American Journal of Respiratory Cell and Molecular Biology

Eosinophils are potent effector cells contributing to allergic inflammation and asthma. The differentiation, recruitment, and effector functions of eosinophils are greatly affected by interleukin (IL)-5. In the eosinophil, signal transduction pathways including Jak–STAT and Ras–Raf–MAP kinase are stimulated by IL-5 and enzymatic activation of tyrosine kinases Jak-2 and Lyn has been demonstrated. The participation of adapter proteins in the responses of the Ras–Raf–MAP kinase pathway has been documented in many cytokine family receptors but the expression and activation of these proteins have not been demonstrated in eosinophils. In these studies, we have found three isoforms of the adapter protein, Shc, to be expressed in eosinophils. One of these isoforms, p52 Shc, was tyrosine phosphorylated following IL-5 treatment of eosinophils. A second adapter protein, Grb2, coimmunoprecipitated with Shc following IL-5 stimulation of eosinophils. Furthermore, p52 Shc was increasingly associated with a cell fraction resistant to detergent solubilization, following IL-5 administration. This cell fraction of limited detergent solubility is a complex mixture of proteins and the adapter protein Grb2, the tyrosine kinases Jak-2 and Lyn, the nucleotide exchange factor Vav, and the serine–threonine kinases p45 MAP kinase, Raf-1, and PKCβ, were distributed either wholly or partially in the same fraction, as were the cytoskeletal proteins actin and vimentin. Only p52 Shc, however, demonstrated discernibly increased association with this fraction following IL-5 stimulation of eosinophils. These data suggest that IL-5 activates a signal transduction pathway utilizing the adapter proteins Shc and Grb2 in the human eosinophil.

Allergic inflammation in asthma is associated with eosinophilia and an increased production of interleukin 5 (IL-5) (1, 2). The biologic activity of eosinophils can have profound effects on the airways (3, 4), suggesting that eosinophils are potent and key effector cells in the pathogenesis of asthma. The proliferation, recruitment, survival, and effector functions of eosinophils are greatly affected by IL-5, as demonstrated by in vivo and in vitro studies, and the relevance of this cytokine to asthma and allergic inflammation has been well documented (5-7). IL-5 promotes eosinophil differentiation (8-10) and, when purified eosinophils are exposed to IL-5 in vitro, a number of the effector functions of the cells are modified, thereby enhancing their inflammatory capacity. For example, integrin-mediated adhesion and the expression of membrane receptors are increased (11-14). Chemotactic responses (15, 16) and the stimulus-induced release of eosinophil-derived mediators such as granule proteins (17, 18), reactive oxygen species (19), and leukotriene C4 (LTC4) (20, 21) are increased following exposure of eosinophils to IL-5. Furthermore, IL-5 enhances eosinophil survival through the suppression of apoptotic cell death (22). Therefore, a large body of evidence supports the role of IL-5 and eosinophils as pivotal mediators in the pathogenesis of asthma and allergic inflammation.

The effects of IL-5 on the human eosinophil are induced following binding to its cell surface receptor, which is composed of two glycoprotein chains: an α chain, which confers ligand specificity to the receptor, and a larger β chain, which is identical to the β-chain subunits of the IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors (23, 24). The human IL-5 receptor is of high affinity and expressed in low numbers on eosinophils (25, 26). The receptor subunits appear to have no intrinsic enzymatic capacity but, after stimulation of eosinophils with ligand, the activation of tyrosine kinases Jak-2 and Lyn has been observed (27-30). Furthermore, IL-5 induces accumulation of GTP-bound Ras, as well as the enzymatic activation of Raf-1 and at least one of the mitogen-activated protein (MAP) kinase isoforms (28, 30). Tyrosine phosphorylation and DNA-binding activity of the transcriptional activator STAT1α have also been documented in human eosinophils following stimulation with IL-5 (27, 29). The STAT proteins, an acronym for signal transducer and activator of transcription, have been previously identified as substrates for the tyrosine kinases of the Janus family, of which Jak-2 is a member (31, 32).

The mechanism by which cytokine family receptors activate downstream signaling pathways such as the Ras–Raf– MAP kinase pathway has been determined in a variety of model systems, and the contribution of adapter proteins has been demonstrated to be of critical significance. These adapter proteins appear to have no enzymatic capacity but function to mediate the assembly of multiprotein complexes that couple cellular responses to receptor-initiated stimuli. One such adapter protein is the Src homologous and collagen homologous protein (Shc). Shc was first identified by screening a cDNA library in search of proteins with Src homology type 2 (SH2) domains, which are protein modules that bind to amino acid motifs containing phosphotyrosine residues in the context of specific amino acid sequences. It was observed that three protein products were generated from the shc gene, namely p46, p52, and p66. The proteins were expressed in a wide range of mammalian cell lines and were tyrosine phosphorylated following stimulation of a variety of receptors (33-37). Structural and sequence analysis of the Shc proteins revealed that they all contained a C-terminal SH2 domain, a domain homologous to human α1 collagen capable of interacting with filamentous actin (33), and an N-terminal phosphotyrosine-binding domain, which mediates Shc interaction with tyrosine-phosphorylated molecules such as the epidermal growth factor receptor (EGFR) (38). The tyrosine phosphorylation of Shc after growth factor stimulation of the cell mediates the interaction of Shc with other SH2 domain-containing proteins including the adapter protein growth factor receptor-binding protein 2 (Grb2) (39). Grb2 has been shown to bind guanine nucleotide exchange factors such as the son of sevenless proteins (SOS) (40-42). The association of this complex with tyrosine-phosphorylated receptors can mediate the accumulation of Ras–GTP and activation of downstream effector pathways including the Raf–MAP kinase pathway (43).

To determine the relevance of these signal transduction mechanisms to the interaction of IL-5 with its receptor on human eosinophils, we investigated the expression of the adapter proteins Shc and Grb2 in human eosinophils. We have identified three isoforms of Shc in human eosinophils and one of these, p52, displays increased association with a detergent-insoluble cell fraction following IL-5 treatment. Immunoprecipitation and immunoblotting experiments demonstrate that, in the human eosinophil, IL-5 induces tyrosine phosphorylation of p52 Shc and its association with Grb2. These observations suggest that, consistent with the mechanism demonstrated for cytokine activation of replicating cells, human eosinophils also activate an IL-5 signal transduction pathway mediated by these adapter proteins.

Reagents

Enhanced chemiluminescence (ECL) reagents were obtained from Amersham (Arlington Heights, IL). Human recombinant IL-5 was purchased from R&D Systems (Minneapolis, MN). Polyvinylidene difluoride (PVDF) membrane for immunoblotting, protein molecular weight standards, dithiothreitol (DTT), and the other remaining protease and phosphatase inhibitors were purchased from Sigma Chemical Co. (St. Louis, MO). Percoll was purchased from Pharmacia (Piscataway, NJ).

Immunochemicals

Anti-phosphotyrosine (anti-P-Tyr) monoclonal antibody (mAb) clone PY20 was acquired from ICN Biomedicals (Costa Mesa, CA). Anti-P-Tyr mAb 4G10 and rabbit antisera to Shc C-terminal peptide, encompassing amino acids 366–473, were purchased from Upstate Biotechnology Inc. (UBI, Lake Placid, NY), as was mAb anti-human Vav, rabbit anti-mouse Jak-2, and rabbit polyclonal anti-human Lyn. UBI also supplied anti-MAP kinase antisera, a pool of rabbit antisera raised against residues 63–98 and residues 333– 367 of rat 43-kD ERK1. Santa Cruz Biotechnology (Santa Cruz, CA) provided rabbit antisera raised against the C-terminal peptide (residues 195–217) of human Grb2, monoclonal anti-human-Shc, and rabbit polyclonal antisera to human Raf-1. Life Technologies (Gaithersburg, MD) was the source of rabbit anti-protein kinase C β (PKCβ). Miltenyi Biotechnology (Auburn, CA) was the source of the anti-CD16 microbeads for the negative selection of neutrophils required for eosinophil purification. Horseradish peroxidase (HRP)-conjugated secondary antibodies, mouse monoclonal anti-vimentin, and rabbit polyclonal anti-actin were purchased from Sigma Chemical Co.

Isolation of Human Eosinophils

Eosinophils were purified from the heparinized peripheral blood of volunteer donors as previously described (30). Blood donors included individuals who were both atopic and nonatopic and eosinophils represented between 2 and 10% of the peripheral blood leukocytes. A granulocyte mixture was obtained from the leukocyte buffy coat after centrifugation through a Percoll solution (density, 1.090 g/ml) and, after lysis of erythrocytes by hypotonic shocks, the suspension was depleted of neutrophils by incubation with anti-CD16-conjugated microbeads and exposure to a magnetic field. The recovered eosinophils were resuspended in Hanks' balanced salt solution (HBSS) supplemented with 0.1% gelatin at a concentration of 107 cells/ml. These cell preparations were at least 95% eosinophils.

Eosinophil Stimulation and Preparation of Cell Lysates

Eosinophils were preincubated at 37°C (5 min) and treated with IL-5 or control buffer, as indicated by each experiment. After this incubation, the cells were diluted with ice-cold buffer A (20 mM Tris, 137 mM NaCl, 1 mM EDTA, 0.1 mM sodium orthovanadate, 10 mM NaF, 2 mM leupeptin, 1 mM DTT, 1% aprotinin) and an aliquot was assessed for viability by trypan blue exclusion. Cells were pelleted, the supernatants discarded, and the cell pellets resuspended in buffer A (in some cases supplemented by detergents).

Cell Fractionation

In some experiments (Figures 1 and 2), eosinophils were divided into detergent-soluble and -insoluble fractions by resuspension of the cells in buffer A supplemented with 1% Triton X-100 (wt/vol) and 0.1% sodium dodecyl sulfate (SDS). After centrifugation, the supernatant soluble fraction was removed and diluted in an equal volume of 2× electrophoresis sample buffer (2×SB) (20 mM Tris, 2 mM EDTA, 2% SDS, 2 mM DTT, 0.02% bromophenol blue, 20% glycerol, 1 mM sodium orthovanadate), and the residual pellet was washed in buffer A. This detergent-insoluble cell fraction was then resuspended in a volume of 2×SB equal to the volume of buffer used for cell lysis.

In other experiments (Figure 3), cytosolic and particulate fractions were acquired. Cytosolic fractions were obtained by five freeze–thaw cycles followed by centrifugation at 4°C (14,000 × g, 10 min). The supernatant liquid from these lysed cells was mixed with an equal volume of 2×SB and called the cytosol. The remaining particulate fraction was washed once in buffer A and resuspended in the original volume of buffer supplemented with detergents. This resuspended particulate fraction was centrifuged, and the soluble material was transferred to a fresh tube and mixed with 2×SB. This residual particulate fraction was washed and resuspended in buffer A supplemented with a higher concentration of detergents. In this way, the particulate fraction was sequentially solubilized and the resulting soluble fractions were mixed with sample buffer for resolution by SDS-polyacrylamide gel electrophoresis (PAGE). After the final solubilization stage, the particulate fraction was diluted in 2×SB and those sample lanes are designated as being soluble in 2% SDS plus 2 mM DTT.

Immunoprecipitation

Clarified lysates of treated eosinophils (2.5 × 107 cells/ml) were incubated for 1–12 h at 4°C with agarose-conjugated antibody 4G10 for anti-P-Tyr immunoprecipitates, or with rabbit antisera, followed by agarose-conjugated protein A. Control immunoprecipitates contained agarose-conjugated antibody 4G10 preincubated with 10 mM P-Tyr or 2 μg of rabbit IgG instead of specific antisera. The agarose beads were washed with six changes of buffer A plus detergents and resuspended in 2×SB for SDS-PAGE and immunoblotting.

SDS-PAGE and Electrophoretic Transfer to Polyvinylidene Difluoride Membrane

Samples were electrophoresed using polyacrylamide slab gels. The lanes were loaded with samples that represented equal numbers of cells. Transfer to PVDF membrane was conducted for 60–150 min at 0.55 A in transblot buffer (25 mM Tris [pH 8.3], 192 mM glycine, and 15% [vol/vol] methanol) (44, 45).

Immunodetection

The PVDF membrane was blocked overnight at room temperature in 10 mM Tris (pH 8.0) and 150 mM NaCl (TBS) containing 0.1% Tween 20 and 0.25% gelatin (TBSTG) or 3.0% nonfat milk (TBSTM). The blocked membrane was incubated in primary antibody for 1–2 h at 37°C, washed with three changes of TBSTG (5 min each), and incubated with HRP-conjugated secondary antibody for 1 h at room temperature. The membrane was washed with six changes of TBS plus 0.1% Tween 20 (TBST) and the labeled proteins were visualized after incubation with ECL substrate reagents and autoluminography. The apparent molecular weight of visualized proteins was determined by interpolation from a semilogarithmic plot of migration of stained protein standards on the PVDF membrane versus their molecular weight. The protein staining of the PVDF membrane also permitted an evaluation of the consistency of the protein loading of the sample lanes. In some experiments, after immunoblotting, PVDF membranes were stripped of bound immunoglobulin by incubation at 50°C for 30 min in 100 mM DTT plus 2% SDS. The membrane was then washed in four changes of TBS and blocked for 2 to 24 h in TBSTM. The membrane was subsequently immunoblotted with an antibody of different specificity. In selected experiments, in which PVDF membranes were stripped and reprobed, the complete removal of immunoglobulin from the first detection was demonstrated by cutting a lane from the PVDF membrane after stripping. This control lane was reprobed with buffer instead of the specific antisera and the subsequent incubation with HRP-conjugated anti-rabbit IgG provided a control for residual immunoglobulin remaining from the first detection. The luminosity detected from these control lanes was always substantially less than the luminosity from the lanes incubated with primary antibody.

Detection of Shc in Eosinophils

As a first step in determining the relationship between Shc activation and IL-5 signaling in eosinophils, Shc proteins were detected in lysates of human eosinophils by immunoblotting with a rabbit antisera or mouse monoclonal antibody raised against a C-terminal peptide sequence of amino acids 366–473. This peptide is part of the Shc SH2 domain and is present in the 46-, 52-, and 66-kD isoforms of the protein (33). In eosinophil lysates, the rabbit antisera detected proteins of 52 and 46 kD (Figure 1A, lane 1) whereas the monoclonal antibody detected one protein of 66 kD (Figure 1C). To determine if the cellular localization of Shc was affected by IL-5, eosinophils were incubated with IL-5 and the cells were disrupted in a lysis buffer containing Triton X-100 (1%, wt/vol) and SDS (0.1%, wt/vol). The distribution of Shc proteins in the detergent-soluble and -insoluble fractions was determined by immunoblotting. An increased association of p52 Shc with the insoluble fraction was seen following treatment with IL-5 concentrations greater than 15 pM (Figure 1A, lanes 8 and 9 and Figure 1B, lane 4). These observations suggest that IL-5 increases the affinity of the association of p52 Shc with components of the cell that are resistant to detergent solubilization. In contrast, the association of p46 Shc with the detergent-insoluble fraction was not affected by IL-5 treatment. The other isoform of Shc, p66 Shc, was detected only in the detergent-soluble fraction (Figure 1C).

We next assessed if other signaling molecules, known to be affected by stimulation of the IL-5 receptor, were also differentially distributed to detergent-insoluble fractions following IL-5 treatment of eosinophils. The MAP kinase isoform, p45 MAP kinase, was observed to be partially distributed to the detergent-insoluble fraction (Figure 1D, lanes 3 and 4). However, p45 MAP kinase did not discernibly increase in its association with the detergent-insoluble fraction following IL-5 treatment. The serine–threonine kinase, Raf-1, was also distributed to both the soluble and insoluble fractions. The apparent reduction in the mass of Raf-1 in the detergent-insoluble fraction following IL-5 treatment (Figure 1E, lane 4) was observed in IL-5 treated eosinophils of three separate donors and may represent a phosphorylation shift of enzymatically active Raf-1 or a reduction in the affinity with which Raf-1 associates with detergent-insoluble cellular elements.

To further characterize this detergent insoluble fraction, sequential solubilization studies were conducted on purified eosinophils following treatment with IL-5 or control buffer. The resulting fractions were immunoblotted with antibodies directed against MAP kinases (Figure 3A), the tyrosine kinases Jak-2 (Figure 3B) and Lyn (Figure 3C), the hematopoietic nucleotide exchange factor Vav (Figure 3D), actin (Figure 3E), Shc (Figure 3F), and PKCβ and vimentin (not shown). All of these molecules were either wholly or partially distributed in the least soluble fraction (Figure 3, lanes 11 and 12), but none demonstrated a discernibly differential localization following IL-5 treatment, as was observed with p52 Shc (Figure 3F, lane 12).

IL-5 Induces Shc Tyrosine Phosphorylation

Shc facilitates downstream signal transduction following tyrosine phosphorylation (33). To determine if Shc was tyrosine phosphorylated following IL-5 treatment, purified eosinophils were treated with 150 pM IL-5 for 0–40 min. Following incubation with IL-5, the cells were lysed and the tyrosine-phosphorylated proteins were immunoprecipitated with anti-P-Tyr antibody 4G10. The immunoprecipitates were immunoblotted with anti-Shc antisera. As demonstrated (Figure 4), greater mass of p52 Shc was evident in the 4G10 immunoprecipitates following IL-5 treatment, reaching maximal levels at 10 min of incubation (Figure 4, lane 7). In addition, we did not observe tyrosine phosphorylation of p46 and p66 Shc (not shown) following IL-5 treatment.

Shc contains a C-terminal SH2 domain and is, therefore, capable of associating with other tyrosine-phosphorylated proteins. Consequently, the presence of p52 Shc in anti-P-Tyr immunoprecipitates could be induced by protein–protein interactions mediated by the SH2 domain of Shc. Therefore, to confirm that Shc is tyrosine phosphorylated, the reverse immunoprecipitation and immunoblotting experiment was performed. Shc proteins were immunoprecipitated from detergent lysates of control or IL-5 treated eosinophils and the captured proteins were immunoblotted with anti-P-Tyr antibodies. In anti-Shc immunoprecipitates of IL-5-treated eosinophils, it appeared that two proteins were labeled (Figure 5, lane 5), which migrated approximately with p52 Shc (Figure 5, lanes 7 and 8). These phosphotyrosine-containing proteins were not present in control-treated eosinophils (Figure 5, lane 6) or in nonspecific immunoprecipitates of IL-5-treated eosinophils (Figure 5, lane 4). Taken together, these data are consistent with the model of signal transduction through cytokine family receptors, which predicts that Shc is tyrosine phosphorylated following IL-5 stimulation of eosinophils.

Shc and Grb2 Coimmunoprecipitate after IL-5 Treatment

Previous studies of Shc in other cell types have demonstrated that, as a consequence of undergoing tyrosine phosphorylation, Shc associates with other proteins, including the adapter protein Grb2, which serve as downstream effectors of signal transduction pathways (34). We determined that Grb2 was expressed in eosinophils, and distributed in both the soluble and insoluble fractions as seen with p52 Shc. There was no discernible redistribution of Grb2 between these fractions following IL-5 treatment of the eosinophils (Figure 2).

To determine if Shc associates with Grb2 following IL-5 treatment, eosinophil anti-Shc immunoprecipitates were immunoblotted with anti-Grb2 antisera. Following IL-5 treatment of eosinophils, greater amounts of Grb2 were detected in anti-Shc immunoprecipitates (Figure 6, lane 5) but not in control immunoprecipitates (Figure 6, lane 4). When immunoprecipitated with anti-Grb2 antisera, the control and IL-5-treated samples were seen to contain similar amounts of the Grb2 protein (Figure 6, lanes 2 and 3). Furthermore, anti-Grb2 immunoprecipitates of eosinophils treated with IL-5 contained the two tyrosine-phosphorylated proteins that appear to migrate with p52 Shc (Figure 5, lane 2). These data confirm that association between p52 Shc and Grb2 is increased after IL-5 treatment.

We have presented data suggesting that IL-5 stimulates a signal transduction pathway that uses the adapter protein p52 Shc to mediate activation of eosinophil effector functions. Following stimulation with IL-5, a greater portion of the p52 Shc of the cells becomes more tightly associated with cellular structures that are soluble only in high concentrations of detergents (Figure 1). This process is evident following treatment of the eosinophils with concentrations of IL-5 greater than 15 pM. These data suggest that Shc may participate in the assembly of multiprotein complexes. Several other signaling and cytoskeletal proteins were found in the same less soluble cellular fraction, including p45 MAP kinase (Figure 1D and Figure 3A), Raf-1 (Figure 1D), Jak-2 (Figure 3B), Lyn (Figure 3C), Vav (Figure 3D), actin (Figure 3E), Grb2 (Figure 2), PKCβ, and vimentin (not shown). However, none of these proteins was demonstrated to be increasingly distributed to this fraction following IL-5 treatment. In addition, immunoprecipitation and immunoblotting demonstrated that, after IL-5 treatment of eosinophils, p52 Shc was tyrosine phosphorylated (Figures 4 and 5) in a time-dependent process maximal sometime after 2 min of incubation (Figure 4). Finally, there was an increase in the association of Shc with the adapter protein Grb2 following IL-5 treatment, as demonstrated by coimmunoprecipitation experiments (Figures 5 and 6).

The increased association of p52 Shc with the eosinophil fraction of low detergent solubility following IL-5 treatment (Figure 1A, lanes 8 and 9; Figure 1B, lane 4; Figure 3F, lane 12) may reflect increased association with cytoskeletal elements or membrane-bound protein complexes. The less soluble cell fraction, with which Shc associates, is a complex mixture of proteins, many of which are distributed in the soluble fraction as well. In eosinophils, this fraction (Figure 3, lanes 11 and 12) contained cytoskeletal proteins such as actin (Figure 3E) and vimentin (not shown), the hematopoietic nucleotide exchange factor Vav (Figure 3D), the adapter protein Grb2 (Figure 2, lanes 3 and 4), and several protein kinases including Lyn (Figure 3C), Jak-2 (Figure 3B), p45 MAP kinase (Figures 1D and 3A), Raf-1 (Figure 1E), and PKCβ (data not shown). Shc redistribution upon growth factor stimulation of other cells has been described previously (46, 47) and is presumably a reflection of the association of Shc with activated growth factor receptors. This association, in many cases, is dependent on the tyrosine phosphorylation of the receptor and mediated through the N-terminal phosphotyrosine-binding domain of Shc (38, 48). This process is a necessary precursor to the activation of the Ras–Raf–MAP kinase pathway and subsequent cell replication in some (33) but not all (49) receptor systems. Because many growth factor receptors are linked to the cytoskeleton (50), the association of Shc with detergent-insoluble cell fractions may reflect this phosphotyrosine-mediated receptor association. Alternatively, Shc may associate directly with the actin cytoskeleton. The direct interaction between Shc and filamentous actin has been reported following exposure of PC12 cells to nerve growth factor (37). Therefore, the observed change in the solubility of Shc following IL-5 stimulation of eosinophils could reflect increased association with the membrane proteins or cytoskeletal fractions or both. Many cellular structures contain both membrane and cytoskeletal components including the plasma membrane, the nucleus, and the granules. All three of these cellular structures are lysed by treatment with solutions containing 1% Triton X-100 plus 0.1% SDS. However, insoluble cytoskeletal components of all three may be present in the less soluble cell fractions seen in Figures 1 and 3. Moreover, this reduced solubility of Shc following IL-5 treatment may reflect an increase in the affinity with which Shc associates with insoluble cell structures rather than its physical translocation from one compartment to another. Such an increased affinity could result from allosteric modifications in the protein structure of Shc induced by tyrosine phosphorylation, or Grb2 interaction, or both.

Immunoprecipitation and immunoblotting experiments demonstrated that Shc became tyrosine phosphorylated following IL-5 treatment of eosinophils. Shc was found in greater amounts in anti-P-Tyr immunoprecipitates of IL-5-treated eosinophils in a time-dependent (Figure 4, lane 7) and dose-dependent (not shown) manner, being maximal at 10 min of incubation and at IL-5 concentrations greater than 15 pM. In anti-Shc immunoprecipitates of eosinophils, two tyrosine-phosphorylated proteins could be identified that approximately comigrated with p52 Shc. The proteins were evident only after IL-5 stimulation (Figure 5, lane 5) and were not seen in control immunoprecipitates (Figure 6, lane 4). The two protein bands could represent a single isoform of Shc that migrates as two bands due to a post-translational modification such as phosphorylation. Shc undergoes both tyrosine and serine phosphorylation (33). Alternatively, there could be a second protein that coimmunoprecipitates and comigrates with p52 Shc and is also tyrosine phosphorylated following IL-5 treatment of eosinophils. Finally, because these two proteins were not distinctly resolved in all experiments, they may represent a feature of our immunoprecipitation/immunoblotting procedure such as variable protein reduction by the sample buffer.

Shc is tyrosine phosphorylated on residue Y316 and a number of different tyrosine kinases can utilize Shc as a substrate (35, 51-53). Two of these, Jak-2 and Lyn, have been previously shown to be activated in response to IL-5 in eosinophils (35-38). Following erythropoietin exposure, Shc will associate with Jak-2 in a process dependent on the tyrosine phosphorylation of Jak-2 and mediated by the SH2 domain of Shc (34, 52). In addition, enzymatic activation of Jak-2, independent of receptor binding and phosphorylation, results in the tyrosine phosphorylation of Shc (54). Furthermore, the tyrosine kinase Lyn, a member of the Src family of tyrosine kinases, has been shown to associate with Shc in some model systems and mediate its tyrosine phosphorylation (35, 36).

The phosphorylation of Shc provides a site for the interaction of Shc with other proteins carrying SH2 domains. One such molecule, Grb2, has been previously shown to bind tyrosine-phosphorylated Shc through the Grb2 SH2 domain (39). Grb2 is expressed in eosinophils and distributed both in the soluble and insoluble fractions following cell lysis in buffers containing 1% Triton X-100 plus 0.1% SDS. This distribution was not discernibly affected by incubation with IL-5 (Figure 6). Following IL-5 treatment of eosinophils, Grb2 was detected in anti-Shc immunoprecipitates (Figure 6, lane 5) but not in control immunoprecipitates (Figure 6, lane 4). Furthermore, this anti-Grb2 immunoprecipitate contained two tyrosine-phosphorylated proteins that comigrated with p52 Shc and the tyrosine-phosphorylated proteins present in anti-Shc immunoprecipitates (Figure 5, lane 2). These phosphotyrosyl proteins were not evident in Grb2 immunoprecipitates of control-treated eosinophils (Figure 5, lane 3) or in control immunoprecipitates after IL-5 treatment (Figure 5, lane 1). These data suggest that in eosinophils, Shc and Grb2 associate following exposure of the cells to IL-5.

The importance of Grb2 to downstream signaling pathways was discovered by virtue of its ability to bind both the EGFR and the Ras guanine nucleotide exchange factor SOS and, therefore, couple the stimulation of tyrosine phosphorylation by growth factor receptors to the stimulation of the low molecular weight G protein, Ras (41, 42). Ras then functions as a molecular switch to mediate effects on cellular growth and differentiation (55). In the stimulation of some receptors, the binding of Grb2 and SOS to the activated receptor was mediated by Shc (56-59). This paradigm was shown to apply to signaling through the IL-5 receptor in murine cell lines (60) and through human IL-3, IL-5, or GM-CSF receptors in myeloid cell lines (57– 59). An important functional readout in many of these studies was the ability of Shc to mediate activation of Ras, the entry into the cell cycle, and cellular replication. Our observations that both Shc tyrosine phosphorylation and association with Grb2 are evident in eosinophils, which are terminally differentiated cells incapable of entering the cell cycle, suggest that this signal transduction pathway may also mediate functions unrelated to cell division. Activation of Ras and MAP kinases by IL-5 has been reported in human eosinophils (28, 30) and the hypothesis that this activation is mediated through Shc and Grb2 is consistent with models of cytokine receptor signal transduction suggested by work in other cells. To rigorously establish the relevance of these signal transduction pathways to eosinophil biology, however, requires the development of effective means of manipulating eosinophils at the molecular level. This capability is currently limited by the resistance of eosinophils to conventional transfection methodologies.

Convincing evidence for the participation of Shc–Grb2 or the Ras–Raf–MAP kinase pathway in specific functional changes induced in eosinophils by IL-5 is not yet available. Numerous studies have demonstrated that activated MAP kinases participate in a large variety of cellular processes, many of which are affected by IL-5 in eosinophils. Among the previously identified substrates of MAP kinases are proteins that affect rates of transcription (61) and translation (43), cytoskeletal organization (62), and arachidonic acid metabolism (63). Therefore, a number of the functional changes induced in eosinophils by IL-5 could conceivably be mediated by the regulation of MAP kinase activity through tyrosine phosphorylation of Shc and its association with Grb2. Furthermore, Shc is capable of affecting other signaling pathways (64). A study of mutant GM-CSFR β-chain constructs demonstrated that, in the absence of detectable Shc recruitment to the receptor, Ras–Raf-1–Map kinase activation was unaffected and proliferation and viability maintenance were unaffected. This study suggests that, in these transfectants, Shc may participate in other signaling pathways and cellular processes (65). The specific signal transduction pathways stimulated in eosinophils by IL-5 through tyrosine phosphorylation of Shc have not been identified and are the subject of current study. The identification of the functional end points of these pathways mediated by Shc phosphorylation will contribute to our understanding of the role of IL-5 in the upregulation of eosinophil phlogistic properties and the pathogenesis of allergic inflammation.

This study was supported by an Eli Lilly Biochemistry Grant, a Shaw Scholar Award, and NIH Grants RO1 GM53271 and AI23181.

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Address correspondence to: Mary Ellen Bates, Department of Medicine (Allergy), H6-355 CSC, 600 Highland Ave., Madison, WI 53792. E-mail:

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