Downstream signaling molecules bind to different phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) peptides of the high affinity IgE receptor.

The cytoplasmic tails of both the β and γ subunits of the high affinity IgE receptor (FcεRI) contain a consensus sequence termed the immunoreceptor tyrosine-based activation motif (ITAM). This motif plays a critical role in receptor-mediated signal transduction. Synthetic peptides based on the ITAM sequences of the β and γ subunits of FcεRI were used to investigate which proteins associate with these motifs. Tyrosine-phosphorylated β and γ ITAM peptides immobilized on beads precipitated Syk, Lyn, Shc, Grb2, and phospholipase C-γ1 from lysates of rat basophilic leukemia RBL-2H3 cells. Syk was precipitated predominantly by the tyrosine-diphosphorylated γ ITAM peptide, but much less by the diphosphorylated β ITAM peptide or by the monophosphorylated peptides. Phospholipase C-γ1, Shc, and Grb2 were precipitated only by the diphosphorylated β ITAM peptide. Non-phosphorylated ITAM peptides did not precipitate these proteins. In membrane binding assays, fusion proteins containing the Src homology 2 domains of phospholipase C-γ1, Shc, Syk, and Lyn directly bound the tyrosine-phosphorylated ITAM peptides. Although the ITAM sequences of the β and γ subunits of FcεRI are similar, once they are tyrosine-phosphorylated they preferentially bind different downstream signaling molecules. Tyrosine phosphorylation of the ITAM of the γ subunit recruits and activates Syk, whereas the β subunit may be important for the Ras signaling pathway.

Aggregation of the high affinity IgE receptors (Fc⑀RI) 1 on basophils and mast cells initiates a cascade of events that results in the release of inflammatory mediators. This pathway includes the activation of several protein-tyrosine kinases such as Lyn, Syk, Btk, and Fak that induce the tyrosine phosphorylation of various proteins (1)(2)(3). There is also stimulation of phospholipase A 2 , C, and D, the mobilization of Ca 2ϩ from intracellular and extracellular sources and activation of serine and threonine kinases (4,5).
The high affinity IgE receptor on mast cells and basophils is a tetrameric structure composed of the IgE binding ␣ chain, a ␤ subunit, and a homodimer of disulfide-linked ␥ chains (6). The COOH-terminal cytoplasmic domain of Fc⑀RI␤ and the cytoplasmic domain of Fc⑀RI␥ appear to be important in receptor-mediated signal transduction (7,8). These transducing subunits contain a cytoplasmic motif with the amino acid sequence (D/E)X 2 YX 2 LX 6 -7 YX 2 (L/I) that is critical for cell activation (9 -11). This motif called ITAM (for Immunoreceptor T yrosinebased Activation Motif) (12) is present in the ␤ and ␥ subunits of Fc⑀RI, in the subunit of the T-cell receptor complex, and in Ig␣ and Ig␤ of the B-cell receptor. This motif contains all the structural information essential for signal transduction and is rapidly tyrosine-phosphorylated after receptor aggregation (11,(13)(14)(15). Phosphorylation of the tyrosine residues appears to be necessary as mutants where the tyrosines are replaced with phenylalanine are inactive (16,17). This phosphorylation is probably due to src family tyrosine kinases that associate with these receptors (11). After receptor aggregation, a Syk/ZAP-70 family protein tyrosine kinase associates with these receptors and is critical for the downstream activation signals (10, 11, 18 -21).
Fusion proteins containing the two Src homology 2 (SH2) domains of Syk bind preferentially to the tyrosine-phosphorylated ␥ subunit of Fc⑀RI, whereas a fusion protein containing the single Lyn SH2 domain binds to tyrosine-phosphorylated Fc⑀RI␤ (22,23). Therefore, to better understand signal transduction from the Fc⑀RI, we used synthetic peptides based on the ITAM sequences of the ␤ and ␥ subunits of Fc⑀RI to investigate the interaction of molecules that may be involved in downstream propagation. We observed an association of Syk predominantly with the tyrosine-phosphorylated ␥ ITAM, whereas Lyn, Shc, Grb2, and PLC␥1 interacted with the ITAM of Fc⑀RI␤. The binding of distinct downstream effector molecules to the different ITAMs may be critical in the activation of distinct signaling pathways from the different receptor subunits.

EXPERIMENTAL PROCEDURES
Materials-Streptavidin coupled to agarose beads were from Pierce (Rockford, IL). Protein A, aprotinin, and Triton X were obtained from Sigma. The materials for electrophoresis were purchased from Novex (San Diego, CA), and the source of other materials was as described previously (20).
Antibodies-Mouse monoclonal anti-PLC␥1 antibody was from Upstate Biotechnology Inc. (Lake Placid, NY), rabbit polyclonal anti-PLC␥2 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA), mouse monoclonal anti-Grb2 antibody, mouse monoclonal, and rabbit * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Synthesis of ITAM Peptide of Fc⑀RI-Peptides based on the sequences of the ␤ and ␥ subunits of Fc⑀RI (6) were synthesized by solid-phase Fmoc chemistry on an Applied Biosystems 430A synthesizer. Phosphotyrosine residues were incorporated by using Fmoc-Tyr(PO 3 H 2 )OH (26). Peptides were also biotinylated on the resins. The purification and characterization of the peptides was as described previously (25).
Cell Activation and Preparation of Cell Lysates-RBL-2H3 cells were cultured and activated with anti-Fc⑀RI␣ antibodies (mAb BC4) as described previously (1,27). After 10 min of stimulation, the monolayers were rinsed twice with ice-cold phosphate-buffered saline and solubilized in lysis buffer (10 mM Tris, pH 7.5, containing 1.0% Triton X-100, 1 mM Na 3 VO 4 , 150 mM NaCl, 50 g/ml leupeptin, 0.5 unit/ml aprotinin, 2 mM pepstatin A, 1 mM phenylmethylsulfonyl fluoride). After leaving the plates on ice for 10 min, the cells were scraped and supernatants were collected after centrifugation for 30 min at 16,000 ϫ g, 4°C.
Precipitation with ITAM Peptides-Lysates from 1.5 ϫ 10 7 cells in 1.0 ml were precleared by mixing for 90 min at 4°C with streptavidin coupled to agarose beads. The lysates were then incubated with 1 nmol of biotinylated ITAM peptides that had been preincubated with 20 l of streptavidin beads. After gentle rotation at 4°C for 90 min, the beads were washed three times with wash buffer (lysis buffer with Triton 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 (22).
For reimmunoprecipitation experiments, proteins were first precipitated with ITAM peptides bound to beads and then eluted by boiling for 10 min in 1% SDS. The proteins were reimmunoprecipitated with antibody prebound to protein A and analyzed by immunoblotting.
In Vitro Kinase Assay-Precipitation was performed as above with the biotinylated ITAM peptides bound to the streptavidin beads. After the four washes, the beads were further washed with kinase buffer (30 mM HEPES, pH 7.5, 10 mM MgCl 2 , and 2 mM MnCl 2 ), and resuspended in 20 l of kinase buffer. The kinase reaction was started by the addition of 1 Ci of [␥-32 P]ATP (ICN) and incubated for 10 min at room temperature. The reaction was stopped by washing once with 1 ml of ice-cold 50 mM Tris, 150 mM NaCl, then by adding sample buffer. After boiling for 5 min, the proteins were separated by SDS-PAGE electrophoresis on 10% Tris glycine gels, transferred to PVDF (polyvinylidene difluoride membranes, Millipore, Bedford, MA) membranes, and 32 P was detected by autoradiography at Ϫ70°C.
Immunoblotting-Samples from the precipitates were separated by SDS-PAGE under reducing conditions and electrotransferred to PVDF membranes. The membranes were incubated overnight in blocking buffer. Tyrosine-phosphorylated proteins were detected with monoclonal antibody PY-20 conjugated to horseradish peroxidase as described previously (28). Lyn, Syk, Shc, PLC␥2, and Fc⑀RI␥ were detected by rabbit antibodies using peroxidase-conjugated donkey antirabbit as a secondary antibody. PLC␥1, Grb2, and Fc⑀RI␤ were detected by mouse antibodies and the secondary antibody was peroxidase-conjugated donkey anti-mouse antibody. In all blots, proteins were visualized using the enhanced chemiluminescence kit from DuPont and Kodak X-Omat radiographic film (Eastman Kodak Co.). In some experiments antibodies were stripped from the membranes according to the protocol of the manufacturer and then membranes reprobed with other antibodies. Scanning densitometry on the film was with a Pharmacia LKB Imagemaster.
Expression of GST Fusion Proteins-The SH2 domains of Lyn and Syk expressed as GST fusion proteins have been described previously (22). The plasmids containing the SH2 domains of PLC␥1 and Shc as fusion proteins were kindly provided by Dr. T. Pawson, Mount Sinai Hospital Research Institute, Toronto, Canada. The proteins were expressed in Escherichia coli, affinity purified on glutathione-Sepharose beads, and characterized as described previously (22).
Membrane Binding Assays-PVDF membranes were used to determine the interaction of GST fusion proteins with the ITAM peptides. Ten pmol and 2 pmol of the GST fusion proteins containing the SH2 domains of PLC␥1, Shc, Syk, and Lyn were spotted onto PVDF membranes. The membranes were then blocked by overnight incubation with 4% bovine serum albumin and for 1 h with 100 g/ml donkey IgG. After extensive washing, membranes were incubated with 1 M biotinylated ITAM peptides for 1 h. The membranes were then washed and incubated with horseradish peroxidase-conjugated streptavidin and the signal was detected by enhanced chemiluminescence.

Phosphorylated ITAM Peptides Precipitated Syk from Lysates of RBL-2H3
Cells-Previously we observed that Syk coprecipitated with Fc⑀RI in activated cells (20) and that fusion proteins containing the SH2 domains of Syk bound to the ␥ subunit of Fc⑀RI (22). To further investigate these interactions we used synthetic peptides that encompass the ITAM motif of both the ␤ and ␥ subunits of Fc⑀RI (Fig. 1). The different ITAM synthetic peptides (both non-phosphorylated and tyrosinephosphorylated) were used to affinity precipitate proteins from both non-stimulated and stimulated RBL-2H3 cells (Fig. 2). The tyrosine-diphosphorylated ␤ and ␥ ITAM peptides precipitated several proteins that were tyrosine-phosphorylated. The most prominent was a 72-kDa protein that was only precipitated from lysates of stimulated RBL-2H3 cells by tyrosinephosphorylated ␥ ITAM peptide. In contrast, ␤ phosphorylated ITAM peptide precipitated 145-and 52-kDa tyrosine-phosphorylated proteins, both of which were present in both non-stimulated and activated cells. However, no tyrosine-phosphorylated proteins were precipitated by the non-phosphorylated ITAM peptides. These results indicate that proteins were selectively precipitated by ITAM peptides and that the ITAM peptides had to be tyrosine-phosphorylated for them to bind these proteins.
Several antibodies were used to examine whether these ITAM-associated proteins were previously identified tyrosinephosphorylated proteins. Immunoblotting identified Syk as the 72-kDa phosphorylated protein (Fig. 2C). The immunoblots also demonstrated that Syk was precipitated by the ␤PP and the monophosphorylated ␥YP peptides. As Syk was approxi- The tyrosines that are probably phosphorylated after receptor activation are in bold. In the nomenclature used here ␤ refers to Fc⑀RI␤ and ␥ to Fc⑀RI␥. A, peptides based on the ITAM of Fc⑀RI␤. ␤YY is unphosphorylated; whereas ␤PP is the diphosphorylated ITAM of Fc⑀RI␤. B, peptides based on the ITAM of Fc⑀RI␥. ␥YY is the un-phosphorylated peptide; ␥PP, is the same peptide with both tyrosines phosphorylated; ␥YP and ␥PY are mono-phosphorylated peptides with phosphorylation of either the first or second tyrosine. mately equally precipitated from both non-stimulated and stimulated cells, tyrosine phosphorylation of Syk is not important for its interaction with the ITAM peptides. On longer exposures we could detect some Syk precipitation by the other monophosphorylated ␥PY peptide, whereas no Syk was precipitated by the non-phosphorylated peptides. Combining the two different monophosphorylated ␥ peptides did not result in increased precipitation of Syk (Fig. 3). Similarly addition of the mono-phosphorylated ␥ peptides (␥PY and ␥YP) to ␤PP did not affect the binding of Syk. The efficiency of precipitating Syk from the cell lysates was ␥PPϾ Ͼ␤PPϾ␥YP (by densitometric analysis the ratios were 100:13:0.5). These results support the reports of the preferential interaction of Syk with the tyrosinephosphorylated ␥ subunit of Fc⑀RI and demonstrate that this binding requires that both tyrosines in the ITAM be phosphorylated. Previously we observed that fusion proteins containing the two SH2 domains of Syk were more efficient than either one of the two SH2 domains alone in interacting with the ␥ subunit of Fc⑀RI. Therefore, the binding of Syk to ␥ requires both SH2 domains of Syk and phosphorylation of both tyrosines of the ITAM.
Activation of RBL-2H3 cells results in tyrosine phosphorylation of several 72-kDa proteins that are not Syk (20,29). We therefore investigated whether Syk was the only 72-kDa tyrosine-phosphorylated protein precipitated with the ␥PP peptide. Syk was depleted from cell lysates by incubation with protein A beads preincubated with anti-Syk antibodies. This removed in parallel the 72-kDa tyrosine-phosphorylated pro-tein precipitated by ␥PP (data not shown). To further confirm that the 72-kDa tyrosine-phosphorylated protein was Syk, the proteins precipitated from activated cell lysates with peptidestreptavidin beads were eluted by boiling in SDS. These were then immunoprecipitated with control or anti-Syk antibodies. Analysis by anti-phosphotyrosine and anti-Syk antibodies confirmed that this 72-kDa tyrosine-phosphorylated protein was Syk (data not shown). Therefore, Syk was the only tyrosinephosphorylated 72-kDa protein precipitated with ␥PP.
In vitro kinase reactions were used to further define proteins that were precipitated with the ITAM peptides (Fig. 4). The major proteins phosphorylated in vitro were 72 kDa in size and were seen after precipitation with the ␥PP peptide. The long exposure of this radioautograph is used here to show that similar bands were detected with ␤PP and ␥YP. Depletion of Syk from lysates with antibodies confirmed that this in vitro kinase activity was due to Syk. As was observed in the immunoblotting experiments, there was good association of the kinase with the ITAM peptides in both stimulated and nonstimulated cells.
Phosphorylated ITAM Peptides of Fc⑀RI␤ Precipitated Lyn, PLC␥1, Shc, and Grb2 from Lysates of RBL-2H3 Cells-Several studies suggest that Lyn associates with Fc⑀RI in both nonstimulated and stimulated cells (30 -33) and that this interaction is preferentially with the ␤ subunit of the receptor (22,34). However, we could not detect Lyn precipitation with the ITAM peptides by immunoblotting or by the more sensitive in vitro kinase reaction (Fig. 2 and 3). There was still no Lyn when ITAM peptides based on the ␥ and ␤ subunits of Fc⑀RI were used together for precipitation (data not shown). Under these conditions Lyn is normally tyrosine-phosphorylated which could result in masking of the SH2 domain. Interestingly, when cell lysates were prepared in the absence of vanadate to allow for dephosphorylation, there was binding of Lyn to the diphosphorylated ␤ ITAM peptide (Fig. 5). In contrast, binding to the ␥-diphosphorylated peptide was weaker. These results suggest that tyrosine phosphorylation of the regulatory site of Lyn controls its binding to Fc⑀RI after cell activation.
Tyrosine-phosphorylated proteins of 145, 52, and 46 kDa were selectively precipitated by the diphosphorylated ␤ ITAM peptide (Fig. 2). Using a panel of antibodies, the 145-kDa protein was identified as PLC␥1. This identification of PLC␥1 was confirmed by reimmunoprecipitation and depletion experiments as was described above for Syk (data not shown). Although PLC␥1 and PLC␥2 are structurally very similar, only PLC␥1 was precipitated by ␤PP (data not shown). PLC␥1 was not precipitated by mono-or diphosphorylated ␥ peptides (Fig.  2). Furthermore, addition to ␤PP of the monophosphorylated ␥PY peptide, but not of the diphosphorylated ␥PP peptide, resulted in a decrease in the precipitation of PLC␥1 (Fig. 3). Altogether this data suggests that PLC␥1 can interact with the tyrosine-phosphorylated ␤ subunit of Fc⑀RI.
The 52-and 46-kDa proteins were identified as Shc by blotting with both monoclonal and polyclonal anti-Shc antibodies (Fig. 2). In longer exposures, Shc could be visualized in the precipitates with ␥PP from both non-stimulated and stimulated cells. The ␤PP peptides still precipitated PLC␥1 from cell lysates depleted of Shc. Therefore, the interaction of PLC␥1 with ␤PP is independent of Shc. There was increased precipitation of Shc from non-stimulated cells, but not from stimulated cells with the mixture of ␥YP and ␤PP peptides (Fig. 3). These results suggest that Shc can preferentially bind to the tyrosine-phosphorylated ␤ ITAM and that this binding may be modulated by the ITAM of the ␥ subunit.
When Shc protein is tyrosine-phosphorylated there is strong SH2-mediated binding of Grb2, an adaptor protein (35). Grb2 was precipitated by the diphosphorylated ␤ subunit of the ITAM peptide (Fig. 6). However, none of the other synthetic tyrosine-phosphorylated and non-phosphorylated peptides precipitated Grb2.
Membrane Binding Assay-A membrane binding assay was used to examine the potential for direct interaction between the SH2 domains of the different proteins with the synthetic ITAM peptides. GST fusion proteins containing the SH2 domains were bound to membranes and then reacted with two concentrations of the synthetic ITAM peptides (Fig. 7). The Syk fusion proteins contained the amino-terminal, carboxyl-terminal, or both SH2 domains expressed in tandem (Fig. 7A). Diphosphorylated ␤ and ␥ ITAM peptides bound to fusion proteins containing the SH2 domains of Syk. By contrast, there was very little binding of ITAM peptides to the NH 2 -terminal SH2 domain of Syk, more to the COOH-terminal SH2 domain, and markedly stronger binding when both SH2 domains were expressed in tandem. Binding by the lower concentrations of the fusion proteins to the diphosphorylated ITAM of the ␥ subunit was better than with ITAM of the ␤ subunit. This suggests that the binding to ␥ ITAM was with higher affinity. The monophosphorylated peptides based on the ␥ ITAM bound much less and this binding was only when both SH2 domains of Syk were expressed as fusion proteins. There was very little selectivity in the binding by the Lyn SH2, with it binding to some extent to all the mono-and diphosphorylated synthetic peptides. Nevertheless, neither the Syk nor the Lyn SH2 fusion proteins bound to the non-phosphorylated ITAM synthetic peptides (not shown).
The SH2 domains of phospholipase C␥1 and Shc were also tested in membrane binding assays (Fig. 7B). The amino-terminal, carboxyl-terminal, and tandemly expressed SH2 domains of phospholipase C-␥1 all strongly bound to the diphosphorylated ␤ ITAM. The protein with both SH2 domains of PLC␥1 bound weakly to the non-phosphorylated ␤ and ␥ ITAM peptides and to the phosphorylated ␥ ITAM peptide. The lack of the binding of the two SH2 domains of PLC␥1 to the monophosphorylated ␥ ITAM peptides indicates the specificity in the binding with this fusion protein. In this membrane binding assay, the SH2 domain of Shc interacted with only the ␤-phosphorylated ITAM peptide. Therefore, the SH2-mediated binding of Syk is to the tyrosine-phosphorylated ITAM of Fc⑀RI␥, whereas PLC␥1 and Shc binding is to the tyrosine-phosphorylated ITAM of Fc⑀RI␤.

FIG. 3. Precipitation of proteins with combination of ITAM peptides.
Precipitation with ITAM peptides was as in the legend of Fig. 2. Different peptides at 1 nmol were added to streptavidinbound beads. The precipitated proteins were analyzed by immunoblotting with anti-PLC␥1 antibody (A), anti-Syk antibody (B), and anti-Shc antibody (C). There was 1.4-fold (average of three experiments) increased precipitation of Shc when ␥YP was added to ␤PP.

FIG. 4. In vitro kinase assay of precipitates with ITAM peptides.
Lysates from 1.5 ϫ 10 7 stimulated (BC4ϩ) or non-stimulated (BC4Ϫ) RBL-2H3 cells were incubated for 90 min at 4°C with 1 nmol of the different biotinylated ITAM peptides that had been prebound to 20 l of streptavidin beads. For controls streptavidin beads were used without any peptides. The precipitates were washed and after in vitro kinase assay the proteins were separated by SDS-PAGE on 10% gels and analyzed by autoradiography. The bands seen at 55 and 40 kDa are digestion products of Syk.
FIG. 5. Lyn was precipitated by diphosphorylated ITAM peptide. The precipitation with ITAM peptides was from stimulated (BC4ϩ) or non-stimulated (BC4Ϫ) cells. Cell lysates were prepared in the absence of vanadate, other details were as described in the legend of Fig. 2. The proteins were analyzed by immunoblotting with anti-Lyn antibodies. There was no detectable Lyn precipitated by non-phosphorylated peptides (data not shown).
FIG. 6. Grb2 was precipitated by diphosphorylated Fc⑀RI␤ ITAM peptide. The precipitation with ITAM peptides was as in the legend of Fig. 2. The proteins were separated by electrophoresis on 10 -20% Tricine gels, transferred to PVDF membranes, and immunoblotted with anti-Grb2 antibodies.

DISCUSSION
One of the earliest events after aggregation of Fc⑀RI is the activation of kinases that result in the rapid phosphorylation on Tyr and Ser of the ␤ subunit of the receptor and on Tyr and Thr of the ␥ subunit (36). The phosphorylation on tyrosines of the ITAM in these subunits then recruits other proteins that are crucial for propagating the intracellular signals. The binding of such proteins is mediated by SH2 domains present on many proteins involved in signal transduction (35). The specificity of SH2 domains for phosphorylated tyrosines (37) would explain the selectivity of the interactions of Syk, Lyn, PLC␥1, and Shc with the ␤ and ␥ subunits of the Fc⑀RI. Binding of these downstream signaling molecules then can result in conformational changes and in their activation (25,38).
The present results further refine the model for Fc⑀RI-induced activation of mast cells. Aggregation of the receptor results in the activation of Lyn which then tyrosine phosphorylates the ␤ and ␥ subunits of the receptor. Syk is then recruited and activated by binding to the ␥ subunit of the receptor. PLC␥1 is recruited to the ␤ subunit of the receptor, bringing it in close proximity to Syk where it could be efficiently tyrosine phosphorylated. Previous experiments suggest that both the ␤ and ␥ subunits of Fc⑀RI contribute to signal transduction. Chimeric proteins have been expressed in RBL-2H3 cells that contain the extracellular and transmembrane domains of CD25 fused with the carboxyl-terminal domain of either the ␥ or ␤ subunits of Fc⑀RI (8,34,39,40). Whereas there is some secretion by aggregation of the chimeric proteins that contain the ␥ domain, there is minimal if any by the ␤ containing chimeras. Furthermore, signaling by the chimeric proteins containing the ␥ sequence is less than through the normal Fc⑀RI, suggesting that the ␤ subunit is important for signal transduction. The present results suggest an explanation for such observations requiring cooperation between the two receptor subunits in inducing secretion in mast cells.
The much stronger binding of Syk with the ␥ than with the ␤ ITAM-diphosphorylated peptides is probably due to the fine structural differences in the sequence of the ITAM of these two molecules. There is also one less amino acid between the two tyrosines in the ␤ motif. By membrane binding studies, the best binding to ITAM peptides was with fusion proteins containing both SH2 domains of Syk expressed in tandem. These results strongly suggest that the two SH2 domains of Syk and phosphorylation of both tyrosines in the ITAMs are necessary for optimal binding. The recently described crystal structure of ZAP-70 associated with the phosphorylated ITAM of T cell receptor supports this model for binding (41). The binding of Syk/ZAP-70 tyrosine kinases to the tyrosine-phosphorylated ITAM peptides results in their activation and tyrosine phosphorylation (25,42,43). The interaction of Syk with the tyrosine-phosphorylated ITAM results in a conformational change (25) and an increase in its kinase activity (42,43). These conformational changes in Syk and increased enzymatic activity are probably critical for downstream propagation of intracellular signals in both mast cells and B cells (20, 21, 44 -46).
Lyn, a member of the Src family of protein-tyrosine kinases, is associated with Fc⑀RI in both non-stimulated and activated teins were detected by enhanced chemiluminescence. A, SH2 containing proteins were: the NH 2 -terminal (SykSH2, N), COOH-terminal (SykSH2, C), both NH 2 -terminal and COOH-terminal in tandem (SykSH2, NC) of Syk and Lyn (Lyn, SH2). GST alone was used as a control (GST). B, the SH2 containing fusion proteins used were the following: the NH 2 -terminal (PLC␥1SH2, N), COOH-terminal (PLC␥1SH2, C), the NH 2 -terminal and COOH-terminal expressed in tandem (PLC␥1SH2, NC) of PLC␥1 and Shc (Shc-SH2).

FIG. 7.
Interaction of ITAM peptides with GST fusion proteins containing the SH2 domains of PLC␥1 and Shc. Ten and 2 pmol of the different GST fusion proteins containing the SH2 domains were spotted on PVDF membranes, blocked, and then incubated with 1 M biotinylated ITAM peptides. The membranes were washed and incubated with horseradish peroxidase-conjugated streptavidin and pro-cells (30 -33). The present precipitation results suggest that after cell activation the interaction of Lyn is predominantly with the ␤ subunit. There is association of Lyn with the COOHterminal cytoplasmic domain of the ␤ subunit which contains the ITAM, although mutation of one of the Tyr to Phe does not decrease this association (34). Furthermore, a fusion protein containing only the SH2 domain of Lyn precipitates and binds with the tyrosine-phosphorylated ␤ subunit and with the phosphorylated ITAM. These data suggest that the SH2 domain of Lyn can potentially bind the phosphorylated ITAM, but in the intact Lyn this SH2 site is not available due to intramolecular interaction with its regulatory domain. Stimulation of cells and activation of Lyn could result in dephosphorylation of these regulatory sites and the release of the SH2 domain for binding to the phosphorylated subunits of Fc⑀RI that are in close proximity. This can explain the increased association of Lyn with Fc⑀RI after cell stimulation (32). However, Lyn is tyrosine phosphorylated to the same extent in non-stimulated compared to stimulated cells, suggesting that there may be rapid rephosphorylation of these regulatory sites. Therefore, Lyn association with Fc⑀RI in activated cells is probably mediated by SH2 domain binding.
Ligand binding to receptor protein-tyrosine kinases, such as the epidermal growth factor receptor or the platelet-derived growth factor receptor, induces receptor oligomerization and autophosphorylation, generating docking sites for PLC␥1 (47). PLC␥1 can also associate with ITAM-containing receptors such as the T cell receptor (48). PLC␥1 is tyrosine-phosphorylated after Fc⑀RI activation (49,50) and translocates to the membrane (51). Tyrosine phosphorylation activates PLC␥1 inducing it to hydrolyze phosphatidylinositol 4,5-bisphosphate to produce two second messengers, inositol trisphosphate and diacylglycerol, these in turn result in the increase in intracellular Ca 2ϩ and the activation of protein kinase C (52). PLC␥1 associates with a tyrosine kinase after Fc⑀RI aggregation (53), and a complex containing Syk, PLC␥1, and a 120-kDa phosphoprotein has been seen in B-cells (54). This 120-kDa protein has been postulated to be a bridge between Syk and PLC␥1. Similarly, fusion proteins containing the SH2 domains of PLC␥1 bind tyrosine-phosphorylated Syk (55). However, the present experiments suggest direct interaction of PLC␥1 with the tyrosine-phosphorylated ITAM of Fc⑀RI␤ on the basis of both precipitation and membrane binding studies. Furthermore, the ␥PP peptide precipitated Syk, but there was no detectable PLC␥1. PLC␥1 has two SH2 domains, one SH3, and two split pleckstrin homology domains. The interaction between PLC␥1 and the phosphorylated Fc⑀RI␤ ITAM is SH2-mediated, whereas the pleckstrin homology domains may play a role in other membrane interactions.
Tyrosine-phosphorylated ␤ ITAM peptide precipitated Shc and Grb2, two members of a family of adaptor molecules that have SH2 domains. Stimulation of T cell and B cell antigen receptors results in tyrosine phosphorylation of Shc and the formation of a complex that contains Shc, Grb2, and Sos (56,57). However, in RBL-2H3 cells Shc is constitutively tyrosinephosphorylated (data not shown and Ref. 58). The single SH2 domain at the carboxyl terminus of Shc probably bound to the tyrosine-phosphorylated ␤ ITAM sequence pYEEL (37). Grb2 is another adaptor molecule with an SH2 domain that binds to tyrosine-phosphorylated Shc. The single SH2 domain of Grb2 is flanked by two SH3 domains. One of the SH3 domains binds Sos1, the Ras guanine nucleotide exchange factor, and thus the complex can activate the Ras pathway (59). Fc⑀RI aggregation results in the activation of the ERK1 and ERK2 (60, 61), which are probably important for regulating nuclear events and for the generation of arachidonic acid (62). Therefore, association of Shc with the ␤ subunit of Fc⑀RI could be important for initiating the activation of the Ras pathway that leads to nuclear events, such as the synthesis of cytokines and for the generation of arachidonic acid.