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Temporal quantitative phosphoproteomics of ADP stimulation reveals novel central nodes in platelet activation and inhibition

Florian Beck, Jörg Geiger, Stepan Gambaryan, Fiorella A. Solari, Margherita Dell’Aica, Stefan Loroch, Nadine J. Mattheij, Igor Mindukshev, Oliver Pötz, Kerstin Jurk, Julia M. Burkhart, Christian Fufezan, Johan W. M. Heemskerk, Ulrich Walter, René P. Zahedi and Albert Sickmann

Data supplements

Article Figures & Data

Figures

  • Figure 1.

    Phosphorylation response to ADP stimulation. Phospho-peptides classified in (A) early, (B) medium, and (C) late signals after ADP treatment of 10 seconds, 30 seconds, and 60 seconds. Early responders were further classified into (D) sustained and (E) short signals.

  • Figure 2.

    Pathways show differential patterns of phospho-regulation. Per time point, regulated phosphoproteins were analyzed for enriched GO-terms, which mainly show a similar trend: (1) strong regulation after 10 s, (2) followed by a clearly reduced regulation over time. The pathways “transmembrane receptor protein tyrosine kinase signaling” and “enzyme linked receptor protein signaling” clearly deviated from this pattern, showing a second strong phase of regulation at 60 seconds.

  • Figure 3.

    Dose-dependent aggregometry data for ADP-induced platelet shape change at different Iloprost concentrations. The half-maximal ADP concentration for platelet shape change increased from 48 nM to 217 nM in the presence of 1 nM Iloprost, whereas the Hill-slope of the dose-response curve increased from 1.05 to 1.92 (means of 4 independent experiments ± standard deviation). Hill coefficients were calculated by nonlinear regression analysis of the raw data.

  • Figure 4.

    Iloprost induces a diverse pattern of responses. (A) Heatmap of 368 phosphorylation sites that were regulated in the ADP and/or ADP+Iloprost (*) data sets and were consistently quantified in both. (B) Sixty-three sites showing a significant decrease in phosphorylation upon Iloprost treatment (*)compared with their corresponding ADP temporal profiles. (C) Sixty-eight sites showing a significant increase in phosphorylation upon Iloprost treatment (*)compared with their corresponding ADP temporal profiles.

  • Figure 5.

    Unsupervised hierarchical clustering of combined ADP and ADP+Iloprost temporal profiles. Color codes reflect log2 ratios compared with unstimulated controls, square sizes indicate relative standard deviations (RSD, see supplemental Methods). Entries are given as: Gene name, phosphorylation site, protein description. (A) Complete node map. All temporal phosphorylation profiles (central gray node) were clustered into different communities according to their similarities, out of which certain examples were selected that indicate functional nodes. (B) Node representing phosphopeptides with early and sustained upregulation, 5 of which are potential PKC targets.26 (C) Node with phosphopeptides not or only slightly affected by ADP but upregulated by Iloprost treatment, consequently potential PKA targets.26 (D) Node with phosphopeptides upregulated by ADP and downregulated by subsequent Iloprost treatment.

  • Figure 6.

    Heatmap summarizing temporal profiles of 23 selected phosphopeptides. PRM (left) and iTRAQ-based (right) temporal phosphorylation profiles are depicted. Phosphopeptides were clustered according to their PRM profiles, the PRM color code represents log2-fold changes compared with controls. In 7 cases, the corresponding quantitative value was missing for the iTRAQ data (black). Notably, the lower precision of iTRAQ-based quantification leads to ratio compression. Therefore, the smaller scale of iTRAQ ratios was adjusted to the PRM measurements to facilitate the visual comparison (ie, iTRAQ log2-ratios ranged from −1.2 to 2.8). In most cases, the overall phosphorylation changes could be confirmed using an alternative quantification method, additional donors, and platelet preparations conducted by different coworkers.

  • Figure 7.

    Reciprocal regulation of selected sites upon ADP and Iloprost treatment. Log2 ratio refers to the changes compared with resting platelets. Iloprost regulation data were taken from our previous study.14

  • Figure 8.

    Western blot validation of inverse GSK3ASer21, RASGRP2Ser567phosphorylation, and RAP1 pull-down after stimulation with ADP, Iloprost, and ADP+Iloprost. (A) Human platelets (3 × 108/mL) were stimulated by ADP (10 μM, 1 minute), Iloprost (2 nM, 1 minute), or both; notably, slightly different than for the phosphoproteomics data. After stimulation, platelets were lysed and subjected to western blot GSK3ASer21, RASGRP2Ser567, and Rap1 pull-down assay. The representative blots of 3 independent experiments are shown. (B) Immunoblots using selected phosphospecific antibodies were done in triplicate and (C) quantified using the ImageJ program. The intensities of the phosphorylation signals were normalized to actin. Data are normalized against controls, results are given as means ± standard error of the mean (n = 3, + P < .05 compared with the control, *P < .05 compared with the Iloprost sample).

Tables

  • Table 1.

    ADP-regulated kinases and Tyr-phosphatases

    Early kinases (10 s)
    FamilyUniprotGene (alternative)SiteRegulation
    AGCQ05655PRKCD (PKDCδ)Ser130↑10ADP
    Thr218↑10ADP
    Ser304↑10ADP
    AGCQ04759PRKCQ (PKCθ)Thr219↑10ADP
    Ser685↑10ADP
    AGCQ13464ROCK1Ser1341↑10ADP
    AGCO75116ROCK2Ser1374↑10ADP
    AGCP23443RPS6KB1 (p70S6K)Ser427↑10ADP
    CAMKO60229KALRN (TRAD)Ser1896↑10ADP
    Ser2261↓10ADP
    Ser1756+1757↑10ADP
    CAMKP49137MAPKAPK2Thr334↑10ADP
    CAMKQ9BUB5MKNK1 (MNK1)Thr385↑10ADP
    CAMKQ7KZI7MARK2Thr596↑10ADP
    CAMKQ15746MYLK (smMLCK)Ser1760↑10ADP
    Ser1776/Ser1779↑10ADP
    CAMKQ13555CAMK2GThr287↑10ADP
    Thr306↑10ADP
    Ser311↑10ADP
    CK1P0C1S8WEE2 (WEE1B)Ser139↑10ADP
    CMGCO94921CDK14 (PFTAIRE1)Ser24↑10ADP
    STEO94804STK10 (LOK)Ser454↑10ADP
    STEQ13043STK4 (MST1)Ser414↑10ADP
    Ser438↑10ADP
    STEQ99683MAP3K5 (ASK)Ser1033↑10ADP
    Ser1240↑10ADP
    Ser1029+Ser1033↑10ADP
    STEQ13177PAK2Ser19/Ser20↑10ADP
    TKP52333JAK3Ser15/17/20/Thr21↑10ADP
    TKQ06187BTKThr184↑10ADP
    TKQ07912TNK2 (ACK)Thr517/Tyr518↑10ADP
    TKLQ8IVT5KSR1Thr268+Thr272↓10ADP
    Q8TD19NEK9Thr333↑10ADP
    Q9H4A3WNK1Ser1261↓10ADP
    Medium (30 s) and late kinases (60 s)
    FamilyUniprotGene (alternative)SiteRegulation
    AGCQ05655PRKCD (PKCδ)Thr43↑30ADP
    CAMKQ9Y2K2SIK3 (QSK)Ser568↓30ADP
    CAMKQ9UEW8STK39 (PASK)Ser385↓30ADP
    STEQ13043STK4 (MST1)Thr340↑60ADP
    STEQ7L7X3TAOK1 (TAO1)Thr440↑30ADP
    STEQ9UKE5TNIK (ZC2)Thr903↑60ADP
    STEQ13233MAP3K1 (MEKK1)Thr20↓60ADP
    STEQ8N4C8MINK1 (MINK/ZC3)Ser701↓60ADP
    STEQ9Y4K4MAP4K5 (KHS1)Thr168↑60ADP
    STEO95747OXSR1 (OSR1)Ser359↓60ADP
    STEQ13177PAK2Ser197↑30ADP
    TKP43405SYKSer297↑30ADP
    TKLP15056BRAFSer151↓60ADP
    Tyrosine phosphatases
    UniprotGeneSiteRegulation
    Q05209PTPN12Thr454↓10ADP
    Q99952PTPN18Ser341↑10ADP
    Q8WYL5SSH1Ser897↑10ADP
    Q8TE77SSH3Ser37↑10ADP
    Q8TF42UBASH3BThr106↑10ADP
    Regulatory subunits of phosphatases
    UniprotGeneSiteRegulation
    O14974PPP1R12ASer478/Ser479↑10ADP
    Ser903↑60ADP
    O95685PPP1R3DSer25↑10ADP
    Q96SB3PPP1R9BSer99/Ser100↑10ADP
    • Kinase phosphorylation sites were grouped into early (10 seconds), medium (30 seconds), and late (60 seconds) responders. ↑10ADP corresponds to upregulation after 10 seconds of ADP stimulation. Involvement of Tyr-phosphatases and regulatory subunits are summarized. If no specific phosphorylation Ser/Thr/Tyr residue could be assigned (eg, JAK3), all possible phospho-residues are listed.

  • Table 2.

    Regulated GAP and GEF proteins belonging to the Rho/Rac pathway

    GDP exchange proteins (GEF)GTPase-activating proteins (GAP)
    AccessionGeneSiteRegulationAccessionGeneSiteRegulation
    Q12802AKAP13Ser1750↑10ADPP98171ARHGAP4Ser860↑10ADP
    Ser2709↑10ADP
    Q92974ARHGEF2Ser121/122↑10ADPO43182ARHGAP6Ser927↑10ADP
    Ser820/Thr821↓60ADP
    Q14155ARHGEF7Ser674/676↑30ADPQ53QZ3ARHGAP15Ser43↑30ADP
    O15013ARHGEF10Ser379↑10ADPA7KAX9ARHGAP32Ser587↑60ADP
    Q9NZN5ARHGEF12Ser22↑10ADPQ92619HMHA1Ser14↑10ADP
    Ser599↑10ADPSer73↑10ADP
    Thr96/Ser99↑10ADP
    Q9H7D0DOCK5Ser1742↑10ADPQ13576IQGAP2Ser16↑10ADP
    Ser1766↑10ADP
    Ser1781↑10ADP
    Q96N67DOCK7Ser182↑10ADPQ13459MYO9BThr1271↑10ADP
    Ser1354↓60ADP
    Thr1492↑10ADP
    Ser1972↑10ADP
    Q5JSL3DOCK11Ser31↑10ADPO60890OPHN1Ser703↑30ADP
    O60229KALRNSer1896↑10ADP
    Ser2261↑10ADP
    Ser1756 + 1757↑10ADP
    Q07890SOS2Ser1315↑10ADP
    P21731TBXA2Ser329↑30ADP
    Q9UKW4VAV3Ser786↑10ADP
    • ↑10ADP corresponds to upregulation after 10 seconds of ADP stimulation. If the phosphorylated site could not be localized to a distinct Ser/Thr/Tyr residue with high confidence (eg, ARHGAP6), all possibly phosphorylated residues are listed, according to the phosphoRS algorithm.