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• W1977619443 abstract "FcγRIIB 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 FcγRIIB 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 FcγRIIB, 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 FcγRIIB 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 FcγRIIB phosphorylation in B cells and mast cells, we found that concentrations of ligands optimal for FcγRIIB phosphorylation failed to induce SHP-1 recruitment. SHP-1 was, however, recruited by FcγRIIB when hyperphosphorylated following cell treatment with pervanadate. Our data suggest that FcγRIIB phosphorylation may not be sufficientin vivo to enable the recruitment of SHP-1 but that (pathological?) conditions that would hyperphosphorylate FcγRIIB might enable SHP-1 recruitment. FcγRIIB 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 FcγRIIB 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 FcγRIIB, 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 FcγRIIB 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 FcγRIIB phosphorylation in B cells and mast cells, we found that concentrations of ligands optimal for FcγRIIB phosphorylation failed to induce SHP-1 recruitment. SHP-1 was, however, recruited by FcγRIIB when hyperphosphorylated following cell treatment with pervanadate. Our data suggest that FcγRIIB phosphorylation may not be sufficientin vivo to enable the recruitment of SHP-1 but that (pathological?) conditions that would hyperphosphorylate FcγRIIB might enable SHP-1 recruitment. immunoreceptor tyrosine-based activation motif immunoreceptor tyrosine-based inhibition motif B cell receptor for antigen bone marrow-derived mast cells bovine serum albumin dinitrophenyl high-affinity receptors for the Fc portion of IgE low-affinity receptors for the Fc portion of IgG goat anti-mouse Ig goat anti-rabbit Ig gluthationeS-transferase horseradish peroxidase mouse anti-rat Ig phosphorylated ITIMs 3-nitro-4-hydroxyphenyl acetic acid rabbit anti-mouse Ig Src homology-2 domains SH2 domain-containing inositol 5-phosphatases SH2 domain-containing protein-tyrosine phosphatases. TNP, trinitrophenyl polyacrylamide gel electrophoresis FcγRIIB are single-chain low-affinity receptors for the Fc portion of IgG antibodies that bind multivalent immune complexes. They exist as two (FcγRIIB1 and B2 in humans) or three (FcγRIIB1, B1′, and B2 in mice) alternatively spliced products of the FcgR2bgene (1Daëron M. Annu. Rev. Immunol. 1997; 15: 203-234Crossref PubMed Scopus (1031) Google Scholar). All murine and human FcγRIIB isoforms were shown to negatively regulate cell activation induced by all receptors bearing intracytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs)1 (2Daëron M. Latour S. Malbec O. Espinosa E. Pina P. Pasmans S. Fridman W.H. Immunity. 1995; 3: 635-646Abstract Full Text PDF PubMed Scopus (385) Google Scholar). FcγRIIB also negatively regulate cell proliferation induced by growth factor receptors with an intrinsic protein tyrosine kinase activity (3Malbec O. Fridman W.H. Daëron M. J. Immunol. 1999; 162: 4424-4429PubMed Google Scholar). Confirming these results, FcγRIIB-deficient mice were found: 1) to exhibit enhanced antibody responses (4Takai T. Ono M. Hikida M. Ohmori H. Ravetch J.V. Nature. 1996; 379: 346-349Crossref PubMed Scopus (726) Google Scholar); 2) to develop exaggerated IgE- (5Ujike A. Ishikawa Y. Ono M. Yuasa T. Yoshino T. Fukumoto M. Ravetch J. Takai T. J. Exp. Med. 1999; 189: 1573-1579Crossref PubMed Scopus (153) Google Scholar) and IgG-dependent anaphylactic reactions (4Takai T. Ono M. Hikida M. Ohmori H. Ravetch J.V. Nature. 1996; 379: 346-349Crossref PubMed Scopus (726) Google Scholar); 3) to have an enhanced susceptibility to experimental murine models of IgG-dependent autoimmune diseases (6Yuasa T. Kubo S. Yoshino T. Ujike A. Matsumura K. Ono M. Ravetch J.V. Takai T. J. Exp. Med. 1999; 189: 187-194Crossref PubMed Scopus (292) Google Scholar, 7Clynes R. Maizes J.S. Guinamard R. Ono M. Takai T. Ravetch J.V. J. Exp. Med. 1999; 189: 179-185Crossref PubMed Scopus (337) Google Scholar, 8Nakamura A. Yuasa T. Ujike A. Ono M. Nukiwa T. Ravetch J.V. Takai T. J. Exp. Med. 2000; 191: 899-905Crossref PubMed Scopus (178) Google Scholar); and 4) to exhibit enhanced antibody-dependent cell-mediated cytotoxic responses to the injection of therapeutic antibodies to tumor antigens (9Clynes R.A. Towers L.T. Presta L.G. Ravetch J.V. Nat. Med. 2000; 6: 443-446Crossref PubMed Scopus (2289) Google Scholar).To inhibit cell activation, FcγRIIB need to be coaggregated with ITAM-bearing receptors by immune complexes or by any extracellular ligand capable of interacting with the two receptors simultaneously (10Sinclair N.R.S.C. Chan P.L. Adv. Exp. Med. Biol. 1971; 12: 609-615Crossref Google Scholar, 11Daëron M. Malbec O. Latour S. Arock M. Fridman W.H. J. Clin. Invest. 1995; 95: 577-585Crossref PubMed Scopus (305) Google Scholar, 12Bléry M. Delon J. Trautmann A. Cambiaggi A. Olcese L. Biassoni R. Moretta L. Chavrier P. Moretta A. Daëron M. Vivier E. J. Biol. Chem. 1997; 272: 8989-8996Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Coaggregation indeed enables FcγRIIB to be tyrosyl-phosphorylated by Lyn (13Malbec O. Fong D. Turner M. Tybulewicz V.L.J. Cambier J., C. Fridman W.H. Daëron M. J. Immunol. 1998; 160: 1647-1658PubMed Google Scholar), a Src family protein tyrosine kinase provided by ITAM-bearing receptors. FcγRIIB isoforms contain a variable number of tyrosine residues in their intracytoplasmic domain, one of which proved to be critical (2Daëron M. Latour S. Malbec O. Espinosa E. Pina P. Pasmans S. Fridman W.H. Immunity. 1995; 3: 635-646Abstract Full Text PDF PubMed Scopus (385) Google Scholar, 14Muta T. Kurosaki T. Misulovin Z. Sanchez M. Nussenzweig M.C. Ravetch J.V. Nature. 1994; 368: 70-73Crossref PubMed Scopus (433) Google Scholar). This tyrosine stands within a 13-amino acid sequence that was found to be necessary (2Daëron M. Latour S. Malbec O. Espinosa E. Pina P. Pasmans S. Fridman W.H. Immunity. 1995; 3: 635-646Abstract Full Text PDF PubMed Scopus (385) Google Scholar, 15Amigorena S. Bonnerot C. Drake J. Choquet D. Hunziker W. Guillet J.G. Webster P. Sautès C. Mellman I. Fridman W.H. Science. 1992; 256: 1808-1812Crossref PubMed Scopus (402) Google Scholar) and sufficient (14Muta T. Kurosaki T. Misulovin Z. Sanchez M. Nussenzweig M.C. Ravetch J.V. Nature. 1994; 368: 70-73Crossref PubMed Scopus (433) Google Scholar) 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 (16Burshtyn D.N. Scharenberg A.M. Wagtmann N. Rajogopalan S. Berrada K. Yi T. Kinet J.-P. Long E.O. Immunity. 1996; 4: 77-85Abstract Full Text Full Text PDF PubMed Scopus (556) Google Scholar, 17Vivier E. Daëron M. Immunol. Today. 1997; 18: 286-291Abstract Full Text PDF PubMed Scopus (326) Google Scholar).A general property of ITIMs is to have an affinity for cytoplasmic SH2 domain-containing phosphatases when tyrosyl- phosphorylated (17Vivier E. Daëron M. Immunol. Today. 1997; 18: 286-291Abstract Full Text PDF PubMed Scopus (326) Google Scholar). 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 (18Tamir I. Dal Porto J.M. Cambier J.C. Curr. Opin. Immunol. 2000; 12: 307-315Crossref PubMed Scopus (100) Google Scholar). SHP-1 is thought to dephosphorylate tyrosines in ITAMs (19Binstadt B.A. Brumbaugh K.M. Dick C.J. Scharenberg A.M. Williams B.L. Colonna M. Lanier L.L. Kinet J.-P. Abraham R.T. Leibson P.J. Immunity. 1996; 5: 629-638Abstract Full Text PDF PubMed Scopus (258) Google Scholar), protein tyrosine kinases and/or adapter proteins such as SLP-76 (20Binstadt B.A. Billadeau D.D. Jevremovic D. Williams B.L. Fang N. Yi T. Koretsky G.A. Abraham R.T. Leibson P.J. J. Biol. Chem. 1998; 273: 27518-27523Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) 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 (21Rohrschneider L.R. Fuller J.F. Wolf I. Liu Y. Lucas D.M. Genes Dev. 2000; 14: 505-520PubMed Google Scholar). 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 (22Bolland S. Pearse R.N. Kurosaki T. Ravetch J.V. Immunity. 1998; 8: 509-516Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). Bruton's tyrosine kinase is mandatory for phospholipase Cγ to be activated and to hydrolyze phosphatidylinositol (4,5)-bisphosphate into inositol (1,4,5)-trisphosphate, which induces a Ca2+ response (23Scharenberg A.M. El-Hillal O. Fruman D.A. Beitz L.O. Li Z. Lin S. Gout I. Cantley L.C. Rawlings D.J. Kinet J.-P. EMBO J. 1998; 17: 1961-1972Crossref PubMed Scopus (386) Google Scholar), 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 (24Tamir I. Stolpa J.C. Helgason C.D. Nakamura K. Bruhns P. Daëron M. Cambier J.C. Immunity. 2000; 12: 347-358Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). 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 FcγRIIB ITIM, bound SHP-1 and SHP-2 (25Daëron M. Vivier E. Curr. Top. Microbiol. Immunol. 1999; 244: 1-12PubMed Google Scholar). Remarkably, the FcγRIIB ITIM also bound SHIP1 (26Ono M. Bolland S. Tempst P. Ravetch J.V. Nature. 1996; 383: 263-266Crossref PubMed Scopus (644) Google Scholar) and SHIP2 (27Muraille E. Bruhns P. Pesesse X. Daëron M. Erneux C. Immunol. Lett. 2000; 72: 7-15Crossref PubMed Scopus (52) Google Scholar). We previously identified two hydrophobic residues, at positions Y-2 and Y+2, that determine the binding of SHPs (28Vély F. Olivero S. Olcese L. Moretta A. Damen J.E. Liu L. Krystal G. Cambier J.C. Daëron M. Vivier E. Eur. J. Immunol. 1997; 27: 1994-2000Crossref PubMed Scopus (122) Google Scholar) and SHIPs (29Bruhns P. Vély F. Malbec O. Fridman W.H. Vivier E. Daëron M. J. Biol. Chem. 2000; 276: 37357-37664Abstract Full Text Full Text PDF Scopus (79) Google Scholar), 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 FcγRIIB were initially reported to recruit SHP-1 in B cells following their coaggregation with B cell receptors (BCR) (30D'Ambrosio D. Hippen K.H. Minskoff S.A. Mellman I. Pani G. Siminovitch K.A. Cambier J.C. Science. 1995; 268: 293-296Crossref PubMed Scopus (507) Google Scholar, 31Sato K. Ochi A. J. Immunol. 1998; 161: 2716-2722PubMed Google Scholar). FcγRIIB, however, were found to recruit selectively SHIP1, when coaggregated with high-affinity IgE receptors (FcεRI) in mast cells (26Ono M. Bolland S. Tempst P. Ravetch J.V. Nature. 1996; 383: 263-266Crossref PubMed Scopus (644) Google Scholar, 32Fong D.C. Malbec O. Arock M. Cambier J.C. Fridman W.H. Daëron M. Immunol. Lett. 1996; 54: 83-91Crossref PubMed Scopus (112) Google Scholar). SHIP1, but not SHP-1, was subsequently demonstrated to be necessary for FcγRIIB-dependent inhibition of cell activation in SHIP1-deficient DT40 B cells (33Ono M. Okada H. Bolland S. Yanagi S. Kurosaki T. Ravetch J.V. Cell. 1997; 90: 293-301Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar) and in SHP-1-deficient mast cells derived from motheaten mice (26Ono M. Bolland S. Tempst P. Ravetch J.V. Nature. 1996; 383: 263-266Crossref PubMed Scopus (644) Google Scholar, 32Fong D.C. Malbec O. Arock M. Cambier J.C. Fridman W.H. Daëron M. Immunol. Lett. 1996; 54: 83-91Crossref PubMed Scopus (112) Google Scholar). These results altogether generated some confusion, and whether FcγRIIB indeed recruit SHP-1in 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 thein vitro binding of phosphatases to ITIM peptides and thein vivo recruitment of phosphatases by FcγRIIB.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 FcγRIIB in mast cells and in B cells. We failed to detect SHP-1 recruitment by FcγRIIB in vivo when coaggregated either with FcεRI in mast cells, or with BCR in B cells. We found that the in vitro binding of SHP-1 required a higher level of FcγRIIB 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 FcγRIIB phosphorylation than SHIP1 recruitment. The level of FcγRIIB phosphorylation that enabled the recruitment of SHP-1 was reached after treating cells with pervanadate, but not following coaggregation of FcγRIIB with BCR or FcεRI, in B cells and mast cells, respectively.DISCUSSIONWe show here that murine FcγRIIB recruit the inositol 5-phosphatase SHIP1, but not the protein-tyrosine phosphatase SHP-1in vivo, although the FcγRIIB ITIM has an affinity for both phosphatases in vitro, because the binding of SHP-1 requires a higher degree of FcγRIIB 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 FcγRIIB precipitated from cells treated with low concentrations of pervanadate, and 4) in vivo, when FcγRIIB was phosphorylated following coaggregation with BCR or FcεRI, in B cells and in mast cells, respectively. Our results suggest that, depending on their level of phosphorylation, FcγRIIB could potentially use the two phosphatases, with different consequences.Evidence that, when tyrosyl phosphorylated, the FcγRIIB ITIM has an affinity for SH2 domain-containing phosphatases was first provided in 1995 by D'Ambrosio et al. (30D'Ambrosio D. Hippen K.H. Minskoff S.A. Mellman I. Pani G. Siminovitch K.A. Cambier J.C. Science. 1995; 268: 293-296Crossref PubMed Scopus (507) Google Scholar) who demonstrated that phosphorylated synthetic peptides containing the FcγRIIB 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 (28Vély F. Olivero S. Olcese L. Moretta A. Damen J.E. Liu L. Krystal G. Cambier J.C. Daëron M. Vivier E. Eur. J. Immunol. 1997; 27: 1994-2000Crossref PubMed Scopus (122) Google Scholar) and SHIP1 (26Ono M. Bolland S. Tempst P. Ravetch J.V. Nature. 1996; 383: 263-266Crossref PubMed Scopus (644) Google Scholar). Similar experiments confirmed D'Ambrosio's results (28Vély F. Olivero S. Olcese L. Moretta A. Damen J.E. Liu L. Krystal G. Cambier J.C. Daëron M. Vivier E. Eur. J. Immunol. 1997; 27: 1994-2000Crossref PubMed Scopus (122) Google Scholar, 32Fong D.C. Malbec O. Arock M. Cambier J.C. Fridman W.H. Daëron M. Immunol. Lett. 1996; 54: 83-91Crossref PubMed Scopus (112) Google Scholar). SHIP2, a second SH2 domain-containing inositol phosphatase was recently found to bind also to phosphorylated FcγRIIB ITIM (27Muraille E. Bruhns P. Pesesse X. Daëron M. Erneux C. Immunol. Lett. 2000; 72: 7-15Crossref PubMed Scopus (52) Google Scholar, 29Bruhns P. Vély F. Malbec O. Fridman W.H. Vivier E. Daëron M. J. Biol. Chem. 2000; 276: 37357-37664Abstract Full Text Full Text PDF Scopus (79) Google Scholar). It follows that phosphorylated FcγRIIB ITIM peptides can bind all four known SH2 domain-containing phosphatases in vitro.In the same 1995 paper, D'Ambrosio et al. (30D'Ambrosio D. Hippen K.H. Minskoff S.A. Mellman I. Pani G. Siminovitch K.A. Cambier J.C. Science. 1995; 268: 293-296Crossref PubMed Scopus (507) Google Scholar) reported that SHP-1 co-precipitated with FcγRIIB bearing an intact ITIM, following coaggregation with BCR in A20 and in IIA1.6 B cells reconstituted with FcγRIIB, and that FcγRIIB-dependent inhibition of B cell proliferation was impaired in B cells from SHP-1-deficient motheaten mice. In 1996, Ono et al. (26Ono M. Bolland S. Tempst P. Ravetch J.V. Nature. 1996; 383: 263-266Crossref PubMed Scopus (644) Google Scholar) reported that SHIP1 co-precipitated with FcγRIIB following coaggregation with FcεRI in BMMCs or with BCR in A20 cells, and that FcγRIIB-dependent inhibition of IgE-induced serotonin release was unaffected in BMMCs derived from motheaten mice. Fonget al. (32Fong D.C. Malbec O. Arock M. Cambier J.C. Fridman W.H. Daëron M. Immunol. Lett. 1996; 54: 83-91Crossref PubMed Scopus (112) Google Scholar) reported that SHIP1, but not SHP-1 or SHP-2, co-precipitated with FcγRIIB following coaggregation with FcεRI in BMMCs. In 1997, Ono et al. (33Ono M. Okada H. Bolland S. Yanagi S. Kurosaki T. Ravetch J.V. Cell. 1997; 90: 293-301Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar) showed that FcγRIIB-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 FcγRIIB following coaggregation with BCR in A20 cells. In 1998, however, Sato et al. (31Sato K. Ochi A. J. Immunol. 1998; 161: 2716-2722PubMed Google Scholar) observed the co-precipitation of both SHIP1 and SHP-1 with FcγRIIB in A20 cells expressing an anti-TNP BCR following coaggregation with intact anti-idiotypic antibodies. Contrasting with the consensus that FcγRIIB 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 FcγRIIB 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 FcγRIIB1 stably expressed by transfecting the same cDNA into the rat mast cells RBL-2H3, and into the two FcγRIIB-deficient mouse lymphoma B cells IIA1.6 and K46μ. The coaggregation of FcγRIIB1 with FcεRI or with BCR induced a comparable tyrosyl phosphorylation of FcγRIIB1 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 FcγRIIB in vivo was lost. Whatever the explanation, we observed no difference between the three cells examined in which FcγRIIB preferentially, if not exclusively, recruited SHIP1. There is therefore a discrepancy between the ability of SHP-1 and SHIP1 to bind in vitro to FcγRIIB pITIM peptides and to co-precipitate with phosphorylated FcγRIIBin 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 FcγRIIB 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 FcγRIIB 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 FcγRIIB 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 (43Famiglietti S. Nakamura K. Cambier J.C. Immunol. Lett. 1999; 68: 35-40Crossref PubMed Scopus (35) Google Scholar). Results obtained in these two sets of experiments are reminiscent of the selective in vivorecruitment of SHIP1 by FcγRIIB.Based on data previously reported by others (43Famiglietti S. Nakamura K. Cambier J.C. Immunol. Lett. 1999; 68: 35-40Crossref PubMed Scopus (35) Google Scholar, 44Pei D. Wang J. Walsh C.T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1141-1145Crossref PubMed Scopus (127) Google Scholar), 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 thein 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-2in vivo, when kept in its original context, failed to recruit this phosphatase, when transposed in the intracytoplasmic domain of FcγRIIB1 (29Bruhns P. Vély F. Malbec O. Fridman W.H. Vivier E. Daëron M. J. Biol. Chem. 2000; 276: 37357-37664Abstract Full Text Full Text PDF Scopus (79) Google Scholar). To answer this question, we compared the ability of GST fusion proteins containing the FcγRIIB ITIM only or the whole intracytoplasmic domain of FcγRIIB1′ 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 FcγRIIB could bind SHP-1 when high enough phosphorylated.Based on the latter result, we searched for experimental conditions that would induce a FcγRIIB 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 FcγRIIB, at various concentrations, in mast cells and in B cells. We found that indeed, the phosphorylation of FcγRIIB varied with the concentrations of antigen and antibody in immune complexes but that ligands which induced a maximal phosphorylation of FcγRIIB 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 FcγRIIB become tyrosyl phosphorylated in vivo upon coaggregation with ITAM-bearing receptors by physiological ligands. If so, we wondered whether FcγRIIB 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 FcγRIIB phosphorylation than coaggregation with FcεRI or BCR, respectively, and under these conditions, not only SHIP1 but also SHP-1 co-precipitated with phosphorylated FcγRIIB. This observation indicates that, in resting cells, FcγRIIB are tyrosyl phosphorylated but that protein-tyrosine phosphatases maintain this phosphorylation below the detection level. This implies that FcγRIIB 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. FcγRIIB phosphorylation observed following their coaggregation with ITAM-bearing receptors results from the additional effect of a Src kinase, brought by activating receptors (13Malbec O. Fong D. Turner M. Tybulewicz V.L.J. Cambier J., C. Fridman W.H. Daëron M. J. Immunol. 1998; 160: 1647-1658PubMed Google Scholar), 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 FcγRIIB 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 FcγRIIB 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 intrans of the two SHP-1 SH2 domains. Supporting this interpretation, the recruitment of SHP-1 was lost before that of SHIP1 when pervanadate-induced FcγRIIB phosphorylation decreased following treatment of cells with decreasing concentrations of pervanadate. Alternatively, the recruitment of SHP-1 after pervanadate treatment may" @default.
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• W1977619443 date "2001-03-01" @default.
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• W1977619443 title "Insufficient Phosphorylation Prevents FcγRIIB from Recruiting the SH2 Domain-containing Protein-tyrosine Phosphatase SHP-1" @default.
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