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

(Received for publication, December 23, 1996, and in revised form, March 11, 1997)

Teruaki Kimura Dagger §, Hiroshi Sakamoto , Ettore Appella and Reuben P. Siraganian Dagger

From the Dagger  Laboratory of Immunology, NIDR and the  Laboratory of Cell Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892-1188

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The SH2 domain-containing inositol-polyphosphate 5-phosphatase, SHIP, associates with Fcgamma RIIB and negatively regulates both B-cell and mast cell function. We report here that SHIP was tyrosine-phosphorylated after high affinity IgE receptor (Fcepsilon RI) 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 beta  but not the gamma subunit of the high affinity IgE receptor. Tyrosine phosphorylation of SHIP and its association with the tyrosine-phosphorylated beta subunit of Fcepsilon RI could play an important role in down-regulating receptor-mediated signal transduction in mast cells. Thus, whereas the activation molecule Syk associates with the gamma  subunit ITAM, the beta  subunit ITAM binds the negative signaling molecule SHIP. Therefore, unlike B cells where the antigen receptor and coreceptors such as Fcgamma RIIB or CD22 each recruits molecules with opposite effects, the Fcepsilon RI contains subunits which recruit molecules that activate and inhibit signal transduction.


INTRODUCTION

Aggregation of the high affinity IgE receptors (Fcepsilon RI)1 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 (1-3). 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 (4, 5). There is also activation of the Ras pathway that may be important for the release of arachidonic acid and its metabolites (6). 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 (7, 8). This complex activates Ras which then, by a pathway that involves Raf1, induces phosphorylation and stimulation of mitogen-activated protein kinase (9, 10). A 145-kDa tyrosine-phosphorylated protein is present in association with Shc and Grb2 (11, 12). Recently this 145-kDa tyrosine-phosphorylated protein was found to be an SH2 domain-containing inositol-polyphosphate 5-phosphatase, which has been called SHIP (13). 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 (13, 14).

The high affinity IgE receptor (Fcepsilon RI) on mast cells and basophils is a tetrameric structure composed of the IgE binding alpha chain, a beta  subunit, and disulfide-linked homodimeric gamma  chains (15). The COOH-terminal cytoplasmic domains of the beta  and the gamma  contain a motif with the amino acid sequence (D/E)X2YX2LX6-7YX2(L/I) that is critical for cell activation (16-18). This immunoreceptor tyrosine-based activation motif (ITAM) is also present in the zeta  subunit of the T-cell receptor complex and in Igalpha and Igbeta of the B-cell receptor and is important for cell activation (19-23). In B cells and mast cells, cross-linking of ITAM-containing receptors to Fcgamma RIIB results in down-regulatory signals (24). The cytoplasmic domain of Fcgamma RIIB contains the immunoreceptor tyrosine-based inhibitory motif that recruits negative regulatory molecules such as SHIP and protein-tyrosine phosphatase SHP-1 (25-27).

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 beta  but not the gamma  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.


EXPERIMENTAL PROCEDURES

Materials

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 (28). The non-phosphorylated and phosphorylated peptides based on the sequences of the beta  and gamma  subunits of the rat Fcepsilon RI (15) have been described previously (28, 29). The sequence of the Fcepsilon RIbeta peptide was KVPDDRLYEELHVYSPIYSALEDTR, and that of the Fcepsilon RIgamma peptide was REKSDAVYTGLNTRNQETYETLKHEK. Some of the peptides were also biotinylated as described previously (29).

Antibodies

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) (13). 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 (28-31).

Cell Culture

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 (1, 32). The Syk negative variant of the RBL-2H3 was described previously (33).

Cell Activation and Preparation of Cell Lysates

RBL-2H3 cells were stimulated essentially as described previously (1, 32). Briefly, for Fcepsilon RI-mediated activation cells were stimulated with either anti-Fcepsilon RIalpha 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 (34, 35). 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 (28). 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 (34).

Immunoprecipitation and Precipitation with ITAM Peptides

For immunoprecipitation, lysates from 107 cells 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 preincubated with 20 µl of beads. After gentle rotation at 4 °C for 90 min, the beads were washed three times with wash buffer (lysis buffer with Triton X-100 concentration decreased to 0.5%), once with 150 mM NaCl, 50 mM Tris, pH 7.4, and the proteins eluted by boiling for 5 min with Laemmli's sample buffer as described previously (28). For precipitation with ITAM peptides, lysates from 1.5 × 107 cells in 1.0 ml were precleared by mixing for 90 min at 4 °C with streptavidin coupled to agarose beads and then were incubated with biotinylated ITAM peptides that had been preincubated with 20 ml of streptavidin beads. The beads were then washed as described above.

Immunoblotting

Samples from the precipitates were separated by SDS-PAGE under reducing conditions and electrotransferred to polyvinylidine difluoride membranes (Millipore, Bedford, MA). Tyrosine-phosphorylated proteins were detected with the monoclonal antibody PY-20 conjugated to horseradish peroxidase as described previously (36). Other proteins were immunoblotted with specific antibodies and then detected using horseradish peroxidase-conjugated protein A, donkey anti-rabbit IgG, or donkey anti-mouse IgG antibodies. In all blots, proteins were visualized using the enhanced chemiluminescence. In some experiments antibodies were stripped from the membranes according to the protocol of the manufacturer and then the membranes were reprobed with other antibodies.


RESULTS

Tyrosine Phosphorylation of Inositol Phosphatase SHIP after Fcepsilon RI Aggregation

Cross-linking the high affinity IgE receptor on mast cells results in activation of protein-tyrosine kinases and rapid changes in inositol phosphates (1-3). We therefore investigated whether the recently described SHIP protein was tyrosine-phosphorylated after receptor aggregation. By immunoblot and immunoprecipitation analysis, SHIP was present in RBL-2H3 cells (Fig. 1). There were proteins of 145, 140, and 105 kDa recognized by the anti-SHIP antibody. The low level tyrosine phosphorylation of the 145-kDa SHIP protein was dramatically enhanced after receptor aggregation. There was also the tyrosine phosphorylation of the 140-kDa protein. Similar data were obtained when Fcepsilon RI was stimulated by preincubating the cells with antigen-specific IgE and then adding antigen (see below). As has been reported previously, immunoblot analysis demonstrated the association of SHIP with Shc and Grb2 (data not shown).


Fig. 1. SHIP was tyrosine-phosphorylated by Fcepsilon RI aggregation. RBL-2H3 cells were either non-stimulated or stimulated with anti-Fcepsilon RIalpha monoclonal antibody for 30 min. Lysates were then immunoprecipitated with either 2 µg of anti-SHIP antibody or normal rabbit IgG prebound to 20 µl of protein A-agarose beads. The precipitates were washed, eluted by boiling, separated by SDS-PAGE on 12% gels, and analyzed by immunoblotting with anti-phosphotyrosine antibody (Anti-PY) followed by anti-SHIP antibody (Anti-SHIP).
[View Larger Version of this Image (31K GIF file)]

To further define the tyrosine phosphorylation of SHIP and its interaction with other molecules, Shc was immunoprecipitated with anti-Shc antibody from non-stimulated or stimulated cells (Fig. 2). As previously reported, Shc was already tyrosine-phosphorylated before stimulation, and there were no changes in its tyrosine phosphorylation after receptor aggregation (37). As expected, SHIP was associated with Shc in these anti-Shc immunoprecipitates. Similar to the previous results, the 145-kDa SHIP protein was tyrosine-phosphorylated in non-stimulated cells, and after receptor aggregation the increase in this phosphorylation was detectable at 3 min with further gradual increase to reach a maximum at 30 min. With stimulation there was also the coprecipitation of the 140- and 105-kDa SHIP proteins. Grb2 was also coimmunoprecipitated with Shc from both non-stimulated and stimulated cells. However, Grb2 did not get tyrosine-phosphorylated, and there was no detectable change in the extent of its association with Shc after receptor aggregation. The SHIP associated with Shc was already tyrosine-phosphorylated in non-stimulated cells (Figs. 2 and 3). In the absence of the tyrosine phosphatase inhibitor, vanadate, there was no coprecipitation of SHIP with Shc (Fig. 3). However, there was still association of Grb2 with Shc. Therefore, SHIP in this mast cell line was tyrosine-phosphorylated in non-stimulated cells, and its tyrosine phosphorylation was enhanced by receptor aggregation. This phosphorylation was important for its association with Shc.


Fig. 2. SHIP was coprecipitated with anti-Shc antibody and tyrosine-phosphorylated after Fcepsilon RI aggregation. RBL-2H3 cells were stimulated with anti-Fcepsilon RIalpha monoclonal antibody for the indicated times, and the cell lysates were immunoprecipitated with anti-Shc antibody (2 µg of anti-Shc monoclonal antibody prebound to 20 µl of rabbit anti-mouse IgG coupled to protein A-agarose beads). The precipitates were separated by SDS-PAGE on 12% gels. After transfer the membrane was blotted with anti-phosphotyrosine antibody (Anti-PY), stripped, cut into three parts, and immunoblotted with anti-SHIP polyclonal antibody (Anti-SHIP), anti-Shc polyclonal antibody (Anti-Shc), and anti-Grb2 monoclonal antibody (Anti-Grb2).
[View Larger Version of this Image (27K GIF file)]


Fig. 3. Tyrosine phosphorylation is required for the association of SHIP with Shc. RBL-2H3 cells were stimulated with anti-Fcepsilon RIalpha monoclonal antibody for 30 min. The cell lysates were prepared in the presence or absence of vanadate and immunoprecipitated with anti-Shc antibody (2 µg of anti-Shc polyclonal antibody prebound to protein A beads). The precipitates were analyzed by immunoblotting with anti-phosphotyrosine antibody (Anti-PY), stripped, cut into three parts, and immunoblotted with anti-SHIP polyclonal antibody (Anti-SHIP), anti-Shc polyclonal antibody (Anti-Shc), and anti-Grb2 monoclonal antibody (Anti-Grb2).
[View Larger Version of this Image (26K GIF file)]

Tyrosine Phosphorylation of SHIP Was a Receptor-mediated Event Not Induced by Calcium Ionophore or by PMA

Some proteins are tyrosine-phosphorylated very early after Fcepsilon RI aggregation whereas others are phosphorylated at later stages after a rise in intracellular calcium and/or after the activation of protein kinase C (2). Stimulation of cells with either IgE and antigen or with anti-Fcepsilon RIalpha antibodies resulted in an increase in SHIP tyrosine phosphorylation (Fig. 4A). However, there was no increase in SHIP tyrosine phosphorylation after direct activation of protein kinase C by the addition of PMA or by the addition of the calcium ionophore A23187. To further define the role of Ca2+ in Fcepsilon RI-mediated tyrosine phosphorylation of SHIP, cells were stimulated in a Ca2+-free medium containing EDTA (Fig. 5). The absence of extracellular Ca2+ did not affect Fcepsilon RI-mediated tyrosine phosphorylation of SHIP. Therefore, the tyrosine phosphorylation of SHIP is an early receptor-mediated event that is upstream of the rise in the intracellular calcium and/or the activation of protein kinase C. 


Fig. 4. Tyrosine phosphorylation of SHIP was a receptor-mediated event not induced by calcium ionophore or PMA and did not require Syk. Cells used were either RBL-2H3 (A) or the Syk negative TB1A2 (B, C) and were either non-stimulated or stimulated for 30 min with 0.5 µM calcium ionophore A23187 (A23187), 40 nM PMA (PMA), anti-Fcepsilon RIalpha monoclonal antibody (@Fcepsilon RI), or IgE antigen (Ag). Lysates were then immunoprecipitated with either anti-phosphotyrosine polyclonal antibody (A, B) or with anti-Shc polyclonal antibody (C). The immunoprecipitates were separated by SDS-PAGE (8%) and analyzed by immunoblotting with anti-SHIP antibody (Anti-SHIP) and anti-phosphotyrosine antibody (Anti-PY). Note that the lower percent PAGE of these gels results in better separation of the anti-SHIP immunoblotted 145- and 140-kDa proteins.
[View Larger Version of this Image (21K GIF file)]


Fig. 5. Tyrosine phosphorylation of SHIP did not require calcium in the medium. RBL-2H3 cells were stimulated with anti-Fcepsilon RIalpha monoclonal antibody for 30 min in the presence or absence of calcium in the medium. Lysates were then immunoprecipitated with anti-Shc antibodies and analyzed by immunoblotting with anti-phosphotyrosine antibody (Anti-PY), anti-SHIP polyclonal antibody (Anti-SHIP), anti-Shc polyclonal antibody (Anti-Shc), and anti-Grb2 monoclonal antibody (Anti-Grb2).
[View Larger Version of this Image (22K GIF file)]

Tyrosine Phosphorylation of SHIP Did Not Require Syk

The protein-tyrosine kinase Syk is essential for Fcepsilon RI-mediated degranulation. Although the beta  and gamma  subunits of Fcepsilon RI are tyrosine-phosphorylated in a Syk-deficient variant of the RBL-2H3 cells, histamine release and phosphorylation of downstream molecules such as phospholipase C are not observed (33). Histamine release and phosphorylation of other substrates were reconstituted by the transfection of Syk into these negative cells (33). These Syk negative cells were used to evaluate the tyrosine phosphorylation of SHIP (Fig. 4B). Cells were stimulated by receptor aggregation, the calcium ionophore A23187, or PMA, and the lysates were then immunoprecipitated with anti-phosphotyrosine antibody. There was some constitutive tyrosine phosphorylation of SHIP in the non-stimulated Syk negative cells. After either IgE-antigen or anti-Fcepsilon RIalpha antibody stimulation there was increased tyrosine phosphorylation of SHIP (Fig. 4B). In some experiments this was stronger after IgE-antigen than with anti-Fcepsilon RIalpha antibodies. Similar results were obtained when SHIP was coimmunoprecipitated with anti-Shc antibody (Fig. 4C). The addition of PMA resulted in dephosphorylation of SHIP and a decrease in its coprecipitation with Shc. Therefore, both the constitutive and the Fcepsilon RI-induced tyrosine phosphorylation of SHIP are independent of the presence of Syk in cells. Furthermore, the receptor-mediated phosphorylation of SHIP was an early event upstream of Syk.

Phosphorylated Peptides Based on the ITAM of Fcepsilon RIbeta but Not the ITAM of Fcepsilon RIgamma Precipitated SHIP from Lysates of RBL-2H3 Cells

In previous studies we observed the association of Shc with tyrosine-phosphorylated peptides based on the ITAM of the beta  subunit of Fcepsilon RI (29). As SHIP coprecipitates with Shc, we investigated whether there was an association of SHIP with Fcepsilon RI. Synthetic phosphorylated and non-phosphorylated peptides based on the ITAM of the beta  and gamma  subunits of Fcepsilon RI were used for precipitation studies (Fig. 6). SHIP was precipitated only by the phosphorylated peptide based on the ITAM of Fcepsilon RIbeta . The immunoblots also suggested that the beta -phosphorylated ITAM peptide was precipitating two isoforms of SHIP with slightly different characteristics in migration in SDS-PAGE. To further define the interaction of SHIP with the ITAM synthetic peptide, we compared the proteins precipitated with anti-Shc or anti-SHIP antibodies with those precipitated by the tyrosine-phosphorylated beta  ITAM peptide (Fig. 7). Anti-SHIP antibodies precipitated 145-, 140-, and 105-kDa proteins; the 145-kDa form was the most strongly tyrosine phosphorylated in these non-stimulated cells (Fig. 7A). This 145-kDa form of SHIP was the major form associated with Shc. In contrast, the beta -phosphorylated ITAM precipitated all the isoforms of SHIP (Fig. 7B). Therefore, the SHIP associated with Shc was predominantly tyrosine phosphorylated, whereas the beta  ITAM precipitated both non-phosphorylated and phosphorylated SHIP. Altogether these data strongly suggest that the precipitation of SHIP by the beta -phosphorylated ITAM is due to direct interaction.


Fig. 6. SHIP was precipitated by diphosphorylated synthetic peptide based on the ITAM of the beta  subunit of Fcepsilon RI. RBL-2H3 cells were either non-stimulated or stimulated with anti-Fcepsilon RIalpha monoclonal antibody, and lysates from 1.5 × 107 were then incubated for 90 min at 4 °C with 1 nmol of the different biotinylated ITAM peptides that had been prebound to 20 µl of streptavidin beads. For controls streptavidin beads were used without any peptide. The precipitates were washed, eluted by boiling, and separated by SDS-PAGE on 10% gels. After transfer the membrane was blotted with anti-SHIP polyclonal antibody (Anti-SHIP). In the nomenclature used here beta  refers to Fcepsilon RIbeta and gamma  to Fcepsilon RIgamma . The beta YY is unphosphorylated, whereas beta PP is the diphosphorylated synthetic peptide based on the ITAM of Fcepsilon RIbeta . Similarly, for the ITAM based on the gamma  subunit gamma YY is the unphosphorylated peptide, gamma PP is the same peptide with both tyrosines phosphorylated, and gamma PY and gamma YP are monophosphorylated peptides with phosphorylation of either the first or second tyrosine.
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Fig. 7. Comparison of SHIP immunoprecipitated by anti-SHIP or anti-Shc antibodies to that precipitated by phosphorylated synthetic beta  ITAM peptide. Lysates from 1.5 × 107 non-stimulated RBL-2H3 cells were precipitated with anti-Shc or anti-SHIP polyclonal antibodies (A), with 3 nmol of biotinylated phosphorylated ITAM of Fcepsilon RIbeta peptide (beta PP), or with anti-Shc polyclonal antibody (B). The precipitates were analyzed by immunoblotting with anti-phosphotyrosine antibody (Anti-PY) followed by anti-SHIP polyclonal antibody (Anti-SHIP).
[View Larger Version of this Image (23K GIF file)]


DISCUSSION

These experiments identified SHIP as one of the substrates that is tyrosine-phosphorylated after Fcepsilon RI aggregation. This tyrosine phosphorylation occurred after receptor stimulation but not when cells were activated with either calcium ionophore or with PMA. Therefore, tyrosine phosphorylation of SHIP was not secondary to the increase of intracellular calcium or the activation of protein kinase C. Interestingly, the phosphorylation of SHIP was present even in Syk negative cells indicating that this is an early event upstream of the activation of Syk.

SHIP hydrolyzes inositol 1,3,4,5-tetraphosphate with the formation of inositol 1,3,4-trisphosphate by a reaction that requires Mg2+ (13). It also catalyzes the hydrolysis of phosphatidylinositol 3,4,5-trisphosphate to phosphatidylinositol-3,4-bisphosphate. Inositol 1,3,4,5-tetraphosphate stimulates the release of intracellular Ca2+, which results in activation of the calcium release-activated Ca2+ channel, Icrac, and the influx of Ca2+ into the cell (38, 39). In mast cells, receptor aggregation activates phospholipase Cgamma 1 and phospholipase Cgamma 2, which results in the formation of inositol phosphates that release Ca2+ from intracellular sources. This is followed by an influx of Ca2+ from extracellular sources mediated by the Icrac channel. Therefore, by decreasing the concentration of inositol 1,3,4,5-tetraphosphate SHIP would limit calcium influx.

The tyrosine phosphorylation of SHIP does not enhance its enzymatic activity (13). However, tyrosine phosphorylation of SHIP and other proteins could result in SH2-mediated interactions and changes in the cellular localization of SHIP to sites where it could play important physiological roles. Thus, the association of SHIP with Shc and with the tyrosine-phosphorylated beta  subunit of Fcepsilon RI would localize it to membrane sites where there is activation of signaling pathways.

The Ras-signaling pathway after Fcepsilon RI aggregation results in the activation of mitogen-activated protein kinase, which is important for the release of arachidonic acid and in regulating nuclear events that result in the synthesis of cytokines (6, 8, 40, 41). In the case of antigen receptors on T and B cells, activation of this pathway is initiated by the tyrosine phosphorylation of Shc which then forms a complex with Grb2-Sos and activates p21ras (7, 8). However, Shc is constitutively tyrosine-phosphorylated in non-stimulated RBL-2H3 cells, and this phosphorylation may be increased after Fcepsilon RI aggregation (37). Shc also associates with the tyrosine-phosphorylated beta  subunit of Fcepsilon RI (29). Therefore, the tyrosine-phosphorylated beta  subunit of Fcepsilon RI could bind SHIP either directly or indirectly by binding mediated by Shc. The extent of the tyrosine phosphorylation of the beta  subunit would determine how much SHIP was recruited to the receptor and thereby regulate and/or limit the extent of the signals generated by the receptor.

In the present model for mast cell signaling, the earliest event after aggregation of Fcepsilon RI is the activation of protein-tyrosine kinase, probably Lyn, which results in tyrosine phosphorylation of the receptor subunits (42). The COOH-terminal cytoplasmic domain of Fcepsilon RIbeta and the cytoplasmic domain of Fcepsilon RIgamma contain ITAMs that once they are tyrosine-phosphorylated preferentially bind different downstream signaling molecules (29). Thus, we recently observed that a synthetic tyrosine-diphosphorylated peptide based on the ITAM sequence of the beta  subunit of Fcepsilon RI precipitated Shc, phospholipase Cgamma 1, and Lyn, whereas the similar peptide based on the ITAM of gamma  did not bind these molecules but was very effective in binding Syk (29). The binding of Syk to the tyrosine-phosphorylated ITAM results in a conformational change in Syk with an increase in its enzymatic activity and the downstream propagation of signals such as the tyrosine phosphorylation of phospholipase Cgamma 1, phospholipase Cgamma 2, and the influx of calcium (28). Therefore, tyrosine phosphorylation of the ITAM of the gamma  subunit recruits Syk, which is critical in downstream activating signals. In contrast the ITAM based on the beta  subunit, once it is phosphorylated, recruits other molecules such as Shc and SHIP, which are important for activating and regulating signaling events.

While these experiments were in progress two reports appeared that suggest that SHIP may play a role in the negative regulation of signaling in B cells or in mast cells (43, 44). The optimal tyrosine phosphorylation of SHIP occurred when the B-cell receptor was coclustered with Fcgamma RIIB (43). Such coclustering results in decreased signaling from the B-cell receptor. In mast cells, SHIP associated with the Fcgamma RIIB that was coclustered with Fcepsilon RI (44). These are conditions that resulted in a decrease in Fcepsilon RI-mediated signaling (45). The coclustering of the immune receptor with Fcgamma RIIB probably results in the tyrosine phosphorylation of the cytoplasmic domain of Fcgamma RIIB and the recruitment of SHIP. In the present experiments we found that in fact the beta  subunit of Fcepsilon RI has a domain that can recruit SHIP to the receptor. SHIP most probably binds to the amino acid sequence SPIYSAL that is similar to the (T/S)XXYXX(L/I) immunoreceptor tyrosine-based inhibitory motif present in Fcgamma RIIB (24, 26). However, unlike this inhibiting motif in Fcgamma RIIB, we could not detect any binding of the protein-tyrosine phosphatase SHP-1 to the beta -phosphorylated ITAM peptide.2 Therefore, unlike B cells where the antigen receptor and coreceptors such as Fcgamma RIIB or CD22 each recruit molecules with opposite effects, the Fcepsilon RI contains subunits that recruit molecules that activate and inhibit signal transduction.


FOOTNOTES

*   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.
§   To whom correspondence should be addressed: Laboratory of Immunology, Bldg. 10, Rm. 1N106, NIDR, NIH, Bethesda, MD 20892-1188. Tel.: 301-496-5105; Fax: 301-480-8328; E-mail: tk51w{at}nih.gov.
1   The abbreviations used are: Fcepsilon RI, 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.
2   T. Kimura, H. Sakamoto, E. Apella, and R. P. Siraganian, unpublished observations.

ACKNOWLEDGEMENTS

We thank Drs. Mark Swieter and Nicholas Ryba for helpful discussions and for reviewing this manuscript. We also thank Greta Bader and Elsa Berenstein for excellent technical help.


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