.36 TLT-1 is part of a family of receptors termed studies in which platelets have been proposed as a model for triggering receptors expressed on myeloid cells (TREMs)37; and neuronal function.44 Of the 215 proteins that compose the platelet although TLT-1 is homologous to TREMs in its V-type immuno- palmitoylome, 75 proteins overlap between our study and the rat globulin-like extracellular domain, TLT-1 has a longer cytoplasmic neuronal palmitoylome (Figure 7). We also find that, of the tail with a proline-rich region and contains an ITIM instead of an 51 known palmitoylated proteins identified in the platelet palmitoy- immunoreceptor tyrosine-based activation motif (Figure 3). It has lome, 37 overlap with the 68 known palmitoylated proteins identified in been shown in vitro that the ITIM region of TLT-1 is capable of the neuronal palmitoylome (Figure 7).17 Of particular interest was our being phosphorylated and can recruit SH2-domain–containing identification of the palmitoylated form of Cdc42. Although Cdc42 null protein tyrosine phosphatases 1 and 2.31,38 platelets display a complex phenotype,45 Cdc42 is implicated in platelet We present evidence here that TLT-1 is a novel palmitoylated cytoskeletal remodeling46,47 and filopodia formation.48 In neurons, the protein and is palmitoylated on Cys196. Full-length TLT-1 has palmitoylated form of Cdc42 induces the formation of dendritic 7 cysteine residues.39 Four of these reside in the extracellular spines.17 It is tempting to speculate that the palmitoylated form of Cdc42 domain and form disulfide bonds.40 Of the 3 intracellular cysteine may play a similar role in megakaryocytes or platelets contributing to residues, Cys196 occurs just after the transmembrane domain. Our proplatelet formation or morphologic changes seen on platelet activa- identification of Cys196 as a putative palmitoylation site is in tion. Studies are currently underway to assess the implications of this agreement with the observation that the likelihood of a cysteine finding. being palmitoylated increases after stretches of hydrophobic amino This study is the first comprehensive description of the platelet acids, such as a transmembrane domains. As expected, the trun- palmitoylome and expands our understanding of the scope of cated 25-kDa TLT-1 splice variant did not incorporate [3H]palmi- palmitoylation in platelets. However, given the importance of tate (Figure 5B), consistent with the lack of intracellular cysteines. palmitoylation in regulating protein function, our findings extend The role that palmitoylation serves in TLT-1 function is unknown. beyond platelet biology as our global characterization of the Many of the palmitoyl proteins that we found in the platelet had platelet palmitoylome resulted in the identification of 103 novel previously been identified in our analysis of palmitoyl proteins in palmitoyl protein candidates. The validation of the palmitoylation lipid rafts (supplemental Table 2). We have found that a fraction of of these candidate proteins will provide opportunities for future TLT-1 incorporates into lipid rafts (supplemental Figure 2). TLT-1 studies aimed at increasing our understanding of the role palmitoyl- palmitoylation could affect its incorporation into rafts, as has been ation plays in platelet function. The platelet palmitoylome will demonstrated for the immunoreceptor tyrosine-based activation establish a platform, providing an essential first step to the motif-containing protein PECAM-1.29 Alternatively, palmitoyl- determination of how palmitoylation alters the function of the ation may influence phosphorylation of TLT-1, as observed in novel palmitoyl protein candidates. tissue factor41 and linker for activation of T cells (LAT).42 Our methodologies and experimental conditions did not demonstrate activation-induced palmitoylation of TLT-1. However, activation could affect palmitate cycling, and presently we cannot separate Acknowledgments palmitoylation and depalmitoylation activities. Therefore, we cannot conclude whether or not activation-induced TLT-1 palmitoyl- The authors thank Valance Washington (University of Puerto Rico– ation occurs. We are currently developing methods to study Mayaguez) for helpful discussions and the Beth Israel Deaconess activation-dependent platelet palmitoylation and determine the Medical Center Blood Bank for providing a source of platelets. significance of TLT-1 palmitoylation in platelets. This work was supported by the National Institutes of Health We compared the platelet palmitoylome with other palmitoyl- (grant HL87203, R.F.) and the United States Army (grant PC093459, ation-specific proteome studies16-18,23,24,43 and identified 103 new M.R.F. and H.S.). L.D. was supported by the National Institutes of palmitoyl protein candidates. In our analysis of the platelet Health (grant T32 HL07917). R.F. is a recipient of an Established palmitoylome, we determined the overlap between it and the rat Investigator Award from the American Heart Association. From www.bloodjournal.org by guest on September 11, 2016. For personal use only.

BLOOD, 29 SEPTEMBER 2011 ⅐ VOLUME 118, NUMBER 13 PALMITOYLATED PLATELET PROTEINS e73

writing the paper; and R.F. conceived of study, analyzed data, and Authorship contributed to writing the paper. Conflict-of-interest disclosure: The authors declare no compet- Contribution: L.D. designed and performed research, analyzed ing financial interests. data, and wrote the paper; W.Y. designed and performed research, Correspondence: Robert Flaumenhaft, Division of Hemostasis analyzed data, and contributed to writing the paper; M.R.F. and Thrombosis, Department of Medicine, Beth Israel Deaconess provided reagents and contributed to writing the paper; H.S. Medical Center, 330 Brookline Ave, Boston, MA 02215; e-mail: facilitated the mass spectrometric analysis and contributed to rfl[email protected]. References

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2011 118: e62-e73 doi:10.1182/blood-2011-05-353078 originally published online August 2, 2011

Proteomic analysis of palmitoylated platelet proteins

Louisa Dowal, Wei Yang, Michael R. Freeman, Hanno Steen and Robert Flaumenhaft

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