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Dysregulated FcRI Signaling and Altered Fyn and SHIP Activities in Lyn-Deficient Mast Cells¹

Valerie Hernandez-Hansen^2,*, Alexander J. Smith^3,*, Zurab Surviladze^*, Alexandre Chigaev^*, Tomas Mazel^*, Janet Kalesnikoff, Clifford A. Lowell, Gerald Krystal, Larry A. Sklar^*, Bridget S. Wilson^* and Janet M. Oliver^*

^* Department of Pathology and Cancer Research and Treatment Center, University of New Mexico School of Medicine, Albuquerque, NM 87131; Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, Canada; and Department of Laboratory Medicine, University of California, San Francisco, CA 94143

	Abstract

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Studies in B cells from Lyn-deficient mice have identified Lyn as both a kinetic accelerator and negative regulator of signaling through the BCR. The signaling properties of bone marrow-derived mast cells from Lyn^–/– mice (Lyn^–/– BMMCs) have also been explored, but their signaling phenotype remains controversial. We confirm that Lyn^–/– BMMCs release more -hexosaminidase than wild-type BMMCs following FcRI cross-linking and show that multiple mast cell responses to FcRI cross-linking (the phosphorylation of receptor subunits and other proteins, the activation of phospholipase C isoforms, the mobilization of Ca²⁺, the synthesis of phosphatidylinositol 3,4,5-trisphosphate, the activation of the ₄₁ integrin, VLA-4) are slow to initiate in Lyn^–/– BMMCs, but persist far longer than in wild-type cells. Mechanistic studies revealed increased basal as well as stimulated phosphorylation of the Src kinase, Fyn, in Lyn^–/– BMMCs. Conversely, there was very little basal or stimulated tyrosine phosphorylation or activity of the inositol phosphatase, SHIP, in Lyn^–/– BMMCs. We speculate that Fyn may substitute (inefficiently) for Lyn in signal initiation in Lyn^–/– BMMCs. The loss of SHIP phosphorylation and activity very likely contributes to the increased levels of phosphatidylinositol 3,4,5-trisphosphate and the excess FcRI signaling in Lyn^–/– BMMCs. The unexpected absence of the transient receptor potential channel, Trpc4, from Lyn^–/– BMMCs may additionally contribute to their altered signaling properties.

	Introduction

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The high affinity IgE receptor, FcRI, of mouse basophils and mast cells is a tetrameric complex comprising an IgE-binding subunit, a four-transmembrane-spanning subunit, and two disulfide-linked subunits. Each subunit plays a distinct role in both the cell surface expression and biological activity of the receptor (1, 2). FcRI cross-linking activates intracellular signaling pathways that lead within minutes to the release of inflammatory mediators by degranulation and within hours to the production of proinflammatory cytokines (1, 3). The mechanism is believed to involve the activation of the tyrosine kinase Lyn, which phosphorylates tyrosines in conserved ITAM motifs found in the FcRI and subunit cytoplasmic tails (4, 5). The phosphorylated ITAMs serve as binding sites for the tandem Src homology 2 (SH2)⁴ domains of a second tyrosine kinase, Syk, resulting in the autophosphorylation and activation of Syk (6, 7, 8). Active Lyn and Syk phosphorylate and/or activate many proteins, including isoforms of phospholipase C (PLC1, PLC2) (9, 10, 11), the guanine nucleotide exchange factor, Vav (12), and others (reviewed in Ref.13). The consequences of these initiating events include the production of inositol-1,4,5-trisphosphate (Ins(1,4,5)P₃) (14), the mobilization of Ca²⁺ (15), and the activation of the MAPK pathway (16).

Recent studies in Lyn^–/– B cells have identified Lyn as both a kinetic accelerator and a negative regulator of signaling though the BCR. Specifically, early responses to BCR cross-linking are delayed in Lyn^–/– B cells, supporting a role for Lyn in initiation of BCR signaling (17, 18, 19). However, Lyn^–/– B cells are also hyperresponsive to anti-IgM-induced proliferation, and, once signaling occurs, show enhanced activation of MAPK and prolonged calcium mobilization (18, 19, 20, 21). These effects suggest that Lyn contributes additionally to the down-regulation of BCR-induced signaling.

The signaling properties of mast cells derived from Lyn^–/– mice have also been explored, but their signaling phenotype remains controversial (22). Hibbs et al. (17) reported that Lyn^–/– mice fail to mediate an anaphylactic response in a passive cutaneous anaphylaxis model, suggesting that FcRI-mediated degranulation is inhibited in vivo in Lyn^–/– mast cells. Studies in bone marrow-derived mast cells (BMMCs) from Lyn^–/– mice have variously reported impaired (23), normal (22), or enhanced (24) FcRI-mediated histamine release, accompanied by absent or severely reduced tyrosine phosphorylation of the FcRI and subunits and of downstream signaling proteins (Syk, linker for activation of T cells, focal adhesion kinase, PLC1, PLC2, Cbl, Vav, HS1, Bruton’s tyrosine kinase, Shc, and others). In addition, studies have reported absent (24, 25) or impaired (22, 23) Ca²⁺ mobilization. Other alterations in FcRI-mediated signaling reported in Lyn^–/– BMMCs include: enhanced secretion of TNF- and IL-2, delayed but persistent production of Ins(1,4,5)P₃, prolonged activation of protein kinase C (PKC) and MAPK (23); and constitutively enhanced Gab2, Akt, and PKC phosphorylation and PI3K activity (24).

In this study, we use BMMCs from Lyn^–/– mice to further explore the role of Lyn in FcRI signaling. We confirm that Lyn^–/– BMMCs release more -hexosaminidase than wild-type (WT) BMMCs, and show that multiple mast cell responses to FcRI cross-linking are slow to initiate in Lyn^–/– BMMCs, but persist far longer than in WT cells. Mechanistic studies link these altered signaling properties to increased activity of the Src kinase, Fyn, and decreased activity of the inositol phosphatase, SHIP, in Lyn^–/– BMMCs. Differences between WT and Lyn^–/– BMMCs in the expression of transient receptor potential channels (Trpc) may partially account for altered Ca²⁺ mobilization in Lyn^–/– BMMCs.

	Materials and Methods

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Abs and chemicals

The monoclonal mouse anti-DNP IgE of Liu et al. (26) was purified from ascites. Biotinylated anti-DNP IgE was prepared using EZ-Link sulfo-normal human serum-biotin (Pierce, Rockford, IL). Biotinylated anti-CD117 (c-kit) mAb and the isotype control biotinylated rat anti-mouse IgG2B were purchased from Caltag Laboratories (Burlingame, CA). Avidin-R-PE, DNP-BSA, fura 2-AM, fura Red-AM, and Pluronic F-127 were from Molecular Probes (Eugene, OR). p-Nitrophenyl-N-acetyl--D-glucosamine was from Sigma-Aldrich (St. Louis, MO). The VLA-4 affinity probe, 4-((N'-2-methylphenyl)ureido)-phenylacetyl-L-leucyl-L--aspartyl-L-valyl-L-prolyl-L-alanyl-L-alanyl-L-lysine (LDV), and its FITC-conjugated analog (LDV-FITC) were synthesized at Commonwealth Biotechnologies (Richmond, VA) and are described elsewhere (27). FITC-conjugated mAb 44H6 (against CD49d, the ₄ subunit of VLA-4) was from Immunotech (Westbrook, ME). Rabbit anti-Gab2 serum was provided by H. Gu and B. Neel (Harvard Institutes of Medicine, Boston, MA), and anti-FcRI subunit mAb (28) was provided by J. Rivera (National Institutes of Health, Bethesda, MD). Rabbit Abs to phospho-Akt, Akt, phospho-PTEN (phosphatase and tensin homologue deleted on chromosome 10), and phospho-PKC (Thr⁵⁰⁵) were from Cell Signaling Technology (Beverly, MA). Mouse anti-PY99, anti-phosphotyrosine (PY)20, and anti-p85 mAbs and rabbit Abs to PTEN, PKC, Syk, PLC1, PLC2, Lyn, Fyn, and PKC were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P₃) mAb was from Echelon Biosciences (Salt Lake City, UT). Rabbit Abs to Trpc1, Trpc3, Trpc4, and Trpc6 were from Alomone Laboratories (Jerusalem, Israel). Rabbit anti-actin Ab was from Sigma-Aldrich. Mouse anti-HRP 4G10, and rabbit Abs to SHIP, Gab2, and FcRI were from Upstate Biotechnology (Lake Placid, NY). Polyclonal Abs against phospho-SHIP were generated by immunizing rabbits with a synthetic phosphopeptide corresponding to residues surrounding Tyr¹⁰²⁰ of human SHIP and purified by affinity chromatography using the immunizing peptide bound to beads. Protein A/G-Sepharose was from Oncogene (Cambridge, MA). Goat anti-IgE and rabbit anti-C-Src kinase (Csk)-binding protein (Cbp) Abs were provided by C. Torigoe (University of New Mexico, Albuquerque, NM). HRP-conjugated goat anti-mouse Ig, goat anti-rabbit Ig, and FITC-conjugated Affinipure F(ab')₂ anti-mouse IgM (µ-chain specific) were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Thapsigargin was from Sigma-Aldrich; wortmannin and ionomycin were from Calbiochem (La Jolla, CA). PtdIns(4,5)bisphosphate (PtdIns(4,5)P₂) was from Boehringer Mannheim (Indianapolis, IN), and Ptd[³H]Ins(4,5)P₂ and [³H]inositol-1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P₄) were from DuPont-NEN (Boston, MA). [-³²P]ATP (Redivue) was from Amersham Biosciences (Piscataway, NJ). AG1-X8 formate powder was from Bio-Rad (Hercules, CA). All cell culture reagents were purchased from Life Technologies (Grand Island, NY), except for FCS (HyClone, Logan, UT).

Animals and cell culture conditions

Lyn knockout mice generated on a C57BL/6 background (19) were bred in specific pathogen-free facilities in the University of New Mexico Animal Research Facility (Albuquerque, NM). WT C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). BMMCs were obtained by culturing bone marrows from 8- to 12-wk-old mice in RPMI 1640 medium supplemented with 2 mM L-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, 10% FCS, 55 µM 2-ME, 1 mM HEPES (complete RPMI 1640), and 30% WEHI-3-conditioned (IL-3-containing) medium, as described (29). By 5 wk, >95% of cells expressed similar levels of FcRI and c-kit, and contained the prominent granules characteristic of differentiated mast cells (29).

The experiments described below were all performed with 5- to 8-wk-old mast cells that had been transferred to complete RPMI 1640 medium without added cytokine and sensitized overnight with 1 µg/ml anti-DNP IgE. Except as noted, activation was with 10 ng/ml DBP-BSA.

Degranulation assays

IgE-primed BMMCs were washed twice with 5 ml of RPMI 1640 medium without Phenol Red and stimulated at 37°C, and degranulation was measured from the release of -hexosaminidase, as described (30). Data are reported as the averages ± SEM of four measurements obtained from two independent experiments. Statistical analysis was performed with Student’s unpaired t test using GraphPad (San Diego, CA) Prism software.

Intracellular Ca²⁺ measurements

IgE-primed BMMCs (10⁵ cells/ml) were suspended in 1 ml of RPMI 1640 medium containing 2–5 µM fura 2-AM and 0.1% Pluronic F-127 for 30 min at room temperature. Cells were washed, resuspended in Phenol Red-free RPMI 1640 or HBSS, plated on fibronectin-coated coverslips, and warmed to 35°C in a stage incubator for 10–20 min. Ratiometric imaging and data analysis were performed, as described (31). Briefly, baseline data were collected for 60 s; then DNP-BSA or thapsigargin (100 nM) was added and data collection continued for 10 min. In some experiments, extracellular Ca²⁺ was removed by the addition of 5 mM EGTA. In some cases of thapsigargin stimulation, baseline data were first collected for 60 s in normal Ca²⁺-containing HBSS, then in HBSS solution without Ca²⁺ for 3 min, followed by the addition of 100 nM thapsigargin in calcium-free solution. Ten minutes after the beginning of the experiment, HBSS (no Ca²⁺) was replaced with normal calcium-containing HBSS. Because BMMCs accumulate significant amounts of Ca²⁺ indicator within their exocytic vesicles, analysis regions were drawn around the nucleus to minimize artifactual signals contributed by this compartmentalized dye. Responses were measured in three to six independent experiments. Data were analyzed and graphed with GraphPad Prism software. Calcium measurements with thapsigargin stimulation were verified by flow cytometry using a FACScan (BD Biosciences, San Jose, CA). For these experiments, cells were loaded with 5 µM fura Red-AM at 37°C for 25 min, washed and resuspended in Tyrode’s buffer, and stimulated in the presence or absence of extracellular Ca²⁺ with 200 nm thapsigargin. A decrease in fura Red emission (excited with a 488-nm Ar laser) at 650 nm corresponds to an increase in free intracellular Ca²⁺ concentration ([Ca²⁺]_i).

Measuring VLA-4 surface expression and ligand-binding affinity

VLA-4 expression levels were quantified by incubating BMMCs with FITC-conjugated anti-CD49d mAb at 1/50 in HBSS for 30 min on ice and analyzing the washed cells by flow cytometry (FACScan; BD Biosciences). Total numbers of Ab-binding sites per cell (Ab-binding capacity) were quantified by comparison with a standard curve generated with Quantum Simply Cellular microspheres (Flow Cytometry Standards, San Juan, Puerto Rico) stained in parallel with the anti-CD49d mAb (27, 32).

VLA-4 affinity was determined from the binding kinetics of the VLA-4 affinity probe, LDV-FITC, as described (27, 32). Briefly, IgE-primed BMMCs (1 x 10⁶ cells/ml) were incubated with 5 nM LDV-FITC for 10 min at 37°C with constant stirring at 500 rpm. Cell-associated fluorescence was measured in the FACScan for 30–120 s to establish a baseline. Stimuli were added, and data acquisition was immediately re-established, losing 5–10 s of the total time course. Data collection continued up to 1000 s. To measure the kinetics of probe dissociation, cells were preincubated with LDV-FITC and stimulated for 6–7 min in its continued presence. A 500-fold excess of unlabeled LDV was then added, and the loss of cell-bound fluorescence was monitored. Data were converted to mean channel fluorescence over time using custom FACSQuery software (courtesy of B. Edwards, University of New Mexico). GraphPad Prism was used for curve fitting and statistics.

Western blot analysis of whole cell lysates

BMMCs were lysed on ice for 15 min in lysis buffer A (50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 1 mM sodium orthovanadate, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml antipain, 10 µg/ml leupeptin), containing either 1% Brij-96 or 1% Triton X-100. Proteins in the clarified lysates were resolved using 7.5, 10, or 12% SDS-PAGE or 4–20% gradient gels; transferred to nitrocellulose membranes; and blocked for 1 h with 5% milk. Membranes were incubated with primary Abs for 2 h at room temperature, then washed, probed with HRP-conjugated goat anti-rabbit or goat anti-mouse IgG, developed with SuperSignal substrate (Pierce), and exposed to film. The blots were stripped with stripping buffer (Pierce) for 15 min at room temperature, washed, and reprobed with different Abs to verify loading.

Western blot analysis of immunoprecipitated proteins

For analyses of FcRI and Cbp phosphorylation, BMMCs were lysed on ice in 0.1% Triton X-100-containing solubilization buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM sodium orthovanadate, 5 mM sodium pyrophosphate, 50 mM NaF, 3 mM iodoacetate, 5 mM EDTA) supplemented with the protease and phosphatase inhibitors listed above. For all other analyses, cells were lysed in lysis buffer A (above) containing 0.5% Nonidet P-40. Proteins in the clarified lysates were immunoprecipitated at 4°C for 2 h with Abs prebound to protein A- or protein G-Sepharose. Immunoprecipitates were washed three times in solubilization buffer, resuspended in 40 µl of 2x Laemmli sample buffer (without 2-ME for anti- and anti-IgE immunoprecipitations to preserve the FcRI dimer), and boiled for 5 min, and the released proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 3% Ig-free BSA (Sigma-Aldrich) for 1 h at room temperature and probed with anti-PY Abs (either HRP-conjugated anti-PY or anti-PY, followed by HRP-conjugated goat anti-mouse IgG). Phosphorylation was detected by ECL, as described above. Blots were stripped and reprobed to verify equal loading.

Fyn in vitro kinase assays

BMMCs (5 x 10⁶ cells/condition) were lysed with 1 ml of ice-cold lysis buffer B (25 mM HEPES, pH 7.2, 150 mM NaCl, 1.0% Brij-96, 0.1 mM EGTA, 1 mM NaVO₃, and protease inhibitors) for 20 min. Clarified lysates were incubated for 2 h at 4°C with anti-Fyn Abs (2 µg/ml) prebound to protein A-Sepharose beads. Beads were washed three times in ice-cold buffer B, twice in the same buffer, but containing 0.1% Brij-96, and once in 25 mM HEPES, pH 7.5. Pelleted beads were resuspended in 40 µl of kinase buffer (25 mM HEPES, 10 mM MnCl₂, pH 7.5) containing 10 µCi of [-³²P]ATP and transferred to a 30°C heat block for 5 min. After washing, proteins were solubilized by boiling in 40 µl of Laemmli sample buffer containing 5% 2-ME, separated by 10% SDS-PAGE, and phosphoproteins were visualized by autoradiography.

PLC activity

PLC activity was measured, as previously described (11). Briefly, BMMCs (5 x 10⁶ per condition) were lysed with 20 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100, 5 mM -glycerophosphate, 0.2 mM sodium orthovanadate, 1 mM EGTA, and 1 µg/ml aprotinin and leupeptin; the clarified lysates were rocked for 1 h with 1 µg of anti-PLC1- or PLC2-specific Abs prebound to 30 µl of protein A/G-Sepharose, and the beads were washed with reaction buffer (35 mM NaH₂PO₄, pH 6.8, 70 mM KCl, 0.8 mM EGTA, 0.8 mM CaCl₂). A 100-µl aliquot of PtdIns(4,5)P₂ (1 mg/ml) plus 30 µl of Ptd[³H]Ins(4,5)P₂ (0.3 µCi) was dried under nitrogen and resolubilized in 50 µl of 50 mM Na phosphate, pH 6.8, 100 mM KCl, plus 50 µl of 5% (80 mM) Triton X-100. A total of 10 µl each of 5x reaction buffer and phospholipid substrate solution was added to beads, followed by incubation at 35°C for 20 min. Reactions were stopped by transfer to an ice bath, and addition of 100 µl of 1% (w/v) BSA and 250 µl of 10% (w/v) TCA. Samples were centrifuged, and levels of [³H]Ins(1,4,5)P₃ in the supernatants were determined by liquid scintillation counting.

Measurement of PI(3,4,5)P₃ levels

BMMCs were fixed with 2% paraformaldehyde and permeabilized with 0.05% Triton X-100 in PBS. Washed cells were incubated sequentially for 1 h with mouse anti-PI(3,4,5)P₃ Ab (5 µg/ml) and with FITC-conjugated Affinipure F(ab')₂ anti-mouse IgM (µ-chain specific) (1/200), then washed, resuspended in PBS, and analyzed by flow cytometry.

SHIP in vitro phosphatase assay

SHIP 5-phosphatase activity was measured, as described by Ono et al. (33). Briefly, BMMCs were activated and lysed in lysis buffer A containing 0.5% Nonidet P-40, and proteins in the clarified supernatants were immunoprecipitated at 4°C for 2 h with anti-SHIP Abs prebound to protein A-Sepharose. Immunoprecipitates were washed and incubated for 30 min at 37°C in reaction buffer (20 µl) consisting of [³H]Ins(1,3,4,5)P₄ in 50 mM Tris and 10 mM MgCl₂ (pH 7.2). Cold 2 mM LiCl was added to stop the reactions. Samples were centrifuged and supernatants were added to columns packed with AG1-X8 formate powder equilibrated with 50 mM NH₄⁺ formate/100 mM formic acid. [³H]Ins(1,3,4)P₃ and [³H]Ins(1,3,4,5)P₄ were eluted with 0.7 M NH₄⁺ formate/100 mM formic acid and 1.5 M NH₄⁺ formate/100 mM formic acid, respectively. Samples were analyzed in a Beckman LS 1801 liquid scintillation counter (Beckman Coulter, Fullerton, CA). SHIP activity was determined from the loss of the original substrate, [³H]Ins(1,3,4,5)P₄, and gain of the product of SHIP activity, [³H]Ins(1,3,4)P₃, in the eluted fractions.

	Results

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Lyn^–/– mast cells degranulate more than WT when stimulated with Ag, stem cell factor (SCF), or ionomycin

Cross-linking the FcRI on mast cells with multivalent Ag induces the release of preformed inflammatory mediators such as histamine and -hexosaminidase by degranulation. In agreement with Parravicini et al. (24), the amount of -hexosaminidase released from Lyn^–/– mast cells over a 30-min assay period is higher at all Ag doses tested than in WT mast cells (Fig. 1A). Time course studies show that -hexosaminidase release from Lyn^–/– cells is initially delayed, but surpasses release from WT BMMCs after 3 min of activation (Fig. 1B). The greater releasibility of Lyn^–/– BMMCs extends to other stimuli. Thus, Lyn^–/– BMMCs degranulate significantly more than WT BMMCs in response to ionomycin (Fig. 1C). Furthermore, only Lyn^–/– mast cells, and not WT cells, degranulate in response to SCF (Fig. 1D). Neither WT nor Lyn^–/– BMMCs release -hexosaminidase to any agonist when extracellular calcium is absent (data not shown).

View larger version (30K): [in this window] [in a new window]   FIGURE 1. Lyn–/– BMMCs are hyperresponsive to stimulation with Ag, ionophore, or SCF. IgE-primed BMMCs were stimulated with increasing concentrations of DNP-BSA (A), with 10 ng/ml DNP-BSA over a time course of 30 min (B), with increasing concentrations of ionomycin (C), and with increasing concentrations of SCF for 30 min (D). Data are represented as the mean ± SEM of two experiments performed with duplicate samples. Means significantly different from WT values are indicated: *, p < 0.05; **, p < 0.01; ***, p < 0.005, Student’s t test.

Previous investigators reported that FcRI cross-linking induces degranulation in Lyn^–/– BMMCs without detectable phosphorylation of FcRI subunits or signaling proteins (22, 23, 24, 25). In contrast, we find that FcRI and subunit phosphorylation does occur in Ag-stimulated Lyn^–/– BMMCs. Fig. 2, A and B, shows two (from a total of five) independent experiments in which FcRI was immunoprecipitated from resting and activated cells, separated on nonreducing gels, and the resolved proteins analyzed by Western blotting. In Fig. 2A (upper panel), anti-PY blotting shows that FcRI cross-linking causes a rapid, but short-lived phosphorylation of both the and subunits in WT BMMCs and a delayed, but persistent phosphorylation of both the and subunits in Lyn^–/– BMMCs. The anti-FcRI blot (lower panel) provides a loading control. In Fig. 2B, anti-PY blotting again shows the rapid phosphorylation of FcRI in WT cells and its slower phosphorylation in Lyn^–/– cells (upper panel). The anti-FcRI blot (lower panel) provides a loading control. This receptor subunit phosphorylation occurs despite the lack of Lyn in immunoblots of whole cell lysates (Fig. 2C; the anti-PKC band provides a loading control).

View larger version (63K): [in this window] [in a new window]   FIGURE 2. Sustained phosphorylation of FcRI and other proteins in Lyn–/– BMMCs. IgE-primed BMMCs were stimulated with 10 ng/ml DNP-BSA for the indicated times. Clarified lysates were immunoprecipitated with 1 µg/ml anti-IgE. Immunoprecipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose, and Western blotted sequentially with anti-PY Ab HRP-4G10 and with anti-FcRI Ab (A) or with anti-FcRI Ab (B). C, Whole cell lysates (5 x 105 cells/lane) were separated by SDS-PAGE and analyzed by immunoblotting with anti-Lyn, anti-PKC, or D, with anti-PY99/PY20 Abs. The arrowhead and arrow correspond to bands containing Syk and Lyn, respectively. *, Denotes the 60-kDa band containing Fyn. Experiments were performed a minimum of three times.

Impaired Ag-induced phosphorylation and delayed, but persistent activation of PLC isoforms in Lyn-deficient mast cells

Western blot analysis confirmed previous evidence (23) that phosphorylation of PLC1 and PLC2 is both delayed and reduced in Lyn^–/– BMMCs in comparison with WT BMMCs (data not shown). To further investigate the properties of PLC isoforms, we measured PLC1 and PLC2 activities in WT and Lyn^–/– BMMCs. Resting activities of PLC1 and PLC2 are comparable between WT and Lyn^–/– BMMCs (Fig. 3). In WT BMMCs, both PLC1 and PLC2 activities peak after 2 min of Ag stimulation, and are lower after 5 and 10 min of stimulation (Fig. 3). In contrast, there is little activity of either PLC1 or PLC2 in Lyn^–/– BMMCs after 2 min of stimulation, but activities of both rise steadily as time of stimulation progresses (Fig. 3). Thus, Lyn^–/– BMMCs support persistent PLC activity even though PLC phosphorylation is impaired.

View larger version (23K): [in this window] [in a new window]   FIGURE 3. Altered PLC activity in Lyn–/– BMMCs. IgE-primed BMMCs were stimulated with 10 ng/ml DNP-BSA for the indicated times. Clarified lysates were immunoprecipitated with 1 µg/ml anti-PLC1 or 1 µg/ml anti-PLC2 Abs. PLC activity was assayed by incubating anti-PLC immunoprecipitates with 200 µM [3H]PI(4,5)P2 for 25 min. Data represent the average of two independent experiments performed with duplicate samples.

Ag-induced Ca²⁺ mobilization was measured in WT and Lyn^–/– BMMCs as a cellular readout of PLC activity and Ins(1,4,5)P₃ production. Responses for groups of three to four typical cells are plotted in Fig. 4. In medium containing Ca²⁺ (Fig. 4, A and B), Ag-stimulated Lyn^–/– BMMCs show a longer lag time to response and a smaller initial peak response than WT BMMCs. In all experiments, the mean response time for WT cells was 24 s, compared with a mean response time of 148 s for Lyn^–/– cells. Despite their delayed responses, Lyn^–/– cells do show a significant plateau phase of elevated intracellular Ca²⁺ (Fig. 4B), consistent with the well-established requirement for Ca²⁺ in the degranulation process.

View larger version (21K): [in this window] [in a new window]   FIGURE 4. Altered calcium responses in Lyn–/– BMMCs. IgE-primed WT (A) or Lyn–/– (B) BMMCs were loaded with fura 2-AM in Ca2+-containing medium, and Ca2+ mobilization induced by the addition of 10 ng/ml DNP-BSA was measured by single cell ratiometric imaging. WT (C) or Lyn–/– (D) was treated as in A and B, but stimulated in the absence of extracellular Ca2+ by addition of 5 mM EGTA plus DNP-BSA. Traces show the responses of three to four typical cells in each group.

Decreased Trpc4 expression in Lyn^–/– BMMCs

Recent studies suggest that the family of Trp proteins are structural components of Ca²⁺ entry channels (reviewed in Ref.34) and are activated in response to Ins(1,4,5)P₃ production or Ca²⁺ stores depletion. However, neither these channels nor their roles have been documented in BMMCs. Western blot analyses show Trpc1, Trpc3, and Trpc6 are all expressed at similar levels in WT and Lyn^–/– BMMCs (Fig. 5A). Trpc5 is also expressed at similar levels in both cell types (data not shown). However, Lyn^–/– BMMCs express much less Trpc4 protein than WT BMMCs (Fig. 5, A and B). Trpc4 protein is also significantly lower in Lyn^–/– peritoneal macrophages than in WT cells (data not shown).

View larger version (37K): [in this window] [in a new window]   FIGURE 5. Decreased Trpc4 protein and impaired store-operated calcium entry in Lyn–/– BMMCs. A, Proteins from clarified BMMC lysates were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and analyzed by immunoblotting with Abs to multiple Trpc. B, BMMCs were stimulated for 5 min with 10 ng/ml DNP-BSA, and clarified lysates were analyzed for Trpc4 and actin (loading control). Experiments were performed more than three times. C, WT or Lyn–/– BMMCs were loaded with fura 2-AM in HBSS in the presence of extracellular calcium and stimulated with 100 nM thapsigargin. D, BMMCs were incubated in calcium-free HBSS and stimulated with 100 nM thapsigargin, as in D. After 600 s, the solution was quickly exchanged for HBSS with calcium. Calcium mobilization was measured by single cell ratiometric imaging, as described for Fig. 4 above. Traces shown represent the responses of 15 typical cells in each experimental group. Calcium mobilization was also measured by flow cytometry (inset) in cells loaded with fura Red-AM and stimulated with 200 nM thapsigargin. A decrease in fura Red emission (excited with a 488-nm Ar laser) at 650 nm corresponds to an increase in free [Ca2+]i.

To evaluate whether altered calcium responses of Lyn^–/– cells might be linked to loss of Ca²⁺ entry channels, we tested the effect of depleting intracellular calcium stores with the sarco-endoplasmic reticulum calcium transport ATPase inhibitor, thapsigargin. Treatment with thapsigargin increases the concentration of intracellular calcium in both WT and Lyn^–/– BMMCs (Fig. 5C). However, the increase in [Ca²⁺]_i is significantly smaller in Lyn^–/– cells than in WT cells in either the presence or absence of calcium (Fig. 5, C and D). Similarly, reintroduction of calcium results in a significantly higher increase in [Ca²⁺]_i in WT cells than in Lyn^–/– cells (Fig. 5D). These data suggest that the capacitative entry of extracellular calcium through store-operated channels, possibly including Trpc4, might be partially impaired in Lyn^–/– BMMCs.

Delayed activation of VLA-4 in Lyn^–/– BMMCs

FcRI cross-linking up-regulates the VLA-4-dependent adhesion of mast cells and basophils, as determined by binding to fibronectin- or VCAM-1-coated surfaces (35, 36, 37), by conjugate formation with VCAM-1-expressing cells (38), and most informatively, by use of the fluorescent probe, LDV-FITC, to directly measure the affinity state of VLA-4 (27). Src kinases are implicated in integrin activation (reviewed in Ref.39). Therefore, we evaluated the FcRI-mediated activation of VLA-4 in WT and Lyn^–/– BMMCs.

To determine the affinity state of the integrin, cells were first treated with 1 mM Mn²⁺ that up-regulates surface VLA-4 through its interaction with the integrin from the outside of the cell (40). As shown in Fig. 6A, addition of Mn²⁺ caused an almost immediate increase in the affinity state of VLA-4 in both WT and Lyn^–/– BMMCs. The increased plateau value of LDV-FITC binding to Lyn^–/– cells is most likely due to the modestly higher levels of VLA-4 in Lyn^–/– BMMCs in this experiment (see inset). Next, changes in LDV-FITC binding in response to Ag were evaluated. Stimulation of WT BMMCs with 10 ng/ml DNP-BSA induced an increase in LDV-FITC binding to a new steady state level that is very close to the level reached in Mn²⁺-treated cells (Fig. 6B). The rate of increase in LDV-FITC binding is slower in Ag-treated than in Mn²⁺-treated cells, reflecting changes in VLA-4 affinity mediated by signals from the inside of the cell. In comparison with WT cells, Lyn^–/– BMMCs display a 25- to 30-s delay in the onset of increased LDV-FITC binding (Fig. 6B). Nevertheless, the plateau level of LDV-FITC binding is comparable to the plateau level for WT cells (Fig. 6B). We note that in the specific experiment shown in this study, the numbers of ₄₁ integrin molecules were slightly higher on Lyn^–/– BMMCs than in WT BMMCs (Fig. 6A, inset). However, the difference was not statistically significant (p = 0.1554) when two other replicate experiments were analyzed.

View larger version (31K): [in this window] [in a new window]   FIGURE 6. Delayed kinetics of LDV-FITC binding to VLA-4 in Lyn–/– BMMCs. A, Baseline fluorescence was measured by flow cytometry after equilibrating BMMCs with 5 nm LDV-FITC for 10 min at 37°C. Mn2+ (1 mM) was added, and measurement of LDV-FITC binding was continued for an additional 7 min. Inset, Expression of VLA-4 integrin on the same BMMCs (in thousands of sites per cell) was determined, as described in Materials and Methods. B, IgE-primed BMMCs were incubated with 5 nM LDV-FITC for 10 min at 37°C. After 60 s of data collection, 10 ng/ml DNP-BSA was added and measurement of LDV-FITC binding was continued for an additional 5 min. C, To measure dissociation of LDV-FITC peptide from VLA-4, cells were treated as in B. After 5 min of Ag stimulation, 2 µM nonfluorescent LDV was added, and dissociation of the LDV-FITC peptide was followed for an additional 5 min. D, BMMCs were treated as in B, but stimulation was with 300 ng/ml rSCF. Inset, Dissociation of LDV-FITC peptide studies was performed in WT and Lyn–/– BMMCs, as described in C. Binding is shown as mean channel fluorescence vs time. Experiments were performed three times with three to six samples per condition.

O’Laughlin-Bunner et al. (41) reported that Lyn^–/– mast cells display impaired chemotaxis in response to SCF. Therefore, we measured VLA-4 activation in response to SCF. Lyn^–/– BMMCs again show a delayed response compared with WT BMMCs (Fig. 6D). Additionally, the overall amplitude of the response is lower, with slightly faster dissociation rates (0.015–0.02 s^–1) for both WT and Lyn^–/– BMMCs compared with FcRI cross-linking. These results suggest that only a fraction of the mast cell population responds to SCF.

Fyn activity is up-regulated in Lyn^–/– BMMCs

Studies of total tyrosine phosphorylation revealed a protein band with an approximate molecular mass of 60 kDa (* in Fig. 2C above), which is constitutively phosphorylated in Lyn^–/–, but not WT BMMCs, and is further phosphorylated upon receptor activation. This band was shown by Western blotting to contain the Src family tyrosine kinase, Fyn (data not shown). Anti-Fyn in vitro kinase assays show that Fyn activity is up-regulated in Lyn^–/– BMMCs (Fig. 7A), despite similar levels of Fyn protein (Fig. 7B) in the two cell types.

View larger version (25K): [in this window] [in a new window]   FIGURE 7. Fyn kinase activity is up-regulated in Lyn–/– BMMCs. A, IgE-primed BMMCs were stimulated with 10 ng/ml DNP-BSA and lysed at the indicated times. Fyn was immunoprecipitated from clarified lysates, and immunoprecipitates were used for in vitro kinase reactions. Phosphoproteins were resolved by SDS-PAGE and detected by autoradiography. Results from one of three similar in vitro kinase assays are shown. B, Lysates from resting and stimulated BMMCs (5 x 105 cells/lane) were separated by SDS-PAGE and Western blotted with anti-Fyn Abs (2 µg/ml). One of three independent experiments is shown.

View larger version (40K): [in this window] [in a new window]   FIGURE 8. Sustained phosphorylation of Cbp in Lyn–/– BMMCs. Clarified lysates from resting and Ag-stimulated (10 ng/ml DNP-BSA) BMMCs were immunoprecipitated with 10 µg/ml anti-Cbp Abs. Immunoprecipitated proteins were separated by electrophoresis and immunoblotted with anti-HRP-4G10 Abs and anti-Cbp Abs. One of three independent experiments is shown.

We confirmed previously published data that basal and Ag-stimulated levels of PI(3,4,5)P₃ are both increased in Lyn^–/– cells (24, 44), and that the PI3K inhibitor, wortmannin, inhibits the FcRI-mediated release of -hexosaminidase (24) in both WT and Lyn^–/– BMMCs (data not shown). Additionally, basal and Ag-stimulated phosphorylation of the PI(3,4,5)P₃-dependent serine kinase, Akt, is increased in Lyn^–/– cells, and pretreatment with wortmannin blocks this phosphorylation (Fig. 9).

View larger version (32K): [in this window] [in a new window]   FIGURE 9. PI(3,4,5)P3 levels are increased in Lyn–/– BMMCs. BMMCs were sensitized overnight with 1 µg/ml anti-DNP IgE and stimulated with 10 ng/ml DNP-BSA in the presence or absence of 100 nM wortmannin for the indicated times. Proteins from total cell lysates from 5 x 105 cells/lane were resolved by SDS-PAGE, transferred onto nitrocellulose membranes, and analyzed by immunoblotting with phospho-specific Abs (Ser473) to Akt. Membranes were stripped and reprobed with anti-Akt to verify equal loading. One of three independent experiments is shown.

View larger version (48K): [in this window] [in a new window]   FIGURE 10. Unaltered tyrosine phosphorylation of Gab2 and PKC in Lyn–/– BMMCs. IgE-primed BMMCs were stimulated with 10 ng/ml DNP-BSA and lysed at the indicated times. A, Clarified lysates were incubated with 4 µg/ml anti-Gab2 rabbit serum bound to protein A-Sepharose, and immunoprecipitated proteins were separated by SDS-PAGE. Blots were probed with anti-HRP-4G10 Abs, followed by anti-Gab2 rabbit serum. The data were quantified by densitometry and are presented as the mean fold induction relative to unstimulated WT cells ± SD (lane 2, 0.794 ± 0.133; lane 3, 0.927 ± 0.023; lane 4, 0.733 ± 0.06, n = 3). B, Proteins from lysates were resolved by SDS-PAGE and Western blotted sequentially with anti-phospho-specific PKC Abs (Thr505) and anti-PKC Abs. Data were quantified by densitometry. One of four independent experiments is shown.

The increased PI(3,4,5)P₃ levels in Lyn^–/– cells could arise from its impaired degradation rather than its increased synthesis. PI(3,4,5)P₃ is degraded primarily by two inositol phospholipid phosphatases, PTEN and SHIP (SH2-containing inositol phospholipid phosphatase). SHIP2 also degrades PI(3,4,5)P₃, but was not studied due to its very low expression levels compared with SHIP in BMMCs (J. Kalesnikoff and G. Krystal, unpublished data).

Fig. 11 shows that the levels (upper panel) and phosphorylation (lower panel) of PTEN are comparable between WT and Lyn^–/– BMMCs. The levels of PI3K, used as a loading control, are also similar between both cell types (Fig. 11, lower panel).

View larger version (42K): [in this window] [in a new window]   FIGURE 11. PTEN levels and phosphorylation are similar in WT and Lyn–/– BMMCs. IgE-primed BMMCs were stimulated with 10 ng/ml DNP-BSA and lysed at the indicated times. Proteins from lysates were resolved by SDS-PAGE and Western blotted with either PTEN Abs or sequentially with anti-phospho-specific PTEN Abs (Ser380/Thr382/Thr383) and anti-p85 Abs. One of three independent experiments is shown for each panel.

View larger version (25K): [in this window] [in a new window]   FIGURE 12. Lyn–/– BMMCs fail to activate SHIP. A, IgE-primed BMMCs were treated with or without 10 ng/ml DNP-BSA, lysed, and immunoprecipitated with 2 µg of anti-SHIP Abs. Immunoprecipitates were resolved by SDS-PAGE and consecutively probed with anti-PY99/20 Abs and anti-SHIP Abs. B, Cells were treated, as described in Fig. 11 above, activated with 200 ng/ml DNP-BSA, and lysed in reducing sample buffer. Whole cell lysates were resolved by SDS-PAGE and Western blotted sequentially with anti-phospho-SHIP Abs and anti-SHIP Abs. Experiments were performed a minimum of three times. C, Immunoprecipitates, prepared as in A, were washed and incubated with [3H]Ins(1,3,4,5)P4 for 30 min at 37°C. Reactions were stopped with cold 2 mM LiCl, supernatants were applied to AG1-X columns, and [3H]Ins(1,3,4)P3 and [3H] Ins(1,3,4,5)P4 were eluted sequentially. Tritium label in eluates was counted with a scintillation counter. Protein A beads + anti-SHIP Abs were used as a negative control. Data are reported as percentage of Ins(1,3,4)P3 produced from two independent experiments performed with duplicate samples.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Previously, we showed that Lyn^–/– bone marrow cells (both progenitors and mature BMMCs) divide faster than WT cells in response to cytokine (IL-3 and SCF) and that Lyn^–/– BMMCs undergo less apoptosis in response to cytokine withdrawal than WT BMMCs. Additionally, Lyn^–/– BMMCs support greater IL-3-mediated phosphorylation of the prosurvival kinase, Akt, and the proliferative kinase, ERK1/2. These results identified Lyn as a negative regulator of murine mast cell survival and proliferation (29). In this study, we use mature Lyn^–/– BMMCs to explore the role of the Src family member, Lyn, in the control of FcRI signaling in mast cells.

Lyn^–/– BMMCs release significantly more -hexosaminidase when stimulated with Ag than WT BMMCs. Although a previous study suggested that Lyn^–/– BMMCs degranulate less than WT BMMCs in response to Ag (23), other studies also find that Ag-stimulated Lyn^–/– BMMCs degranulate as much or more (22, 24) than WT BMMCs. Like SHIP^–/– BMMCs (48, 49), Lyn^–/– BMMCs also secrete -hexosaminidase when stimulated with SCF. In contrast, WT BMMCs do not degranulate when stimulated with SCF.

Intriguingly, the Ca²⁺ ionophore, ionomycin, also induces more degranulation in Lyn^–/– BMMCs than in WT BMMCs. The mechanism is unknown. However, circumstantial evidence suggests that the increased sensitivity of Lyn^–/– BMMCs to ionomycin could be a consequence of dysregulated PKC activity. Ionophore-induced secretion from mast cells correlates with PKC-mediated myosin L chain phosphorylation (50); ionomycin does not degranulate PKC-deficient BMMCs (51); and Lyn^–/– BMMCs are reported to have lower basal activities, but higher peak Ag-stimulated activities, of the Ca²⁺-dependent PKC isoforms, PKC and PKCII, than WT BMMCs (23, 52).

We found that the increased degranulation in the Lyn^–/– cells correlates with a delayed onset, but greater duration of key events in the FcRI signaling pathway, including the tyrosine phosphorylation of the FcRI and subunits and of multiple other proteins, including Syk and Akt, the activation of PLC isoforms, the synthesis of PI(3,4,5)P₃, and the mobilization of Ca²⁺. Some of these results are consistent with previously published results. For example, Kawakami et al. (23) and Parravicini et al. (24) showed increased Ins(1,4,5)P₃ and increased PI(3,4,5)P₃ levels in Lyn^–/– BMMC. However, some of our results are substantially different from previous groups. In particular, Kovarova et al. (25) reported Ag-induced degranulation in the absence of detectable FcRI subunit phosphorylation in Lyn^–/– BMMCs. Their cells were stimulated for a single time point, and delayed subunit phosphorylation may have been missed. Kawakami et al. (23) also reported very low FcRI phosphorylation in anti- immunoprecipitates from Lyn^–/– BMMCs. In this case, the difference may reflect the choice of immunoprecipitating Abs. We detected significantly less FcRI phosphorylation in mouse anti- immunoprecipitates than in goat anti-IgE immunoprecipitates from both WT and Lyn^–/– BMMCs. This was true even when using 50 µg/ml anti- Abs and twice as many cells per immunoprecipitation than in experiments in which we immunoprecipitated with anti-IgE Abs (data not shown). The relative inefficiency of anti- Abs for immunoprecipitation was noted previously by Miller et al. (53).

Additionally, Kovarova et al. (25) and Parravicini et al. (24) detected no Ag-induced Ca²⁺ mobilization in Lyn^–/– BMMCs. One group (24) suggested that Lyn^–/– BMMCs may bypass the well-established link between Ca²⁺ influx and secretion by instead hyperactivating the Ca²⁺-independent PKC isoform, PKC. We were unable to confirm the basal hyperphosphorylation of PKC. In contrast, like Nishizumi and Yamamoto (22), we were able to measure Ag-induced Ca²⁺ mobilization in Lyn^–/– BMMCs by ratio-imaging microscopy of fura 2-loaded single cells. Our success is most likely due to use of ratio imaging. This method is more sensitive and accurate than fluorometric or flow cytometric analysis, that use low-affinity calcium reporters such as Fluo-4 that do not record unsynchronized oscillations occurring among individual cells in the population.

Overall, the Ca²⁺ responses of Ag-stimulated WT BMMCs are less robust than those of other mast cell model systems, particularly the extensively studied RBL-2H3 mast cell line (15). This is partially explained by sparse amounts of Ca²⁺ maintained in the intracellular stores of BMMCs, revealed in this study by treatment with thapsigargin treatment in the absence of extracellular calcium (Fig. 5D).

Lyn^–/– BMMCs show a delayed, but readily measurable calcium response, consistent with slower Ag-stimulated activation of PLC. However, we also made the unexpected observation that Lyn^–/– cells lack Trpc4, a member of the large Trp family that includes candidates for both store-operated and 1,2-diacylglycerol-activated Ca²⁺ entry (reviewed in Refs.34 and 54). Previous studies suggest that Trpc4 may regulate both store-operated Ca²⁺ currents and receptor-coupled cation channels (55, 56, 57). A role for Trpc4 in store-operated Ca²⁺ influx of BMMCs is suggested in this study by the observation that Lyn^–/– cells transport significantly less extracellular calcium when sarco-endoplasmic reticulum calcium transport ATPase pumps are inhibited and stores are passively emptied through the leak pathway. Thus, it seems likely that the altered calcium responses in Lyn^–/– cells are at least partially independent of Lyn’s role in receptor signaling and instead attributed to a Lyn-dependent role in maintaining basal expression of Trpc4. We speculate that this role is normally upstream of SHIP, because SHIP^–/– BMMC also have reduced levels of Trpc4 (G. Krystal, unpublished observations). It is intriguing that the presence of Trpc4 could potentially modulate other aspects of mast cell signaling, including degranulation and PLC activity, based upon the interaction of its C-terminal sequence with Postsynaptic density 95/Drosophila Discs large/Zonula occludens 1 motifs found in scaffolding and cytoskeletal proteins (58, 59).

A new indicator of FcRI-mediated mast cell and basophil activation, the up-regulation of the binding affinity of the integrin, VLA-4, also occurs with delayed kinetics, but normal amplitude in Lyn^–/– BMMCs. Up-regulation of integrin affinity is generally considered to involve changes in integrin conformation mediated in part by Src or by Src kinase family members. In this case, we suppose that the delay in FcRI-mediated VLA-4 up-regulation occurs at the level of FcRI signal initiation and that another Src kinase expressed in BMMCs translates the delayed signal to a normal integrin response.

The increased levels of PI(3,4,5)P₃ that we observe are consistent with previous reports (23, 24). However, we propose a different mechanism. Parravicini et al. (24) hypothesized that increased levels of PI(3,4,5)P₃ result from increased PI3K activity, an effect attributed in turn to increased Gab2 phosphorylation; these data were consistent with Gab2 as the primary mechanism for PI3K activation (45). We failed to confirm Gab2 hyperphosphorylation in Lyn-deficient BMMCs, even when using multiple sources of anti-Gab2 Abs for immunoprecipitation. Alternative pathways to PI3K activation, possibly through Syk coupled to linker for activation of T cells or other scaffold proteins, may also exist in BMMCs. We therefore propose that the increased levels of PI(3,4,5)P₃ in Lyn^–/– cells result primarily from its impaired degradation by inositol phospholipid phosphatases.

The key phosphatase appears to be SHIP. Whereas SHIP that is immunoprecipitated from resting and activated WT BMMCs shows substantial in vitro phosphatase activity, SHIP that is immunoprecipitated from Lyn^–/– BMMCs is essentially inactive. Furthermore, Ag stimulation induces substantial phosphorylation of SHIP in WT cells, whereas SHIP phosphorylation is only detectable in Lyn^–/– BMMCs when stimulation is with very high concentrations of Ag. In Lyn^–/– BMMCs stimulated with SCF, SHIP tyrosine phosphorylation is absent in resting cells and is only weakly phosphorylated after 15 min of activation (data not shown). It remains to be determined whether the reduced SHIP phosphorylation accounts for SHIP inactivity in Lyn^–/– BMMCs. Previous studies suggest that SHIP is constitutively phosphorylated to some extent in mast cells (46) and that cytokine stimulation increases this tyrosine phosphorylation with no apparent affect on its phosphatase activity (46, 47, 60). In agreement with those studies, we found that Ag stimulation increased the tyrosine phosphorylation of SHIP in WT BMMCs, but had no apparent effect on its phosphatase activity. Others have reported that tyrosine phosphorylation of SHIP decreases its phosphatase activity (46). Phee et al. (60) found Lyn, but not Syk, capable of efficiently phosphorylating SHIP in vitro and in vivo, but also concluded that SHIP activation depends most importantly on its translocation to the plasma membrane. Lyn has also been implicated in the regulation of SHIP in many cell types (61, 62, 63, 64). Phosphorylation of SHIP is significantly reduced in Lyn^–/– macrophages (61) and in Lyn^–/– B cells (62). SHIP activity is impaired in stimulated Lyn^–/– neutrophils (63). Recently, Lyn was shown to associate with the SH2 domain of SHIP, thereby promoting the localization of SHIP to the plasma membrane (64). Consistent with these studies (63, 64), we found impaired membrane localization of SHIP in resting and Ag-stimulated Lyn^–/– BMMCs (data not shown). We therefore propose a strict requirement for Lyn in SHIP activation, but by a mechanism that is not yet defined clearly.

Decreased SHIP activity could increase signaling in several ways. The most obvious, based on evidence that PLC1 and PLC2 both depend on PI(3,4,5)P₃ for activity (11, 65), is by persistent stimulation of PLC isoforms leading to persistent Ins(1,4,5)P₃ synthesis and Ca²⁺ mobilization. Supporting this, Ca²⁺ mobilization is sustained for several minutes in SHIP^–/– BMMCs (48, 49). In addition, tyrosine-phosphorylated SHIP has been reported to bind tyrosine-phosphorylated (66) and (46) subunits of FcRI, thus down-regulating receptor-mediated signal transduction in mast cells.

Our data raise the possibility that Fyn, whose phosphorylation is higher in resting and activated Lyn^–/– BMMCs than in WT BMMCs, substitutes, albeit inefficiently, for Lyn to initiate FcRI signaling. The mechanism of increased Fyn activation in Lyn^–/– cells remains to be discovered. We tested the hypothesis that Lyn promotes Csk activation and the phosphorylation of Src family members on negative regulatory sites via the phosphorylation of Cbp (67, 68). We found that Ag-induced Cbp phosphorylation is delayed, but not absent in Lyn^–/– BMMCs. Thus, impaired Cbp phosphorylation is not a satisfactory explanation for the excess Fyn activity in Lyn-deficient cells. An unidentified high m.w. protein coprecipitated with Cbp in WT BMMCs, but not in Lyn^–/– BMMCs, and might, when characterized, provide new insight into the regulation of Csk/Cbp in BMMCs.

Although dysregulated activities of both Fyn and SHIP most likely contribute to the signaling properties of Lyn^–/– cells, other possibilities may exist. For instance, Lyn may be involved in Syk-dependent activation of negative regulatory proteins. Elegant studies by Hong et al. (69) suggest that Lyn phosphorylates a critical residue in the linker region of Syk (Tyr³¹⁷), resulting in decreased Ins(1,4,5)P₃ production and a dampened Ca²⁺ signal in B cells. Mutation of Tyr³¹⁷ in RBL-2H3 mast cells results in enhanced FcRI-mediated tyrosine phosphorylation of PLC and degranulation (70). Thus, decreased phosphorylation of Tyr³¹⁷ may also contribute to the increased signaling properties of Lyn^–/– mast cells.

There is one other hypothesis for the prolonged signaling in Lyn^–/– cells. Ma et al. (71) showed in a transfected DT40 (chicken) B cell system that Lyn is not required for the recruitment of Syk to the cross-linked BCR, but is required for the internalization of the clustered BCR complexes. These results raised the possibility that Lyn may negatively regulate BCR signaling in part by stimulating receptor internalization. Consistent with these results (71), we found that the internalization of cross-linked FcRI, measured by flow cytometry, is delayed in Lyn^–/– BMMCs (V. Hernandez-Hansen, unpublished results). However, early signaling responses (FcRI and subunit phosphorylation, Ca²⁺ mobilization) terminate in WT cells before endocytosis is evident, suggesting that signaling and endocytosis are not closely linked processes in the case of the FcRI. A proposed model highlighting the roles of Lyn as an initiator and negative regulator of FcRI signaling is shown in Fig. 13.

View larger version (22K): [in this window] [in a new window]   FIGURE 13. Summary of Lyn’s role as a positive and negative regulator of FcRI signaling. FcRI cross-linking activates Lyn, Fyn, and Syk. Both Syk and Fyn activate the PI3K pathway that produces PI(3,4,5)P3, resulting in activation of downstream targets PLC and Btk. Lyn may either positively regulate Syk or negatively inhibit signaling by phosphorylating Tyr317. Lyn positively regulates SHIP activity that opposes PI3K by hydrolyzing PI(3,4,5)P3. The role of Fyn in phosphorylation of FcRI or Syk is unknown.

	Acknowledgments

 
We thank Drs. Bruce Edwards and Rebecca Lee, technical directors of the University of New Mexico Flow Cytometry and Fluorescence Microscopy Facilities, for help with the experiments reported in this work, and the University of New Mexico Cancer Research and Treatment Center for its support of these facilities. We also thank Janet Pfeiffer and Marina Martinez for skilled technical assistance, Dr. Benjamin Neel and Dr. Haihua Gu for generous gifts of rabbit anti-Gab2 serum, Dr. Juan Rivera for providing anti- Abs, and Dr. Chikako Torigoe for gifts of anti-Cbp and anti-IgE Abs and helpful discussions.

	Footnotes

 
¹ This work was supported in part by National Institutes of Health Grants RO1 GM49814, RO1 AI051575, RO1 DK58066, and P50 HL56384, and by a minority graduate fellowship from the National Institutes of Health/National Institute of General Medical Sciences (to V.H.-H.). The Cytometry and Fluorescence Microscopy Facilities are supported by National Cancer Institute Grant R24 CA88339 and the University of New Mexico Cancer Research and Treatment Center.

² Address correspondence and reprint requests to Dr. Valerie Hernandez-Hansen, Department of Pathology, University of New Mexico, School of Medicine, CRF 205, 2325 Camino De Salud NE, Albuquerque, NM 87131.

³ Current address: Department of Neurosciences, University of New Mexico School of Medicine, Albuquerque, NM 87131.

⁴ Abbreviations used in this paper: SH2, Src homology 2; BMMC, bone marrow-derived mast cell; [Ca²⁺]_i, intracellular Ca²⁺ concentration; Cbp, Csk-binding protein; Csk, C-Src kinase; Ins(1,3,4)P₃, inositol-1,3,4-trisphosphate; Ins(1,4,5)P₃, inositol-1,4,5-trisphosphate; Ins(1,3,4,5)P₄, inositol-1,3,4,5-tetrakisphosphate; LDV, 4-((N'-2-methylphenyl)ureido)-phenylacetyl-L-leucyl-L--aspartyl-L-valyl-L-prolyl-L-alanyl-L-alanyl-L-lysine; PI(3,4,5)P₃, phosphatidylinositol 3,4,5-trisphosphate; PKC, protein kinase C; PLC, phospholipase C; PtdIns(4,5)P₂, PtdIns(4,5)bisphosphate; PTEN, phosphatase and tensin homolog deleted on chromosome 10; PY, phosphotyrosine; SCF, stem cell factor; Trp, transient receptor protein; Trpc, Trp channel; WT, wild type.

Received for publication September 4, 2003. Accepted for publication April 23, 2004.

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