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The Journal of Immunology, 2002, 168: 4430-4439.
Copyright © 2002 by The American Association of Immunologists

Downstream of Kinase, p62dok, Is a Mediator of Fc{gamma}RIIB Inhibition of Fc{epsilon}RI Signaling1

Vanessa L. Ott*, Idan Tamir2,*, Masaru Niki{dagger}, Pier Paolo Pandolfi{dagger} and John C. Cambier3,*

* Integrated Department of Immunology, National Jewish Medical and Research Center and University of Colorado Health Sciences Center, Denver, CO 80206; and {dagger} Molecular Biology Program and Department of Pathology, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, Graduate School of Medical Sciences, Cornell University, New York, NY 10021


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The low-affinity receptor for IgG, Fc{gamma}RIIB, is expressed widely in the immune system and functions to attenuate Ag-induced immune responses. In mast cells, coaggregation of Fc{gamma}RIIB with the high-affinity IgE receptor, Fc{epsilon}RI, leads to inhibition of Ag-induced degranulation and cytokine production. Fc{gamma}RIIB inhibitory activity requires a conserved motif within the Fc{gamma}RIIB cytoplasmic domain termed the immunoreceptor tyrosine-based inhibition motif. When coaggregated with an activating receptor (e.g., Fc{epsilon}RI, B cell Ag receptor), Fc{gamma}RIIB is rapidly phosphorylated on tyrosine and recruits the SH2 domain-containing inositol 5-phosphatase (SHIP). However, the mechanisms by which SHIP mediates Fc{gamma}RIIB inhibitory function in mast cells remain poorly defined. In this report we demonstrate that Fc{gamma}RIIB coaggregation with Fc{epsilon}RI stimulates enhanced SHIP tyrosine phosphorylation and association with Shc and p62dok. Concurrently, enhanced p62dok tyrosine phosphorylation and association with RasGAP are observed, suggesting that SHIP may mediate Fc{gamma}RIIB inhibitory function in mast cells via recruitment of p62dok and RasGAP. Supporting this hypothesis, recruitment of p62dok to Fc{epsilon}RI is sufficient to inhibit Fc{epsilon}RI-induced calcium mobilization and extracellular signal-regulated kinase 1/2 activation. Interestingly, both the amino-terminal pleckstrin homology and phosphotyrosine binding domains and the carboxyl-terminal proline/tyrosine-rich region of p62dok can mediate inhibition, suggesting activation of parallel downstream signaling pathways that converge at extracellular signal-regulated kinase 1/2 activation. Finally, studies using gene-ablated mice indicate that p62dok is dispensable for Fc{gamma}RIIB inhibitory signaling in mast cells. Taken together, these data suggest a role for p62dok as a mediator of Fc{gamma}RIIB inhibition of Fc{epsilon}RI signal transduction in mast cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mast cells have long been recognized as the critical tissue-based effector cells that mediate IgE-dependent allergic responses. Aggregation of the high-affinity IgE receptor, Fc{epsilon}RI, expressed on the surface of mast cells stimulates a complex series of signaling events leading to the release of proinflammatory mediators that contribute to both acute and late phase allergic responses (1). Importantly, Fc{epsilon}RI-induced mast cell activation is subject to potent negative regulation by members of a growing family of structurally related receptors termed the inhibitory receptor superfamily (2). The most characterized member of this family is the low-affinity IgG receptor, Fc{gamma}RIIB, which inhibits cell activation when coaggregated with a variety of activating receptors including the B cell Ag receptor (BCR),4 TCR, activating FcRs (e.g., Fc{epsilon}RI, Fc{gamma}RIIA, and Fc{gamma}RIII) and cytokine receptors (e.g., c-Kit) (3). All members of the inhibitory receptor superfamily have cytoplasmic domains that contain at least one copy of a conserved sequence (I/VXYXXL) termed the immunoreceptor tyrosine-based inhibition motif (ITIM). Coaggregation with activating receptors (e.g., Fc{epsilon}RI and BCR) leads to rapid phosphorylation of the ITIM tyrosine, providing a docking site for SH2 domain-containing protein tyrosine phosphatase (SHP)-1 and SHP-2, and SH2 domain-containing inositol polyphosphate 5-phosphatase (SHIP; also known as SHIP-1) and SHIP-2. While all known ITIM-containing receptors bind SHP-1 and/or SHP-2 (4), only Fc{gamma}RIIB binds SHIP in vivo (5, 6, 7). This selectivity is attributable to the Y + 2 leucine located within the Fc{gamma}RIIB ITIM, which confers the ability to recruit SHIP and SHIP-2, but not SHP-1 or SHP-2 (8).

SHIP is an inositol phosphatase that dephosphorylates phosphoinositides and inositol polyphosphates at their 5 position (9). The major substrate for SHIP is phosphatidylinositol (3, 4, 5)-trisphosphate, which is dephosphorylated by SHIP to yield PI(3, 4)P2. In mast cells cleavage of phosphatidylinositol (3, 4, 5)-trisphosphate could prevent Fc{epsilon}RI-induced recruitment and activation of proximal signaling molecules, including phospholipase C{gamma}1 and Btk, thereby blocking downstream signaling events required for Fc{epsilon}RI-mediated degranulation and gene expression. In addition to its enzymatic activity, SHIP contains NPXY and proline-rich motifs through which it may bind effectors containing phosphotyrosine binding (PTB) and SH3 domains, respectively. Studies performed using both hemopoietic and nonhemopoietic cell types indicate that SHIP can bind a number of signaling molecules, including Grb2, Shc, and p62dok, in response to a variety of stimuli (9, 10).

Originally identified as a 62-kDa tyrosine phosphorylated protein associated with RasGAP in v-Abl or p210bcr-abl-transformed hemopoietic cells (11, 12), p62dok is the prototype of a family of adapter molecules that also includes p56dok-2 (13) (also called FRIP (14) or Dok-R (15)) and dok-3 (16) (also called DOKL (17)). A role for p62dok as an adapter molecule is suggested by its domain structure, which includes amino-terminal pleckstrin homology (PH) and PTB domains, and a carboxyl-terminal region containing numerous PXXP motifs and potential tyrosine phosphorylation sites (11, 18). Enhanced tyrosine phosphorylation of p62dok has been observed following activation of a wide variety of receptors including, among others, epidermal growth factor (EGF) receptor (19), IL-4R (14), BCR (20), and Fc{gamma}RIIB (21). Once phosphorylated, p62dok binds a number of signaling molecules, including RasGAP (19), Nck (22), and Csk (21).

Coaggregation of Fc{gamma}RIIB and Fc{epsilon}RI on the surface of mast cells occurs by the simultaneous binding of specific Ag complexed with IgG to surface-bound IgE (through antigenic epitopes) and Fc{gamma}RIIB (through Ig Fc determinants). When coaggregated with Fc{epsilon}RI, Fc{gamma}RIIB is rapidly phosphorylated on tyrosine, recruits SHIP, and inhibits Fc{epsilon}RI-induced calcium mobilization, degranulation, and cytokine production (5, 6, 7, 23). However, the mechanisms by which SHIP mediates Fc{gamma}RIIB inhibitory function in mast cells remain poorly defined. In contrast to B and T cells, which exhibit restricted expression of p62dok family members, mast cells express p62dok, p56dok-2, and dok-3 at the mRNA level (16), suggesting that these molecules play an important role in mast cell biology. In this report we demonstrate enhanced SHIP tyrosine phosphorylation and association with Shc and p62dok in mast cells following Fc{gamma}RIIB coaggregation with Fc{epsilon}RI. Similar to our recently published studies using B cells, p62dok recruitment to Fc{epsilon}RI is sufficient to inhibit Ag-induced calcium mobilization and extracellular signal-regulated kinase (Erk)1/2 phosphorylation. Interestingly, both the amino-terminal PH + PTB domains and the carboxyl-terminal proline/tyrosine-rich region of p62dok encode structural information sufficient to inhibit Fc{epsilon}RI-induced Erk1/2 phosphorylation. This is in contrast to B cells in which only the proline/tyrosine-rich region of p62dok can mediate this effect. Finally, studies using mast cells derived from p62dok-deficient mice indicate that p62dok is dispensable for Fc{gamma}RIIB inhibition of Fc{epsilon}RI-induced calcium mobilization and mitogen-activated protein kinase (MAPK) activation. Together, these data indicate that p62dok functions as a mediator of Fc{gamma}RIIB inhibition of Fc{epsilon}RI signal transduction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and cell culture

Rat basophilic leukemia (RBL-2H3) cells (24) and RBL-2H3 cells expressing murine Fc{gamma}RIIB1 (RBL-mFc{gamma}RIIB) or derivatives of Fc{gamma}RIIB, were cultured in IMDM supplemented with 5% heat-inactivated FCS (HyClone Laboratories, Logan, UT), 100 IU/ml penicillin, and 100 µg/ml streptomycin at 37°C in 7% CO2. All culture reagents were from Life Technologies (Gaithersburg, MD). Bone marrow-derived mast cells (BMMC) were obtained by culturing bone marrow cells from wild-type or p62dok-deficient mice in IMDM (Cellgro, Herndon, VA) supplemented with 10% heat-inactivated FCS, 100 IU/ml penicillin, and 100 µg/ml streptomycin. These cultures were supplemented with conditioned medium (1/100) from J558L cells transfected with murine IL-3 (provided by Dr. A. Rolink, Basel Institute for Immunology, Basel, Switzerland) (25), as a source of IL-3, and CHO-KL cells (provided by Dr. G. M. Keller, Mount Sinai School of Medicine, New York, NY), as a source of c-Kit ligand. Bone marrow cultures were maintained for 3–4 wk at 37°C in 7% CO2 and were confirmed to contain >95% mast cells by surface staining for Fc{epsilon}RI and c-Kit and analysis by flow cytometry. The p62dok-deficient mice have been described previously (26).

Abs and reagents

The rat anti-mouse Fc{gamma}RIIB/IIIA mAb 2.4G2 was affinity purified from hybridoma culture supernatants using protein G-Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ). F(ab')2 of mAb 2.4G2 were generated by pepsin (Sigma-Aldrich, St. Louis, MO) cleavage at pH 4.5 and 37°C for 24 h using standard protocols. F(ab')2 were purified by size exclusion chromatography using a Superdex 200 column (Amersham Pharmacia Biotech). Purified F(ab')2 were resolved by SDS-PAGE and analyzed by protein staining with SYPRO Red (Molecular Probes, Eugene, OR) and scanning on a Storm PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Trinitrophenol (TNP)-conjugated F(ab')2 of donkey anti-rat IgG (H and L chains) were generated using standard methods. Briefly, F(ab')2 of donkey anti-rat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) were dialyzed against 0.28 M cacodylate buffer, pH 7.2, and incubated with 2,4,6-trinitrobenzene sulfonic acid (TNBSA; Pierce, Rockford, IL) at 400 µg TNBSA/1 mg F(ab')2. After 1 h at room temperature, 1 M lysine was added to a final concentration of 10 mM to bind unreacted TNBSA. TNP-conjugated F(ab')2 were dialyzed against PBS to remove the TNP-Lys.

Polyclonal anti-Fc{gamma}RIIB1 Abs were generated by immunizing rabbits with the cytoplasmic tail of murine Fc{gamma}RIIB1 (27). Polyclonal anti-SHIP and anti-p62dok Abs were generated by immunizing rabbits with a peptide composed of aa residues 909–959 of murine SHIP or full-length murine p62dok, respectively (10). All polyclonal Abs were purified on an Ag-coupled Sepharose 4B column. Murine anti-Fc{epsilon}RI{beta} mAbs were provided by Dr. A. M. Scharenberg (Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA). IgE anti-OVA (28) was provided by Dr. E. W. Gelfand (National Jewish Medical and Research Center, Denver, CO). Purified F(ab')2 of rabbit anti-mouse IgG (H and L chains) and whole rabbit anti-mouse IgG (RAMIG) were purchased from Zymed Laboratories (South San Francisco, CA). Purified mouse IgE anti-TNP was obtained from BD PharMingen (San Diego, CA). Other Abs include phosphotyrosine-specific mAb Ab-2 (Calbiochem, La Jolla, CA), rabbit polyclonal anti-Erk1 and anti-Erk2 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal anti-phospho-p44/42 MAPK (Erk1/2), anti-phospho-c-Jun N-terminal kinase (anti-phospho-JNK), anti-JNK, anti-phospho-p38, and anti-p38 (Cell Signaling, Beverly, MA), pan-anti-Erk, anti-RasGAP, and anti-signal regulatory protein (SIRP) {alpha}1 (Transduction Laboratories, Lexington, KY), and anti-Shc (Upstate Biotechnology, Lake Placid, NY). Unless otherwise noted, all other reagents were purchased from Sigma-Aldrich.

DNA constructs and expression and GST fusion proteins

GST fusion proteins. Murine p62dok cDNA (12) was a gift from Dr. Y. Yamanashi (University of Tokyo, Tokyo, Japan). To generate GST-Dok, GST-Dok1–258, and GST-Dok259–482 fusion proteins, cDNA fragments corresponding to full-length murine p62dok (aa 1–482), the PH + PTB domains of p62dok (aa 1–258), and the proline/tyrosine-rich region of p62dok (aa 259–482) were amplified by PCR and cloned into pGEX-5X (Amersham Pharmacia Biotech) as described previously (10). GST-Dok fusion proteins were expressed in bacterial DH5{alpha} cells and purified using glutathione-Sepharose beads (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. GST-Dok fusion proteins bound to glutathione-Sepharose beads were used directly in in vitro binding experiments.

To generate SHIP-SH2 and SHP-2(SH2)2 peptides, the SH2 domain of SHIP and the two SH2 domains of SHP-2 were amplified by PCR and cloned into pGEX-5X and pGEX-3X (Amersham Pharmacia Biotech), respectively, as described previously (27, 29). GST-SHIP-SH2 and GST-SHP-2(SH2)2 fusion proteins were expressed in bacterial BL21 cells and purified using glutathione-Sepharose beads according to the manufacturer’s instructions. SHIP-SH2 and SHP-2(SH2)2 peptides were cleaved from GST using factor Xa protease (Roche, Indianapolis, IN) and covalently coupled to cyanogen bromide-activated Sepharose 4B (Amersham Pharmacia Biotech) at 1 mg/ml according to the manufacturer’s instructions.

Fc{gamma}RIIB mutant and Fc{gamma}RIIB-Dok chimeric receptors. The Fc{gamma}RIIB tailless and Fc{gamma}RIIB-Dok chimeric receptors were generated by PCR from mouse Fc{gamma}RIIB1 (accession no. M16367) and cloned into pMXI-egfp (a gift from Dr. A. Mui, DNAX, Palo Alto, CA) as previously described (10). The pMXI-egfp vector is a derivative of the pMX retroviral vector (30) into which an internal ribosomal entry site and the coding sequence for enhanced green fluorescent protein (EGFP) were cloned downstream of the gene of interest. The proviral DNA is transcribed as a single polycistronic mRNA, allowing EGFP expression to function as a direct measure of expression of the gene of interest.

Preparation of retrovirus and infection of RBL-2H3 cells

Retroviruses containing Fc{gamma}RIIB tailless and Fc{gamma}RIIB-Dok chimeric receptors were produced using the amphotropic Phoenix packaging cell line (31, 32) (American Type Culture Collection, Manassas, VA) and used to infect RBL-2H3 cells as described previously (33). Surface expression of Fc{gamma}RIIB and its derivatives were determined by flow cytometry using the anti-Fc{gamma}R mAb 2.4G2. Cells expressing equivalent levels of receptors were sorted and propagated for use in experiments.

Cell stimulation and lysis

In most experiments cells were incubated with IgE anti-OVA (8 µg/20 x 106 cells/1 ml) in complete medium for 2 h at room temperature. Cells were collected by centrifugation, washed, and resuspended in serum-free IMDM (20 x 106 cells/1 ml). Cells were stimulated with equimolar concentrations of F(ab')2 of rabbit anti-mouse IgG (20 µg/ml) or RAMIG (30 µg/ml) and incubated at 37°C for the indicated amounts of time. Cells were collected by centrifugation and lysed in 1x lysis buffer (1% Nonidet P-40, 150 mM NaCl, 10 mM Tris (pH 7.5), containing 10 mM sodium pyrophosphate, 2 mM Na3VO4, 10 mM NaF, 0.4 M EDTA, 1 mM PMSF, and 1 µg/ml each of aprotinin, anti-trypsin, and leupeptin) for 30 min on ice. Insoluble material was removed by centrifugation at 14,000 rpm for 5 min. In some experiments cells were incubated with IgE anti-TNP (8 µg/20 x 106 cells/1 ml) and/or F(ab')2 of rat anti-Fc{gamma}R mAb 2.4G2 (1 µg/20 x 106 cells/1 ml) in complete medium for 2 h at room temperature. Cells were collected by centrifugation, washed, and resuspended in incomplete IMDM as described above. Cells were stimulated with 30 µg/ml TNP-conjugated F(ab')2 of anti-rat IgG for the indicated amounts of time. Cells were collected by centrifugation and lysed as described above.

Immunoprecipitation and immunoblot analysis

For immunoprecipitation, cell lysates (2 x 107 cell equivalents) were incubated with either 12 µg anti-Fc{gamma}R mAb 2.4G2 covalently coupled to Sepharose 4B beads at 4°C for 2 h or with 10 µg anti-SHIP, anti-p62dok, or anti-Shc polyclonal Abs at 4°C, followed by 25 µl protein A-Sepharose (Amersham Pharmacia Biotech). Beads were collected by centrifugation, washed, and resuspended in 1x Laemmli sample buffer. Samples were resolved on a 10% Laemmli gel and transferred to polyvinlylidene difluoride membrane (NEN Life Science Products, Boston, MA). For analysis of MAPK activation, cell lysates (1–5 x 106 cell equivalents) were loaded directly on a 10% Laemmli gel and transferred to a polyvinylidene difluoride membrane. Membranes were blocked in 5% BSA in TBS containing 0.05% Tween 20 at 4°C overnight. Membranes were probed with anti-phosphotyrosine, pan-anti-Erk, anti-RasGAP, anti-Fc{epsilon}RI{beta}, anti-SIRP{alpha}1, anti-Fc{gamma}RIIB, anti-SHIP, anti-phospho-p44/42 MAPK (Erk1/2), anti-phospho-JNK, anti-phospho-p38, anti-p38, anti-Shc, or anti-p62dok Abs, followed by alkaline phosphatase-conjugated anti-mouse IgG or alkaline phosphatase-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories). Blots were developed using Vistra ECF reagent (Amersham Pharmacia Biotech) and scanned on a Storm PhosphorImager (Molecular Dynamics). In some experiments blots were probed with HRP-conjugated anti-mouse IgG1 or HRP-conjugated rec-protein A (Zymed Laboratories) and developed using ECL reagent (NEN Life Science Products) and autoradiography.

In vitro binding experiments

Cells (~1 x 107 cells/dish) were stimulated with pervanadate (3 mM H2O2 and 0.1 mM vanadate) for 10 min at 37°C. Cells were placed on ice, washed twice with cold PBS, and lysed in 1 ml 1x lysis buffer (1% Triton X-100, 10 mM Tris (pH 7.5), 150 mM NaCl, 0.4 mM EDTA, 10 mM NaF, 2 mM Na3VO4, and 1 µg/ml each of aprotinin, anti-trypsin, and leupeptin) for 10 min on ice. Insoluble material was removed by centrifugation at 14,000 rpm for 5 min. Cell lysates were incubated with 25 µl (packed) glutathione-Sepharose beads bound with GST-Dok fusion proteins or 25 µg SHIP-SH2 or SHP-2(SH2)2 peptide covalently coupled to Sepharose 4B beads at 4°C for 2 h to overnight. Beads were collected by centrifugation, washed, and resuspended directly in 1x Laemmli sample buffer. Samples were resolved on an 8/15% Laemmli step gel, transferred to a polyvinylidene difluoride membrane, and probed as described above.

Measurement of intracellular free calcium concentration

Cells were sensitized with IgE anti-OVA, or IgE anti-TNP and F(ab')2 of rat anti-Fc{gamma}R mAb 2.4G2 as described above. Cells were collected by centrifugation, washed twice with IMDM containing 2.5% FCS, pH 7, and resuspended at 5 x 106 cells/ml in IMDM and 2.5% FCS, pH 7, containing 10 µM indo-1/AM (34) and an equal volume of Pluronic F-127 (20% in DMSO; Molecular Probes) according to the manufacturer’s instructions. Cells were placed at 37°C for 30 min followed by the addition of an equal volume of IMDM and 2.5% FCS, pH 7.4, and incubation at 37°C for an additional 30 min. Cells were collected by centrifugation, washed twice with IMDM and 2.5% FCS, pH 7.2, and resuspended in the same medium at 1–5 x 106 cells/ml. For each sample, 5 x 106 cells were prewarmed at 37°C for 3 min and stimulated with F(ab')2 of rabbit anti-mouse IgG (20 µg/ml), RAMIG (30 µg/ml), or TNP-conjugated F(ab')2 of anti-rat IgG (15 µg/ml). The intracellular calcium concentration was monitored for the indicated period of time by excitation of 365 nm and measurement of the ratio of fluorescence emissions at 490 vs 390 nm using an LSR flow cytometer (BD Biosciences, San Jose, CA). The data shown are representative of two or three experiments in which populations of cells were independently derived (for BMMC) or sorted for equivalent Fc{gamma}R levels (for RBL cells expressing Fc{gamma}R-Dok chimeras).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Coaggregation of Fc{gamma}RIIB and Fc{epsilon}RI stimulates Fc{gamma}RIIB tyrosine phosphorylation, recruitment of SHIP, and inhibition of Fc{epsilon}RI-induced activation of MAPK

We and others have previously shown that when coaggregated with Fc{epsilon}RI, Fc{gamma}RIIB inhibits Fc{epsilon}RI-mediated mast cell degranulation and cytokine production (5, 6, 7, 23). Coaggregation of Fc{gamma}RIIB and Fc{epsilon}RI stimulates rapid Fc{gamma}RIIB tyrosine phosphorylation and recruitment of SHIP, suggesting that SHIP plays a role in Fc{gamma}RIIB inhibitory signaling in mast cells. To begin to explore whether SHIP mediates Fc{gamma}RIIB inhibitory signaling in mast cells, we analyzed signaling events using the rat basophilic leukemia cell line transfected with the murine Fc{gamma}RIIB1 (RBL-mFc{gamma}RIIB). These cells have been used extensively to study the signaling mechanisms underlying IgE-mediated mast cell activation. Furthermore, mouse Fc{gamma}RIIB has previously been shown to inhibit Fc{epsilon}RI-induced mast cell activation when expressed in these cells (7, 23). In Fig. 1Go RBL-mFc{gamma}RIIB cells were sensitized with IgE, and stimulated with either F(ab')2 of rabbit anti-mouse IgG, to aggregate Fc{epsilon}RI alone, or RAMIG, to coaggregate Fc{gamma}RIIB and Fc{epsilon}RI. Confirming previous reports, coaggregation of Fc{gamma}RIIB and Fc{epsilon}RI stimulated rapid tyrosine phosphorylation of Fc{gamma}RIIB (Fig. 1GoA). As expected, Fc{gamma}RIIB was not phosphorylated in unstimulated cells or in cells stimulated through Fc{epsilon}RI alone. In addition, Fc{gamma}RIIB was not phosphorylated following stimulation with RAMIG in the absence of IgE (data not shown). In positive controls, Fc{gamma}RIIB was heavily phosphorylated on tyrosine following treatment with pervanadate, a potent inhibitor of tyrosine phosphatases that stimulates the rapid accumulation of phosphotyrosine on cellular proteins (35).



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  		FIGURE 1. Fc{gamma}RIIB tyrosine phosphorylation and recruitment of SHIP are associated with inhibition of Erk, JNK, and p38 phosphorylation. RBL-mFc{gamma}RIIB cells were cultured with or without sensitizing IgE and stimulated with F(ab')2 of rabbit anti-mouse IgG, RAMIG, or pervanadate (PV) as indicated. A, Cells were stimulated for 4 min before lysis, and Fc{gamma}RIIB was immunoprecipitated with anti-Fc{gamma}R mAb 2.4G2. Immune complexes were resolved by SDS-PAGE, transferred electrophoretically to membranes, and analyzed by immunoblotting with anti-phosphotyrosine Abs. Membranes were stripped and reprobed with anti-Fc{gamma}RIIB or anti-SHIP Abs. B, Cells were stimulated for the indicated amounts of time. Lysates of total cellular proteins were prepared and resolved by SDS-PAGE, transferred to membranes, and analyzed by immunoblotting with anti-phospho-Erk1/2, anti-phospho-JNK, anti-phospho-p38, pan-anti-Erk, or anti-p38 Abs as indicated.

 
Previous studies have demonstrated that Fc{gamma}RIIB recruits SHIP following coaggregation with Fc{epsilon}RI in vivo (5, 6, 7). In the system described above SHIP was detected in anti-Fc{gamma}RIIB immune complexes from cells following coaggregation of Fc{gamma}RIIB and Fc{epsilon}RI (Fig. 1GoA). As expected, SHIP was not detected in Fc{gamma}RIIB-containing immune complexes from unstimulated cells or from cells previously stimulated by Fc{epsilon}RI alone. However, SHIP was readily detected in anti-Fc{gamma}RIIB immune complexes isolated from pervanadate-treated cells. Under these conditions tyrosine phosphorylation of SHIP was also evident. It is likely that SHIP was also phosphorylated on tyrosine following Fc{gamma}RIIB coaggregation with Fc{epsilon}RI (see Fig. 2Go), but the level of phosphorylation was below the limits of detection in this assay.



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  		FIGURE 2. Tyrosine phosphorylation and association of SHIP and Shc following Fc{gamma}RIIB coaggregation with Fc{epsilon}RI. RBL-mFc{gamma}RIIB cells were cultured with or without sensitizing IgE and stimulated with F(ab')2 of rabbit anti-mouse IgG, RAMIG, or PV for 4 min as indicated. Lysates of total cellular proteins were prepared and subjected to immunoprecipitation with anti-SHIP (A) or anti-Shc (B) Abs. Immune complexes were resolved by SDS-PAGE, transferred to membranes, and analyzed by immunoblotting with anti-phosphotyrosine Abs. Membranes were stripped and reprobed with anti-SHIP or anti-Shc Abs as indicated.

 
When expressed in RBL-2H3 cells and coaggregated with Fc{epsilon}RI, Fc{gamma}RIIB inhibits Fc{epsilon}RI-induced calcium mobilization, degranulation, and cytokine production (7, 23). Aggregation of Fc{epsilon}RI also stimulates the activation of several MAPK family members, including Erk1/2, JNK, and p38, in the mouse mast cell line MC/9 (36, 37) and in BMMC (38). To determine whether Fc{gamma}RIIB coaggregation with Fc{epsilon}RI inhibits Fc{epsilon}RI-induced activation of MAPK, cells were stimulated as described above, and cell lysates were analyzed by immunoblotting with Abs specific for phosphorylated Erk1/2 (phospho-Erk1/2), phospho-JNK, or phospho-p38 (Fig. 1GoB). As expected, aggregation of Fc{epsilon}RI alone stimulated rapid phosphorylation, and presumably activation, of Erk1 and Erk2. To confirm that equivalent amounts of protein were loaded in each lane, membranes were reprobed with a pan-anti-Erk Ab that preferentially recognizes Erk2 (Transduction Laboratories). Enhanced phosphorylation of JNK and p38 was also detected within 1 min following aggregation of Fc{epsilon}RI. Reprobing with an anti-p38 Ab confirmed that equivalent amounts of protein were loaded in each lane. Additionally, p38 appeared to migrate at a higher m.w. when phosphorylated; this was especially evident at 15 and 30 min. Attempts to detect total JNK protein using commercially available Abs were not successful. Further analysis indicated that Fc{epsilon}RI-mediated activation of Erk1/2, JNK, and p38 is attenuated following Fc{gamma}RIIB coaggregation with Fc{epsilon}RI. In addition, the p38 band that presumably represents phosphorylated p38 was not detected following Fc{gamma}RIIB coaggregation with Fc{epsilon}RI. Inhibition of all MAPK was observed as early as 1 min following receptor coaggregation and was maintained for 30 min poststimulation.

Coaggregation of Fc{gamma}RIIB and Fc{epsilon}RI stimulates enhanced tyrosine phosphorylation of SHIP and its association with Shc

In a recent report SHIP was shown to be indispensable for Fc{gamma}RIIB-mediated inhibition of BCR-induced Erk phosphorylation, suggesting that it plays a critical role in Fc{gamma}RIIB inhibition of the Ras/MAPK signaling pathway (39). To further explore the role of SHIP in the regulation of mast cell function, SHIP was immunoprecipitated from RBL-mFc{gamma}RIIB cells following stimulation as described in Fig. 1Go. Immunoblot analysis using anti-phosphotyrosine Abs demonstrated that SHIP is constitutively phosphorylated at a low level in RBL-mFc{gamma}RIIB cells, and phosphorylation is enhanced following aggregation of Fc{epsilon}RI alone (Fig. 2GoA). However, an even greater increase in SHIP phosphorylation was detected following Fc{gamma}RIIB coaggregation with Fc{epsilon}RI. Phosphorylated SHIP migrated as a single anti-phosphotyrosine-reactive band on SDS-PAGE; however, an additional band was detected by immunoblotting with anti-SHIP Abs. The lower, more prominent, band corresponded to tyrosine-phosphorylated SHIP. Multiple bands are often detected by immunoblotting with this Ab; however, it is unclear whether the upper band represents an alternatively spliced isoform of SHIP (9) or a nonspecific band.

Two additional phosphoproteins that migrate at ~46 and ~52 kDa were detected in SHIP immunoprecipitates from unstimulated cells (Fig. 2GoA). Based on published reports indicating that Shc is constitutively phosphorylated on tyrosine and associates with SHIP in these cells, it seemed likely that these phosphoproteins were 46- and 52-kDa isoforms of Shc (40, 41). Immunoblot analysis using anti-Shc Abs failed to confirm the identity of these phosphoproteins due to cross-reactivity with the precipitating Ig H chain. Therefore, to confirm their identity, the reciprocal experiment was performed (Fig. 2GoB). Cells were stimulated as described in Fig. 1Go, and Shc was isolated by immunoprecipitation using anti-Shc Abs. Immunoblot analysis confirmed that small amounts of Shc are constitutively phosphorylated on tyrosine and associated with SHIP in unstimulated cells. When the membrane shown in Fig. 2GoB was overexposed, it was evident that the coimmunoprecipitated SHIP was phosphorylated on tyrosine (data not shown). Aggregation of Fc{epsilon}RI alone did not affect the level of Shc tyrosine phosphorylation, consistent with published reports (41). However, coaggregation of Fc{gamma}RIIB and Fc{epsilon}RI resulted in a significant increase in Shc phosphorylation and enhanced association with SHIP. Taken together, these data demonstrate that coaggregation of Fc{gamma}RIIB and Fc{epsilon}RI results in enhanced tyrosine phosphorylation of SHIP and Shc and increased association of the two molecules.

Coaggregation of Fc{gamma}RIIB and Fc{epsilon}RI stimulates enhanced tyrosine phosphorylation of p62dok and its association with SHIP and RasGAP

In addition to its ability to bind Shc, SHIP has been shown to associate with the RasGAP-binding protein p62dok. This association is constitutive in hemopoietic cells expressing Bcr-Abl (42, 43) and is induced by coaggregation of the BCR and Fc{gamma}RIIB in B cells (10). When phosphorylated on tyrosine, p62dok binds to RasGAP (12, 20, 44), which, in turn, negatively regulates Ras function by enhancing its intrinsic GTPase activity (45). Therefore, SHIP may affect Ras/MAPK signaling in mast cells at least in part via association with p62dok. To explore this hypothesis, p62dok was immunoprecipitated from cell lysates following stimulation as described above (Fig. 3GoA). Immunoblot analysis using anti-phosphotyrosine Abs demonstrated that p62dok is constitutively phosphorylated on tyrosine at a low level in unstimulated cells, and phosphorylation is enhanced following aggregation of Fc{epsilon}RI alone. However, similar to SHIP, tyrosine phosphorylation of p62dok was further enhanced following coaggregation of Fc{gamma}RIIB and Fc{epsilon}RI. In addition, RasGAP was found to associate with p62dok following Fc{gamma}RIIB coaggregation with Fc{epsilon}RI. Subsequent immunoblot analysis using anti-SHIP Abs revealed that a low level of SHIP protein coimmunoprecipitated with p62dok from unstimulated cells, and this remained unchanged following aggregation of Fc{epsilon}RI alone. In contrast, increased SHIP protein was detected in anti-p62dok immune complexes following Fc{gamma}RIIB coaggregationwith Fc{epsilon}RI. Together, these data demonstrate that coaggregation of Fc{gamma}RIIB and Fc{epsilon}RI stimulates enhanced p62dok tyrosine phosphorylation and association with SHIP and RasGAP.



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  		FIGURE 3. p62dok tyrosine phosphorylation and association with RasGAP and SHIP following Fc{gamma}RIIB coaggregation with Fc{epsilon}RI. A, RBL-mFc{gamma}RIIB cells were cultured with sensitizing IgE and stimulated with F(ab')2 of rabbit anti-mouse IgG or RAMIG for 4 min as indicated. Lysates of total cellular proteins were prepared and subjected to immunoprecipitation with anti-p62dok Abs. Immune complexes were resolved by SDS-PAGE, transferred to membranes, and analyzed by immunoblotting with anti-phosphotyrosine Abs. Membranes were stripped and reprobed with anti-p62dok, anti-RasGAP, or anti-SHIP Abs as indicated. RBL-2H3 cells (B and C) or J774A macrophage cells (D) were either not treated (-) or treated (+) with PV for 10 min. B, Lysates of total cellular proteins were prepared and incubated with GST fusion proteins containing full-length p62dok (Dok-FL), the amino-terminal PH + PTB domains of p62dok (Dok1–258), or the carboxyl-terminal proline/tyrosine-rich region of p62dok (Dok259–482) as indicated. Protein complexes were resolved by SDS-PAGE, transferred to membranes, and analyzed by immunoblotting with anti-phosphotyrosine or anti-SHIP Abs as indicated. C and D, Lysates of total cellular proteins were prepared and incubated with bead-conjugated peptides corresponding to the two SH2 domains of SHP-2 (SHP-2(SH2)2) or the SH2 domain of SHIP (SHIP-SH2) as indicated. Protein complexes were resolved by SDS-PAGE, transferred to membranes, and analyzed by immunoblotting with anti-Fc{gamma}RIIB, anti-Fc{epsilon}RI{beta}, and anti-p62dok Abs (C) or anti-SIRP{alpha}1 Abs (D) as indicated.

 
To map the site(s) within p62dok that mediates its association with SHIP in this model, GST fusion proteins consisting of either full-length Dok (GST-Dok), the amino-terminal PH + PTB domains (GST-Dok1–258), or the carboxyl-terminal proline/tyrosine-rich region (GST-Dok259–482) of p62dok (10, 12) were incubated with cell lysates prepared from pervanadate-treated RBL-2H3 cells. One prominent phosphoprotein bound to GST-Dok and GST-Dok1–258, but not to GST-Dok259–482 (Fig. 3GoB). The identity of this phosphoprotein was confirmed to be SHIP by immunoblotting with specific Abs. In lysates prepared from untreated cells, SHIP failed to bind p62dok, suggesting that tyrosine phosphorylation of SHIP is required for its association with p62dok. These data suggest that SHIP and p62dok associate via the p62dok PTB domain binding to phosphorylated tyrosine residues within SHIP, consistent with other recent published reports (10, 42, 43).

Results from other studies suggest that optimal SHIP association with p62dok also requires the SHIP SH2 domain (42). Supporting this contention, the data presented in Fig. 3GoC demonstrate that, when immobilized, a peptide corresponding to the SH2 domain of SHIP (SHIP-SH2), but not the two SH2 domains of SHP-2 (SHP-2(SH2)2), can bind p62dok from cell lysates prepared from pervanadate-treated RBL-2H3 cells. In this experiment the anti-p62dok Ab detected a less prominent band that migrates at or below p62dok. This band was detected variably in different experiments and appears to be a nonspecific band, because it is present in samples containing beads alone. Additionally, SHIP-SH2, but not SHP-2(SH2)2, bound p62dok from cell lysates prepared from cells following coaggregation of Fc{gamma}RIIB and Fc{epsilon}RI (data not shown). In controls, SHIP-SH2 also bound Fc{gamma}RIIB and the Fc{epsilon}RI {beta} subunit from lysates prepared from pervanadate-treated cells (Fig. 3GoC). In contrast, SHP-2(SH2)2 failed to bind either Fc{gamma}RIIB or Fc{epsilon}RI{beta}; however, SHP-2(SH2)2, but not SHIP-SH2, bound the ITIM-containing receptor SIRP{alpha}1 from lysates prepared from pervanadate-treated J774A macrophage cells (Fig. 3GoD). The in vitro interaction between SHIP and Fc{epsilon}R1{beta} has been described previously (40, 41). Although it has yet to be confirmed in vivo, this interaction as well as the observation that SHIP is phosphorylated in response to Fc{epsilon}RI aggregation alone (Fig. 2GoA) suggests that SHIP may regulate Fc{epsilon}RI signal transduction via an Fc{gamma}RIIB-independent mechanism.

p62dok recruitment to the plasma membrane is sufficient for p62dok tyrosine phosphorylation

The molecular interactions described above suggest a model in which SHIP functions as an adapter molecule to recruit p62dok to the Ag-receptor complex following Fc{gamma}RIIB coaggregation with Fc{epsilon}RI. To test this hypothesis and to determine whether recruitment of p62dok to Fc{epsilon}RI is sufficient to inhibit Fc{epsilon}RI-induced signal transduction, we used chimeric receptors containing the extracellular and transmembrane domains of mouse Fc{gamma}RIIB (aa 1–240) fused to full-length p62dok (Fc{gamma}R-Dok), the amino-terminal PH + PTB domains (Fc{gamma}R-Dok1–259), or the carboxyl-terminal proline/tyrosine-rich region (Fc{gamma}R-Dok260–482) of p62dok (10). These chimeric receptors as well as wild-type (Fc{gamma}R-wt) and tailless (Fc{gamma}R-tl) Fc{gamma}RIIB were expressed in RBL-2H3 cells. Using mAb 2.4G2, which recognizes the extracellular domain of mouse, but not rat, Fc{gamma}RIIB, cells expressing equivalent levels of these receptors were isolated by FACS. Fc{gamma}RIIB-wt as well as Fc{gamma}RIIB-Dok chimeric receptors were isolated by immunoprecipitation using mAb 2.4G2. The position of each receptor on SDS-PAGE is indicated by an asterisk (Fig. 4Go) and was determined by immunoblotting for tyrosine-phosphorylated receptors following treatment with pervanadate (data not shown), which stimulated enhanced tyrosine phosphorylation of all Fc{gamma}RIIB-Dok chimeric receptors.



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  		FIGURE 4. Fc{gamma}RIIB-p62dok chimeric receptor tyrosine phosphorylation and association with SHIP. RBL-2H3 cells expressing Fc{gamma}R-wt or Fc{gamma}RIIB chimeric receptors in which the Fc{gamma}RIIB cytoplasmic domain is replaced with full-length p62dok (Fc{gamma}R-Dok), the PH + PTB domains of p62dok (Fc{gamma}R-Dok1–259), or the proline/tyrosine-rich region of p62dok (Fc{gamma}R-Dok260–482), were sensitized with IgE and either not stimulated or stimulated with F(ab')2 of rabbit anti-mouse IgG or RAMIG for 4 min before lysates were prepared. Fc{gamma}RIIB-wt and Fc{gamma}RIIB-p62dok chimeric receptors were immunoprecipitated with anti-Fc{gamma}R mAb 2.4G2. Immune complexes were resolved by SDS-PAGE, transferred to membranes, and analyzed by immunoblotting with anti-phosphotyrosine or anti-SHIP Abs as indicated. Asterisks denote the positions of Fc{gamma}RIIB and Fc{gamma}R-Dok chimeras.

 
Coaggregation of wild-type Fc{gamma}RIIB and Fc{epsilon}RI stimulated enhanced Fc{gamma}RIIB tyrosine phosphorylation and recruitment of SHIP as expected (Fig. 4Go). Interestingly, Fc{gamma}R-Dok was constitutively phosphorylated on tyrosine and associated with SHIP when expressed in RBL-2H3 cells. These data indicate that localization of p62dok to Fc{gamma}RIIB is sufficient to facilitate a low level of p62dok phosphorylation. However, tyrosine phosphorylation of Fc{gamma}R-Dok and its association with SHIP were enhanced following Fc{gamma}R-Dok coaggregation with Fc{epsilon}RI. In contrast, Fc{gamma}R-Dok1–259 was not constitutively phosphorylated when expressed in RBL-2H3 cells, and only a very low level of phosphotyrosine was detected following coaggregation with Fc{epsilon}RI. However, Fc{gamma}R-Dok1–259 readily associated with SHIP following coaggregation with Fc{epsilon}RI. This result was not surprising, because Fc{gamma}R-Dok1–259 contains the p62dok PTB domain, which has been implicated in binding to phosphorylated SHIP (see Fig. 3GoB). Additionally, a 65-kDa phosphoprotein coimmunoprecipitated with Fc{gamma}R-Dok1–259 following coaggregation with Fc{epsilon}RI; however, the identity of this phosphoprotein is unknown.

Further analysis demonstrated that similar to full-length Fc{gamma}R-Dok, Fc{gamma}R-Dok260–482 is constitutively phosphorylated on tyrosine when expressed in RBL-2H3 cells, confirming that the majority of the tyrosine phosphorylation sites reside within the carboxyl-terminal proline/tyrosine-rich region of the molecule. When coaggregated with Fc{epsilon}RI, enhanced tyrosine phosphorylation of Fc{gamma}R-Dok260–482 was observed. However, no association with SHIP was found (Fig. 4Go) even following treatment with pervanadate (data not shown). These data suggest that although the SHIP SH2 domain can bind to phosphorylated p62dok in vitro (see Fig. 3GoC), the primary interaction between the two molecules is mediated by the PTB domain of p62dok and phosphorylated tyrosine residues within SHIP. It is possible that SHIP SH2 domain binding to phosphorylated p62dok functions to stabilize the interaction after the primary association is made. In addition to SHIP, a phosphoprotein migrating at about 120 kDa is also detected in Fc{gamma}R-Dok and Fc{gamma}R-Dok260–482 immune complexes following treatment with pervanadate (data not shown). Immunoblot analysis indicated that this 120-kDa phosphoprotein is RasGAP (data not shown), which is consistent with published reports that RasGAP binds p62dok via tyrosine residues located within the carboxyl-terminal region (46, 47).

p62dok recruitment to Fc{epsilon}RI is sufficient to inhibit Fc{epsilon}RI-induced MAPK activation and calcium mobilization

Next, we determined whether recruitment of p62dok to Fc{epsilon}RI is sufficient to inhibit Fc{epsilon}RI-induced signal transduction by using Fc{gamma}RIIB-Dok chimeras. This analysis was complicated by the fact that RBL-2H3 cells express a low level of endogenous rat Fc{gamma}RIIB. Therefore, we developed a protocol in which ectopically expressed mouse Fc{gamma}RIIB chimeric receptors, but not endogenous rat Fc{gamma}RIIB, could be coaggregated with Fc{epsilon}RI. Briefly, cells were incubated with IgE anti-TNP and F(ab')2 of rat anti-Fc{gamma}R mAb 2.4G2 and stimulated with TNP-conjugated F(ab')2 of donkey anti-rat IgG. As mentioned above, mAb 2.4G2 recognizes mouse, but not rat, Fc{gamma}RIIB. To aggregate either Fc{epsilon}RI or the Fc{gamma}RIIB chimeric receptors alone, cells were incubated with IgE anti-TNP or F(ab')2 of rat anti-Fc{gamma}R mAb 2.4G2, respectively, and stimulated with TNP-conjugated F(ab')2 of donkey anti-rat IgG.

To demonstrate the effectiveness of this protocol, RBL-2H3 cells expressing mouse Fc{gamma}R-wt were stimulated as described above, and Erk1/2 activation was assayed using phospho-specific anti-Erk1/2 Abs. As shown in Fig. 5GoA, aggregation of Fc{epsilon}RI alone stimulated enhanced phosphorylation of Erk1/2 (panel 1, lane 2). As expected, activation of Erk1/2 was not detected in cells treated with TNP-conjugated F(ab')2 of donkey anti-rat IgG alone (Fig. 5GoA, lane 1) or following coaggregation of Fc{gamma}RIIB alone (Fig. 5GoA, lane 3). However, Fc{epsilon}RI-induced activation of Erk1/2 was reduced following Fc{gamma}RIIB coaggregation with Fc{epsilon}RI (Fig. 5GoA, lane 4). The Fc{gamma}RIIB-mediated inhibition of Erk1/2 activation shown here is less robust than in previous experiments (see Fig. 1GoB). This is probably the result of less efficient coaggregation of Fc{gamma}RIIB and Fc{epsilon}RI using this assay. As a negative control, a tailless Fc{gamma}RIIB mutant that lacks its cytoplasmic domain (Fc{gamma}R-tl) failed to inhibit Fc{epsilon}RI-induced Erk1/2 activation (panel 5).



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  		FIGURE 5. p62dok mediates inhibition of Fc{epsilon}RI-induced Erk1/2 activation and calcium mobilization. RBL-2H3 cells expressing Fc{gamma}R-wt, Fc{gamma}R-Dok, Fc{gamma}R-Dok1–259, Fc{gamma}R-Dok260–482, or tailless Fc{gamma}RIIB (Fc{gamma}R-tl) were not treated or were pretreated with IgE anti-TNP, F(ab')2 of rat anti-Fc{gamma}R mAb 2.4G2, or both and stimulated with TNP-conjugated F(ab')2 of anti-rat IgG. A, Cells were stimulated for 15 min and lysed. Cell lysates were resolved by SDS-PAGE, transferred to membranes, and analyzed by immunoblotting with anti-phospho-Erk1/2 followed by pan-anti-Erk Abs as indicated. B, Cells were loaded with indo-1/AM, and a baseline calcium concentration was determined as described in Materials and Methods. Cells were stimulated as described above, and the change in the intracellular free calcium concentration was measured as described in Materials and Methods. Represented are the calcium responses of cells pretreated with IgE anti-TNP alone (solid lines) or with IgE anti-TNP and F(ab')2 of rat anti-Fc{gamma}R mAb 2.4G2 (dashed lines) and stimulated with TNP-conjugated F(ab')2 of anti-rat IgG. Cells that were either untreated or pretreated with F(ab')2 of rat anti-Fc{gamma}R mAb 2.4G2 alone showed no calcium response following stimulation (data not shown).

 
Further analysis demonstrated that when coaggregated with Fc{epsilon}RI, Fc{gamma}R-Dok and Fc{gamma}R-Dok260–482 inhibited Fc{epsilon}RI-induced activation of Erk1/2 (Fig. 5GoA, panels 2 and 4). This is an expected result, because both of these chimeric receptors bind RasGAP (10). Surprisingly, when coaggregated with Fc{epsilon}RI, Fc{gamma}R-Dok1–259 also inhibited Fc{epsilon}RI-induced Erk1/2 activation (panel 3). The mechanism by which the amino-terminal PH + PTB region of p62dok mediates this effect is not known; however, it may reflect the ability of Fc{gamma}R-Dok1–259 to recruit endogenous SHIP and possibly endogenous p62dok as well. Supporting this hypothesis, SHIP phosphorylation was enhanced ~9-fold following coaggregation of either Fc{gamma}R-wt or Fc{gamma}R-Dok1–259 with Fc{epsilon}RI (data not shown). In contrast, SHIP phosphorylation was enhanced only 1.5-fold following Fc{epsilon}RI coaggregation with Fc{gamma}R-Dok260–482. Furthermore, a protein fragment corresponding to the p62dok PH + PTB domains was found to bind endogenous SHIP and p62dok from B cell lysates following coaggregation of the BCR and Fc{gamma}RIIB (I. Tamir and J. C. Cambier, unpublished observations).

When coaggregated with Fc{epsilon}RI, Fc{gamma}RIIB also inhibits Fc{epsilon}RI-induced calcium mobilization in both RBL-2H3 (7) and BMMC (5). We therefore determined whether p62dok recruitment to Fc{epsilon}RI is sufficient to mediate Fc{gamma}RIIB inhibition of Fc{epsilon}RI-induced calcium mobilization. When coaggregated with Fc{epsilon}RI using the protocol described above, Fc{gamma}R-wt inhibited Fc{epsilon}RI-induced calcium mobilization (Fig. 5GoB). No calcium mobilization was observed in the absence of IgE or following aggregation of Fc{gamma}RIIB alone (data not shown). Using the same stimulation conditions, calcium mobilization was not inhibited in RBL-2H3 cells expressing tailless Fc{gamma}RIIB (Fc{gamma}R-tl; Fig. 5GoB) or in parental cells that do not express Fc{gamma}RIIB (data not shown). Interestingly, when coaggregated with Fc{epsilon}RI, Fc{gamma}R-Dok and Fc{gamma}R-Dok260–482 effectively inhibited Fc{epsilon}RI-induced calcium mobilization. These data suggest that colocalization of p62dok via Fc{gamma}RIIB is sufficient to mediate Fc{gamma}RIIB inhibition of Fc{epsilon}RI-induced calcium mobilization. Although the mechanism(s) by which p62dok causes this effect is not known, these data suggest that it involves effector interactions with the carboxyl-terminal proline/tyrosine-rich region of the molecule. In contrast, Fc{gamma}R-Dok1–259 showed a reduced, although significant, ability to inhibit Fc{epsilon}RI-induced calcium mobilization. This is an unexpected result, because Fc{gamma}R-Dok1–259 binds SHIP (Fig. 4Go) and stimulates its tyrosine phosphorylation (data not shown), suggesting that membrane recruitment and tyrosine phosphorylation of SHIP may not be sufficient for optimal activation of SHIP function.

p62dok is not required for Fc{gamma}RIIB-mediated inhibition of Fc{epsilon}RI-induced signal transduction

The data presented in Figs. 1–5GoGoGoGoGo suggest that Fc{gamma}RIIB-mediated recruitment of p62dok to Fc{epsilon}RI is sufficient to inhibit Fc{epsilon}RI-induced Erk1/2 activation and calcium mobilization. To determine whether p62dok is required for Fc{gamma}RIIB inhibitory signaling, we assessed whether Fc{gamma}RIIB can inhibit Fc{epsilon}RI-induced signal transduction in BMMC derived from p62dok-deficient mice. BMMC derived from p62dok-deficient mice express similar levels of cell surface Fc{epsilon}RI, c-Kit, and Fc{gamma}RIIB compared to their wild-type counterparts, as determined by immunofluorescence staining and analysis by flow cytometry (data not shown). To determine whether p62dok is required for Fc{gamma}RIIB-mediated inhibition of Fc{epsilon}RI-induced calcium mobilization, BMMC were sensitized with IgE and stimulated with either F(ab')2 of rabbit anti-mouse IgG, to aggregate Fc{epsilon}RI alone, or with RAMIG, to coaggregate Fc{gamma}RIIB and Fc{epsilon}RI, as described in Fig. 1Go. Consistent with previous findings (5), aggregation of Fc{epsilon}RI alone stimulated a robust calcium response in BMMC, which was inhibited by coaggregation with Fc{gamma}RIIB (Fig. 6GoA). Surprisingly, Fc{gamma}RIIB also inhibited Fc{epsilon}RI-induced calcium mobilization in p62dok-deficient BMMC. These data suggest that either a redundant mechanism exists that can compensate for p62dok deficiency or that SHIP activity is sufficient for Fc{gamma}RIIB-mediated inhibition of Fc{epsilon}RI-induced calcium mobilization.



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  		FIGURE 6. p62dok is not required for Fc{gamma}RIIB inhibition of Fc{epsilon}RI-induced calcium mobilization and MAPK activation. A, BMMC derived from wild-type (upper panel) or p62dok-deficient (lower panel) mice were sensitized with IgE and loaded with indo-1/AM as described in Materials and Methods. Cells were stimulated with F(ab')2 of rabbit anti-mouse IgG (solid lines) or RAMIG (dashed lines), and the change in intracellular calcium concentration was determined as described in Fig. 5Go. Cells not sensitized with IgE showed no calcium mobilization following stimulation (data not shown). B, Wild-type and p62dok-deficient BMMC were left untreated or were sensitized with IgE and either not stimulated or stimulated with F(ab')2 of rabbit anti-mouse IgG or RAMIG for the indicated amounts of time. Lysates of total cellular proteins were resolved by SDS-PAGE, transferred to membranes, and analyzed by immunoblotting with anti-phopsho-Erk1/2, anti-phospho-JNK, anti-phospho-p38, anti-Erk1 and -Erk2, or anti-p38 Abs as indicated.

 
To determine whether p62dok is required for Fc{gamma}RIIB-mediated inhibition of Fc{epsilon}RI-induced MAPK activation, BMMC derived from wild-type or p62dok-deficient mice were stimulated as described above, and cell lysates were analyzed by immunoblotting with Abs specific for phosphorylated Erk1/2, JNK, or p38. Consistent with previous findings (38), aggregation of Fc{epsilon}RI alone stimulated enhanced phosphorylation of Erk1/2, JNK, and p38 in wild-type BMMC (Fig. 6GoB). Fc{epsilon}RI-induced activation of Erk1/2, JNK, and p38 was also observed in p62dok-deficient BMMC, demonstrating that p62dok is not required for Fc{epsilon}RI-induced activation of MAPK. When coaggregated with Fc{epsilon}RI, Fc{gamma}RIIB inhibited Fc{epsilon}RI-induced Erk1/2, JNK, and p38 activation in both wild-type and p62dok-deficient BMMC with similar kinetics. Inhibition was detected as early as 5 min poststimulation and was maximal by 15 min. Together with the data presented in Fig. 5Go, these results suggest that p62dok recruitment to Fc{epsilon}RI is sufficient, but not required, for Fc{gamma}RIIB-mediated inhibition of Fc{epsilon}RI-induced signal transduction.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
When coaggregated with Fc{epsilon}RI, Fc{gamma}RIIB is rapidly phosphorylated on tyrosine and inhibits Fc{epsilon}RI-induced calcium mobilization, degranulation, and cytokine production (5, 6, 7, 23). SHIP is the major effector for Fc{gamma}RIIB in vivo (5, 6), suggesting that SHIP plays an important role in Fc{gamma}RIIB-mediated inhibitory signaling. However, the mechanism(s) by which SHIP mediates these effects in mast cells remains poorly defined. Initial studies seeking to define the role of SHIP in Fc{gamma}RIIB inhibitory signaling in mast cells showed that SHIP phosphorylation and association with Shc is dramatically enhanced following Fc{gamma}RIIB coaggregation with Fc{epsilon}RI (Fig. 2Go). Similar observations have been made in B cells following Fc{gamma}RIIB coaggregation with the BCR (48, 49). In B and T cells, Ag receptor aggregation stimulates enhanced tyrosine phosphorylation of Shc and the formation of a Shc/Grb2/Sos complex (50, 51), resulting in p21ras activation (52). In B cells Fc{gamma}RIIB-mediated SHIP association with Shc has been proposed to recruit Shc away from the Grb2/Sos complex, thereby blocking Ag receptor-mediated activation of p21ras (53). In this model SHIP and Grb2 compete for binding to phosphorylated Shc. This model is supported by in vitro binding studies in which SHIP and Shc associate via a bi-dentate interaction characterized by the SHIP SH2 domain binding to phosphorylated Shc, and the Shc PTB domain binding phosphorylated NPXY motifs within SHIP (54).

In contrast to B and T cells, in which Shc phosphorylation and association with SHIP occur only in response to Ag receptor aggregation, SHIP and Shc are constitutively phosphorylated at a low level in unstimulated RBL cells, and Shc phosphorylation remains unchanged following Fc{epsilon}RI aggregation (Fig. 2Go). This result suggests that Shc is not a major target for phosphorylation downstream of Fc{epsilon}RI, and thus it seems unlikely that SHIP could inhibit Fc{epsilon}RI signaling by competing for Shc.

In contrast, SHIP phosphorylation is enhanced following Fc{epsilon}RI aggregation alone. The implications of this observation are not known; however, we and others have shown that SHIP binds directly to the Fc{epsilon}RI {beta} chain in vitro (Fig. 3Go) (40, 41), suggesting that SHIP also regulates Fc{epsilon}RI signal transduction via an Fc{gamma}RIIB-independent mechanism. In support of this, Fc{epsilon}RI-induced calcium mobilization and MAPK activation are enhanced in BMMC derived from SHIP-deficient mice compared to wild-type mice (55) (data not shown).

In B cells SHIP has also been shown to associate with the RasGAP binding protein p62dok in response to Fc{gamma}RIIB coaggregation with the BCR (10), and p62dok has been implicated in Fc{gamma}RIIB-mediated inhibition of BCR-induced proliferation and MAPK activation (10, 18). We therefore determined whether p62dok functions similarly in mast cells. The data presented in Fig. 3GoA demonstrate that SHIP’s association with p62dok is enhanced following Fc{gamma}RIIB coaggregation with Fc{epsilon}RI. This is accompanied by enhanced p62dok phosphorylation and recruitment of RasGAP. These biochemical data suggest that SHIP-mediated recruitment of p62dok may play an important role in Fc{gamma}RIIB inhibitory activity. Interestingly, p62dok tyrosine phosphorylation is also enhanced following Fc{epsilon}RI aggregation alone, suggesting that p62dok may regulate Fc{epsilon}RI signaling independently of Fc{gamma}RIIB. At the present time the significance of this observation is not known.

Supporting this hypothesis, when coaggregated with Fc{epsilon}RI, a chimeric receptor in which the cytoplasmic domain of mouse Fc{gamma}RIIB was replaced with p62dok inhibited Fc{epsilon}RI-induced Erk1/2 activation (Fig. 4Go). Similarly, an Fc{gamma}RIIB-Dok chimeric receptor containing the carboxyl-terminal proline/tyrosine-rich region (aa 260–482) of p62dok inhibited Fc{epsilon}RI-induced Erk1/2 activation. This presumably occurs via recruitment of RasGAP, because this region of p62dok recruits RasGAP in B cells (10) and mast cells (data not shown). Surprisingly, an Fc{gamma}RIIB-Dok chimeric receptor containing the amino-terminal PH + PTB domains (aa 1–259) of p62dok also inhibited Fc{epsilon}RI-induced Erk1/2 activation. This is in contrast to findings in B cells, in which only the carboxyl-terminal region of p62dok showed inhibitory activity toward Erk1/2 (10). Interestingly, when coaggregated with Fc{epsilon}RI, Fc{gamma}RIIB-Dok chimeric receptors containing either the PH + PTB domains or the proline/tyrosine-rich region of p62dok are also sufficient to inhibit Fc{epsilon}RI-induced activation of JNK and p38 MAPK (data not shown). The mechanism by which the amino-terminal PH + PTB domains of p62dok mediate inhibition of MAPK is unknown, but may involve a RasGAP-independent pathway. Consistent with this hypothesis, a recently described p62dok homolog, designated p56dok-2/frip/dok-R, inhibited IL-2- and EGF receptor-induced MAPK activation when overexpressed in cell lines (14, 56). However, overexpression of a mutant p56dok-2 that is unable to bind RasGAP still attenuated EGF receptor-induced MAPK activation (56). Similarly, overexpression of dok-3, a third p62dok homolog that does not bind RasGAP, resulted in attenuated BCR-induced NFAT activation and cytokine production, providing further support for a RasGAP-independent inhibitory mechanism (16).

In a recent study p62dok was found to be essential for Fc{gamma}RIIB-mediated inhibition of BCR-induced proliferation (18). However, p62dok was dispensable for Fc{gamma}RIIB-mediated inhibition of BCR-induced calcium mobilization. Consistent with these studies, we showed using p62dok-deficient BMMC that p62dok is also dispensable for Fc{gamma}RIIB-mediated inhibition of Fc{epsilon}RI-induced calcium mobilization and activation of MAPK (Fig. 6Go). Supporting these biochemical data, Fc{gamma}RIIB inhibits Fc{epsilon}RI-induced degranulation in p62dok-deficient BMMC (data not shown). Together, these data suggest the existence of a redundant mechanism(s) or molecule(s) in mast cells that functions to compensate for p62dok deficiency. Two possibilities include p56dok-2 and dok-3, which have both been detected in mouse mast cell lines at the mRNA level (16). Similar to p62dok, dok-3 is tyrosine phosphorylated and associates with SHIP in response to BCR aggregation. Furthermore, overexpression of a dok-3 mutant that is unable to bind SHIP and the tyrosine kinase Csk enhances B cell responsiveness. Together, these data suggest that p56dok-2 and/or dok-3 may function to mediate Fc{gamma}RIIB inhibitory signaling in p62dok-deficient BMMC.


   Acknowledgments
 
We thank Francis Crawford for technical assistance.


   Footnotes
 
1 This work was supported by grants from the National Institutes of Health. V.L.O. is supported by a National Research Service Award from the National Institutes of Health. J.C.C. is an Ida and Cecil Green endowed Professor of Cell Biology. Back

2 Current address: QBI Enterprises Ltd., Nes Ziona, Israel. Back

3 Address correspondence and reprint requests to Dr. John C. Cambier, Integrated Department of Immunology, National Jewish Medical and Research Center and University of Colorado Health Sciences Center, 1400 Jackson Street, Denver, CO 80206. E-mail address: cambierj{at}njc.org Back

4 Abbreviations used in this paper: BCR, B cell Ag receptor; BMMC, bone marrow-derived mast cells; EGF, epidermal growth factor; EGFP, enhanced green fluorescent protein; Fc{gamma}R-wt, wild-type mouse Fc{gamma}RIIB; ITIM, immunoreceptor tyrosine-based inhibition motif; JNK, c-Jun N-terminal kinase; Erk, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; PH, pleckstrin homology; PTB, phosphotyrosine binding; RAMIG, whole rabbit anti-mouse IgG; SHIP, SH2 domain-containing inositol polyphosphate 5-phosphatase; SHP, SH2 domain-containing protein tyrosine phosphatase; TNBSA, 2,4,6-trinitrobenzene sulfonic acid; TNP, trinitrophenol; PV, pervanadate; SIRP, signal regulatory protein. Back

Received for publication November 14, 2001. Accepted for publication February 25, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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