Quantitative Time-Resolved Phosphoproteomic Analysis of Mast Cell Signaling¹

Mast cells are regarded as crucial effector cells in allergic reactions and IgE-associated immune responses (1). Activation of mast cells, a critical feature of type I hypersensitive reactions, leads to the release of a wide range of chemical mediators and cytokines that recruit inflammatory cells and regulate inflammatory responses, such as mucus secretion, vasodilation, and bronchoconstriction (2). During activation, the mast cell high-affinity IgE receptor FcεRI is cross-linked by allergens through bound IgE, leading to a cascade of signaling events and the release of preformed inflammatory mediators localized in specialized granules, the de novo synthesis and secretion of proinflammatory lipid mediators, and the synthesis and secretion of cytokines and chemokines (3).

Some aspects of FcεRI-mediated mast cell activation and inhibition pathways have been described previously (4) (Fig. 1). FcεRI expressed on mast cells is comprised of three subunits: an IgE-binding α subunit, a signal-amplifying β subunit, and two disulfide-linked signal-initiating γ subunits (5). Following FcεRI aggregation, the activation of β subunit-bound protein tyrosine kinase Lyn and, subsequently, tyrosine phosphorylation of ITAM regions in the β and γ subunits of FcεRI, initiates a complex series of intracellular signaling events (6, 7, 8, 9, 10). Phosphorylated ITAMs provide docking sites for Src homology 2 (SH2)³ domain-containing cytoplasmic tyrosine kinases such as Syk (11), Lyn, and Fyn (12), leading to the activation of Syk (11). The subsequent phosphorylation of linker for activation of T cells (LAT) results in the recruitment and activation of several proteins, including Grb2-related adaptor protein 2, phospholipase C γ (PLCγ), SH2 domain-containing leukocyte protein of 65 kDa (SLP76), VAV, growth factor receptor-bound protein 2 (Grb2), SH2 domain-containing transforming protein C (SHC), and son of sevenless (Sos), leading ultimately to mast cell degranulation, cytokine gene transcription, and synthesis of lipid mediators (4). The phosphorylation of non-T cell activation linker might also participate in mast cell signaling by binding Grb2 (4). In mast cells, coaggregation of FcεRI with the low-affinity receptor for IgG (FcγRIIb) leads to the inhibition of Ag-induced mast cell degranulation and cytokine production (Fig. 1 c) (13). Inhibitory pathways triggered by FcεRI/FcγRIIb coaggregation include the Lyn kinase-mediated phosphorylation of ITIM tyrosine residues in FcγRIIb leading to recruitment of SHIP, followed by its association with SHC and Dok1, eventually culminating in inactivation of Ras (3).

Recent advances in the area of phosphorylation analysis, using emerging mass spectrometric proteomic technologies, have created the possibility of simultaneous, quantitative monitoring in total cellular lysates of large numbers of phosphorylation sites following receptor stimulation (14, 15, 16). To facilitate the quantitative analysis of tyrosine phosphorylation sites in FcεRI-activated mast cells, peptide phosphotyrosine immunoprecipitation (14, 17) was used to enrich complex cellular lysates for tyrosine-phosphorylated peptides before immobilized metal affinity chromatography (IMAC) and liquid chromatography-mass spectrometry (LC-MS) analysis using a label-free quantitation method (18) (Fig. 2).

A plethora of quantitative proteomic methods have recently been developed using the incorporation of isotopic labels into proteins or through label-free methodologies (19). Highly reproducible chromatographic enrichment and detection of cell-derived phosphopeptides are essential for the successful implementation of a label-free quantitation method. To provide the necessary chromatographic performance and sensitive detection of phosphopeptides, an automated phosphoproteomic platform for the enrichment of phosphotyrosine-immunoprecipitated peptides was used (20). This system employs automated desalting, IMAC enrichment, and reversed-phase separation of peptides in a highly reproducible fashion because all column elutions and loading steps are precisely replicated with computer controls. This system provides highly reproducible retention times and peak areas as well as highly sensitive detection of cell-derived phosphopeptides, as described previously (20).

FcεRI-mediated mast cell signaling pathways, including many components, their interactions, and their phosphorylation sites, have been intensively studied. However, a systematic, quantitative analysis of global mast cell phosphorylation has never been performed. To generate a comprehensive map of mast cell phosphorylation events, a quantitative proteomic analysis of mast cell signaling through FcεRI was performed. This study provides a starting foundation for the detailed examination of the biological role of these newly discovered phosphorylation sites through mutagenesis, leading ultimately to a better molecular understanding of the structure of the mast cell signaling pathway.

The mouse bone marrow-derived mast cell line MCP5 was cultured in RPMI 1640 (Sigma-Aldrich) supplemented with 10% heat-inactivated FBS (Sigma-Aldrich), 2 mM l-glutamine, 100 U/ml penicillin G, 100 μg/ml streptomycin (Mediatech), and 5% conditioned medium from mouse IL-3-expressing D11 fibroblasts grown in 5% CO₂ at 37°C. Bone marrow cells from C57BL/6 mice were cultured in IL-3-containing medium for 4–6 wk to generate immature mast cells (bone marrow-derived mast cells (BMMC)) with >95% purity (c-Kit⁺ and FcεRI⁺ by flow cytometry). Animal experiments were approved by the Brown University Institutional Animal Care and Use Committee and performed in accordance with the guidelines of the National Institutes of Health. MCP5 cells and BMMC were sensitized with 0.5 μg/ml mouse monoclonal anti-DNP IgE (clone SPE-7; Sigma-Aldrich) overnight at a density of 2 × 10⁶ cells/ml in a CO₂ incubator at 37°C. Cells were then washed with Tyrode’s buffer and treated at 2.5 × 10⁷ cells/ml in Tyrode’s buffer with 100 ng/ml DNP[27]-BSA (Biosearch Technologies) at 37°C, for varying durations. The reaction was stopped by the addition of a final concentration of 8 M urea, 1 mM Na₃VO₄, 100 mM NH₄HCO₃ (pH 8.0) lysis buffer. After incubation on ice for 20 min, lysates from all time points were centrifuged at 14,000 × g for 15 min at 4°C. Lysate protein concentration was measured by the DC Protein Assay (Bio-Rad).

Proteins were reduced with 10 mM DTT for 1 h in a 56°C water bath, followed by alkylation with 55 mM indoacetamide for 1 h at room temperature in the dark. Cell lysates were diluted five times with 100 mM NH₄HCO₃ (pH 8.9). Proteins were digested with affinity-purified, l-1-Tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (Promega) at a trypsin:protein ratio 1:100 (w/w) overnight at 37°C. Tryptic peptides were desalted using Sep-Pak C18 Cartridges (Waters), as described (21), and dried in a Speed Vac plus (Thermo Savant).

Dry peptides from each time point (1 × 10⁸ cells/time point) were reconstituted in 1 ml of cold immunoprecipitation buffer (30 mM Tris, 30 mM NaCl, and 0.3% Nonidet P-40 (pH 7.4)), and 10 pmol of synthetic peptide LIEDAEpYTAK was added to each time point as a control for label-free quantitation, accompanying the cellular phosphopeptides through the peptide immunoprecipitation, subsequent peptide purification steps, and reversed-phase elution of peptides into the mass spectrometer. Bead-conjugated anti-phosphotyrosine Ab was prepared by coupling anti-phosphotyrosine mAb clone pTyr¹⁰⁰ (Cell Signaling Technology) to protein G agarose beads (Roche) noncovalently at 2 mg/ml overnight at 4°C. Anti-phosphotyrosine beads were then added at 15 μl of resin/ 1 × 10⁸ cells overnight at 4°C with gentle shaking. Beads were washed and eluted, as described (14).

Tryptic peptides were analyzed by a fully automated phosphoproteomic technology platform incorporating peptide desalting via reversed-phase chromatography, gradient elution to an Fe³⁺-loaded IMAC column, reversed-phase separation of peptides, followed by tandem mass spectrometry with static peak parking, as described previously (20). Briefly, tryptic peptides were loaded onto a desalting reversed-phase column (360 μm OD × 200 μm ID fused silica (Polymicro Technologies), fritted with an inline microfilter (M520; Upchurch Scientific), packed on a pressure bomb (GNF Commercial Systems) with 12-cm SelfPack POROS 10 R2 resin (Applied Biosystems)). Peptides were rinsed with 0.1 M acetic acid/MilliQ water (solvent A) at a flow rate of 10 μl/min and then eluted to an Fe³⁺-activated IMAC column (360 μm OD × 200 μm ID fused silica, bomb packed with 15-cm SelfPack POROS 20 MC resin (Applied Biosystems)) with an HPLC gradient of 0–70% solvent B (0.1 M acetic acid/acetonitrile) in 17 min at a flow rate of 1.8 μl/min. The column was washed with ∼40 μl of a 25:74:1 acetonitrile/water/acetic acid mixture containing 100 mM NaCl, followed by 4-min solvent A at a flow rate of 0.035 ml/min. Enriched phosphopeptides were eluted to a precolumn (360 μm OD × 75 μm ID) containing 2 cm of 5 μm Monitor C18 resin (Column Engineering) with 40 μl of 25 mM potassium phosphate (pH 9.0) and rinsed with solvent A for 30 min. Peptides were eluted into the mass spectrometer (LTQ-FT; Thermo Electron) through an analytical column (360 μm OD × 75 μm ID fused silica with 12 cm of 5 μm Monitor C18 particles with an integrated ∼4 μm ESI emitter tip fritted with 3 μm silica; Bangs Laboratories) with an HPLC gradient (0–70% solvent B in 30 min). Static peak parking was performed via flow rate reduction from the initial 200 nl/min to ∼20 nl/min when peptides began to elute as judged from a BSA peptide scouting run, as described previously (20). The electrospray voltage of 2.0 kV was applied in a split flow configuration, as described (20). Spectra were collected in positive ion mode and in cycles of one full MS scan in the FT (m/z: 400-1800) (∼1 s each), followed by MS/MS scans in the LTQ (∼0.3 s each) sequentially of the five most abundant ions in each MS scan with charge state screening for +1, +2, and +3 ions and dynamic exclusion time of 30 s. The automatic gain control was 1,000,000 for the FTMS MS scan and 10,000 for the ion trap MS (ITMS) scans. The maximum ion time was 100 ms for the ITMS scan and 500 ms for the FTMS full scan. FTMS resolution was set at 100,000.

MS/MS spectra were automatically searched against the mouse National Center for Biotechnology Information nonredundant protein database using the SEQUEST algorithm provided with Bioworks 3.2SR1 (22). Search parameters specified a differential modification of phosphorylation (+79.9663 Da) on serine, threonine, and tyrosine residues and a static modification of carbamidomethylation (+57.0215 Da) on cysteine. Before manual spectral validation, SEQUEST results were filtered by Xcorr (+1 > 1.5; +2 > 2.0; +3 > 2.5), precursor mass error (<20 ppm), minimum repetition of five of nine total time points containing an MS/MS spectra for each individual peptide, nonredundant within each time point, and phosphotyrosine containing. For all the peptides exceeding these thresholds (labeled “high stringency” throughout this study), SEQUEST peptide sequence assignments and the position of the phosphorylation site were manually validated, as described previously (22). Manually validated spectra for all reported phosphopeptides are available (http://mastcellpathway.com).

Relative quantitation was performed on all manually validated peptides via calculation of selected ion chromatogram (SIC) peak areas, normalization with the SIC peak area of the copurified synthetic peptide LIEDAEpYTAK, and generation of a heatmap representation. SIC peak areas were calculated using newly developed software programmed in Microsoft Visual Basic 6.0 based on Xcalibur Development Kit 2.0 SR1 (Thermo Electron). Manually validated SIC peak areas for all reported phosphopeptides are available (supplemental material 1).⁴ Peak areas determined with this tool were consistent with areas determined manually through the manufacturer’s software, Xcalibur. Peak areas were represented as heatmaps. Phosphopeptide abundance was represented as black when a peptide’s peak area was approximately the geometric mean for that peptide across all time points. A blue color represented abundance less than this average, and a yellow color represented abundance more than this average. The magnitude of change of the heatmap color was based on the log of the fold change of each individual peptide peak area compared with the geometric mean for that peptide across all time points. All heatmap representations were based upon SIC peak areas normalized in each time point to LIEDAEpYTAK control peptide that was spiked in an equal amount to each time point sample and copurified and analyzed with the cell-derived peptides. Blanks in the heatmap indicated that both a manually validated MS/MS spectra and SIC peak were not observed for that phosphopeptide in that time point.

Peptide peak areas were normalized and clustered according to their temporal profiles. Peptide peak areas were normalized to have a mean of 0 and a SD of 1 to capture the temporal profile of each peptide. Peptides with missing data across nine time points or whose maximum fold change was <4-fold were discarded before clustering. Normalized peak areas were clustered using a fuzzy k-means clustering algorithm called MFuzz (23). MFuzz assigns a membership value to each peptide for every cluster, allowing partial membership of a peptide to more than one group. Peptides with <70% membership to the best cluster were removed. Because the number of clusters, k, was specified as a parameter to the algorithm, the optimal number of clusters was determined by running the clustering algorithm 100 times with k = 3, … , 9. The best run was chosen for each k as the smallest sum of all proteins to their assigned cluster center. Phosphoprotein sequences were searched against the Minmotif Miner dataset for identification of sequence motifs containing phosphorylated tyrosine residues (24).

To clearly define the temporal dimension of mast cell signaling as revealed through protein phosphorylation, a phosphoproteomic time course experiment was conducted (Fig. 2). Anti-DNP IgE-sensitized mouse BMMC and a mast cell line MCP5 were stimulated through DNP-BSA cross-linking. Within the mast cell time course experiment described in this study, the automated chromatographic system performed as expected with highly reproducible retention times and peak areas for each phosphopeptide among the various time points. The LIEDAEpYTAK phosphopeptide normalization standard accompanied the true cellular phosphopeptides through peptide immunoprecipitation, desalt, IMAC, and reversed-phase elution and detection in the mass spectrometer. The average SIC peak area of this peptide standard was 2.4 × 10⁷ with a SD of 0.7 × 10⁷. Additionally, the SD of retention times of each cellular phosphopeptide was compared with the peptide’s average half-maximal peak width among the nine time points. The magnitude of the variation of retention time among the time points for each detected phosphopeptide was well within the width of the peaks on average, indicating coelution of phosphopeptides in the separate time point analyses. The average SD of 6.3 s for peptide retention time among the nine time points, the average maximal variation in retention time of 19 s among the nine time points, and the average half-maximal SIC peak width of 22 s indicated the highly reproducible acquisition of data.

For every phosphopeptide, spectra were manually validated for both the correct sequence assignment as well as the position of the phosphorylation site as illustrated for the FcεRIγ ITAM-containing phosphopeptide ADAVpYTGLNTR (Fig. 3^,a). Although 549 unique tyrosine phosphorylation sites on 426 proteins in MCP5 cells and 450 unique tyrosine sites on 342 proteins in BMMC were uncovered with high quality sequence assignments (Xcorr +1 > 1.5; +2 > 2.0; +3 > 2.5; precursor mass error <20 ppm), only peptides with repeated observations of MS/MS spectra in at least five of the nine time points in the MCP5 dataset and two of the three time points in BMMC dataset were manually validated and quantified (high stringency). We also defined a medium stringency threshold that was identical with the high stringency threshold with only the removal of the requirement for repeated observations of MS/MS spectra in separate time points. Medium stringency threshold was used only in the comparison of BMMC to MCP5 data sets (Fig. 4^,c only). The quantitation for the representative FcεRIγ ITAM-containing phosphopeptde ADAVpYTGLNTR is shown (Fig. 3 b). After manual validation and quantitation, the temporal variation of 171 phosphorylation sites on 121 proteins was revealed in FcεRI-stimulated MCP5 cells, whereas 179 phosphorylation sites on 117 proteins were revealed in stimulated BMMC (supplemental material 1).⁴

Dynamic tyrosine phosphorylation of proteins associated with the canonical mast cell signaling pathway was observed (Figs. 1 and 4) (17, 21, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49). Quantitative analysis of tyrosine phosphorylation of proteins involved in the known mast cell signaling pathway for both MCP5 cells (Fig. 4,a) and BMMC (Fig. 4,b) is shown for comparison. The high similarity of tyrosine-phosphorylated proteins observed between the MCP5 dataset and the BMMC dataset (89% identical; Fig. 4,c) argues for the validity of the MCP5 cell line as a model system for mast cell activation through FcεRI. All of the remaining three proteins observed in BMMC, but absent from the MCP5 dataset, were actually observed in the MCP5 dataset at medium stringency. Furthermore, 59% of identical phosphorylation sites were observed both in MCP5 and BMMC (Fig. 4 c). Of the 41% of sites observed only in BMMC, over one-half of those missing sites were also observed in MCP5 at medium stringency.

The small differences in specific phosphorylation sites observed in MCP5 compared with BMMC in the known mast cell signaling pathway could be explained by subtle variations in the absolute abundance of each phosphopeptide, or variations in the level of induction of phosphorylation after FcεRI activation. For example, it is quite possible that sites observed in BMMC, but seemingly absent in MCP5, are actually present at levels slightly below the sensitivity limit of our proteomic methodology. Also, the MCP5 cell line was derived from AD12-SV40-infected BMMC from BALB/c mice, whereas the BMMC analyzed in this experiment were obtained from C57BL6 mice (50). The subtle differences observed in specific phosphorylation sites could also arise from the different strains of mice compared in this experiment.

Lyn binds to FcεRIβ in resting mast cells, and once activated following receptor aggregation, it phosphorylates the ITAM tyrosine residues on FcεRIβ and FcεRIγ (6, 7, 8, 9, 10). Phosphorylation on both FcεRIβ and FcεRIγ was extremely rapid, with increased phosphorylation as early as 10 s and substantial phosphorylation at 1 min. FcεRIγ phosphorylation was sustained at a high level from 1 to 5 min, whereas FcεRIβ phosphorylation declined rapidly after reaching its maximum at 1 min. All ITAM phosphorylation sites on FcεRI were observed in BMMC with the exception of FcεRIγ Tyr⁷⁶.

The phosphorylation of ITAMs on FcεRI leading to the recruitment and activation of SH2 domain-containing proteins, such as Syk, results in a cascade of phosphorylation of downstream signaling molecules, including LAT, Grb2-associated binding protein 2 (Gab2), PI3K, Bruton’s tyrosine kinase (Btk)/Tec, PLCγ, and SLP76 (4). We observed novel phosphorylation sites (MCP5 Tyr¹⁹⁴, BMMC Tyr¹⁹³) within the SH2 domain of Lyn and the known kinase activation site (MCP5 Tyr⁴¹⁶) within the tyrosine kinase domain of Fyn (51). Mutations of both tyrosine residues (Tyr³⁴²Tyr³⁴⁶) of Syk are known to induce a significant reduction in FcεRI-mediated mast cell degranulation and calcium flux (25). We observed an increase in phosphorylation of Syk at 1 min postreceptor stimulation, followed by maximal phosphorylation increase of 15-fold overall at 5 min on the two autophosphorylation sites (Tyr³⁴²Tyr³⁴⁶) in MCP5. A 6-fold induction of phosphorylation at 1 min on the autophosphorylation sites Tyr³¹⁷, Tyr⁵¹⁹, and Tyr⁵²⁰ of Syk was observed in BMMC. Novel phosphorylation sites (Tyr⁵⁴⁰, Tyr⁶²³, and Tyr⁶²⁴) on Syk were also observed.

Dynamic phosphorylation of known signaling proteins was observed beyond the initial phosphorylation of the receptor and receptor-associated kinases. Adaptor protein Gab2 can be phosphorylated by Fyn tyrosine kinase, creating binding sites for Grb2, PI3K, SH2-containing protein tyrosine phosphatase 2 (SHP2), and Crkl (26). We observed strong induction of phosphorylation on the known Crkl binding sites in MCP5 and BMMC (Tyr²⁶³, Tyr²⁹⁰) on Gab2. Phosphorylation of biologically uncharacterized sites with strong Scansite (52) motifs for SH2 interaction with the Src kinase Fyn and phosphorylation by Src kinase was observed on the PI3K p85 regulatory subunit β (Tyr⁴⁵⁸) and the p85 regulatory subunit α (Tyr⁴⁶⁷) in MCP5 cells. Formation of phosphatidylinositol-3,4,5-triphosphate (PIP3) from phosphatidylinositol-4,5-bisphosphate (PIP2), through the action of PI3K, leads to activation of Btk and Tec tyrosine kinases, which results in the phosphorylation and activation of PLCγ (4). Syk- or Lyn-mediated phosphorylation of Btk (Tyr⁵⁵¹ observed in both BMMC and MCP5) leads to activation of Btk tyrosine kinase activity and phosphorylation of PLCγ (27). The location of novel phosphorylation sites observed within the pleckstrin homology domain (Tyr⁴⁰) and SH2 domain (Tyr³⁴⁴) of Btk in MCP5 suggests that these sites might play a regulatory role in binding of Btk to PIP3 or interactions with other tyrosine-phosphorylated signaling molecules. Similarly, phosphorylation of the Btk homologue Tec was observed in both BMMC and MCP5 (Tyr⁴¹⁵, homologous to Tyr⁵⁵¹ of Btk) within its tyrosine kinase domain. Both Tyr⁷⁷¹ and Tyr⁷⁷⁵ on PLCγ1 are known to be activation-induced tyrosine phosphorylation sites, and Tyr⁷⁷⁵ is established as a critical phosphorylation site for PLCγ1 activation (28, 53). Phosphorylation of these two tyrosine sites on PLCγ1 in MCP5 (Tyr⁷⁷¹ and Tyr⁷⁷⁵) and in BMMC (Tyr⁷⁷¹) was observed. Phosphorylation of Tyr⁷⁵³ on PLCγ2, which has been previously characterized to regulate the lipase activity of PLCγ2 (54), was highly elevated upon receptor stimulation in both MCP5 and BMMC. PLCγ2 phosphorylation on Tyr¹²¹⁷ and on the biologically uncharacterized site Tyr¹²⁴⁵ was also observed in both MCP5 and BMMC (55). Ras can be activated through Syk-mediated phosphorylation of SHC and binding of SHC to Grb2 and Sos (56). We observed strong and rapid induction of phosphorylation of SHC on the Syk-phosphorylated residue Tyr⁴²³ in both MCP5 and BMMC (57). The largest change in phosphorylation after receptor aggregation was observed on the autophosphorylation sites Thr²⁰³/Tyr²⁰⁵ of ERK1 (202-fold induced in MCP5 and 238-fold induced in BMMC). Strong elevation of ERK2 autophosphorylation on Thr¹⁸³/Tyr¹⁸⁵ was also observed in both BMMC and MCP5 (58).

Dynamic tyrosine phosphorylation was observed on inhibitory signaling molecules that participate in the regulation of mast cell signaling after coaggregation of FcεRI and FcγRIIB. Although IgG was not added in our experiment, IgE also binds and activates FcγRIIB signaling in mouse mast cells (59). There was rapid and strong induction of tyrosine phosphorylation at previously uncharacterized sites on SHIP (Ser⁹³⁴/Tyr⁹⁴⁴, Tyr⁸⁶⁷, Tyr⁹⁴⁴) in both BMMC and MCP5, an enzyme whose activation leads to cleavage of PIP3 and inhibition of Btk-mediated Ca²⁺ release (3). Following receptor coaggregation, studies have shown that phosphorylated SHIP associates with SHC and Dok1 through their phosphotyrosine binding domains (13). Phosphorylation of Dok1 at Tyr³⁶¹ (induced in both BMMC and MCP5), the major binding site of NCK and Ras GTPase-activating proteins (GAP), leads to its association with RasGAP and inhibition of FcεRI-induced ERK1/2 activation and calcium mobilization (13). Induction of phosphorylation on biologically uncharacterized sites was also observed on Dok1 in BMMC (Tyr⁴⁰⁸, Tyr⁴⁵⁰). We observed a profound increase in tyrosine phosphorylation of the Src kinase-regulated site Tyr³¹¹ of protein kinase C δ (in both MCP5 and BMMC), a negative regulator of Ag-induced mast cell degranulation (60, 61). Clathrin-mediated endocytic recycling of activated FcεRI occurs through interaction between the receptor and the tyrosine-phosphorylated scaffold/E3 ubiquitin ligase Cbl and CIN85 complex (62). Strong induction of tyrosine phosphorylation was observed on Cbl in MCP5 after receptor cross-linking (Ser⁶⁶⁶Tyr⁶⁷²).

In mast cells, actin cytoskeleton is crucially involved in the processes of cytosolic granule exocytosis and receptor endocytosis. Before cell stimulation, granules are kept apart from their fusion sites by the cytoskeletal barrier composed predominantly of actin and myosin. In response to stimulation, the actin cytoskeleton serves as a scaffold for signaling molecule recruitment and provides pathways for vesicle transportation (63). A dramatic reorganization of the cytoskeleton is essential for mast cell granule exocytosis (64). Many phosphorylated components of the actin cytoskeleton were observed in FcεRI-stimulated MCP5 mast cells (Fig. 5) (17, 20, 21, 40, 55, 65, 66, 67, 68, 69, 70). Although phosphorylation on cytoskeleton-associated proteins was also observed in BMMC (and available in supplemental material 1),⁴ the remaining discussion is concentrated on the MCP5 dataset. Phosphorylation of the cytoskeletal components actin (Tyr⁹¹), beta tubulin (Tyr³⁴⁰), advillin (Tyr⁸⁵, Tyr⁷⁴⁸), cofilin 1 (Tyr⁶⁸, Tyr ¹⁴⁰, Tyr⁸⁹), plectin (Tyr26, Tyr³⁶⁴⁹), and vimentin (Tyr⁵³) was induced after mast cell receptor activation. Conversely, a rapid reduction of phosphorylation on a previously undescribed phosphorylation site Tyr³²³ of an actin-associated tyrosine-phosphorylated protein was observed immediately following receptor aggregation.

Dynamic cytoskeletal rearrangements are also regulated through the action of receptor tyrosine kinases such as the Ephrin receptors. There is now much evidence that Ephrin receptor activation leads to signaling pathways regulating cell adhesion and plasticity of the actin cytoskeleton (66). We detected phosphorylation of the previously established autophosphorylation sites within the tyrosine kinase domain of EphA3 at Tyr⁷⁷⁹ as well as a novel site at Tyr⁹³⁷, adjacent to a known SH2 interaction site critical in regulating downstream signaling events via interactions with cytosolic proteins (71, 72). One of the cytosolic adaptor molecules that interact with phosphorylated Ephrin receptors is NCK. NCK proteins modulate actin cytoskeletal dynamics by linking proline-rich effector molecules to tyrosine kinases or phosphorylated signaling intermediates (73). A strong elevation of phosphorylation was observed on NCK2/Grb4 SH2/SH3 adaptor at Tyr¹¹⁰.

The Rho family of GTPases, including Rac, is critically important in the regulation of dynamic processes within the actin cytoskeleton downstream of receptor protein tyrosine kinases in mast cells (74). We observed phosphorylation of sites on a collection of proteins that regulate the activity of Rho GTPases. A collection of GAPs showed elevated tyrosine phosphorylation as a result of receptor activation in mast cells. ARAP3 is a dual GAP for RhoA and Arf6 that controls dynamic actin rearrangements and vesicular trafficking events (75). Elevation of tyrosine phosphorylation was observed on ARAP3 at a site (Tyr¹⁴⁰⁴) that was previously shown to negatively regulate cell spreading through inhibition of ARAP3 activation of Rho GTPases (65). Another RhoGAP, p190RhoGAP, stably associates with RasGAP through a Src-mediated phosphorylation site at Tyr⁹⁴³ (76). An increase in phosphorylation was observed on this critical tyrosine residue in stimulated mast cells. The RhoGAP family member, RhoGAP12, also became tyrosine phosphorylated after mast cell receptor activation (Ser²³⁸/Tyr²⁴¹). ELMO2 forms a ternary complex with RhoG and Crk2 to activate Rac1 (77, 78). A strong elevation of ELMO2 tyrosine phosphorylation was observed on a novel site Tyr⁷¹⁵ after mast cell receptor aggregation.

The activation of Rho family GTPases after mast cell receptor activation triggers exocytosis of histamine-containing cytoplasmic granules within seconds of stimulation (79). We observed rapid tyrosine phosphorylation of many proteins associated with exocytosis after receptor aggregation. The interaction between Wiskott-Aldrich syndrome protein (WASP) and Cdc42 induces a conformational change, stimulating Arp2/3 protein complex to nucleate actin polymerization (74). Arp2/3 protein is associated with secretory granules at the point of contact between the vesicle and the cellular membrane, providing an actin structure that enhances the efficiency of the exocytotic process (80). In B cells, WASP phosphorylation by Src-type kinases at Tyr²⁹³ stimulates actin polymerization indepedently of Cdc42 (81). We observed a rapid increase in tyrosine phosphorylation at this site on WASP. A rapid increase in tyrosine phosphorylation was also observed on Arp2/3 subunit p21 after mast cell receptor stimulation (Tyr⁴⁷). The motor protein kinesin has been proposed to transport intracellular organelles and vesicles to the cell periphery preceding exocytosis of secretory vesicles (82). We detected rapid phosphorylation at a novel tyrosine site on kinesin 2 in stimulated mast cells (Tyr⁴⁴⁸). Rapid tyrosine phosphorylation of annexin VI, a negative regulator of membrane exocytosis (83), was also observed at a novel site Tyr²⁰¹.

Phosphorylation of proteins previously associated with exocytosis was also observed on a collection of proteins late after receptor stimulation. Scinderin, a Ca²⁺-dependent actin filament-severing protein, regulates exocytosis by affecting the organization of the microfilament network proximal to the plasma membrane (84). We identified a novel tyrosine phosphorylation site Tyr⁵⁹⁹ on scinderin. AHNAK/Desmoyokin activates PLCγ1 through protein kinase C α-mediated arachidonic acid release and is known to be associated with neural exocytotic vesicles (85, 86). We observed strong induction of tyrosine phosphorylation on AHNAK (Tyr¹²², Lyn kinase ITAM-like motif D/E-X-X-Y-X-X-I/L).

We observed phosphorylation of proteins that are known to play roles in regulating receptor internalization after stimulation of mast cells. Mast cell FcεRI endocytosis occurs through clathrin-coated pits (87). Intersectin 2 functions cooperatively with WASP and cdc42 to link the clathrin endocytic machinery to WASP-mediated actin polymerization and receptor endocytosis (88). Phosphorylation of two biologically undefined sites was induced after mast cell receptor activation on intersectin 2 (Tyr⁵⁵³ and Tyr⁹²¹). Beta arrestin regulates receptor internalization of G protein-coupled receptors as well as receptor protein tyrosine kinases such as epidermal growth factor receptor (89). We observed a decrease in phosphorylation of β arrestin on a previously undescribed site Tyr⁴⁷. Our experiment revealed strong induction of phosphorylation on DAPP1/Bam32 at the Src family kinase-mediated phosphorylation site Tyr¹³⁹, as well as a previously uncharacterized site Ser¹⁴¹ (90). Although DAPP1/Bam32 has never been previously described in mast cells, it is known to regulate BCR internalization through activation of Rac1 and actin reorganization subsequent to pleckstrin homology domain-mediated interaction with PIP2 produced through SHIP cleavage of PIP3 (90).

We detected phosphorylation of many known signaling proteins not previously associated with the canonical pathway in stimulated mast cells (Fig. 6) (17, 40, 55, 67, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101). In receptor-activated mast cells, we observed rapid induction of tyrosine phosphorylation on MAPK activator with WD repeats (MAWD) at a novel site (Tyr³⁴²). MAWD protein is a scaffolding, signaling molecule that activates ERK signaling (102). Rapid Lyn-mediated phosphorylation of Fps and Fer tyrosine kinases causes phosphorylation of PECAM-1 leading to activation of SHP1 and SHP2, limiting mast cell activation (103). Tyrosine phosphorylation at a previously undescribed site Tyr⁴⁰² was observed on Fer kinase. Fps phosphorylation was also observed at a novel phosphorylation site Tyr⁷¹¹.

We saw strong induction of tyrosine phosphorylation on the established Lyn kinase phosphorylation site Tyr⁶⁹ of MIST/Clnk (104). The SLP76 homologue MIST/Clnk is tyrosine phosphorylated by Lyn and Fyn kinase through a complex of SKAP55 and FYB/SLAP130 leading to binding of PLCγ, VAV, Grb2, and LAT (105, 106). Tyrosine phosphorylation on other members of this protein complex was observed (SKAP55R Tyr75, Tyr¹⁵¹, and Tyr²⁶⁰; FYB/SLAP130 Tyr⁵⁵⁹, and Ser⁵⁶¹) (107).

A collection of protein tyrosine phosphorylation was revealed in our quantitative proteomic analysis on proteins not previously associated with any signaling pathway (Fig. 7) (17, 40), including a group of metabolic enzymes, RNA-interacting proteins, unnamed proteins, and proteins associated with ubiquitination. None of these phosphorylation sites have yet been characterized biologically. With a few exceptions, the magnitude of change in phosphopeptide abundance within this group of proteins after receptor stimulation was relatively small compared with the actin cytoskeleton-associated proteins, canonical mast cell signaling proteins, and signaling proteins not associated with mast cell groups.

The largest change in phosphopeptide abundance among this group of proteins was observed on the mast cell adhesion glycoprotein CD34 (108). CD34 is expressed both on hemopoietic stem cells and mature BMMC (108). We observed strong phosphorylation of two tyrosine residues (Tyr³²⁶, Tyr³³⁶) within the short cytosolic portion of CD34 after FcεRI aggregation. Induction of phosphorylation was also observed on the chloride intracellular channel 1 Tyr²³³, hypothetical proteins XP_150108 Tyr²⁷⁸, LOC134492 Tyr¹⁴⁵, RIKEN cDNA 2810441C07 Tyr¹⁵², RIKEN cDNA 9930117H01 Tyr¹⁶⁰, and the ubiquitin-activating enzyme E1 Tyr⁵⁵.

A comprehensive molecular understanding of mast cell signaling will allow the development of improved therapies for treatment of acute allergic disorders, such as anaphylaxis and asthma, as well as a fundamental understanding of the associated cellular pathways. Our study for the first time provides a view, in unprecedented detail, of the dynamic tyrosine phosphoproteome triggered after FcεRI-mediated mast cell activation.

The temporal arrangement of phosphorylation events observed after mast cell receptor stimulation is suggestive of the placement of these events within signaling pathways, as illustrated by the sequential phosphorylation at known sites observed among proteins in the canonical pathway (Fig. 1). A rapid increase in phosphorylation was observed at 10 s after receptor cross-linking on all expected ITAM phosphorylation sites of FcεRIβ and FcεRIγ. Phosphorylation of FcεRIβ was transient, whereas FcεRIγ phosphorylation was sustained for 5 min. The timing of phosphorylation of SYK at previously established autophosphorylation sites Tyr³⁴²/Tyr³⁴⁶ paralleled phosphorylation on FcεRIγ, suggesting rapid activation of Syk following FcεRIγ phosphorylation. The phosphorylation of Btk on Tyr⁵⁵¹ was increased later at 2 min, further replicating the expected structure of the canonical mast cell signaling pathway. PLCγ2 is known to be phosphorylated by Btk, Lyn, and Fyn (54, 109). Phosphorylation of PLCγ2 at the established Btk phosphorylation site Tyr¹²¹⁷ and the Btk or Lyn phosphorylation site Tyr⁷⁵³ increased concurrently with Btk activation, suggesting rapid phosphorylation of these sites by Btk or phosphorylation of these sites by another kinase such as Lyn or Fyn. ERK1 and ERK2 phosphorylation was maximal very late at 5 min, consistent with the placement of these downstream signaling proteins within the known canonical mast cell pathway.

It is clear from the observation of overlapping pattern of phosphorylation of some proteins that the timing of phosphorylation alone is insufficient for defining the precise placement of proteins within the signaling cascade. First, phosphorylation is a rapid modification requiring a high density of time points to define the precise sequence of phosphorylation. Second, examination of phosphorylation in individual cells such as with recently developed FACS techniques will be necessary to eliminate errors introduced by heterogeneities within bulk populations of cells necessary for proteomic analysis (110). Third, the mast cell signaling pathway has many branch points, and similar patterns of phosphorylation are not necessarily indicative of the coincident placement of proteins within a pathway. Nonetheless, a general indication of the relative pathway placement of newly discovered phosphorylation sites is afforded by this temporal phosphoproteomic analysis. Also, an indication of whether a phosphorylation event is an active participant in the signaling pathway is provided quantitatively by the magnitude of change in its phosphorylation. Phosphorylation sites that have been previously characterized to be biologically meaningful tended to have a higher fold change of phosphorylation (compare Figs. 4 and 7). There was also clear evidence of signal amplification in this dataset because downstream signaling proteins such as ERK and PLCγ displayed greater induction of phosphorylation (>100-fold maximally induced) compared with FcεRI (<10-fold maximally induced).

Phosphorylation of proteins previously associated with exocytosis, such as ARP2/3 p21 subunit, annexin VI, and kinesin 2, was observed to be induced rapidly after receptor cross-linking. Conversely, phosphorylation was also observed on the exocytosis-associated protein AHNAK late after receptor activation. Although the precise role that these early and late phosphorylation events play in exocytosis remains to be determined, the timing is suggestive of a role in initiation or completion of exocytosis.

Phosphorylation of proteins previously associated with receptor endocytosis occurred late after receptor activation. Intersectin 2 is a known participant in mast cell receptor endocytosis, linking WASP-mediated actin polymerization and the clathrin endocytic machinery. Highly induced phosphorylation of biologically uncharacterized sites (Tyr⁵⁵³ and Tyr⁹²¹) was observed to be maximally induced at 5 min after receptor activation on intersectin 2. Tyrosine phosphorylation at Tyr¹³⁹ was maximally elevated on the receptor endocytosis-related protein DAPP1/Bam32 at 5 min.

Clustering of quantitative proteomic data allows for the identification of groups of peptides with similar temporal responses (Fig. 8). Although it is not possible to infer the structure of the signaling pathway directly from these clusters, they provide a general indication of the placement of signaling proteins within the pathway, as described in Results. Cluster 1 contains the earliest and most transient phosphorylation events, including phosphorylation of the ITAM motif of FcεRIβ. Cluster 2, including all the FcεRIγ ITAM-phosphorylated peptides, displays a similar pattern of early phosphorylation, but this phosphorylation is sustained for a longer period of time. The binding of Syk to the FcεRIγ ITAM-phosphorylated residues may protect these phosphorylation sites from dephosphorylation by a phosphatase. Clusters 4 and 5 display the late induction of phosphorylation and include a large number of previously established downstream signaling molecules within the mast cell pathway.

Cluster 3 displays a bimodal pattern of phosphorylation. Included within cluster 3 is a tyrosine phosphorylation site at Tyr²¹⁶ contained within the linker region of the FcεRIβ ITAM motif (YXXL-X₆-YXXL). Although the canonical ITAM phosphorylation sites at Tyr²¹⁰ and Tyr²²⁰ of FcεRIβ show rapid and transient phosphorylation, singly phosphorylated Tyr²¹⁶ within the ITAM linker region displays a remarkable bimodal pattern. This noncanonical ITAM tyrosine phosphorylation site at Tyr²¹⁶ has been suggested to play an inhibitory role in mast cell signaling, probably through its association with the inositol phosphatase SHIP and the protein tyrosine phosphatase SHP1/SHP2 (35). The bimodal pattern of phosphorylation on FcεRIβ at Tyr²¹⁶ is shared by a collection of inhibitory mast cell signaling proteins, including SHIP, SHC, and Dok1, possibly indicating phosphorylation by a common kinase. A bimodal pattern of phosphorylation could indicate the presence of two different kinases that could alter the phosphorylation of a single site and/or the existence of two separate pools of signaling proteins within the cell. Tyr⁴²³ of SHC is phosphorylated by an assortment of different tyrosine kinases, including Lck, Fyn, and Syk in T cells (111, 112). In mast cells, it is possible that this site is phosphorylated initially by Lyn and then by newly activated Syk.

The results described in this study offer many encouraging leads for further detailed analysis of the mast cell signaling pathway. The elucidation of the role of each phosphorylation site will require the use of traditional biochemical techniques, such as creation of site-directed mutants and signaling protein disruptions. The traditional paradigm for unraveling the structure of signaling pathways by the sequence motif-inspired creation of site-directed mutants is greatly enhanced by knowledge of true cellular phosphorylation sites before creation of the site-directed mutants, minimizing the futile creation of mutants of sites that do not exist in mast cells. Quantitative phosphoproteomic profiling of mast cells harboring site-directed mutants of newly discovered phosphorylation sites could greatly complement traditional phenotypic assays of mast cell activation, such as release of histamine, Western blotting for specific proteins or specific sites, or cytokine release assays.

We thank Dr. Laurent Brossay and Dr. Karsten Sauer for reviewing this manuscript.

The authors have no financial conflict of interest.