Insufficient Phosphorylation Prevents Fcgamma RIIB from Recruiting the SH2 Domain-containing Protein-tyrosine Phosphatase SHP-1*

Renaud LesourneDagger, Pierre Bruhns, Wolf H. Fridman, and Marc Daëron§

From the Laboratoire d'Immunologie Cellulaire et Clinique, INSERM U.255, Institut Curie, 75005 Paris, France

Received for publication, July 21, 2000, and in revised form, November 13, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fcgamma RIIB are IgG receptors that inhibit immunoreceptor tyrosine-based activation motif (ITAM)-dependent cell activation. Inhibition depends on an immunoreceptor tyrosine-based inhibition motif (ITIM) that is phosphorylated upon Fcgamma RIIB coaggregation with ITAM-bearing receptors and recruits SH2 domain-containing phosphatases. Agarose bead-coated phosphorylated ITIM peptides (pITIMs) bind in vitro the single-SH2 inositol 5-phosphatases (SHIP1 and SHIP2) and the two-SH2 protein tyrosine phosphatases (SHP-1 and SHP-2). Phosphorylated Fcgamma RIIB, however, recruit selectively SHIP1/2 in vivo. We aimed here at explaining this discordance. We found that beads coated with low amounts of pITIM bound in vitro SHIP1, but not SHP-1, i.e. behaved as phosphorylated Fcgamma RIIB in vivo. The reason is that SHP-1 requires its two SH2 domains to bind on adjacent pITIMs. Consequently, the binding of SHP-1, but not of SHIP1, increased with pITIM density on beads. When trying to increase Fcgamma RIIB phosphorylation in B cells and mast cells, we found that concentrations of ligands optimal for Fcgamma RIIB phosphorylation failed to induce SHP-1 recruitment. SHP-1 was, however, recruited by Fcgamma RIIB when hyperphosphorylated following cell treatment with pervanadate. Our data suggest that Fcgamma RIIB phosphorylation may not be sufficient in vivo to enable the recruitment of SHP-1 but that (pathological?) conditions that would hyperphosphorylate Fcgamma RIIB might enable SHP-1 recruitment.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fcgamma RIIB are single-chain low-affinity receptors for the Fc portion of IgG antibodies that bind multivalent immune complexes. They exist as two (Fcgamma RIIB1 and B2 in humans) or three (Fcgamma RIIB1, B1', and B2 in mice) alternatively spliced products of the FcgR2b gene (1). All murine and human Fcgamma RIIB isoforms were shown to negatively regulate cell activation induced by all receptors bearing intracytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs)1 (2). Fcgamma RIIB also negatively regulate cell proliferation induced by growth factor receptors with an intrinsic protein tyrosine kinase activity (3). Confirming these results, Fcgamma RIIB-deficient mice were found: 1) to exhibit enhanced antibody responses (4); 2) to develop exaggerated IgE- (5) and IgG-dependent anaphylactic reactions (4); 3) to have an enhanced susceptibility to experimental murine models of IgG-dependent autoimmune diseases (6-8); and 4) to exhibit enhanced antibody-dependent cell-mediated cytotoxic responses to the injection of therapeutic antibodies to tumor antigens (9).

To inhibit cell activation, Fcgamma RIIB need to be coaggregated with ITAM-bearing receptors by immune complexes or by any extracellular ligand capable of interacting with the two receptors simultaneously (10-12). Coaggregation indeed enables Fcgamma RIIB to be tyrosyl-phosphorylated by Lyn (13), a Src family protein tyrosine kinase provided by ITAM-bearing receptors. Fcgamma RIIB isoforms contain a variable number of tyrosine residues in their intracytoplasmic domain, one of which proved to be critical (2, 14). This tyrosine stands within a 13-amino acid sequence that was found to be necessary (2, 15) and sufficient (14) for inhibition. Related sequences subsequently found in a large number of transmembrane molecules with negative regulatory properties provided the molecular basis for the definition of an immunoreceptor tyrosine-based inhibition motif (ITIM) having the (I/V/L)xYxxL consensus sequence (16, 17).

A general property of ITIMs is to have an affinity for cytoplasmic SH2 domain-containing phosphatases when tyrosyl- phosphorylated (17). Phosphatases that are recruited to the membrane antagonize with activation signals transduced by ITAM-bearing receptors. ITIM-bearing receptors were found to recruit two classes of phosphatases which exert markedly different effects: protein-tyrosine phosphatases and inositol 5-phosphatases. Protein-tyrosine phosphatases are SHP-1 and SHP-2. They have two SH2 domains and their substrates are tyrosyl-phosphorylated proteins (18). SHP-1 is thought to dephosphorylate tyrosines in ITAMs (19), protein tyrosine kinases and/or adapter proteins such as SLP-76 (20) whose phosphorylation is critical for activation signals. SHP-1 thus stops the initial steps of transduction. The possible role of SHP-2 is not clear, as both positive and negative effects have been assigned to this phosphatase. Inositol 5-phosphatases are SHIP1 and SHIP2. They have a single SH2 domain and they remove 5'-phosphate groups in inositol phosphates and phosphatidylinositol phosphates that are 3'-phosphorylated (21). The preferred substrate of SHIP1 is phosphatidylinositol (3,4,5)-trisphosphate which enables the membrane translocation of the Bruton's tyrosine kinase via its pleckstrin homology domain (22). Bruton's tyrosine kinase is mandatory for phospholipase Cgamma to be activated and to hydrolyze phosphatidylinositol (4,5)-bisphosphate into inositol (1,4,5)-trisphosphate, which induces a Ca2+ response (23), and diacylglycerol, which activates protein kinase C. SHIP1 was recently shown to inhibit the Ras pathway by acting as an adapter molecule in B cells. When phosphorylated by Lyn, SHIP1 recruits Dok by its protein tyrosine-binding domain. Dok is phosphorylated and recruits RasGAP which inactivates Ras by exchanging GTP for GDP on the latter molecule (24). SHIP1 therefore arrests the propagation of intracellular signals leading to the Ca2+ response and to the activation of the Ras pathway. The possible role of SHIP2 is not yet known.

The in vitro binding specificity of ITIMs was analyzed using phosphorylated synthetic peptides to precipitate phosphatases from cell lysates. Under these conditions, all known ITIMs, including the Fcgamma RIIB ITIM, bound SHP-1 and SHP-2 (25). Remarkably, the Fcgamma RIIB ITIM also bound SHIP1 (26) and SHIP2 (27). We previously identified two hydrophobic residues, at positions Y-2 and Y+2, that determine the binding of SHPs (28) and SHIPs (29), respectively. The in vivo recruitment of phosphatases by ITIM-bearing receptors was analyzed by co-precipitation, following their tyrosyl phosphorylation upon coaggregation with ITAM-bearing receptors. Tyrosyl-phosphorylated Fcgamma RIIB were initially reported to recruit SHP-1 in B cells following their coaggregation with B cell receptors (BCR) (30, 31). Fcgamma RIIB, however, were found to recruit selectively SHIP1, when coaggregated with high-affinity IgE receptors (Fcepsilon RI) in mast cells (26, 32). SHIP1, but not SHP-1, was subsequently demonstrated to be necessary for Fcgamma RIIB-dependent inhibition of cell activation in SHIP1-deficient DT40 B cells (33) and in SHP-1-deficient mast cells derived from motheaten mice (26, 32). These results altogether generated some confusion, and whether Fcgamma RIIB indeed recruit SHP-1 in vivo remains unclear. Depending on the answer, the following two issues may be addressed: 1) if they do, do they recruit SHP-1 exclusively in B cells or also in other cell types, and 2) if they do not, how can one reconcile the apparent discordance between the in vitro binding of phosphatases to ITIM peptides and the in vivo recruitment of phosphatases by Fcgamma RIIB.

In the present work, we aimed at clarifying these questions by analyzing the conditions required for SHP-1 to bind to phosphorylated ITIM-coated beads in vitro and to be recruited by phosphorylated Fcgamma RIIB in mast cells and in B cells. We failed to detect SHP-1 recruitment by Fcgamma RIIB in vivo when coaggregated either with Fcepsilon RI in mast cells, or with BCR in B cells. We found that the in vitro binding of SHP-1 required a higher level of Fcgamma RIIB phosphorylation than SHIP1 binding. Indeed, the two SH2 domains of SHP-1 were required to bind phosphorylated ITIMs and, as a consequence, SHP-1 binding, but not SHIP1 binding, depended on the density of phosphorylated ITIMs. In vivo, SHP-1 recruitment also required a higher level of Fcgamma RIIB phosphorylation than SHIP1 recruitment. The level of Fcgamma RIIB phosphorylation that enabled the recruitment of SHP-1 was reached after treating cells with pervanadate, but not following coaggregation of Fcgamma RIIB with BCR or Fcepsilon RI, in B cells and mast cells, respectively.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Cells-- The rat mast cells RBL-2H3 (34) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. Fcgamma RIIB-deficient murine lymphoma B cells K46µ, expressing an anti-NP BCR (35), and IIA1.6 (36) were cultured in RPMI supplemented with 10% fetal calf serum, 0.5 µM 2-mercaptoethanol, 2 mM sodium pyruvate, 100 IU/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and 20 mM Hepes. Bone marrow-derived mast cells (BMMC) were obtained from BALB/c bone marrow cells as described (3). After 4 weeks, cultures contained more than 90% mast cells. Culture reagents were from Life Technologies, Inc. (Paisley, Scotland, United Kingdom).

Transfectants-- Clones of RBL-2H3 cells, stably transfected with cDNA encoding murine Fcgamma RIIB1 (37) or Fcgamma RIIB1' (38) and clones of IIA1.6 cells, stably transfected with cDNA encoding Fcgamma RIIB1 (15) were described previously. cDNA encoding Fcgamma RIIB1, inserted in a NT-neo vector (39), was stably transfected in K46µ cells by electroporation. Transfectants were selected with neomycin (Cayla, Toulouse, France) and by cell sorting using a FACSCalibur (Becton Dickinson, Mountain View, CA). Fcgamma RIIB1-transfected K46µ cells were cloned as described (37). The expression of recombinant receptors on clones remained stable over the duration of experiments.

Antibodies-- The mouse IgE mAb 2682-I was used as culture supernatant of a subclone of DNP-H1-epsilon -26 hybridoma cells (40). The rat anti-mouse Fcgamma RIIB 2.4G2 mAb (41) was purified by affinity chromatography on Protein G-Sepharose from ascitic fluid of nude mice inoculated with 2.4G2 hybridoma cells intraperitoneally. F(ab')2 fragments were obtained by pepsin digestion for 48 h. The purity of IgG and F(ab')2 fragments was assessed by SDS-PAGE analysis. The mouse IgG1 mAb anti-DNP was provided by Dr. Jacques Couderc (Institut Curie, Paris, France). The rabbit polyclonal IgG anti-DNP were purchased from Sigma. F(ab')2 fragments of polyclonal mouse anti-rat Ig (MAR) were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). MAR F(ab')2 were trinitrophenylated by incubation for 1 h at room temperature with trinitrobenzene sulfonic acid (Eastman Kodak, Rochester, NY) in borate-buffered saline, pH 8.0. TNP10-MAR F(ab')2 were obtained after purification on Sephadex G-25 (Amersham Pharmacia Biochemicals, Uppsala, Sweden). Polyclonal rabbit anti-mouse immunoglobulins (RAM) IgG and F(ab')2 were purchased from Cappel Laboratories (West Chester, PA). Rabbit antibodies against soluble recombinant extracellular domains of Fcgamma RIIB and mouse mAb anti-GST were gifts from Prof. Catherine Sautès-Fridman and Dr. Jean-Luc Teillaud (Institut Curie, Paris, France), respectively. Horseradish peroxidase (HRP)-conjugated mouse monoclonal anti-phosphotyrosine antibodies (pY20) were purchased from Chemicon (Temecula, CA), mouse monoclonal anti-SHP-1 from Transduction Laboratories (Lexington, KY), anti-SHIP1 antibodies from Upstate Biotechnology (Lake Placid, NY), and HRP-conjugated goat anti-rabbit and goat anti-mouse immunoglobulin antibodies from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-SHP-1 antibodies were a gift from Dr. John Cambier (National Jewish Medical and Research Center, Denver, CO).

ITIM Peptides and in Vitro Binding of Phosphatases-- Nonphosphorylated or tyrosyl-phosphorylated Fcgamma RIIB ITIM-biotinylated peptides with the amino acid sequence KTAENTITYSLLK were synthesized by Neosystems. Biotinylated ITIM peptides were coupled to streptavidin-coated agarose beads (Pierce, Rockford, IL). Beads were saturated with 1 mg/ml D-biotin (Sigma), washed in lysis buffer containing 10 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1 mM Na3VO4, 5 mM NaF, 5 mM sodium pyrophosphate, 0.4 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride (lysis buffer) and incubated for 2 h at 4 °C with lysates from 1 × 107 RBL-2H3 cells or with SH2 domain-containing GST fusion proteins. Cell lysates were prepared by incubating 1 × 107 RBL-2H3 cells for 10 min on ice in lysis buffer. Post-nuclear supernatants were collected after centrifugation at 12,000 × g for 20 min at 4 °C, and used as a source of phosphatases.

GST Fusion Proteins-- cDNA encoding the intracytoplasmic domain of Fcgamma RIIB1' (ICIIB1') was generated by polymerase chain reaction using as a template cDNA encoding murine Fcgamma RIIB1' (38). The following primers were used: sense 5'-CGTAGGATCCAAGAAAAAGCAGGTTCCA-3'; antisense 5'-TACGGTCGACCTAAATGTGGTTCTGGTA-3'. Nucleotides corresponding to the cDNA sequence encoding amino acids 273-285 of Fcgamma RIIB1', i.e. the same 13 amino acids as synthetic ITIM peptides, were purchased from Amersham Pharmacia Biotech (Little Chalfont, Buckinghamshire). cDNA encoding the SH2 domain of SHIP1 was amplified by polymerase chain reaction, using as a template cDNA generated from RNA extracted from RBL-2H3 cells. The following primers were used: sense 5'-CTGACCCAGTCTAGAGGATCCATGCCTGCCATGGTCCCTG-3'; antisense 5'-GACACCTCGAGCTCTCAGGGAGGCAGCTCA-3'. The sequence was checked on the two strands by dideoxynucleotide sequencing. ICIIB1' cDNA, ITIM-encoding nucleotides, and SHIP1 SH2 domain cDNA were inserted into the pGEX-4T-2 vector (Amersham Pharmacia Biotech) and transfected into DH5alpha Escherichia coli. Bacteria producing SHP-1 SH2 domain-containing GST fusion proteins were a gift from Dr. Eric Vivier (Center d'Immunologie de Marseille-Luminy, Marseille, France). All GST fusion proteins were produced in DH5alpha E. coli following isopropohy-1-thio-beta -D-galactopyranoside induction, purified on glutathione-agarose (Sigma), and analyzed by SDS-PAGE. Soluble SH2 domain-containing GST fusion proteins were eluted from glutathione-agarose beads with a solution of 50 mM Tris and 25 mM glutathione, pH 8.0.

In Vitro Phosphorylation of GST-ITIM and GST-ICIIB1' and in Vitro Binding of Phosphatases-- Ten µl of glutathione-agarose beads coated with GST-ITIM or GST-ICIIB1' were washed in kinase buffer containing 100 mM Tris-HCl, pH 7.4, 125 mM MgCl2, 2 mM EGTA, 0.25 mM Na3VO4, and 2 mM dithiothreitol, and incubated for the indicated periods at 30 °C with 20 µl of kinase buffer containing 2 units of the Src kinase Lyn (Chemicon) and 100 µM ATP. Kinase reaction was stopped on ice; beads were immediately washed in lysis buffer, and incubated for 2 h at 4 °C with lysates from 1 × 107 RBL-2H3 cells.

Cell Stimulation and Immunoprecipitation-- RBL transfectants, resuspended at 5 × 106/ml, were incubated or not for 1 h at 37 °C with IgE anti-DNP (culture supernatant diluted 1/10) and with 3 µg/ml 2.4G2 F(ab')2, washed, and resuspended at 1 × 107 cells/ml. Cells were challenged or not for 5 min or for the indicated periods of time at 37 °C with 10 µg/ml TNP-MAR F(ab')2. Unless otherwise specified, IIA1.6 and K46µ transfectants, resuspended at 5 × 107/ml, were stimulated at 37 °C for 5 min with 0.3 µM RAM IgG (45 µg/ml) or RAM F(ab')2 (30 µg/ml). BMMCs were sensitized as RBL-2H3 cells with the indicated dilutions of IgE anti-DNP, and resuspended at 1 × 107/ml. BMMCs were stimulated at 37 °C for 5 min with preformed immune complexes made with the indicated final concentrations of DNP25-BSA and mAb mouse IgG1 anti-DNP. K46µ transfectants, resuspended at 5 × 107/ml, were stimulated with preformed immune complexes made with the indicated final concentrations of NP9-BSA-TNP12 and of polyclonal rabbit IgG anti-DNP. Immune complexes were preformed at 37 °C for 10 min immediately before stimulation.

Pervanadate was generated by mixing 1 ml of 20 mM Na3VO4 with 330 µl of 30% H2O2 followed by a 5-min incubation at room temperature, yielding a solution of 6 mM pervanadate. Unless otherwise specified, cells were incubated with a final concentration of 100 µM pervanadate for 15 min at 37 °C.

Following stimulation, cells were lysed as described above, and cell lysates were used for immunoprecipitation. Protein G-Sepharose (Amersham Pharmacia Biotech) was used to precipitate 2.4G2-bound Fcgamma RIIB in lysates from RBL transfectants and 2.4G2-coated Sepharose beads were used to precipitate Fcgamma RIIB in lysates from BMMCs or IIA1.6 and K46µ transfectants.

Western Blot Analysis-- Adsorbents were washed in lysis buffer and boiled for 3 min in sample buffer. Eluted material was fractionated by SDS-PAGE and transferred onto Immobilon-P membranes (Millipore, Bedford, MA). In most experiments, immunoprecipitates were fractionated and transferred onto two membranes. These were used to assess: 1) tyrosyl phosphorylation and Fcgamma RIIB or GST on one membrane, and 2) SHIP1 and SHP-1 phosphatases on the other membrane. Membranes were saturated with either 5% BSA (Sigma) or 5% skimmed milk (Régilait, Saint-Martin-Belle-Roche, France) diluted in 10 mM Tris buffer, pH 7.4, containing 0.5% Tween 20 (Merck, Schuchardt, Germany). Membranes Western blotted with HRP-conjugated anti-phosphotyrosine antibodies were stripped and reblotted with anti-Fcgamma RIIB or anti-GST antibodies followed by HRP-conjugated GAR or GAM. In all experiments, the same membrane was used for blotting with anti-SHIP1 and anti-SHP-1 antibodies after having been cut into two pieces. The upper part, containing molecules with a molecular mass higher than 100 kDa was hybridized with anti-SHIP1, and the lower part, containing molecules with a molecular mass lower than 100 kDa was hybridized with anti-SHP-1 antibodies. mAb anti-SHP-1 was used for mast cells analysis while polyclonal antibodies was used for B cells analysis. They were revealed with HRP-conjugated GAM or GAR, respectively. Labeled antibodies were detected using the Amersham ECL kit.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Tyrosyl-phosphorylated Fcgamma RIIB Recruit SHIP1 but Not SHP-1 Both in Mast Cells and in B Cells-- Because phosphatases were reported to be differentially recruited by Fcgamma RIIB, when coaggregated with BCR in B cells or with Fcepsilon RI in mast cells, we compared the ability of Fcgamma RIIB1 to recruit SHP-1 and SHIP1 in a rat mast cell line, RBL-2H3, and in two Fcgamma RIIB-deficient murine B cell lines, IIA1.6 cells and K46µ. The three cell lines were stably transfected with murine Fcgamma RIIB1. Fcgamma RIIB1 were coaggregated with Fcepsilon RI, that are constitutively expressed in RBL cells, by challenging transfectants, previously sensitized with mouse IgE anti-DNP and coated with F(ab')2 fragments of the rat anti-Fcgamma RIIB mAb 2.4G2, using TNP-MAR F(ab')2. Fcgamma RIIB1 were coaggregated with BCR, that are constitutively expressed in IIA1.6 cells and K46µ cells, using RAM IgG. Fcgamma RIIB1 were immunoprecipitated, their phosphorylation was assessed by Western blotting with anti-phosphotyrosine antibodies, and phosphatases co-precipitated with Fcgamma RIIB1 were examined by Western blotting with anti-SHP-1 and anti-SHIP1 antibodies. As previously observed, Fcgamma RIIB1 became tyrosyl phosphorylated following coaggregation with Fcepsilon RI or with BCR, in mast cells and in B cells, respectively. SHIP1, but not SHP-1, co-precipitated with tyrosyl-phosphorylated Fcgamma RIIB1 in RBL cells, but also in IIA1.6 cells and K46µ cells (Fig. 1A). Because the recruitment of SHIP1 and SHP-1 may not have identical kinetics, we repeated the experiment at various time points in RBL transfectants. The induced phosphorylation of Fcgamma RIIB1 was constant over the time range studied, and comparable amounts of SHIP1 co-precipitated with phosphorylated Fcgamma RIIB1. No co-precipitation of SHP-1 was detected between 15 s and 15 min (Fig. 1B). SHIP1, but not SHP-1, was therefore detectably recruited by tyrosyl-phosphorylated Fcgamma RIIB1, and we found no differential in vivo recruitment of phosphatases in a mast cell line and in two B cell lines.



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Fig. 1.   Recruitment of SHIP1, but not SHP-1, by Fcgamma RIIB1 in mast cells and in B cells after coaggregation with Fcepsilon RI or BCR, respectively. A, co-precipitation of phosphatases with Fcgamma RIIB1 in RBL-2H3, IIA1.6 and K46µ transfectants. 6.5 × 107 RBL-2H3 transfectants expressing Fcgamma RIIB1 were incubated with 2.4G2 F(ab')2, sensitized or not with IgE anti-DNP, and challenged or not with TNP-MAR F(ab')2 for 5 min. 6.5 × 107 IIA1.6 and K46µ transfectants expressing Fcgamma RIIB1 were stimulated with 0.3 µM RAM F(ab')2 or RAM IgG for 5 min. B, kinetics of phosphatase recruitment by Fcgamma RIIB1 in RBL transfectants. 6.5 × 107 RBL-2H3 transfectants expressing Fcgamma RIIB1, incubated with 2.4G2 F(ab')2 and sensitized or not with IgE anti-DNP, were challenged or not with TNP-MAR F(ab')2 for the indicated periods of time. Cells were lysed and Fcgamma RIIB were immunoprecipitated. Immunoprecipitated material was fractionated by SDS-PAGE, transferred onto Immobilon, and Western blotted with anti-Fcgamma RIIB, anti-Tyr(P), anti-SHIP1, and anti-SHP-1 antibodies. Whole cell lysates (WCL) corresponding to 5 × 105 cells were used as positive controls for Western blotting with anti-phosphatase antibodies.

In Vitro Binding of SHP-1 Requires a Higher Phosphorylation Level of Fcgamma RIIB ITIM Than Binding of SHIP1-- Such a selective in vivo recruitment of phosphatases by tyrosyl-phosphorylated Fcgamma RIIB1 was in marked contrast with the ability of phosphorylated synthetic peptides corresponding to the Fcgamma RIIB ITIM to bind in vitro not only to the two SHIPs, but also to the two SHPs, when bound to agarose beads. In an attempt to understand this discrepancy, we reasoned that, among other differences between the two types of experiments, the proportion of Fcgamma RIIB1 whose ITIM was phosphorylated in vivo was unknown, whereas all ITIM peptides used to coat agarose beads for in vitro experiments were phosphorylated. To evaluate the possibility that variations in the intensity of ITIM phosphorylation might differentially affect the affinity for SHIPs and for SHPs, we coated beads with a constant amount of ITIM peptides, in which the proportion of phosphorylated peptides (pITIM) varied from 100 to 0%. These were incubated with cell lysate from RBL-2H3 cells, and the binding of SHIP1 and SHP-1 was examined by Western blotting. As previously observed, no phosphatase precipitated with beads coated with nonphosphorylated ITIM (0% pITIM) and both phosphatases precipitated with beads coated with 100% pITIM. SHIP1 precipitation did not detectably decrease with the proportion of pITIM until beads were coated with less than 12% pITIM, and a detectable amount of phosphatase remained precipitated by beads coated with 6% pITIM. By contrast, SHP-1 precipitation progressively decreased with the proportion of pITIM coated to beads and it was not detected when beads were coated with less than 25% pITIM (Fig. 2A). Beads coated with ITIM peptides, a small proportion of which were phosphorylated, therefore bound in vitro SHIP1 but not SHP-1, i.e. displayed the same selectivity for SHIP1 as tyrosyl-phosphorylated Fcgamma RIIB in vivo.



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Fig. 2.   Differential in vitro binding of SHP-1 and SHIP1 to phosphorylated Fcgamma RIIB ITIM. A, SHIP1 and SHP-1 binding to beads coated with Fcgamma RIIB ITIM peptides in which the proportion of phosphorylated peptides (pITIM) varied. 12.5-µl agarose beads were coated with 3.2 nmol of a mixture of nonphosphorylated and phosphorylated ITIM peptides (pITIM), in variable proportions. Beads were incubated with RBL-2H3 cell lysate corresponding to 1 × 107 cells. Precipitated material was fractionated by SDS-PAGE, transferred onto Immobilon and Western blotted with anti-SHIP1 and anti-SHP-1 antibodies. B, SHIP1 and SHP-1 binding to GST-ITIM and GST-ICIIB1' phosphorylated in vitro by Lyn for increasing periods of times. Agarose beads coated with GST fusion proteins containing either the same ITIM containing 13 amino acids as synthetic peptides (GST-ITIM) or the whole intracytoplasmic domain of the Fcgamma RIIB1' isoform (GST-ICIIB1') were incubated for increasing periods of time with Lyn. Beads were incubated with RBL-2H3 cell lysate corresponding to 1 × 107 cells. Precipitated material and whole cell lysate (WCL) were fractionated by SDS-PAGE, transferred onto Immobilon, and Western blotted with anti-GST, anti-Tyr(P), anti-SHIP1, and anti-SHP-1 antibodies.

Another difference between the two types of experiments is that intact receptors are used for in vivo recruitment whereas 13-amino acid peptides are used for in vitro binding. One must therefore consider the possibility that flanking sequences might affect the recruitment of phosphatases by the phosphorylated ITIM. To examine this possibility, we constructed GST fusion proteins containing either the whole intracytoplasmic domain of the Fcgamma RIIB1' isoform (GST-ICIIB1') or only the same ITIM-containing 13 amino acids as synthetic peptides (GST-ITIM). Purified proteins were in vitro phosphorylated by purified Lyn for various periods of time, incubated in RBL lysate, and their ability to precipitate SHIP1 and SHP-1 was assessed by Western blotting. When submitted to in vitro kinase assay with Lyn, GST alone failed to be phosphorylated and recruited no phosphatase (data not shown). GST-ICIIB1' and GST-ITIM were phosphorylated by Lyn, and phosphorylation increased as a function of time. No phosphatase was precipitated by nonphosphorylated proteins. Both GST-ITIM and GST-ICIIB1' precipitated SHIP1 and SHP-1 when heavily phosphorylated, but SHIP1 only when less phosphorylated (Fig. 2B). A tyrosyl-phosphorylated intact Fcgamma RIIB intracytoplasmic domain could therefore bind not only SHIP1 but also SHP-1 in vitro. SHP-1 binding, however, required a higher degree of phosphorylation than SHIP1 binding. The degree of ITIM phosphorylation seems therefore to determine the in vitro binding for SHP-1 rather than the presence or absence of sequences flanking the phosphorylated Fcgamma RIIB ITIM.

The Density of Phosphorylated ITIM Critically Determines the Cooperative Binding of the Two SHP-1 SH2 Domains-- A major difference between SHIP1 and SHP-1 is that the phosphatidylinositol phosphatase contains one SH2 domain whereas the protein tyrosine phosphatase contains two tandem SH2 domains. We investigated the possible role of this difference in the differential binding of these phosphatases to pITIM-coated beads. Agarose beads were coated with increasing concentrations of Fcgamma RIIB pITIM and incubated either with RBL cell lysates or with purified SH2 domain-containing GST fusion proteins. These were the SH2 domain of SHIP1 (GST-SH2 SHIP1), the two SH2 domains of SHP-1 (GST-SH2 (N+C) SHP-1), the N-terminal SH2 domain of SHP-1 (GST-SH2 (N) SHP-1), or the C-terminal SH2 domain of SHP-1 (GST-SH2 (C) SHP-1). Phosphatases precipitated from cell lysates were identified by Western blotting with anti-SHIP1 and anti-SHP-1 antibodies. GST fusion proteins precipitated were identified by Western blotting with anti-GST antibodies. SHIP1 precipitation was detected with beads coated with as little as 0.2 nmol of pITIM and slowly increased with the amount of pITIM coated to beads. SHP-1 precipitation became detectable with beads coated with 0.4 nmol of pITIM and rapidly increased with the amount of pITIM. Parallel variations in binding were observed for GST-SH2 SHIP and GST-SH2 (N+C) SHP-1. No binding of GST-SH2 (N) SHP-1 was detectable, whatever the amount of pITIM coated to beads, and a faint binding of GST-SH2 (C) SHP-1 was observed for beads coated with high amounts of pITIM only (Fig. 3A). The differential binding of phosphatases, in a cell lysate, can therefore be reproduced using GST-SH2 fusion proteins. The single SHIP1 SH2 domain bound readily to ITIM-coated beads, but not isolated SHP-1 SH2 domains. The two SHP-1 SH2 domains, however, bound with a similar pattern as SHP-1 in a cell lysate, indicating the requirement for a cooperative binding between the two SHP-1 SH2 domains.



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Fig. 3.   Cooperative binding of the two SH2 domains of SHP-1 and effect of the density of pITIMs on beads. A, binding of SHIP1 and SHP-1 SH2 domains to beads coated with variable amounts of pITIM. 12.5-ml agarose beads were coated with increasing amounts of pITIM. pITIM-coated beads were incubated in the same experiment either with RBL-2H3 cell lysate or with SH2 domain-containing GST fusion proteins. These were the SH2 domain of SHIP1 (GST-SH2 SHIP), the two SH2 domains of SHP-1 (GST-SH2 (N+C) SHP-1), the N-terminal SH2 domain of SHP-1 (GST-SH2 (N) SHP-1), or the C-terminal SH2 domain of SHP-1 (GST-SH2 (C) SHP-1). Precipitated material was fractionated by SDS-PAGE and transferred onto Immobilon. Precipitates from cell lysates and whole cell lysate (WCL) were Western blotted with anti-SHIP1 and anti-SHP-1. Precipitates from fusion proteins were Western blotted with anti-GST antibodies. Aliquots of GST fusion proteins used for incubation were electrophoresed and Western blotted with anti-GST antibodies to control the relative amounts of fusion proteins. B, binding of SHIP1 and SHP-1 to beads coated with Fcgamma RIIB pITIM at variable densities. 12.5 ml or 50 ml of agarose beads were incubated with the same amounts of pITIM. Beads were incubated with RBL-2H3 cell lysates corresponding to 1 × 107 cells. Precipitated material and WCL were fractionated by SDS-PAGE, transferred onto Immobilon, and Western blotted with anti-SHIP1 and anti-SHP-1 antibodies.

When using increasing amounts of pITIM to coat beads, one increases not only the quantity of pITIM bound to beads, but also the density of pITIM on beads. To investigate whether pITIM density may determine the binding of the two SH2 domain-containing SHP-1, the same amounts of pITIM were used to coat different amounts of beads. These were incubated in RBL cell lysate, and phosphatase binding was then examined as above. For a given amount of pITIM, the binding of SHIP1 did not vary with the amount of beads. By contrast, for a given amount of pITIM, the binding of SHP-1 dramatically decreased when the amount of beads increased (Fig. 3B). SHP-1 binding, but not SHIP1 binding, therefore depends on the density of pITIM coated to beads.

Increasing Fcgamma RIIB Phosphorylation by Increasing the Concentration of Immune Complexes Does Not Enable SHP-1 to Be Recruited-- Based on the above results, we wondered 1) whether the intensity of Fcgamma RIIB phosphorylation would increase with the concentration of extracellular ligands used to coaggregate Fcgamma RIIB with Fcepsilon RI in mast cells, or with BCR in B cells, and 2) whether phosphorylation levels induced under these conditions might enable Fcgamma RIIB to recruit SHP-1 in vivo.

We first tried to increase Fcgamma RIIB phosphorylation by stimulating Fcgamma RIIB1-transfected IIA1.6 cells with increasing concentrations of RAM IgG and, as negative controls, with the same molar concentrations of RAM F(ab')2. Fcgamma RIIB1 were immunoprecipitated, their phosphorylation was assessed by Western blotting with anti-phosphotyrosine antibodies, and co-precipitated phosphatases were examined by Western blotting with anti-phosphatase antibodies. Fcgamma RIIB1 phosphorylation induced by a RAM IgG concentration as high as 2.4 µM (360 µg/ml) was not higher than Fcgamma RIIB1 phosphorylation induced by 0.3 µM (45 µg/ml) RAM IgG. Comparable amounts of SHIP1 co-precipitated with phosphorylated Fcgamma RIIB1, whatever the concentration of RAM IgG, but no detectable SHP-1 (Fig. 4).



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Fig. 4.   Absence of co-precipitation of SHP-1 with Fcgamma RIIB1 in IIA1.6 transfectants stimulated with increasing concentrations of RAM IgG. 6.5 × 107 IIA1.6-Fcgamma RIIB1 cells were stimulated with increasing concentrations of RAM F(ab')2 or IgG. Cells were lysed and Fcgamma RIIB were immunoprecipitated. Immunoprecipitated material and whole cell lysate (WCL) were fractionated by SDS-PAGE, transferred onto Immobilon, and Western blotted with anti-Fcgamma RIIB, anti-Tyr(P), anti-SHIP1, and anti-SHP-1 antibodies.

This negative result led us to use IgG immune complexes, which are the natural ligands of Fcgamma RIIB, to coaggregate these receptors with ITAM-bearing receptors in mast cells and B cells. Fcepsilon RI and Fcgamma RIIB expressed constitutively in BMMCs (42) were coaggregated by challenging cells, sensitized with increasing concentrations of mouse anti-DNP IgE, with immune complexes made with increasing concentrations of DNP-BSA and of a monoclonal mouse IgG1 anti-DNP antibody. Fcgamma RIIB phosphorylation increased with the concentration of IgE used for sensitization and with the concentration of IgG in immune complexes. It also varied with the concentration of DNP-BSA in immune complexes, and optimal concentrations depended on the concentration of IgG antibodies (Fig. 5A). Likewise, Fcgamma RIIB were coaggregated with BCR in Fcgamma RIIB1-transfected K46µ B cells, which express an anti-NP BCR, by incubating cells with immune complexes made of increasing concentrations of polyclonal rabbit IgG anti-DNP and of NP-BSA-TNP. Fcgamma RIIB1 phosphorylation varied with the concentration of IgG antibody and antigen: it peaked with higher concentrations of antigen as the antibody concentrations increased. Immune complexes that induced the highest Fcgamma RIIB1 phosphorylation were made of 10 µg/ml IgG and 1 µg/ml NP-BSA-TNP (Fig. 5B).



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Fig. 5.   Absence of co-precipitation of SHP-1 with Fcgamma RIIB in cells stimulated with increasing concentrations of immune complexes. A, Fcgamma RIIB phosphorylation in BMMCs stimulated with increasing concentrations of ligands. 1 × 107 BMMCs were sensitized with a constant (1/10; left panel) or various dilutions of mouse IgE anti-DNP supernatant (right panel). Cells were then stimulated for 5 min with preformed immune complexes made with a constant (20 µg/ml; right panel) or increasing concentrations of DNP-BSA (left panel) and with increasing concentrations of monoclonal mouse IgG1 anti-DNP antibody. B, Fcgamma RIIB phosphorylation in K46µ-Fcgamma RIIB1 stimulated with increasing concentrations of ligands. 1 × 107 K46µ transfectants expressing both an anti-NP BCR and Fcgamma RIIB1 (K46µ-Fcgamma RIIB1) were stimulated for 5 min with preformed immune complexes made with increasing concentrations of NP-BSA-TNP and with increasing concentrations of rabbit polyclonal IgG anti-DNP. C, SHIP1 and SHP-1 co-precipitation when Fcgamma RIIB is optimally phosphorylated by immune complexes. 6.5 × 107 BMMCs sensitized with mouse IgE anti-DNP supernatant (diluted 1/10) were stimulated or not with immune complexes made with 5 µg/ml DNP-BSA and 50 µg/ml mouse IgG1 anti-DNP (final concentrations). K46µ-Fcgamma RIIB1 cells were stimulated for 5 min with 10 µg/ml rabbit IgG anti-DNP or with immune complexes made with 1 µg/ml NP-BSA-TNP and 10 µg/ml rabbit IgG anti-DNP (final concentrations). For comparison, K46µ transfectants were also stimulated with 0.3 µM RAM IgG or F(ab')2. BMMCs and K46µ transfectants were lysed after stimulation, and Fcgamma RIIB were immunoprecipitated. Immunoprecipitated materials and whole cell lysate from K46µ transfectants (WCL) were fractionated by SDS-PAGE, transferred onto Immobilon, and Western blotted with anti-Fcgamma RIIB, anti-Tyr(P) (A, B, and C), anti-SHIP1 and anti-SHP-1 antibodies (C).

The co-precipitation of phosphatases was next examined in BMMCs and in Fcgamma RIIB1-transfected K46µ cells stimulated with concentrations of antigen and antibodies in the range of those which induced maximal Fcgamma RIIB phosphorylation. For comparison, K46µ transfectants were also stimulated with RAM IgG or F(ab')2 under the same conditions as in Fig. 1. In both cell types, Fcgamma RIIB were markedly phosphorylated following stimulation with immune complexes. Fcgamma RIIB phosphorylation was of comparable magnitude in K46µ cells stimulated with immune complexes or with RAM IgG. SHIP1 co-precipitated with phosphorylated Fcgamma RIIB both in mast cells and in B cells, but not SHP-1 (Fig. 5C). The above results altogether indicate that even when using concentrations of ligands that were optimal for Fcgamma RIIB phosphorylation, SHIP1 but not SHP-1 co-precipitation was detectable, both in mast cells and in B cells.

Fcgamma RIIB Phosphorylation following Pervanadate Treatment Enables SHP-1 Recruitment-- A possible reason explaining the absence of detectable recruitment of SHP-1 by Fcgamma RIIB phosphorylated following stimulation with high concentrations of extracellular ligands was that Fcgamma RIIB phosphorylation levels reached under these conditions were not high enough. We therefore compared the effect of coaggregating Fcgamma RIIB with Fcepsilon RI, in RBL transfectants, or with BCR, in IIA1.6 and K46µ transfectants, and of treating the same three cells with pervanadate.

Fcgamma RIIB1'-expressing RBL cells were sensitized with mouse IgE anti-DNP and incubated with 2.4G2 F(ab')2, treated or not treated with pervanadate and challenged or not with TNP-MAR-F(ab')2. Likewise, Fcgamma RIIB1-expressing IIA1.6 and K46µ cells were treated or not with pervanadate and challenged with RAM F(ab')2 or IgG. Fcgamma RIIB phosphorylation and phosphatase recruitment were assessed as in previous experiments. As expected, Fcgamma RIIB phosphorylation was induced by coaggregating Fcgamma RIIB1' with Fcepsilon RI in RBL transfectants, and by coaggregating Fcgamma RIIB1 with BCR in IIA1.6 and K46µ transfectants. In all three cells, pervanadate treatment alone induced a much higher level of Fcgamma RIIB phosphorylation that did not further increase by coaggregating Fcgamma RIIB with Fcepsilon RI or with BCR. SHIP1 co-precipitated with Fcgamma RIIB phosphorylated following their coaggregation with Fcepsilon RI or with BCR. SHIP1 co-precipitated also with Fcgamma RIIB phosphorylated following treatment of cells with pervanadate (in higher amounts than following coaggregation of Fcgamma RIIB with Fcepsilon RI, in RBL cells, or with BCR, in K46µ cells). SHP-1 did not co-precipitate with Fcgamma RIIB phosphorylated following their coaggregation with Fcepsilon RI or BCR. SHP-1, however, co-precipitated with Fcgamma RIIB phosphorylated following pervanadate treatment in all three cells (Fig. 6A). Treating Fcgamma RIIB1-expressing RBL transfectants with decreasing concentrations of pervanadate induced a dose-dependent tyrosyl phosphorylation of Fcgamma RIIB1. Interestingly, as Fcgamma RIIB1 phosphorylation decreased, the co-precipitation of SHP-1 was lost before that of SHIP1 (Fig. 6B). Treating cells with pervanadate, but not coaggregating Fcgamma RIIB with ITAM-bearing receptors, could therefore induce a phosphorylation of Fcgamma RIIB that was high enough to enable the recruitment of SHP-1.



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Fig. 6.   Co-precipitation of SHP-1 with Fcgamma RIIB phosphorylated following pervanadate treatment. A, co-precipitation of phosphatases with Fcgamma RIIB1 in mast cell and B cell transfectants. 6.5 × 107 Fcgamma RIIB1'-expressing RBL cells were sensitized with mouse IgE anti-DNP and incubated with 2.4G2 F(ab')2 before they were treated or not with 100 µM pervanadate for 15 min. During the last 5 min of pervanadate treatment, cells were stimulated (coaggregation +) or not (coaggregation -) with TNP-MAR F(ab')2. 6.5 × 107 IIA1.6-Fcgamma RIIB1 or K46µ-Fcgamma RIIB1 transfectants were treated or not with pervanadate and stimulated (coaggregation +) or not (coaggregation -) with RAM IgG, under the same conditions as RBL cells. B, dose-dependent co-precipitation of phosphatases with Fcgamma RIIB1 in mast cell transfectants treated with pervanadate. 9.5 × 107 Fcgamma RIIB1-expressing RBL cells were treated or not with the indicated concentrations of pervanadate for 15 min. Following stimulation, cells were lysed, and Fcgamma RIIB were immunoprecipitated. Immunoprecipitated material from RBL, IIA1.6, and K46µ transfectants were fractionated by SDS-PAGE, transferred onto Immobilon, and Western blotted with anti-Fcgamma RIIB, anti-Tyr(P), anti-SHIP1, and anti-SHP-1 antibodies.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We show here that murine Fcgamma RIIB recruit the inositol 5-phosphatase SHIP1, but not the protein-tyrosine phosphatase SHP-1 in vivo, although the Fcgamma RIIB ITIM has an affinity for both phosphatases in vitro, because the binding of SHP-1 requires a higher degree of Fcgamma RIIB phosphorylation than the binding of SHIP1. The same phosphorylation-dependent preference for SHIP1 was observed 1) in vitro using beads coated with suboptimal concentrations of pITIM, 2) in vitro using GST-ICIIB1' phosphorylated by Lyn for short periods of time, 3) in vivo using phosphorylated Fcgamma RIIB precipitated from cells treated with low concentrations of pervanadate, and 4) in vivo, when Fcgamma RIIB was phosphorylated following coaggregation with BCR or Fcepsilon RI, in B cells and in mast cells, respectively. Our results suggest that, depending on their level of phosphorylation, Fcgamma RIIB could potentially use the two phosphatases, with different consequences.

Evidence that, when tyrosyl phosphorylated, the Fcgamma RIIB ITIM has an affinity for SH2 domain-containing phosphatases was first provided in 1995 by D'Ambrosio et al. (30) who demonstrated that phosphorylated synthetic peptides containing the Fcgamma RIIB ITIM precipitated several molecular species from [35S]methionine-labeled cell lysates, one of which was identified as SHP-1. Other molecules precipitated by these peptides were subsequently shown to be SHP-2 (28) and SHIP1 (26). Similar experiments confirmed D'Ambrosio's results (28, 32). SHIP2, a second SH2 domain-containing inositol phosphatase was recently found to bind also to phosphorylated Fcgamma RIIB ITIM (27, 29). It follows that phosphorylated Fcgamma RIIB ITIM peptides can bind all four known SH2 domain-containing phosphatases in vitro.

In the same 1995 paper, D'Ambrosio et al. (30) reported that SHP-1 co-precipitated with Fcgamma RIIB bearing an intact ITIM, following coaggregation with BCR in A20 and in IIA1.6 B cells reconstituted with Fcgamma RIIB, and that Fcgamma RIIB-dependent inhibition of B cell proliferation was impaired in B cells from SHP-1-deficient motheaten mice. In 1996, Ono et al. (26) reported that SHIP1 co-precipitated with Fcgamma RIIB following coaggregation with Fcepsilon RI in BMMCs or with BCR in A20 cells, and that Fcgamma RIIB-dependent inhibition of IgE-induced serotonin release was unaffected in BMMCs derived from motheaten mice. Fong et al. (32) reported that SHIP1, but not SHP-1 or SHP-2, co-precipitated with Fcgamma RIIB following coaggregation with Fcepsilon RI in BMMCs. In 1997, Ono et al. (33) showed that Fcgamma RIIB-dependent inhibition of Ca2+ responses and of NF-AT activity was abolished in SHIP1-deficient, but not in SHP-1-deficient, DT40 chicken B cells, and that SHIP1, but not SHP-1, was detectably co-precipitated with Fcgamma RIIB following coaggregation with BCR in A20 cells. In 1998, however, Sato et al. (31) observed the co-precipitation of both SHIP1 and SHP-1 with Fcgamma RIIB in A20 cells expressing an anti-TNP BCR following coaggregation with intact anti-idiotypic antibodies. Contrasting with the consensus that Fcgamma RIIB recruit SHIP1 both in B cells and in mast cells, their ability to recruit SHP-1 in vivo therefore remains controversial.

That SHP-1 was found by two groups to co-precipitate with Fcgamma RIIB in B cells, but not in mast cells, suggested the possibility that some discrepancies might be accounted for by a cell type-specific differential in vivo recruitment. To address this issue, we examined the co-precipitation of SHP-1 with recombinant Fcgamma RIIB1 stably expressed by transfecting the same cDNA into the rat mast cells RBL-2H3, and into the two Fcgamma RIIB-deficient mouse lymphoma B cells IIA1.6 and K46µ. The coaggregation of Fcgamma RIIB1 with Fcepsilon RI or with BCR induced a comparable tyrosyl phosphorylation of Fcgamma RIIB1 and the co-precipitation of SHIP1, but not of SHP-1, in all three cells. The same result, observed at 5 min in the three cell lines, was also observed between 15 s and 15 min in RBL cells. Failure to detect SHP-1 co-precipitation cannot be accounted for an insufficient sensitivity of Western blotting because traces of SHP-1 could be seen on overexposed films, but in equal amounts in unstimulated and in stimulated cells (data not shown). Due to experimental conditions inherent to the co-precipitation technique, however, we cannot exclude that SHP-1, possibly recruited by Fcgamma RIIB in vivo was lost. Whatever the explanation, we observed no difference between the three cells examined in which Fcgamma RIIB preferentially, if not exclusively, recruited SHIP1. There is therefore a discrepancy between the ability of SHP-1 and SHIP1 to bind in vitro to Fcgamma RIIB pITIM peptides and to co-precipitate with phosphorylated Fcgamma RIIB in vivo.

Due to the different experimental conditions used for the two assay systems many differences can possibly explain this discrepancy. One difference could bear on the level of ITIM phosphorylation. Indeed, all ITIMs are phosphorylated on beads used for in vitro binding assay whereas an unknown proportion of Fcgamma RIIB are phosphorylated following coaggregation with ITAM-bearing receptors. To explore the possible role of quantitative differences in ITIM phosphorylation, we studied the binding of SHIP1 and SHP-1 to beads coated with Fcgamma RIIB ITIMs phosphorylated in varying proportions and incubated in a cell lysate. We found that SHP-1 binding decreased more sharply with the proportion of pITIM than SHIP1 binding, so that beads coated with 12% pITIM bound selectively SHIP1. Comparable results were obtained when incubating beads coated with increasing concentrations of pITIM with GST fusion proteins containing the SH2 domain of SHIP1 or the two SH2 domains of SHP-1. The use of SH2 domains permitted comparison between the binding of the two molecules using the same anti-GST antibodies for blotting. It also excluded that phosphatase binding was mediated by unknown intermediates present in cell lysates. Supporting these results, the Fcgamma RIIB pITIM was reported to have a higher affinity for the SH2 domain of SHIP1 than for the two SH2 domains of SHP-1, when measured by Biacore analysis (43). Results obtained in these two sets of experiments are reminiscent of the selective in vivo recruitment of SHIP1 by Fcgamma RIIB.

Based on data previously reported by others (43, 44), the binding of SHP-1 is likely to involve the two tandem SH2 domains of this phosphatase. Compared with GST fusion proteins containing the two SH2 domains of SHP-1, no GST fusion proteins containing the N-terminal domain of SHP-1 and minute amounts of GST fusion proteins containing the C-terminal SH2 domain of SHP-1 bound to pITIM-coated beads, whatever the concentration of peptides on beads. This suggests that, since there is one tyrosine only in pITIM, GST fusion proteins containing the two SHP-1 SH2 domains bound to adjacent pITIMs on the same bead. The same holds for the binding of SHP-1 when incubating pITIM-coated beads with a cell lysate. If so, variations in the affinity of SHP-1 to beads coated with increasing amounts of pITIM might depend on the density of peptides coated to beads. To examine this possibility, we used a constant amount of pITIM to coat variable numbers of beads that were incubated in a cell lysate, and we compared the ability of these beads to bind SHIP1 and SHP-1. The binding of SHIP1 was proportional to the amount of pITIM on beads, and did not vary with the pITIM density. By contrast, the binding of SHP-1 depended not only on the amount of pITIM but also, critically, on the density of pITIM bound to beads. The in vitro binding of SHP-1 therefore requires a cooperative binding of its two SH2 domains to two adjacent pITIMs in trans, and this feature explains that pITIMs need to be closer to each other for enabling the binding of SHP-1 than for enabling the binding of SHIP1.

Another difference that might explain the discrepancy between the in vitro binding and the in vivo recruitment of SHP-1 is that isolated ITIMs are used in vitro whereas whole receptors are used in vivo. One cannot exclude that the recruitment of SHP-1 might be hampered by non-ITIM sequences or by molecules that could possibly bind to these sequences. Supporting this possibility, the N-terminal KIR2DL3 ITIM that could recruit SHP-2 in vivo, when kept in its original context, failed to recruit this phosphatase, when transposed in the intracytoplasmic domain of Fcgamma RIIB1 (29). To answer this question, we compared the ability of GST fusion proteins containing the Fcgamma RIIB ITIM only or the whole intracytoplasmic domain of Fcgamma RIIB1' to bind SHIP1 and SHP-1, when incubated with a cell lysate, following their phosphorylation with Lyn for various periods of time. No difference was observed between the two fusion proteins and, like the isolated ITIM, the intracytoplasmic domain of Fcgamma RIIB could bind SHP-1 when high enough phosphorylated.

Based on the latter result, we searched for experimental conditions that would induce a Fcgamma RIIB phosphorylation sufficient to enable them to recruit SHP-1 in vivo. To this aim, we used several extracellular ligands including IgG immune complexes that are the physiological ligands of Fcgamma RIIB, at various concentrations, in mast cells and in B cells. We found that indeed, the phosphorylation of Fcgamma RIIB varied with the concentrations of antigen and antibody in immune complexes but that ligands which induced a maximal phosphorylation of Fcgamma RIIB readily induced the recruitment of SHIP1 but failed to induce a detectable recruitment of SHP-1. Based on our results of in vitro binding with pITIM-coated beads, this suggests that a small proportion (less than 12%?) of Fcgamma RIIB become tyrosyl phosphorylated in vivo upon coaggregation with ITAM-bearing receptors by physiological ligands. If so, we wondered whether Fcgamma RIIB phosphorylation would reach a level high enough to enable the recruitment of SHP-1 following treatment of cells with pervanadate.

In both mast cells and B cells, pervanadate treatment indeed induced a higher degree of Fcgamma RIIB phosphorylation than coaggregation with Fcepsilon RI or BCR, respectively, and under these conditions, not only SHIP1 but also SHP-1 co-precipitated with phosphorylated Fcgamma RIIB. This observation indicates that, in resting cells, Fcgamma RIIB are tyrosyl phosphorylated but that protein-tyrosine phosphatases maintain this phosphorylation below the detection level. This implies that Fcgamma RIIB are the substrates of both protein-tyrosine kinases and phosphatases and that, under resting conditions, the effect of phosphatases is dominant over that of kinases. Fcgamma RIIB phosphorylation observed following their coaggregation with ITAM-bearing receptors results from the additional effect of a Src kinase, brought by activating receptors (13), leading to a displacement of the balance so that the effect of kinases becomes dominant over that of phosphatases. It should be emphasized that the higher intensity of Fcgamma RIIB phosphorylation induced by pervanadate, compared with phosphorylation induced by coaggregation, may be due to the phosphorylation of a higher number of receptors and/or to the phosphorylation of a higher number of tyrosine residues in each receptor. The recruitment of SHP-1 by Fcgamma RIIB phosphorylated after pervanadate treatment may indeed simply be explained by a quantitatively different phosphorylation, resulting in an increased density of phosphorylated ITIMs that might permit the binding in trans of the two SHP-1 SH2 domains. Supporting this interpretation, the recruitment of SHP-1 was lost before that of SHIP1 when pervanadate-induced Fcgamma RIIB phosphorylation decreased following treatment of cells with decreasing concentrations of pervanadate. Alternatively, the recruitment of SHP-1 after pervanadate treatment may be explained by a qualitatively different phosphorylation of Fcgamma RIIB, enabling SHP-1 to be recruited through the binding in cis of its two SH2 domains to two tyrosines borne by the same receptor. The intracytoplasmic domain of Fcgamma RIIB1 contains four tyrosine residues. Supporting this possibility, the recruitment of SHP-1 by KIR2DL3 was found to require the conservation of its two ITIMs (39) and all ITIM-bearing receptors that were shown to recruit SHPs in vivo bear more than one ITIM. Finally, SHP-1 may co-precipitate with Fcgamma RIIB phosphorylated following pervanadate treatment because, under these conditions, the enzymatic activity of the phosphatase is inhibited. Several ITIM-bearing receptors were shown both to recruit and to be the substrates of SHP-1 (45) or SHP-2 (46, 47). If recruited by phosphorylated Fcgamma RIIB under physiological conditions, SHP-1 might thus decrease Fcgamma RIIB phosphorylation, thereby giving an advantage for the recruitment of SHIP over that of SHP-1.

The latter interpretation of the effect of pervanadate may explain our failure to co-precipitate SHP-1 with Fcgamma RIIB phosphorylated upon coaggregation with ITAM bearing receptors. This hypothesis would also endow Fcgamma RIIB with additional regulatory properties. These receptors could indeed transiently recruit SHP-1 which could dephosphorylate not only ITIMs, but also ITAMs and other signaling molecules whose phosphorylation is critical for positive signaling. This would have important consequences. By displacing the balance between kinases and phosphatases recruited to the receptor complex in favor of the latter, it would increase the signaling threshold and/or dampen activation signals. Fcgamma RIIB might thus use two different mechanisms, i.e. SHIP-mediated and SHP-mediated, to adjust negative regulation to the intensity of extracellular signals. By decreasing positive signals, SHP-1 would in turn decrease ITIM phosphorylation bringing Fcgamma RIIB back to conditions under which they recruit SHIPs. It remains to be determined whether Fcgamma RIIB could be phosphorylated enough to recruit SHP-1 under physiological or pathological situations such as diseases associated with exaggerated antibody responses or immune complexes.


    ACKNOWLEDGEMENTS

We thank Dr. John C. Cambier for K46µ cells expressing an anti-NP BCR, NP-BSA-TNP, and polyclonal anti-C-terminal sequences of SHP-1 antibodies, Prof. Catherine Sautès-Fridman for polyclonal antibodies to the extracellular domains of Fcgamma RIIB, Odile Malbec (Institut Curie, Paris, France) for GST-ICIIB1', Dr. Eric Vivier for SHP-1 SH2 domain-containing GST fusion proteins, and Dr. Jean-Luc Teillaud for monoclonal anti-GST antibodies. We acknowledge the expert assistance of Zosia Maciorowski for cells sorting.


    FOOTNOTES

* This work was supported in part by the Institut National de la Santé et de la Recherche Médicale, the Institut Curie, and the Association pour la Recherche sur le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of a fellowship from the Ministère de l'Enseignement Supérieur et de la Recherche.

§ To whom correspondence should be addressed: Laboratoire d'Immunologie Cellulaire et Clinique, INSERM U. 255, Institut Curie, 26 rue d'Ulm, 75005 Paris, France. Tel.: 33-1-4432-4223; Fax: 33-1-4051-0420; E-mail: Marc.Daeron@curie.fr.

Published, JBC Papers in Press, November 30, 2000, DOI 10.1074/jbc.M006537200


    ABBREVIATIONS

The abbreviations used are: ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibition motif; BCR, B cell receptor for antigen; BMMCs, bone marrow-derived mast cells; BSA, bovine serum albumin; DNP, dinitrophenyl; Fcepsilon RI, high-affinity receptors for the Fc portion of IgE; Fcgamma RIIB, low-affinity receptors for the Fc portion of IgG; GAM, goat anti-mouse Ig; GAR, goat anti-rabbit Ig; GST, gluthatione S-transferase; HRP, horseradish peroxidase; MAR, mouse anti-rat Ig; pITIMs, phosphorylated ITIMs; NP, 3-nitro-4-hydroxyphenyl acetic acid; RAM, rabbit anti-mouse Ig; SH2, Src homology-2 domains; SHIPs, SH2 domain-containing inositol 5-phosphatases; SHPs, SH2 domain-containing protein-tyrosine phosphatases. TNP, trinitrophenyl; PAGE, polyacrylamide gel electrophoresis.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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