The Negative Signaling Molecule SH2 Domain-containing Inositol-Polyphosphate 5-Phosphatase (SHIP) Binds to the Tyrosine-phosphorylated β Subunit of the High Affinity IgE Receptor

The SH2 domain-containing inositol-polyphosphate 5-phosphatase, SHIP, associates with FcγRIIB and negatively regulates both B-cell and mast cell function. We report here that SHIP was tyrosine-phosphorylated after high affinity IgE receptor (FcεRI) aggregation in rat basophilic leukemia RBL-2H3 cells. The tyrosine phosphorylation of SHIP was an early event after receptor aggregation and was present in cells deficient in the protein-tyrosine kinase Syk. Furthermore it was not secondary to the increase of intracellular calcium or the activation of protein kinase C. SHIP was precipitated by immobilized phosphorylated synthetic peptides based on the immunoreceptor tyrosine-based activation motif (ITAM) of the β but not the γ subunit of the high affinity IgE receptor. Tyrosine phosphorylation of SHIP and its association with the tyrosine-phosphorylated β subunit of FcεRI could play an important role in down-regulating receptor-mediated signal transduction in mast cells. Thus, whereas the activation molecule Syk associates with the γ subunit ITAM, the β subunit ITAM binds the negative signaling molecule SHIP. Therefore, unlike B cells where the antigen receptor and coreceptors such as FcγRIIB or CD22 each recruits molecules with opposite effects, the FcεRI contains subunits which recruit molecules that activate and inhibit signal transduction. The SH2 domain-containing inositol-polyphosphate 5-phosphatase, SHIP, associates with FcγRIIB and negatively regulates both B-cell and mast cell function. We report here that SHIP was tyrosine-phosphorylated after high affinity IgE receptor (FcεRI) aggregation in rat basophilic leukemia RBL-2H3 cells. The tyrosine phosphorylation of SHIP was an early event after receptor aggregation and was present in cells deficient in the protein-tyrosine kinase Syk. Furthermore it was not secondary to the increase of intracellular calcium or the activation of protein kinase C. SHIP was precipitated by immobilized phosphorylated synthetic peptides based on the immunoreceptor tyrosine-based activation motif (ITAM) of the β but not the γ subunit of the high affinity IgE receptor. Tyrosine phosphorylation of SHIP and its association with the tyrosine-phosphorylated β subunit of FcεRI could play an important role in down-regulating receptor-mediated signal transduction in mast cells. Thus, whereas the activation molecule Syk associates with the γ subunit ITAM, the β subunit ITAM binds the negative signaling molecule SHIP. Therefore, unlike B cells where the antigen receptor and coreceptors such as FcγRIIB or CD22 each recruits molecules with opposite effects, the FcεRI contains subunits which recruit molecules that activate and inhibit signal transduction. Aggregation of the high affinity IgE receptors (FcεRI) 1The abbreviations used are: FcεRI, high affinity IgE receptor; ITAM, immunoreceptor tyrosine-based activation motif; SHIP, SH2 domain-containing inositol-polyphosphate 5-phosphatase; PMA, phorbol 12-myristate 13-acetate; PAGE, polyacrylamide gel electrophoresis. on basophils and mast cells initiates a cascade of events that results in the release of inflammatory mediators. This pathway involves the activation of several protein-tyrosine kinases including Lyn, Syk, Btk, and Fak that induce the tyrosine phosphorylation of various proteins (1Benhamou M. Gutkind J.S. Robbins K.C. Siraganian R.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5327-5330Google Scholar, 2Benhamou M. Siraganian R.P. Immunol. Today. 1992; 13: 195-197Google Scholar, 3Hamawy M.M. Mergenhagen S.E. Siraganian R.P. Cell. Signalling. 1995; 7: 535-544Google Scholar). This results in the stimulation of phospholipase A2, C, and D, mobilization of Ca2+ from intracellular and extracellular sources, and activation of serine and threonine kinases (4Beaven M.A. Metzger H. Immunol. Today. 1993; 14: 222-226Google Scholar, 5Siraganian R.P. Middleton E.J. Reed C.E. Ellis E.F. Adkinson N.F.J. Yunginger J.W. Busse W.W. Allergy. Principles and Practice. Mosby-Year Book, Inc., St. Louis, MO1993: 105-134Google Scholar). There is also activation of the Ras pathway that may be important for the release of arachidonic acid and its metabolites (6Rider L.G. Hirasawa N. Santini F. Beaven M.A. J. Immunol. 1996; 157: 2374-2380Google Scholar). In the Ras pathway, best elucidated in non-mast cells, stimulation of growth factor receptors or antigen receptors on T and B cells results in the tyrosine phosphorylation of Shc and the formation of a complex containing Shc, the Grb2 adapter protein, and the nucleotide exchange factor Sos (7Pronk G.J. De Vries-Smits A.M.M. Buday L. Downward J. Maassen J.A. Medema R.H. Bos J.L. Mol. Cell. Biol. 1994; 14: 1575-1581Google Scholar,8Aronheim A. Engelberg D. Li N. Al-Alawi N. Schlessinger J. Karin M. Cell. 1994; 78: 949-961Google Scholar). This complex activates Ras which then, by a pathway that involves Raf1, induces phosphorylation and stimulation of mitogen-activated protein kinase (9Meyer S. LaBudda K. McGlade J. Hayman M.J. Mol. Cell. Biol. 1994; 14: 3253-3262Google Scholar, 10Cazaubon S.M. Ramos-Morales F. Fischer S. Schweighoffer F. Strosberg A.D. Couraud P. J. Biol. Chem. 1994; 269: 24805-24809Google Scholar). A 145-kDa tyrosine-phosphorylated protein is present in association with Shc and Grb2 (11Kavanaugh W.M. Williams L.T. Science. 1994; 266: 1862-1865Google Scholar, 12Liu L. Damen J.E. Cutler R.L. Krystal G. Mol. Cell. Biol. 1994; 14: 6926-6935Google Scholar). Recently this 145-kDa tyrosine-phosphorylated protein was found to be an SH2 domain-containing inositol-polyphosphate 5-phosphatase, which has been called SHIP (13Damen J.E. Liu L. Rosten P. Humphries R.K. Jefferson A.B. Majerus P.W. Krystal G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1689-1693Google Scholar). In addition to the SH2 domain, SHIP has several tyrosine phosphorylation sites that may interact with other SH2-containing proteins such as Shc and the Syk protein-tyrosine kinase (13Damen J.E. Liu L. Rosten P. Humphries R.K. Jefferson A.B. Majerus P.W. Krystal G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1689-1693Google Scholar, 14Crowley M.T. Harmer S.L. DeFranco A.L. J. Biol. Chem. 1996; 271: 1145-1152Google Scholar). The high affinity IgE receptor (FcεRI) on mast cells and basophils is a tetrameric structure composed of the IgE binding α chain, a β subunit, and disulfide-linked homodimeric γ chains (15Metzger H. Immunol. Rev. 1992; 125: 37-48Google Scholar). The COOH-terminal cytoplasmic domains of the β and the γ contain a motif with the amino acid sequence (D/E)X2YX2LX6–7YX2(L/I) that is critical for cell activation (16Alber G. Miller L. Jelsema C.L. Varin-Blank N. Metzger H. J. Biol. Chem. 1991; 266: 22613-22620Google Scholar, 17Reth M. Nature. 1989; 338: 383-384Google Scholar, 18Chan A.C. Desai D.M. Weiss A. Annu. Rev. Immunol. 1994; 12: 555-592Google Scholar). Thisimmunoreceptor tyrosine-basedactivation motif (ITAM) is also present in the ζ subunit of the T-cell receptor complex and in Igα and Igβ of the B-cell receptor and is important for cell activation (19Letourneur F. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8905-8909Google Scholar, 20Romeo C. Amiot M. Seed B. Cell. 1992; 68: 889-897Google Scholar, 21Irving B.A. Chan A.C. Weiss A. J. Exp. Med. 1993; 177: 1093-1103Google Scholar, 22Cambier J.C. Pleiman C.M. Clark M.R. Annu. Rev. Immunol. 1994; 12: 457-486Google Scholar, 23Gauen L.K.T. Zhu Y. Letourneur F. Hu Q. Bolen J.B. Matis L.A. Klausner R.D. Shaw A.S. Mol. Cell. Biol. 1994; 14: 3729-3741Google Scholar). In B cells and mast cells, cross-linking of ITAM-containing receptors to FcγRIIB results in down-regulatory signals (24Muta T. Kurosaki T. Misulovin Z. Sanchez M. Nussenzweig M.C. Ravetch J.V. Nature. 1994; 368: 70-73Google Scholar). The cytoplasmic domain of FcγRIIB contains the immunoreceptor tyrosine-based inhibitory motif that recruits negative regulatory molecules such as SHIP and protein-tyrosine phosphatase SHP-1 (25D'Ambrosio D. Hippen K.L. Minskoff S.A. Mellman I. Pani G. Siminovitch K.A. Cambier J.C. Science. 1995; 268: 293-297Google Scholar, 26Thomas M.L. J. Exp. Med. 1995; 181: 1953-1956Google Scholar, 27DeFranco A.L. Law D.A. Science. 1995; 268: 263-264Google Scholar). We report here evidence that SHIP is tyrosine-phosphorylated after IgE stimulation in rat basophilic leukemia cell line (RBL-2H3). This phosphorylation was an early event after receptor aggregation but was not secondary to the increase of intracellular calcium or to the activation of protein kinase C. Furthermore the phosphorylation of SHIP did not depend on protein kinase Syk. SHIP was precipitated by immobilized tyrosine-phosphorylated synthetic peptides based on the ITAM of the β but not the γ subunit of the high affinity IgE receptor. Tyrosine phosphorylation of SHIP and its association with the receptor may play an important role in down-regulating receptor-mediated signal transduction in mast cells. Protein A, aprotinin, and Triton X-100 were from Sigma. GammaBind plus Sepharose 4B was from Pharmacia Biotech Inc. Streptavidin coupled to agarose beads was from Pierce. The materials for electrophoresis were purchased from Novex (San Diego, CA), and the source of other materials was as described previously (28Kimura T. Sakamoto H. Appella E. Siraganian R.P. Mol. Cell. Biol. 1996; 16: 1471-1478Google Scholar). The non-phosphorylated and phosphorylated peptides based on the sequences of the β and γ subunits of the rat FcεRI (15Metzger H. Immunol. Rev. 1992; 125: 37-48Google Scholar) have been described previously (28Kimura T. Sakamoto H. Appella E. Siraganian R.P. Mol. Cell. Biol. 1996; 16: 1471-1478Google Scholar, 29Kimura T. Kihara H. Bhattacharyya S. Sakamoto H. Appella E. Siraganian R.P. J. Biol. Chem. 1996; 271: 27962-27968Google Scholar). The sequence of the FcεRIβ peptide was KVPDDRLYEELHVYSPIYSALEDTR, and that of the FcεRIγ peptide was REKSDAVYTGLNTRNQETYETLKHEK. Some of the peptides were also biotinylated as described previously (29Kimura T. Kihara H. Bhattacharyya S. Sakamoto H. Appella E. Siraganian R.P. J. Biol. Chem. 1996; 271: 27962-27968Google Scholar). Mouse monoclonal anti-Grb2 antibody, mouse monoclonal anti-Shc antibody (S14620), and rabbit polyclonal anti-Shc antibody (S14630) were from Transduction Laboratories (Lexington, KY). Rabbit polyclonal anti-Shc antibody (06-203) was from Upstate Biotechnology Inc. (Lake Placid, NY). Rabbit polyclonal anti-SHIP was kindly provided by Dr. Gerald Krystal (Terry Fox Laboratory, Vancouver, Canada) (13Damen J.E. Liu L. Rosten P. Humphries R.K. Jefferson A.B. Majerus P.W. Krystal G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1689-1693Google Scholar). The affinity-purified polyclonal rabbit anti-phosphotyrosine antibodies were coupled to Sepharose 4B beads as recommended by the manufacturer. All other antibodies have been described previously (28Kimura T. Sakamoto H. Appella E. Siraganian R.P. Mol. Cell. Biol. 1996; 16: 1471-1478Google Scholar, 29Kimura T. Kihara H. Bhattacharyya S. Sakamoto H. Appella E. Siraganian R.P. J. Biol. Chem. 1996; 271: 27962-27968Google Scholar, 30Benhamou M. Ryba N.J.P. Kihara H. Nishikata H. Siraganian R.P. J. Biol. Chem. 1993; 268: 23318-23324Google Scholar, 31Basciano L.K. Berenstein E.H. Kmak L. Siraganian R.P. J. Biol. Chem. 1986; 261: 11823-11831Google Scholar). The RBL-2H3 cells were maintained as monolayer cultures in Eagle's minimum essential medium (BioWhittaker, Walkersville, MD) supplemented with 15% heat-inactivated fetal bovine serum (Life Technologies, Inc.), penicillin, and streptomycin (1Benhamou M. Gutkind J.S. Robbins K.C. Siraganian R.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5327-5330Google Scholar, 32Barsumian E.L. Isersky C. Petrino M.G. Siraganian R.P. Eur. J. Immunol. 1981; 11: 317-323Google Scholar). The Syk negative variant of the RBL-2H3 was described previously (33Zhang J. Berenstein E.H. Evans R.L. Siraganian R.P. J. Exp. Med. 1996; 184: 71-79Google Scholar). RBL-2H3 cells were stimulated essentially as described previously (1Benhamou M. Gutkind J.S. Robbins K.C. Siraganian R.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5327-5330Google Scholar, 32Barsumian E.L. Isersky C. Petrino M.G. Siraganian R.P. Eur. J. Immunol. 1981; 11: 317-323Google Scholar). Briefly, for FcεRI-mediated activation cells were stimulated with either anti-FcεRIα monoclonal antibody (CA5) or with antigen after overnight culture with antigen-specific IgE. In some experiments cells were stimulated with either 0.5 μm calcium ionophore or 40 nm PMA as described previously (34Benhamou M. Stephan V. Robbins K.C. Siraganian R.P. J. Biol. Chem. 1992; 267: 7310-7314Google Scholar, 35Hamawy M.M. Mergenhagen S. Siraganian R.P. J. Biol. Chem. 1993; 268: 6851-6854Google Scholar). After stimulation, the monolayers were rinsed twice with ice-cold phosphate-buffered saline and solubilized by adding lysis buffer (10 mm Tris, pH 7.5, containing 1.0% Triton X-100, 1 mm Na3VO4, 150 mm NaCl, 50 mg/ml leupeptin, 0.5 unit/ml aprotinin, 2 mm pepstatin A, 1 mm phenylmethylsulfonyl fluoride). The plates were left on ice for 10 min. The cells were then scraped, and the supernatants were collected and centrifuged for 30 min at 16,000 × g at 4 °C as described previously (28Kimura T. Sakamoto H. Appella E. Siraganian R.P. Mol. Cell. Biol. 1996; 16: 1471-1478Google Scholar). In experiments designed to deplete extracellular Ca2+, the monolayers were washed with calcium-free Eagle's minimal essential medium containing 10 mm EDTA and stimulated in this medium (34Benhamou M. Stephan V. Robbins K.C. Siraganian R.P. J. Biol. Chem. 1992; 267: 7310-7314Google Scholar). For immunoprecipitation, lysates from 107cells in 1.0 ml were precleared by mixing for 90 min at 4 °C with protein A-agarose beads or GammaBind plus Sepharose 4B beads. The lysates were then incubated with each antibody that had been with of beads. After at 4 °C for 90 the beads were washed with buffer buffer with Triton X-100 to with 150 mm NaCl, 50 mm Tris, pH and the proteins by for min with buffer as described previously (28Kimura T. Sakamoto H. Appella E. Siraganian R.P. Mol. Cell. Biol. 1996; 16: 1471-1478Google Scholar). For with ITAM lysates from × cells in 1.0 ml were precleared by mixing for 90 min at 4 °C with coupled to agarose beads and then were incubated with biotinylated ITAM peptides that had been with ml of beads. The beads were then washed as described from the were by and to proteins were with the monoclonal antibody to as described previously V. M. Gutkind J.S. Robbins K.C. Siraganian R.P. J. Biol. Chem. 1992; 267: Scholar). proteins were with antibodies and then protein A, or In proteins were the In some experiments antibodies were from the to the of the and then the were with other the high affinity IgE receptor on mast cells results in activation of protein-tyrosine kinases and in (1Benhamou M. Gutkind J.S. Robbins K.C. Siraganian R.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5327-5330Google Scholar, 2Benhamou M. Siraganian R.P. Immunol. Today. 1992; 13: 195-197Google Scholar, 3Hamawy M.M. Mergenhagen S.E. Siraganian R.P. Cell. Signalling. 1995; 7: 535-544Google Scholar). We the described SHIP protein was tyrosine-phosphorylated after receptor and SHIP was present in RBL-2H3 cells There were proteins of and by the anti-SHIP The tyrosine phosphorylation of the 145-kDa SHIP protein was after receptor There was also the tyrosine phosphorylation of the were FcεRI was stimulated by the cells with antigen-specific IgE and then adding antigen has been the association of SHIP with Shc and Grb2 not the tyrosine phosphorylation of SHIP and its with other Shc was with anti-Shc antibody from or stimulated cells previously Shc was tyrosine-phosphorylated stimulation, and were in its tyrosine phosphorylation after receptor aggregation H. K. J. D.A. J. Biol. Chem. 1995; Scholar). SHIP was with Shc in anti-Shc to the the 145-kDa SHIP protein was tyrosine-phosphorylated in cells, and after receptor aggregation the increase in this phosphorylation was at min with increase to a at 30 min. stimulation was also the of the and SHIP Grb2 was also with Shc from both and stimulated cells. Grb2 did not and was in the of its association with Shc after receptor The SHIP with Shc was tyrosine-phosphorylated in cells 2 and In the of the tyrosine phosphatase was of SHIP with Shc was association of Grb2 with Therefore, SHIP in this mast cell line was tyrosine-phosphorylated in cells, and its tyrosine phosphorylation was by receptor This phosphorylation was important for its association with phosphorylation is for the association of SHIP with RBL-2H3 cells were stimulated with anti-FcεRIα monoclonal antibody for 30 min. The cell lysates were in the or of and with anti-Shc antibody of anti-Shc polyclonal antibody to protein A The were by with anti-phosphotyrosine antibody and with anti-SHIP polyclonal antibody anti-Shc polyclonal antibody and anti-Grb2 monoclonal antibody Some proteins tyrosine-phosphorylated early after FcεRI aggregation whereas phosphorylated at after a in intracellular calcium after the activation of protein kinase M. Siraganian R.P. Immunol. Today. 1992; 13: 195-197Google Scholar). of cells with either IgE and antigen or with anti-FcεRIα antibodies in an increase in SHIP tyrosine phosphorylation 4 was increase in SHIP tyrosine phosphorylation after activation of protein kinase by the addition of PMA or by the addition of the calcium ionophore the role of Ca2+ in FcεRI-mediated tyrosine phosphorylation of SHIP, cells were stimulated in a medium containing EDTA The of extracellular not FcεRI-mediated tyrosine phosphorylation of SHIP. Therefore, the tyrosine phosphorylation of SHIP is an early receptor-mediated event that is of the in the intracellular calcium the activation of protein kinase phosphorylation of SHIP did not calcium in the RBL-2H3 cells were stimulated with anti-FcεRIα monoclonal antibody for 30 min in the or of calcium in the were then with anti-Shc antibodies and by with anti-phosphotyrosine antibody anti-SHIP polyclonal antibody anti-Shc polyclonal antibody and anti-Grb2 monoclonal antibody The protein-tyrosine kinase Syk is essential for FcεRI-mediated the β and γ subunits of FcεRI tyrosine-phosphorylated in a variant of the RBL-2H3 cells, release and phosphorylation of molecules such as phospholipase not (33Zhang J. Berenstein E.H. Evans R.L. Siraganian R.P. J. Exp. Med. 1996; 184: 71-79Google Scholar). release and phosphorylation of other were by the of Syk negative cells (33Zhang J. Berenstein E.H. Evans R.L. Siraganian R.P. J. Exp. Med. 1996; 184: 71-79Google Scholar). Syk negative cells were used to the tyrosine phosphorylation of SHIP were stimulated by receptor the calcium ionophore or PMA, and the lysates were then with anti-phosphotyrosine There was some tyrosine phosphorylation of SHIP in the Syk negative cells. After either or anti-FcεRIα antibody stimulation was tyrosine phosphorylation of SHIP In some experiments this was after with anti-FcεRIα results were SHIP was with anti-Shc antibody The addition of PMA in of SHIP and a in its with Therefore, both the and the tyrosine phosphorylation of SHIP of the of Syk in cells. the receptor-mediated phosphorylation of SHIP was an early event of Syk. In the association of Shc with tyrosine-phosphorylated peptides based on the ITAM of the β subunit of FcεRI (29Kimura T. Kihara H. Bhattacharyya S. Sakamoto H. Appella E. Siraganian R.P. J. Biol. Chem. 1996; 271: 27962-27968Google Scholar). SHIP with Shc, was an association of SHIP with phosphorylated and non-phosphorylated peptides based on the ITAM of the β and γ subunits of FcεRI were used for SHIP was precipitated by the phosphorylated peptide based on the ITAM of The also that the ITAM peptide was of SHIP with in in the of SHIP with the ITAM synthetic the proteins precipitated with anti-Shc or anti-SHIP antibodies with precipitated by the tyrosine-phosphorylated β ITAM peptide antibodies precipitated and the 145-kDa was the tyrosine phosphorylated in cells This 145-kDa of SHIP was the with In the ITAM precipitated the of SHIP Therefore, the SHIP with Shc was tyrosine whereas the β ITAM precipitated both non-phosphorylated and phosphorylated SHIP. that the of SHIP by the ITAM is to of SHIP by anti-SHIP or anti-Shc antibodies to that precipitated by phosphorylated synthetic β ITAM from × RBL-2H3 cells were precipitated with anti-Shc or anti-SHIP polyclonal antibodies with of biotinylated phosphorylated ITAM of FcεRIβ peptide or with anti-Shc polyclonal antibody The were by with anti-phosphotyrosine antibody by anti-SHIP polyclonal antibody experiments SHIP as of the that is tyrosine-phosphorylated after FcεRI This tyrosine phosphorylation after receptor stimulation but not cells were with either calcium ionophore or with Therefore, tyrosine phosphorylation of SHIP was not secondary to the increase of intracellular calcium or the activation of protein kinase C. the phosphorylation of SHIP was present in Syk negative cells that this is an early event of the activation of Syk. SHIP with the formation of by a that (13Damen J.E. Liu L. Rosten P. Humphries R.K. Jefferson A.B. Majerus P.W. Krystal G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1689-1693Google Scholar). also the of to the release of intracellular Ca2+, which results in activation of the calcium and the of Ca2+ the cell M. Nature. 1992; Scholar, C. M. J. Biol. Chem. 1993; 268: Scholar). In mast cells, receptor aggregation activates phospholipase and phospholipase which results in the formation of that release Ca2+ from intracellular This is by an of Ca2+ from extracellular by the Therefore, by the of SHIP calcium The tyrosine phosphorylation of SHIP not its (13Damen J.E. Liu L. Rosten P. Humphries R.K. Jefferson A.B. Majerus P.W. Krystal G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1689-1693Google Scholar). tyrosine phosphorylation of SHIP and other proteins could in and in the of SHIP to sites where it could play important Thus, the association of SHIP with Shc and with the tyrosine-phosphorylated β subunit of FcεRI it to sites where is activation of signaling The pathway after FcεRI aggregation results in the activation of mitogen-activated protein which is important for the release of arachidonic acid and in events that in the of (6Rider L.G. Hirasawa N. Santini F. Beaven M.A. J. Immunol. 1996; 157: 2374-2380Google Scholar, A. Engelberg D. Li N. Al-Alawi N. Schlessinger J. Karin M. Cell. 1994; 78: 949-961Google Scholar, N. Santini F. Beaven M.A. J. Immunol. 1995; Scholar, S. E. G. J. Immunol. 1994; Scholar). In the of antigen receptors on T and B cells, activation of this pathway is by the tyrosine phosphorylation of Shc which then a complex with and activates (7Pronk G.J. De Vries-Smits A.M.M. Buday L. Downward J. Maassen J.A. Medema R.H. Bos J.L. Mol. Cell. Biol. 1994; 14: 1575-1581Google Scholar, A. Engelberg D. Li N. Al-Alawi N. Schlessinger J. Karin M. Cell. 1994; 78: 949-961Google Scholar). Shc is tyrosine-phosphorylated in RBL-2H3 cells, and this phosphorylation may be after FcεRI aggregation H. K. J. D.A. J. Biol. Chem. 1995; Scholar). Shc also associates with the tyrosine-phosphorylated β subunit of FcεRI (29Kimura T. Kihara H. Bhattacharyya S. Sakamoto H. Appella E. Siraganian R.P. J. Biol. Chem. 1996; 271: 27962-27968Google Scholar). Therefore, the tyrosine-phosphorylated β subunit of FcεRI could SHIP either or by binding by The of the tyrosine phosphorylation of the β subunit SHIP was to the receptor and the of the signals by the receptor. In the present for mast cell the event after aggregation of FcεRI is the activation of protein-tyrosine Lyn, which results in tyrosine phosphorylation of the receptor subunits Nature. 1991; Scholar). The COOH-terminal cytoplasmic domain of FcεRIβ and the cytoplasmic domain of FcεRIγ contain that tyrosine-phosphorylated signaling molecules (29Kimura T. Kihara H. Bhattacharyya S. Sakamoto H. Appella E. Siraganian R.P. J. Biol. Chem. 1996; 271: 27962-27968Google Scholar). Thus, that a synthetic peptide based on the ITAM sequence of the β subunit of FcεRI precipitated Shc, phospholipase and Lyn, whereas the peptide based on the ITAM of γ did not molecules but was in binding Syk (29Kimura T. Kihara H. Bhattacharyya S. Sakamoto H. Appella E. Siraganian R.P. J. Biol. Chem. 1996; 271: 27962-27968Google Scholar). The binding of Syk to the tyrosine-phosphorylated ITAM results in a in Syk with an increase in its and the of signals such as the tyrosine phosphorylation of phospholipase phospholipase and the of calcium (28Kimura T. Sakamoto H. Appella E. Siraganian R.P. Mol. Cell. Biol. 1996; 16: 1471-1478Google Scholar). Therefore, tyrosine phosphorylation of the ITAM of the γ subunit recruits Syk, which is critical in In the ITAM based on the β subunit, it is recruits other molecules such as Shc and SHIP, which important for and signaling experiments were in that that SHIP may play a role in the negative of signaling in B cells or in mast cells S. Damen J.E. Liu L. Krystal G. J. Immunol. 1996; 157: Scholar, M. S. P. Ravetch J.V. Nature. 1996; Scholar). The tyrosine phosphorylation of SHIP the B-cell receptor was with FcγRIIB S. Damen J.E. Liu L. Krystal G. J. Immunol. 1996; 157: Scholar). results in signaling from the B-cell receptor. In mast cells, SHIP with the FcγRIIB that was with FcεRI M. S. P. Ravetch J.V. Nature. 1996; Scholar). that in a in FcεRI-mediated signaling M. S. E. P. S. 1995; Scholar). The of the receptor with FcγRIIB results in the tyrosine phosphorylation of the cytoplasmic domain of FcγRIIB and the of SHIP. In the present experiments found that in the β subunit of FcεRI has a domain that recruit SHIP to the receptor. SHIP binds to the amino acid sequence that is to the immunoreceptor tyrosine-based inhibitory motif present in FcγRIIB (24Muta T. Kurosaki T. Misulovin Z. Sanchez M. Nussenzweig M.C. Ravetch J.V. Nature. 1994; 368: 70-73Google Scholar, 26Thomas M.L. J. Exp. Med. 1995; 181: 1953-1956Google Scholar). unlike this motif in could not binding of the protein-tyrosine phosphatase SHP-1 to the ITAM H. E. and P. Therefore, unlike B cells where the antigen receptor and coreceptors such as FcγRIIB or CD22 each recruit molecules with opposite effects, the FcεRI contains subunits that recruit molecules that activate and inhibit signal transduction. We and Ryba for and for this We also and Berenstein for