Phosphoproteome of Resting Human Platelets René P. Zahedi,† Urs Lewandrowski,† Julia Wiesner,† Stefanie Wortelkamp,†,‡ Jan Moebius,† Claudia Schütz,† Ulrich Walter,‡ Stepan Gambaryan,‡,§ and Albert Sickmann*,† Rudolf Virchow Center/DFG Research Center for Experimental Biomedicine, University of Würzburg, Protein Mass Spectrometry and Functional Proteomics Group, Würzburg, D-97078 Germany, Institute of Clinical Biochemistry and Pathobiochemistry, University of Würzburg, Würzburg, D-97080 Germany, and Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, 194223 Russia Received July 4, 2007; Accepted November 7, 2007
Beside their main physiological function in hemostasis, platelets are also highly involved in pathological processes, such as atherothrombosis and inflammation. During hemostasis, binding of adhesive substrates to tyrosine-kinase-linked adhesion receptors and/or soluble agonists to G-protein coupled receptors leads to a cascade of intracellular signaling processes based on substrate (de)phosphorylation. The same mechanisms are involved in platelet activation at sites of atherosclerotic plaque rupture, contributing to vessel occlusion and consequently to pathologic states, such as myocardial infarction, stroke, or peripheral artery disease. To gain a deeper insight into platelet function, we analyzed the phosphoproteome of resting platelets and identified 564 phosphorylation sites from more than 270 proteins, of which many have not been described in platelets before. Among those were several unknown potential protein kinase A (PKA) and protein kinase G (PKG) substrates. Because platelet inhibition is tightly regulated especially by PKA and PKG activity, these proteins may represent important new targets for cardiovascular research. Thus, our finding that GPIbR is phosphorylated at Ser603 in resting platelets may represent a novel mechanism for the regulation of one of the most important platelet receptor (GPIb-IX-V) mediated signaling pathways by PKA/PKG. Keywords: Phosphorylation • platelets • mass spectrometry
Introduction Platelets are small anucleate blood cells derived from megakaryocytes, which circulate in the human blood for 8–10 days as monitors of vascular integrity. Upon encountering sites of endothelial lesion and interaction with subendothelial matrix proteins, platelets respond by plug formation, thereby preventing the organism from bleeding. In this context, the initial steps of platelet activation are induced by binding of adhesive substrates to tyrosine-kinaselinked adhesion receptors and/or soluble agonists to G-protein coupled receptors, leading to a cascade of downstream intracellular signaling processes based on the phosphorylation and/ or dephosphorylation of specific substrates. Consequently, platelets undergo a characteristic shape change, and receptors are translocated to the surface and activated, while vasoactive substances are secreted. Thus, platelets adhere to the site of * To whom correspondence should be addressed: Rudolf Virchow Center/ DFG Research Center for Experimental Biomedicine, University of Würzburg, Protein Mass Spectrometry and Functional Proteomics Group, Room 411, Versbacher Strasse 9, 97078 Würzburg, Germany. Telephone: +49-(0)-931201-48730. Fax: +49-(0)-931-201-48123. E-mail:
[email protected]. † Rudolf Virchow Center/DFG Research Center for Experimental Biomedicine, University of Würzburg. ‡ Institute of Clinical Biochemistry and Pathobiochemistry, University of Würzburg. § Russian Academy of Sciences.
526 Journal of Proteome Research 2008, 7, 526–534 Published on Web 12/19/2007
vascular injury, aggregate, and finally build a stable thrombus. In addition, the same mechanisms underlie the pathological activation of platelets at sites of atherosclerotic plaque rupture and therefore contribute to vessel occlusion and consequently to myocardial infarction, stroke, or peripheral artery disease. Their involvement in the pathogenesis and progression of cardiovascular diseases, which are the main cause of death in industrialized countries, renders platelets highly relevant for biomedical and clinical research. Hence, an increasing number of proteome studies have emerged in recent years, aiming mainly at the characterization of platelet subproteomes, which might be important for platelet function.1–5 Furthermore, several proteome studies dealing with platelet phosphorylation have been published.6–9 These are all based on protein separation by two-dimensional electrophoresis (2DE) with subsequent tandem mass spectrometry (MS/MS) and were therefore restricted with regard to (a) the inherent limitations of 2DE in resolving hydrophobic, alkaline, or very small/large proteins and (b) the mere identification of highly abundant phosphorylation sites. Thus, a comprehensive analysis of the platelet phosphoproteome will lead to the identification of novel target proteins involved in platelet activation or inhibition and furthermore may reveal new signaling pathways that are important for physiological as well as pathophysiological processes. 10.1021/pr0704130 CCC: $40.75
2008 American Chemical Society
research articles
Phosphoproteome of Resting Human Platelets
as a result of peptide redundancy, 15 ambiguous proteins, of which many have not been described in platelets before. To our knowledge, this study represents the first large-scale phosphoproteomic data of primary human cells, comprising several hundred phosphorylation sites identified via MS/MS. Furthermore, this is the first proteomic phosphorylation data set of human platelets that is not based on gel-electrophoretic methods including all of their inherent drawbacks.
Materials and Methods
Figure 1. Strategy applied for the characterization of the phosphoproteome of resting human platelets. Platelets were isolated from fresh blood. After lysis and tryptic digestion, phosphopeptides were enriched by means of IMAC and SCX. Preceding SCX enrichment, samples were furthermore treated with PNGase F to reduce interfering elution of glycosylated peptides in the early fractions. Phosphopeptide-enriched samples were analyzed via MS/MS or precursor ion scanning. PIS ) precursor ion scanning.
To address this issue, we analyzed the human platelet phosphoproteome by a two-pronged strategy based on the enrichment of phosphopeptides by immobilized metal-ionaffinity chromatography (IMAC)10 and strong cation-exchange chromatography (SCX) according to Beausoleil et al.,11 coupled with subsequent analysis by nano-liquid chromatographytandem mass spectrometry (nano-LC-MS/MS) or precursor ion scanning (see Figure 1). Currently, IMAC and SCX represent the most powerful tools for large-scale phosphopeptide enrichment, enabling the identification of up to several hundreds or even thousands of phosphorylation sites.11–15 Whereas IMAC is based on the ionic interactions of anionic phosphate groups and cationic metal ions under acidic conditions, SCX phosphopeptide enrichment is obtained by separating phosphorylated from nonphosphorylated tryptic peptides at a defined pH of 2.7. Specificity and yield of IMAC phosphopeptide enrichment can vary dramatically depending upon the applied conditions for binding, washing, and elution, as well as resin/sample ratios, and frequently, severe copurification of acidic, nonphosphorylated peptides can be observed. Although this issue can be addressed by optimizing sample processing, oftentimes methylation of acidic carboxylate groups with HCl-saturated, dried methanol preceding IMAC enrichment is used to reduce copurification. However, side reactions, e.g., partial deamidation and subsequent methylation of asparagine and glutamine residues, might occur, leading to an increase in sample complexity and consequently a decrease in MS sensitivity. On the contrary, phosphopeptide enrichment by SCX can yield a copurification of otherwise net charge-reduced peptides, i.e., carboxy-terminal and amino-terminal acetylated or glycosylated peptides.16 Furthermore, phosphopeptides containing missed cleavage sites or His residues might escape netcharge-based enrichment. Using this two-pronged approach, we were able to identify a total of 564 phosphorylation sites from 278 unambiguous and,
Materials. Sequencing-grade modified trypsin was purchased from Promega, Madison, WI. PNGaseF (Flavobacterium meningosepticum) was supplied by Roche, Mannheim, Germany. Phos-Select iron affinity gel and phosphatase inhibitor cocktails 1 and 2 were obtained from Sigma Aldrich, Steinheim, Germany. All other chemicals including ultrapure high-performance liquid chromatography (HPLC) solvents were from Merck, Darmstadt, Germany. Platelet Isolation and Purification. Fresh blood from healthy donors was collected in 1/5 volume citrate buffer at pH 6.5 [100 mM sodium citrate, 7 mM citric acid, 140 mM glucose, and 15 mM ethylenediaminetetraacetic acid (EDTA)] and centrifuged for 20 min at 300g and room temperature. The obtained platelet-rich plasma was centrifuged once again. Afterward, platelet-rich plasma was adjusted to pH 6.4 with 0.3 M citric acid and centrifuged for 20 min at 400g and room temperature. The supernatant was discarded, and obtained cell pellets were washed twice with 2× N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) buffer at pH 7.2 (152 mM NaCl, 4 mM KCl, 10 mM HEPES, and 3 mM EDTA) and centrifuged for 20 min at 400g and room temperature. A total of 10 mg of purified platelets was lysed in 1 mL of 50 mM Tris, 150 mM NaCl, 1% sodium dodecyl sulfate (SDS; w/v), and 1% phosphatase inhibitor cocktails 1 and 2 (v/v) at pH 7.8. Disulfide bonds were reduced by the addition of 10 mM dithiothreitol (DTT) for 30 min at 56 °C, and free cysteines were carbamidomethylated with 30 mM iodoacetic acid (IAA) for 30 min in darkness at room temperature. Afterward, the platelet lysate was dialyzed against 50 mM NH4HCO3 at pH 7.8 and digested with trypsin (1:100) at 37 °C overnight. IMAC. A total of 5 mg of platelet digest was added to 5 mL binding buffer [250 mM acetic acid and 30% acetonitrile (ACN) at pH 2.3] and 250 µL of Phos-Select iron affinity gel. After incubation at room temperature for 2 h, IMAC beads were washed once with binding buffer and twice with water. Finally, enriched phosphopeptides were recovered by consecutive elution with 300 µL of 200 and 400 mM NH4OH, respectively. Both eluates were immediately adjusted to pH 3.0 with 1% formic acid. For further reduction of sample complexity, enriched phosphopeptides were separated by a conventional SCX according to Sickmann et al.17 Briefly, samples were separated using a binary gradient (solvent A, 50 mM KH2PO4 at pH 3.0; solvent B, 50 mM KH2PO4, 0.25 M NaCl, and 25% ACN at pH 5.5) and a PL-SCX column (2.1 mm i.d. × 15 cm length, 1000 Å pore size, 8 µm particle size, Polymer Laboratories, Darmstadt, Germany) on an Ultimate HPLC system (Dionex, Idstein, Germany), at a flow rate of 220 µL/min. Fractions were collected every minute by a ProteineerFCfraction collector (Bruker Daltonik, Bremen, Germany). SCX for Phosphopeptide Enrichment. A total of 5 mg of platelet digest was incubated with 5 units/100 µL PNGaseF in 50 mM NH4HCO3 at pH 7.8 overnight at 37 °C. Afterward, Journal of Proteome Research • Vol. 7, No. 2, 2008 527
research articles ultrafiltration was accomplished using Amicon filter devices with a 5000 molecular-weight cutoff (Millipore, Schwalbach, Germany). Next, samples were diluted 10-fold with 5 mM NaH2PO4 at pH 2.7, and phosphopeptides were enriched according to Beausoleil et al.,11 with slight modifications. A 2.1 mm i.d. × 15 cm length column (PolySULFOETHYL Aspartamide, 200 Å pore size, 5 µm particle size, Chromatographic Technologies, Basel, Switzerland) was used in combination with an inert Ultimate HPLC system. For chromatographic separation, a binary buffer system consisting of 5 mM NaH2PO4 at pH 2.7 (buffer A) and 5 mM NaH2PO4, 15% acetonitrile, and 500 mM NaCl at pH 2.7 (buffer B) was used, at a flow rate of 150 µL/min. Fractions were collected minute by minute using a ProteineerFC-fraction collector. Nano-LC-MS/MS. Nano-LC-MS/MS analyses were accomplished on a QstarXL, a Qtrap 4000 (both from Applied Biosystems, Darmstadt, Germany) or an LTQ (Thermo Electron, Dreieich, Germany) mass analyzer, coupled to inert Ultimate or Ultimate 3000 nano-HPLC systems. Briefly, peptides were preconcentrated in 0.1% trifluoroacetic acid (TFA) on a 100 µm i.d. RP trapping column (Synergi HydroRP, 4 µm particle size, 80 Å pore size, 2 cm length, Phenomenex, Aschaffenburg, Germany)18 and, afterward, separated on a 75 µm i.d. RP (Synergi HydroRP, 2 µm particle size, 80 Å pore size, 15 cm length, Phenomenex) main column by applying a 40 min binary gradient (solvent A, 0.1% FA; solvent B, 0.1% FA and 84% ACN) ranging from 5 to 50% of solvent B in 40 min and from 50 to 95% B in 1 min, at a flow rate of 270 nL/min. For the QstarXL and Qtrap 4000 mass analyzers, full MS scans from m/z 350 to 2000 were acquired and the four (Qtrap 4000) or two (QstarXL) most intensive signals were subjected to MS/MS, taking into account a dynamic exclusion. To address the low frequency of phosphotyrosines in MS-based studies, SCX-derived fractions were furthermore analyzed by precursor ion scanning for the pTyr immonium ion at m/z 216.01 on the Qtrap 4000 system.19 LTQ samples were analyzed by data-dependent neutral loss MS3 in multistage mode. Therefore, full MS scans from m/z 350 to 2000 were acquired, and MS3 scanning was triggered upon encountering a neutral loss of m/z 98, 49, or 32 within MS/MS spectra of the 10 most intensive peaks using dynamic exclusion. Manual MS Data Interpretation. MS data were transformed into .mgf format using the following software and respective parameters: Qtrap 4000, Analyst 1.4 with the mascot.dll plugin 1.6b5 (http://www.matrixscience.com/help/instruments_ analyst.html). Spectra with less than 10 ions were omitted. Peaks with intensities 0.1% below the base peak were removed, and data were centroided. QstarXL, Analyst 1.1 QS with the mascot.dll plug-in 1.6b7. Spectra with less than 10 signals were omitted. Precursor mass tolerance for grouping was set to 0.05 Da, and the maximum number of cycles between groups was set to 3. Peaks with intensities 0.1% below the base peak were removed. LTQ, extract_msn.exe (December 2005) on the basis of Xcalibur 1.4 and 2.0 format as a plug-in to the Mascot Daemon version 2.1.6. Precursor charge was set to auto, and the grouping tolerance was set to 1.4 Da. Minimum scans per group and intermediate scans were set to 1. Generated peak lists were searched against the Swiss-Prot database (February 2006, 206 586 entries) using Mascot20 version 2.1.03. Taxonomy was set to Homo sapiens (13 447 sequences), and carbamidomethylation of Cys residues (+57.02 528
Journal of Proteome Research • Vol. 7, No. 2, 2008
Zahedi et al.
Figure 2. Representative phosphopeptide MS/MS spectra. (A) The 4-fold phosphorylated peptide pSLDpTIpTLpSGDER from the membrane-targeting tandem C2 domain-containing protein 1 (Q8N9U0). (B) The singly phosphorylated peptide VIYSQPpSAR from the junctional adhesion molecule A precursor (Q9Y624).
Da) as fixed and oxidation of Met (+15.99 Da) as well as phosphorylation of Ser, Thr, and Tyr (+79.96 Da) residues as variable modifications were taken into account. Trypsin with a maximum of two missed cleavage sites was chosen as the enzyme, and peptide and MS/MS tolerances in daltons were selected depending upon the respective mass analyzers (0.2/ 0.5 for the QstarXL, 0.4/0.4 for the Qtrap 4000, and 1.5/1.5 for the LTQ). Only MS/MS spectra of phosphopeptides with more than 6 and less than 30 amino acids as well as Mascot scores above the p < 0.05 threshold value were considered for further data interpretation (QstarXL > 32, Qtrap 4000 > 33, and LTQ > 36). All phosphopeptide spectra were validated manually and, if necessary, verified by comparing extensive MS-Product fragment ion m/z predictions (http://prospector.ucsf.edu/prospector/4.0.7/html/msprod.htm) with raw MS data (see Figure 2). Target/Decoy MS Data Interpretation. A human subset of the Swiss-Prot database was generated, and a reversed decoy database was appended according to Elias et al.,21 using the decoy.pl script on the Mascot website (http://www. matrixscience.com/help/decoy_help.html). All QstarXL, Qtrap4000, and LTQ-derived .mgf data were merged into three different files, respectively, and searched
Phosphoproteome of Resting Human Platelets against the generated target/decoy database using the same parameters as before. Interpretation of data was accomplished with the help of the mass-spectrometry-oriented LIMS project software (http:// genesis.ugent.be/ms_lims/). False-positive rates were determined for each solution state separately. Classification of Data. To classify the obtained data, an inhouse platelet proteome database was used, providing information regarding the protein name, function, localization, and molecular weight based on the Swiss-Prot database (http:// www.expasy.org/; January 2007), as well as the number of predicted transmembrane domains calculated by the TMHMM version 2.0 algorithm (http://www.cbs.dtu.dk/services/ TMHMM/). All identified phosphorylation sites were analyzed for protein kinase A (PKA) and protein kinase G (PKG) consensus sequences (R/K|R/K|X|S/T).
research articles
Figure 3. (A) Distribution of the identified 564 phosphorylation sites. For 4 sites, an unambiguous identification was not possible between pSer and pThr. (B) Number of phosphorylation sites (p sites) per peptide.
Results Although phosphopeptide enrichment by SCX has provided the most extensive phosphorylation data from proteome studies thus far, in the current study, we focused on a combination of SCX and IMAC enrichment of phosphopeptides, because in our experience from other proteome studies, applying different approaches generally results in a higher number of protein and peptide identifications.17,22 In this context, SCX-based phosphopeptide enrichment is biased against phosphopeptides with increased net charges because of missed tryptic cleavage sites or the presence of His residues within peptide sequences. In our hands, the usage of SCX for the enrichment of platelet phosphopeptides leads to a strong accumulation of nonphosphorylated peptides in the early fractions, severely hampering phosphopeptide identification by MS/MS. These nonphosphorylated peptides not only contained amino-terminal acetylated or carboxy-terminal peptides, as already described by Beausoleil et al.,11 but also, as we have recently shown, high numbers of glycosylated peptides.16 To compensate for the latter, we introduced a deglycosylation step with PNGaseF23 prior to SCX enrichment, because numerous abundant platelet membrane proteins are extensively N-glycosylated.2 When the sugar moieties were removed, peptide net charges were increased, resulting in a reduction of background noise and an improvement of phosphopeptide recovery in the early SCX fractions. Whereas Hunter et al. determined the ratio between the three types of O-phosphorylation as 1800:200:1 pSer/pThr/pTyr by phosphoamino acid analysis,24 in the largest phosphoproteome study to date, Olsen et al. obtained a ratio of 43:6:1 in HeLa cells from cell culture.14 In their opinion, this deviance is mostly due to the predominant occurrence of pTyr on less abundant proteins compared to pSer and pThr, as well as the lower stability of pTyr in phosphoamino acid analysis, both contributing to the under-representation within the Hunter data compared to their own study. Therefore, we decided to include pTyr-specific precursor ion scanning in our study to improve the identification of tyrosinephosphorylated peptides, still posing a major obstacle even in samples enriched for phosphopeptides because of dynamic range limitations. Altogether, we were able to identify a total of 564 phosphorylation sites derived from 278 unambiguous and 15 ambiguous proteins (see Supplementary Tables 1 and 2 in the Supporting Information) from the Swiss-Prot database (February 2006). From those 564 phosphorylation sites, 457 are pSer, 77 are pThr, and 26 are pTyr with high confidence,
whereas 4 sites are either pSer or pThr, which is not distinguishable from the obtained spectra. This leads to a ratio of approximately 35:6:2 in the present study, which, in comparison to the ratio determined by Olsen et al., represents a 2-fold higher share of phosphotyrosines. All 494 phosphopeptide spectra were manually validated and represent high-confident mass spectrometric data, comprising 388 monophosphorylated, 89 doubly phosphorylated, 10 triply phosphorylated, 5 4-fold phosphorylated, and 2 5-fold phosphorylated peptides (see Figure 3). Comparison with Target/Decoy-Database Results. To compare the quality of manually validated data with automatic data processing based on the assessment of false-positive rates,21 all MS data were searched against a target/decoy database generated from the human subset of the Swiss-Prot database with appending reversed sequences. Thus, a calculated falsepositive rate (FPR) of 1% corresponds to a total of 630 phosphopeptides and 674 sites, compared to 494 phosphopeptides and 564 sites from the manual interpretation, equivalent to an increase of 17 and 14%, respectively. The overlap between manual and 1% FPR data interpretation only accounts for 356 phosphopeptides. Consequently, 28% of the manually validated phosphopeptides are lost within the 1% FPR data interpretation because of Mascot scores below the 1% FPR threshold, whereas on the contrary, 38% of the 1% FPR hits are lost during manual interpretation because of moderate spectrum quality. Thus, in this study, a 1% FPR data interpretation leads to a total of 221 phosphopeptides, which, although not compellingly bad, did not pass the manual validation because of (a) low number or coverage of annotated b/y ions, (b) bad signal/noise or annotation of ions within noise, and (c) presence of several not explainable (i.e., neither neutral losses of H3PO4, H2O, NH3, nor b and y ions and internal fragments) but very prominent signals. To optimize MS data quality, we therefore recommend, especially for mass analyzers with medium mass accuracy on the MS/MS or MS and MS/MS level, using the target/decoy strategy as a first filtering criteria, which should be combined with manual validation, as long as the generated data set remains manageable. To understand whether the comparably low number of 674 nonredundant phosphorylation sites from the 1% FPR data interpretation might be related to the usage of the relatively small Swiss-Prot database, an additional search against a concatenated target/decoy database of the human IPI version Journal of Proteome Research • Vol. 7, No. 2, 2008 529
research articles 3.23 (November 2006) with 133 238 sequences was accomplished. Using a 1% FPR, 916 nonredundant phosphorylation sites (from 844 peptides) could be identified, accounting for a 36% increase compared to Swiss-Prot-derived data. However, mostly because of the presence of a high number of protein isoforms, 70% of the identified phosphopeptides were not unique within the database, thus complicating data interpretation tremendously. Unique Peptide Assignment. When large data sets are presented, Mascot occasionally groups redundant peptides and unique peptides into an unambiguous protein hit. Thereby, single peptides might be misinterpreted as unique for a given protein. In this study, for instance, the phosphopeptide R.LIEDNEpYTAR.Q was assigned to the Swiss-Prot entry P06241 (Proto-oncogene tyrosine-protein kinase Fyn), as was the peptide R.DGpSLNQSSGYR.Y. However, whereas the latter represents a sequence that is unique for P06241, the former peptide is redundant, also being present within the proteins P12931 (Proto-oncogene tyrosine-protein kinase Src), P07947 (Proto-oncogene tyrosine-protein kinase Yes), and P06239 (Proto-oncogene tyrosine-protein kinase LCK). When both peptides were grouped into the unambiguous protein hit P06241, Mascot presented the redundant peptide also as a unique P06241-derived sequence. In the same way, the sequences R.GApSQAGMTGYGMPR.Q and R.NFpSDNQLQEGK.N were assigned to P37802 (Transgelin-2), although only the latter is unique for the respective protein. To address this important issue, which might induce false-positive phosphorylation site assignment, we looked for the unambiguousness of all identified phosphopeptide sequences within the used database and thereby could identify a total of 17 peptide sequences (3.4%), which were not unique, although originally assigned to single protein hits together with unique peptide sequences. We decided to leave these peptides within the here-presented data set; however, we labeled them as nonunique in Supplementary Table 2 in the Supporting Information, which includes all identified phosphopeptides. Classification of Phosphoproteins. According to the SwissProt database, 37% of the 278 unambiguously identified phosphoproteins are of cytoplasmic origin, whereas 16% are localized to the plasma membrane, 7% to the nucleus, 3% to mitochondria, and for 30%, the Swiss-Prot database did not provide any localization data (see Figure 4A). Although, on the first instance, the share of nuclear proteins might appear unexpectedly high for an anuclear cell, Martens et al. observed even 20% nuclear proteins in their peptide-centric proteome study of thrombocytapheresis-enriched platelets.5 Major reasons for these high amounts of nuclear proteins might be (a) residual proteins from megakaryocytes, (b) multiple localizations of proteins, for instance, because of the shuttling between organelles, and (c) presence of minor contaminations, mainly leukocytes. In contrast to the study by Martens et al., we focused on the analysis of platelets prepared from blood donations, which corresponds to an elevated risk of erythrocyte or leukocyte contamination compared to platelet concentrates. A closer look at protein functions revealed that 32% of the identified proteins comprise signaling members, i.e., receptors, kinases, and phosphatases, as well as members of the Ras and Rho family. A total of 9% are related to vesicles/trafficking/ transport, whereas 3% contribute to cell adhesion. A very interesting share of 22% interacts with actin or is involved in cytoskeletal (re)organization (see Figure 4B) and, therefore, is 530
Journal of Proteome Research • Vol. 7, No. 2, 2008
Zahedi et al.
Figure 4. (A) Localization of phosphoproteins according to the Swiss-Prot database. The 7% share of nuclear proteins might be explained by (a) residual proteins from megakaryocytes, (b) multiple localizations of proteins, for instance, because of shuttling between organelles, and (c) presence of minor contaminations, mainly leukocytes and erythrocytes. (B) Functional classification of identified platelet phosphoproteins according to the Swiss-Prot database. A total of 22% of the identified proteins interact with actin or are involved in cytoskeleton (re)organization, which might be of high interest in the context of platelet function.
of the highest interest in the context of platelet function and morphology. Moreover, 34 proteins contain at least one predicted transmembrane domain based on TMHMM version 2.0 prediction.
Discussion The current study, characterizing 564 high-confidence phosphorylation sites from 278 unambiguous and 15 ambiguous proteins represents the most comprehensive data set on platelet phosphorylation yet. Although originally assigned to single protein hits by the Mascot algorithm, because of the occurrence of further unambiguous peptide hits, 15 phosphopeptides corresponding to 21 phosphorylation sites were revealed as redundant within the used database. Analyzing phosphopeptide-enriched samples by precursor ion scanning for the pTyr immonium ion in addition to MS/MS led to a pSer/ pThr/pTyr ratio of 35:6:2, representing a 2-fold higher share of phosphotyrosines in comparison to the most comprehensive phosphoproteome study to date by Olsen et al.14 From the 494 phosphopeptides, 40% were identified exclusively after IMAC enrichment and 38% exclusively after SCX, whereas the overlap between the two approaches accounts for 22% (Figure 5). Data interpretation using a concatenated target/decoy database of the human Swiss-Prot subset and a FPR of 1% led to the identification of 674 phosphorylation sites, corresponding to a 20% increase, of which many did not pass manual spectrum validation because of inferior spectrum quality. Further data interpretation using a 1% FPR and a concatenated target/decoy database of the human IPI led to the identification of 916 nonredundant phosphorylation sites from 844 peptides. Especially with regard to mass analyzers of medium mass accuracy on the MS/MS or both the MS and MS/MS level, we therefore recommend a combination of target/decoy search strategies and additional manual validation. We therefore decided to base this study on manual interpretation to focus especially on the unambiguousness of the here-presented phosphorylation sites, accepting a decrease in the number of
Phosphoproteome of Resting Human Platelets
Figure 5. Comparison of IMAC and SCX enrichment. The overlap between IMAC and SCX only accounts for 22% of the identified phosphopeptides. Beside general differences in the specificity of phosphopeptide enrichment strategies,25 this small overlap can be partially explained by the presence of phosphopeptides containing missed cleavage sites and His residues, which could be identified almost exclusively after IMAC enrichment. During SCX enrichment, these peptides elute with the majority of nonphosphorylated peptides and, thus, analysis is severely hampered.
identifications but, on the contrary, a general increase in spectrum quality. To our knowledge, the here-presented data represent the largest phosphoproteomic study on native human cells, which were not derived from cell culture, thus far. However, in comparison to recently published large-scale phosphoproteome studies of nuclear cells,13,14 the number of here-identified phosphorylation sites is relatively low, still after exclusion of nuclear phosphopeptide hits from these studies. Whereas the usage of high mass accuracy MS systems might lead to an increase of phosphopeptide identifications by reducing the occurrence of false positives, a distinct localization of phosphorylation sites without assigning ions on the noise level remains challenging, especially in the context of required high mass accuracy MS/MS spectra. Identification of Novel Putative PKA and PKG Substrates. The presented platelet phosphoproteomic data were analyzed with respect to the kinases that may phosphorylate revealed phosphorylation sites. Several Ser/Thr and Tyr protein kinases, including PKC, PKB, and MAP kinases, Src family kinases, and others, are involved in platelet activation, while platelet inhibition is tightly regulated especially by PKA and PKG activity. Therefore, here, we focused on the analysis of platelet phosphoproteins, which are phosphorylated by PKA and PKG. These two kinases have an overlapping consensus sequence (R/K|R/K|X|S/T), and in many cases, they can phosphorylate the same sites of a protein. Accordingly, the kinase (PKA or PKG) responsible for phosphorylation of the respective sites cannot be predicted and needs to be validated by further experiments. Therefore, we analyzed all detected phosphopeptides for specific PKA and PKG phosphorylation sites and found 23 that theoretically can be phosphorylated by these kinases (see Table 1). Several known PKA and PKG substrates, including IRAG26 Rap1-GAP2,27 and PDE5,28 were not detected within our study, while for others, spectra did not pass manual validation; e.g., VASP29 and HSP 2730 are present in 1% FPR results. A reason might be missing detection via MS and low spectrum quality, respectively, or that phosphorylation of these sites occurs only after stimulation of the respective kinases. In resting human platelets ex vivo and in the absence of activators of the cAMP/cGMP signaling pathways, the equilibrium be-
research articles tween phosphorylation and dephosphorylation is shifted toward protein phosphatase activity.31 Among the 23 phosphoproteins with putative PKA and PKG phosphorylation sites, only three (GPIbβ chain, LASP1, and PDE3A) are known to be phosphorylated by these two kinases.30,32,33 A total of 12 of them (see Table 1) have not been identified by platelet proteomic approaches before. Among those proteins already known to be expressed in platelets, GPIbR chain (P07359), PDE3A (Q14432), protein tyrosine kinase 2 β (Q14289), and G6b (O95866) play significant roles in platelet function. GPIbR and GPIbβ are covalently linked through a disulfide bond to form GPIb, a central part of the von Willebrand factor (vWF) binding GPIb-IX-V receptor complex, which mediates initial platelet adhesion and activation.34 Platelet activation induced by vWF and other agonists is known to be inhibited by PKA,35,36 and GPIbβ is one of the major substrates of PKA in platelets.37,38 Phosphorylation of the intracellular domain of GPIbβ by PKA at Ser191 (also found in our study) negatively regulates vWF binding to the GPIb-IX-V receptor complex33 and inhibits receptor-mediated actin polymerization.38 Up to now, there was no indication in the literature that GPIbR may also be a substrate for PKA. However, GPIbR was shown to be phosphorylated at Ser625 (Ser609 without a signal sequence), and thus, the GPIb-IX interaction with 14-3-3 protein is regulated.31 However, the kinase responsible for Ser625 phosphorylation is not known yet, and concluding from its consensus sequence (RYSGHpSL), it is no PKA/PKG site. Our finding that the PKA/PKG consensus sequence VAGRRPpSAL was phosphorylated at Ser603 in resting platelets may represent a novel mechanism for the regulation of one of the most important platelet receptor (GPIb-IX-V) mediated signaling pathways in platelets by PKA/PKG. PDE3A (Q14432) is one of the major phosphodiesterases that regulate platelet cAMP levels.39,40 In platelets and smoothmuscle cells, PDE3A activity is regulated by PKA phosphorylation;41–43 however, the distinct PKA phosphorylation site at Ser312 has not been described before. PDE3B, another major PDE3 isoform has at least three PKA phosphorylation sites (Ser73, Ser296, and Ser318). Phosphorylation of Ser318 increases PDE activity and promotes 14-3-3 protein binding to PDE3B.44 Within the sequence of PDE3A, there are five putative PKA/PKG phosphorylation sites (Ser62, Ser292, Ser312, Ser438, and Ser654). However, in our phosphopeptide data from resting platelets, only Ser312 was phosphorylated, probably because phosphorylation of further sites may only be in the detection range after PKA stimulation. Protein tyrosine kinase 2 β (Pyk2) is a member of the focal adhesion kinase (FAK) family of protein-tyrosine kinases that are expressed in platelets and play a central role in signaling events via integrin activation.45 In our data, we found that Pyk2 is phosphorylated at Ser375; however, there is no indication in the literature concerning PKA-mediated regulation of Pyk2 signaling yet. Only recently, G6b was identified as a novel platelet receptor by proteomic approaches.1,3,46 The G6b gene encodes for a total of six isoforms (from A to F), and at least four of them were detected within platelets.47 The PKA/PKG phosphorylation site at Ser226 (RPRRLpSTAD) found in our study is present only in isoforms B and C, whereas isoforms A and B have a transmembrane domain and are expressed on the platelet surface.3,47 The G6b-B isofom contains multiple tyrosine residues in its immunoreceptor tyrosine-based inhibitory motif (ITIM) and is Journal of Proteome Research • Vol. 7, No. 2, 2008 531
research articles
Zahedi et al. a
Table 1. Putative PKA/PKG Sites Identified in This Study prim acc
protein name
p site
known in platelets?
O75791 O94929 O43561 O43318 O95866 O00559 P35236 P07359 P13224 P12931 Q5T5C0 Q96PM5 Q12982 Q14289 Q9UK76 Q9Y2L6 Q9NQ75 Q8ND76 Q14847 Q99501 Q9UDY2 Q14432 Q9UNZ2
GRB2-related adapter protein 2 actin-binding LIM protein 3 linker for activation of T-cells family member 1 mitogen-activated protein kinase kinase kinase 7 G6b protein receptor-binding cancer antigen expressed on SiSo cells tyrosine-protein phosphatase nonreceptor type 7 platelet glycoprotein 1b R chain platelet glycoprotein 1b β chain proto-oncogen tyrosine protein kinase Src syntaxin-binding protein 5 RING finger and CHY zinc finger domain-containing protein 1 BCL2/adenovirus E1B 19 kDa protein-interacting protein 2 protein tyrosine kinase 2 β hematological and neurological expressed 1 protein FERM domain-containing protein 4B HEF-like protein cyclin-fold protein 1 LASP1_HUMAN GAS2-like protein tight junction protein ZO-2 cGMP-inhibited 3′,5′ cyclic phosphodiesterase A NSFL1 cofactor p47
T262 S372 S101 S439 S226 S36 S44 S603 S191 S16 S759 S257 S114 S375 S86 S574 S305 S324 S146 S352 S966 S312 S114
b unknown c unknown c unknown unknown known known c unknown unknown unknown known unknown unknown b unknown known unknown b known unknown
a Prim acc, Swiss-Prot accession identifier; p site, phosphorylated site. b Identified in a microparticle proteome study (Garcia, B. A.; Smalley, D. M.; Cho, H.; Shabanowitz, J.; Ley, K.; Hunt, D. F. The platelet microparticle proteome. J. Proteome Res. 2005 4, 1516-1521). c Identified in a platelet membrane proteome study.1
Figure 6. Immunodetection with a phospho-specific (pSer146) anti-LASP antibody. Platelets were incubated with 5 µM of forskolin (activator of PKA) or 5 µM of sodium nitroprusside (SNP, activator of PKG) for 2 min. In contrast to other PKA/PKG substrates, such as VASP, LASP is prephosphorylated at Ser146 in resting human platelets and the phosphorylation further increases with the activation of PKA and PKG. The specificity of the used antibody has been previously described in detail.48
factor activated kinase 1, TAK1) is known to be phosphorylated by PKA on the same site (Ser439) in RAW264.7 cells. Phosphorylation of TAK1 in these cells enhances TNFR-induced activation of p38 MAP kinase and is involved in NF-kB activation by stimulation of IkBR degradation.49 Both p38 MAP kinase and the NF-kB/IkB complex are expressed in platelets and activated in agonist-stimulated platelets.50,51 Because the function of these signaling pathways in platelets is not yet clear, phosphorylation of TAK1 by PKA may represent a new regulating mechanism of platelet activity. Several other unknown platelet proteins that were found to be phosphorylated on PKA/PKG consensus sites including actin-binding LIM protein 3 (O94929), tyrosine-protein phosphatase nonreceptor type 7 (P35236), and FERM domaincontaining protein 4B (Q2Y2L6) are listed in Table 1 and might be involved in platelet inhibitory pathways.
Conclusions constitutively associated with nonreceptor protein tyrosine phosphotase SHP-1.3,47 G6b-B receptor activity in platelets is probably associated with GPVI and FcγRIIA receptor functions and plays an inhibitory role in platelets.3,47 Future work is necessary to determine the role of PKA/PKG phosphorylation of G6b-B within platelets. LASP1 was identified as a specific substrate for PKA and PKG in intact human platelets. Phosphorylation of LASP1 at the identified Ser146 site leads to a redistribution of the actinbound protein from the tips of the cell membrane to the cytosol.48 With a phospho-specific (pSer146) anti-LASP antibody, we confirmed that LASP in intact human platelets is prephosphorylated at Ser146 and that the phosphorylation further increases with the activation of PKA and PKG (see Figure 6). Among unknown proteins in platelets, mitogen-activated protein kinase kinase kinase 7 (O43318, transforming growth 532
Journal of Proteome Research • Vol. 7, No. 2, 2008
Clearly, some of the novel phosphorylation sites identified in the current work have to be studied with respect to their functional relevance. Furthermore, this phosphoproteome of resting human platelets needs to be compared to the phosphoproteome of platelets stimulated by platelet agonists and/ or inhibitors, which is the topic of future investigations. In this context, the phosphoproteome of resting human platelets presented here serves as a basis and source for such studies and the identification of novel proteins involved in platelet stimulation/inhibition processes regulated by phosphorylation events, e.g., regarding the here-identified novel GPIbR phosphorylation site at Ser603.
Acknowledgment. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 688 and FZT-82). The authors thank Anja Thiessen for taking care of our Platelet Proteome Database, Lennart Martens for his help
Phosphoproteome of Resting Human Platelets with the mass-spectrometry-oriented LIMS project software, and Elke Butt for LASP antibodies and discussion.
Supporting Information Available: List of the unambiguously identified phosphoproteins (Supplementary Table 1) and identified phosphopeptides (Supplementary Table 2). This material is available free of charge via the Internet at http:// pubs.acs.org. References (1) Moebius, J.; Zahedi, R. P.; Lewandrowski, U.; Berger, C.; Walter, U.; Sickmann, A. The human platelet membrane proteome reveals several new potential membrane proteins. Mol. Cell. Proteomics 2005, 4 (11), 1754–1761. (2) Lewandrowski, U.; Moebius, J.; Walter, U.; Sickmann, A. Elucidation of N-glycosylation sites on human platelet proteins: A glycoproteomic approach. Mol. Cell. Proteomics 2006, 5 (2), 226– 233. (3) Senis, Y. A.; Tomlinson, M. G.; Garcia, A.; Dumon, S.; Heath, V. L.; Herbert, J.; Cobbold, S. P.; Spalton, J. C.; Ayman, S.; Antrobus, R.; Zitzmann, N.; Bicknell, R.; Frampton, J.; Authi, K.; Martin, A.; Wakelam, M. J.; Watson, S. P. A comprehensive proteomics and genomics analysis reveals novel transmembrane proteins in human platelets and mouse megakaryocytes including G6b-B, a novel ITIM protein. Mol. Cell. Proteomics 2007, 6 (3), 548–564. (4) Garcia, A.; Senis, Y. A.; Antrobus, R.; Hughes, C. E.; Dwek, R. A.; Watson, S. P.; Zitzmann, N. A global proteomics approach identifies novel phosphorylated signaling proteins in GPVI-activated platelets: Involvement of G6f, a novel platelet Grb2-binding membrane adapter. Proteomics 2006, 6 (19), 5332–5343. (5) Martens, L.; Van Damme, P.; Van Damme, J.; Staes, A.; Timmerman, E.; Ghesquiere, B.; Thomas, G. R.; Vandekerckhove, J.; Gevaert, K. The human platelet proteome mapped by peptidecentric proteomics: A functional protein profile. Proteomics 2005, 5 (12), 3193–3204. (6) Marcus, K.; Immler, D.; Sternberger, J.; Meyer, H. E. Identification of platelet proteins separated by two-dimensional gel electrophoresis and analyzed by matrix assisted laser desorption/ ionization-time of flight-mass spectrometry and detection of tyrosine-phosphorylated proteins. Electrophoresis 2000, 21 (13), 2622–2636. (7) Marcus, K.; Moebius, J.; Meyer, H. E. Differential analysis of phosphorylated proteins in resting and thrombin-stimulated human platelets. Anal. Bioanal. Chem. 2003, 376 (7), 973–993. (8) Immler, D.; Gremm, D.; Kirsch, D.; Spengler, B.; Presek, P.; Meyer, H. E. Identification of phosphorylated proteins from thrombinactivated human platelets isolated by two-dimensional gel electrophoresis by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) and liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). Electrophoresis 1998, 19 (6), 1015–1023. (9) Garcia, A.; Prabhakar, S.; Hughan, S.; Anderson, T. W.; Brock, C. J.; Pearce, A. C.; Dwek, R. A.; Watson, S. P.; Hebestreit, H. F.; Zitzmann, N. Differential proteome analysis of TRAP-activated platelets: Involvement of DOK-2 and phosphorylation of RGS proteins. Blood 2004, 103 (6), 2088–2095. (10) Ficarro, S. B.; McCleland, M. L.; Stukenberg, P. T.; Burke, D. J.; Ross, M. M.; Shabanowitz, J.; Hunt, D. F.; White, F. M. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat. Biotechnol. 2002, 20 (3), 301–305. (11) Beausoleil, S. A.; Jedrychowski, M.; Schwartz, D.; Elias, J. E.; Villen, J.; Li, J.; Cohn, M. A.; Cantley, L. C.; Gygi, S. P. Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc. Natl. Acad. Sci. U.S.A. 2004, 101 (33), 12130–12135. (12) Li, X.; Gerber, S. A.; Rudner, A. D.; Beausoleil, S. A.; Haas, W.; Villen, J.; Elias, J. E.; Gygi, S. P. Large-scale phosphorylation analysis of R-factor-arrested Saccharomyces cerevisiae. J. Proteome Res. 2007, 6 (3), 1190–1197. (13) Villen, J.; Beausoleil, S. A.; Gerber, S. A.; Gygi, S. P. Large-scale phosphorylation analysis of mouse liver. Proc. Natl. Acad. Sci. U.S.A. 2007, 104 (5), 1488–1493. (14) Olsen, J. V.; Blagoev, B.; Gnad, F.; Macek, B.; Kumar, C.; Mortensen, P.; Mann, M. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 2006, 127 (3), 635–648. (15) Chi, A.; Huttenhower, C.; Geer, L. Y.; Coon, J. J.; Syka, J. E.; Bai, D. L.; Shabanowitz, J.; Burke, D. J.; Troyanskaya, O. G.; Hunt, D. F. Analysis of phosphorylation sites on proteins from Saccharomyces cerevisiae by electron transfer dissociation (ETD) mass spectrometry. Proc. Natl. Acad. Sci. U.S.A. 2007, 104 (7), 2193–2198.
research articles (16) Lewandrowski, U.; Zahedi, R. P.; Moebius, J.; Walter, U.; Sickmann, A. Enhanced N-glycosylation site analysis of sialoglycopeptides by strong cation exchange prefractionation applied to platelet plasma membranes. Mol. Cell. Proteomics 2007, 6 (11), 1933–1941. (17) Sickmann, A.; Reinders, J.; Wagner, Y.; Joppich, C.; Zahedi, R.; Meyer, H. E.; Schonfisch, B.; Perschil, I.; Chacinska, A.; Guiard, B.; Rehling, P.; Pfanner, N.; Meisinger, C. The proteome of Saccharomyces cerevisiae mitochondria. Proc. Natl. Acad. Sci. U.S.A. 2003, 100 (23), 13207–13212. (18) Mitulovic, G.; Smoluch, M.; Chervet, J. P.; Steinmacher, I.; Kungl, A.; Mechtler, K. An improved method for tracking and reducing the void volume in nano HPLC-MS with micro trapping columns. Anal. Bioanal. Chem. 2003, 376 (7), 946–951. (19) Steen, H.; Kuster, B.; Fernandez, M.; Pandey, A.; Mann, M. Detection of tyrosine phosphorylated peptides by precursor ion scanning quadrupole TOF mass spectrometry in positive ion mode. Anal. Chem. 2001, 73 (7), 1440–1448. (20) Perkins, D. N.; Pappin, D. J.; Creasy, D. M.; Cottrell, J. S. Probabilitybased protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 1999, 20 (18), 3551–3567. (21) Elias, J. E.; Haas, W.; Faherty, B. K.; Gygi, S. P. Comparative evaluation of mass spectrometry platforms used in large-scale proteomics investigations. Nat. Methods 2005, 2 (9), 667–675. (22) Reinders, J.; Zahedi, R. P.; Pfanner, N.; Meisinger, C.; Sickmann, A. Toward the complete yeast mitochondrial proteome: Multidimensional separation techniques for mitochondrial proteomics. J. Proteome Res. 2006, 5 (7), 1543–1554. (23) Tarentino, A. L.; Gomez, C. M.; Plummer, T. H., Jr. Deglycosylation of asparagine-linked glycans by peptide:N-glycosidase F. Biochemistry 1985, 24 (17), 4665–4671. (24) Hunter, T. The Croonian Lecture 1997. The phosphorylation of proteins on tyrosine: Its role in cell growth and disease. Philos. Trans. R. Soc. London, Ser. B 1998, 353 (1368), 583–605. (25) Bodenmiller, B.; Mueller, L. N.; Mueller, M.; Domon, B.; Aebersold, R. Reproducible isolation of distinct, overlapping segments of the phosphoproteome. Nat. Methods 2007, 4 (3), 231–237. (26) Antl, M.; von Bruhl, M. L.; Eiglsperger, C.; Werner, M.; Konrad, I.; Kocher, T.; Wilm, M.; Hofmann, F.; Massberg, S.; Schlossmann, J. IRAG mediates NO/cGMP-dependent inhibition of platelet aggregation and thrombus formation. Blood 2007, 109 (2), 552–559. (27) Schultess, J.; Danielewski, O.; Smolenski, A. P. Rap1GAP2 is a new GTPase-activating protein of Rap1 expressed in human platelets. Blood 2005, 105 (8), 3185–3192. (28) Rybalkin, S. D.; Rybalkina, I. G.; Feil, R.; Hofmann, F.; Beavo, J. A. Regulation of cGMP-specific phosphodiesterase (PDE5) phosphorylation in smooth muscle cells. J. Biol. Chem. 2002, 277 (5), 3310– 3317. (29) Butt, E.; Abel, K.; Krieger, M.; Palm, D.; Hoppe, V.; Hoppe, J.; Walter, U. cAMP- and cGMP-dependent protein kinase phosphorylation sites of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) in vitro and in intact human platelets. J. Biol. Chem. 1994, 269 (20), 14509–14517. (30) Butt, E.; Immler, D.; Meyer, H. E.; Kotlyarov, A.; Laass, K.; Gaestel, M. Heat shock protein 27 is a substrate of cGMP-dependent protein kinase in intact human platelets: Phosphorylation-induced actin polymerization caused by HSP27 mutants. J. Biol. Chem. 2001, 276 (10), 7108–7113. (31) Eigenthaler, M.; Nolte, C.; Halbrugge, M.; Walter, U. Concentration and regulation of cyclic nucleotides, cyclic-nucleotide-dependent protein kinases and one of their major substrates in human platelets. Estimating the rate of cAMP-regulated and cGMPregulated protein phosphorylation in intact cells. Eur. J. Biochem. 1992, 205 (2), 471–481. (32) Han, S. J.; Vaccari, S.; Nedachi, T.; Andersen, C. B.; Kovacina, K. S.; Roth, R. A.; Conti, M. Protein kinase B/Akt phosphorylation of PDE3A and its role in mammalian oocyte maturation. EMBO J. 2006, 25 (24), 5716–5725. (33) Bodnar, R. J.; Xi, X.; Li, Z.; Berndt, M. C.; Du, X. Regulation of glycoprotein Ib-IX-von Willebrand factor interaction by cAMPdependent protein kinase-mediated phosphorylation at Ser166 of glycoprotein Ibβ. J. Biol. Chem. 2002, 277 (49), 47080–47087. (34) Lopez, J. A.; Li, C. Q.; Weisman, S.; Chambers, M. The glycoprotein Ib-IX complex-specific monoclonal antibody SZ1 binds to a conformation-sensitive epitope on glycoprotein IX: Implications for the target antigen of quinine/quinidine-dependent autoantibodies. Blood 1995, 85 (5), 1254–1258. (35) Salzman, E. W. Cyclic AMP and platelet function. N. Engl. J. Med. 1972, 286 (7), 358–363. (36) Coller, B. S. Inhibition of von Willebrand factor-dependent platelet function by increased platelet cyclic AMP and its prevention by cytoskeleton-disrupting agents. Blood 1981, 57 (5), 846–855.
Journal of Proteome Research • Vol. 7, No. 2, 2008 533
research articles (37) Fox, J. E.; Reynolds, C. C.; Johnson, M. M. Identification of glycoprotein Ibβ as one of the major proteins phosphorylated during exposure of intact platelets to agents that activate cyclic AMP-dependent protein kinase. J. Biol. Chem. 1987, 262 (26), 12627–12631. (38) Wardell, M. R.; Reynolds, C. C.; Berndt, M. C.; Wallace, R. W.; Fox, J. E. Platelet glycoprotein Ibβ is phosphorylated on serine 166 by cyclic AMP-dependent protein kinase. J. Biol. Chem. 1989, 264 (26), 15656–15661. (39) Feijge, M. A.; Ansink, K.; Vanschoonbeek, K.; Heemskerk, J. W. Control of platelet activation by cyclic AMP turnover and cyclic nucleotide phosphodiesterase type-3. Biochem. Pharmacol. 2004, 67 (8), 1559–1567. (40) Manns, J. M.; Brenna, K. J.; Colman, R. W.; Sheth, S. B. Differential regulation of human platelet responses by cGMP inhibited and stimulated cAMP phosphodiesterases. Thromb. Haemostasis 2002, 87 (5), 873–879. (41) Shakur, Y.; Holst, L. S.; Landstrom, T. R.; Movsesian, M.; Degerman, E.; Manganiello, V. Regulation and function of the cyclic nucleotide phosphodiesterase (PDE3) gene family. Prog. Nucleic Acid Res. Mol. Biol. 2001, 66, 241–277. (42) Murthy, K. S.; Zhou, H.; Makhlouf, G. M. PKA-dependent activation of PDE3A and PDE4 and inhibition of adenylyl cyclase V/VI in smooth muscle. Am. J. Physiol. 2002, 282 (3), C508–C517. (43) Bender, A. T.; Beavo, J. A. Cyclic nucleotide phosphodiesterases: Molecular regulation to clinical use. Pharmacol. Rev. 2006, 58 (3), 488–520. (44) Palmer, D.; Jimmo, S. L.; Raymond, D. R.; Wilson, L. S.; Carter, R. L.; Maurice, D. H. Protein kinase A phosphorylation of human phosphodiesterase 3B promotes 14-3-3 protein binding and inhibits phosphatase-catalyzed inactivation. J. Biol. Chem. 2007, 282 (13), 9411–9419.
534
Journal of Proteome Research • Vol. 7, No. 2, 2008
Zahedi et al. (45) Schlaepfer, D. D.; Hunter, T. Integrin signalling and tyrosine phosphorylation: Just the FAKs. Trends Cell Biol. 1998, 8 (4), 151– 157. (46) Macaulay, I. C.; Tijssen, M. R.; Thijssen-Timmer, D. C.; Gusnanto, A.; Steward, M.; Burns, P.; Langford, C. F.; Ellis, P.; Dudbridge, F.; Zwaginga, J. J.; Watkins, N. A.; van der Schoot, C. E.; Ouwehand, W. H. Comparative gene expression profiling of in vitro differentiated megakaryocytes and erythroblasts identifies novel activatory and inhibitory platelet membrane proteins. Blood 2006, 109 (8), 3260–3269. (47) Newland, S. A.; Macaulay, I. C.; Floto, R. A.; de Vet, E. C.; Ouwehand, W. H.; Watkins, N. A.; Lyons, P. A.; Campbell, R. D. The novel inhibitory receptor G6B is expressed on the surface of platelets and attenuates platelet function in vitro. Blood 2007, 109 (11), 4806–4809. (48) Butt, E.; Gambaryan, S.; Gottfert, N.; Galler, A.; Marcus, K.; Meyer, H. E. Actin binding of human LIM and SH3 protein is regulated by cGMP- and cAMP-dependent protein kinase phosphorylation on serine 146. J. Biol. Chem. 2003, 278 (18), 15601–15607. (49) Kobayashi, Y.; Mizoguchi, T.; Take, I.; Kurihara, S.; Udagawa, N.; Takahashi, N. Prostaglandin E2 enhances osteoclastic differentiation of precursor cells through protein kinase A-dependent phosphorylation of TAK1. J. Biol. Chem. 2005, 280 (12), 11395–11403. (50) Begonja, A. J.; Geiger, J.; Rukoyatkina, N.; Rauchfuss, S.; Gambaryan, S.; Walter, U. Thrombin stimulation of p38 MAP kinase in human platelets is mediated by ADP and thromboxane A2 and inhibited by cGMP/cGMP-dependent protein kinase. Blood 2007, 109 (2), 616–618. (51) Liu, F.; Morris, S.; Epps, J.; Carroll, R. Demonstration of an activation regulated NF-κB/I-κBR complex in human platelets. Thromb. Res. 2002, 106 (4–5), 199–203.
PR0704130
