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The Journal of Immunology, 1999, 163: 6023-6034.
Copyright © 1999 by The American Association of Immunologists

High Affinity IgG Receptor Activation of Src Family Kinases Is Required for Modulation of the Shc-Grb2-Sos Complex and the Downstream Activation of the Nicotinamide Adenine Dinucleotide Phosphate (Reduced) Oxidase1

Rae-Kil Park{dagger},{ddagger}, Anat Erdreich-Epstein{dagger}, Ming Liu{dagger}, Kayvon D. Izadi* and Donald L. Durden2,*

* Herman B. Wells Center for Pediatric Research, Department of Pediatrics and Biochemistry, Indiana University School of Medicine, Indianapolis, IN 46202; {dagger} Childrens Hospital, Los Angeles Research Institute, Los Angeles, CA 90027; and {ddagger} Department of Microbiology and Immunology, Wonkwang University School of Medicine, Iksan Jeonbuk, Korea


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We used the U937 cell line to examine the modulation of adaptor protein interactions (Shc, Grb2, and Cbl) after high affinity IgG receptor (Fc{gamma}RI) cross-linking, leading to the formation of the Grb2-Sos complex, the activation of Ras, and the regulation of the respiratory burst. Cross-linking of Fc{gamma}RI induced the conversion of GDP-Ras to GTP-Ras reaching a maximum 5 min after stimulation. Concomitant with Ras activation, Sos underwent an electrophoretic mobility shift and the Sos-Grb2 association was increased (6-fold). The Grb2-Sos complex was present only in the membrane fraction and was augmented after Fc{gamma}RI stimulation. Tyrosine-phosphorylated Shc, mainly the p52 isoform, was observed to transiently onload to the membrane Grb2-Sos complex on Fc{gamma}RI stimulation. Cross-linking of Fc{gamma}RI induces the tyrosine phosphorylation of Cbl, which forms a complex with Grb2 and Shc via the Cbl C terminus. Kinetic experiments confirm that Cbl-Grb2 is relatively stable, whereas Grb2-Sos, Grb2-Shc, and Cbl-Shc interactions are highly inducible. The Src family tyrosine kinase inhibitor, PP1, was shown to completely inhibit Shc tyrosine phosphorylation, the Shc-Grb2 interaction, and the Fc{gamma}R-induced respiratory burst. Our results provide the first evidence that the upstream activation of Src kinases is required for the modulation of the Shc-Grb2 interaction and the myeloid NADPH oxidase response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Antigenic stimulation of the high affinity Fc receptor for IgG (Fc{gamma}RI),3 on monocytes and macrophages results in the activation of a number of important biological functions such as the generation of free radicals, production of cytokines, and phagocytosis (1). Cross-linking of Fc{gamma}RI receptors induces the tyrosine phosphorylation of their {gamma}-chains containing specific consensus sequences, YXXL, called immunoreceptor tyrosine-based activation motifs (ITAM) (2). Signaling through Fc{gamma}RI, like other immunoreceptors that lack intrinsic tyrosine kinase activity, is initiated by tyrosine phosphorylation of the ITAM. This results in the recruitment and activation of nonreceptor tyrosine kinases including Hck and Lyn and creates a binding site for Src homology (SH2)-containing proteins such as Syk, phospholipase C{gamma}1 (PLC{gamma}1), Ras GTPase-activating protein (GAP), Shc, Grb2, or p85 subunit of phosphoinositol 3-kinase (PI-3 kinase), etc. (3, 4, 5). The receptor-associated adaptor protein complex likely couples these upstream events to the downstream activation of Ras, Raf-1, mitogen-activated protein kinase (MAP kinase), PI-3 kinase, AKT/PKB protein kinase B, and other effectors of myeloid signaling (6).

The small GTPase, p21ras, is a key molecule in the signaling pathway of a number of tyrosine kinase receptors as well as nonreceptor protein tyrosine kinases such as Src family kinases (7). In the growth factor signaling pathways, GTP-Ras catalyzes the activation of Raf-1, which induces the activation of MAP kinases, in turn regulating many cellular components including phospholipase A2 and nuclear transcription factors such as c-fos or c-jun (8). Other effectors of Ras include the PI-3 kinase/Akt kinase pathway. The activation of Ras occurs predominantly via Grb2-Sos complex, either alone or with Shc, via the formation of Shc-Grb2-Sos complexes (9). Grb2 directly interacts with autophosphorylated receptor tyrosine kinases such as epidermal growth factor, insulin, or platelet-derived growth factor receptors through its Src homology 2 (SH2) domain and at the same time associates with the proline-rich region of Ras guanine nucleotide-releasing factor (GNRF) Sos through its SH3 domains (10). Thus, Grb2 bound to Sos helps to translocate Sos to the receptor complexes associated with the cytoplasmic face of the plasma membrane, resulting in the conversion of Ras from its inactive GDP-bound to its active GTP-bound state (10, 11). Alternatively, Grb2-Sos complex can also interact with phosphorylated Shc via Grb2-SH2 domain binding to Tyr317 in the collagen homology (CH) region of Shc (12). Shc action is complex and not completely understood as suggested by the existence of three Shc isoforms of 45, 52, or 66 kDa and the multiple proteins to which Shc binds in the cell. Although Shc does not have apparent catalytic activity, it can directly interact with many signaling molecules via two phosphotyrosine (Tyr(p))-binding motifs, a phosphotyrosine binding (PTB) domain at N-terminus and a SH2 domain at C-terminus. Shc also has two phosphorylation sites in the {alpha}1-CH region (12, 13) and contains phospholipid binding sites. Shc is a known substrate for Src family kinases in v-Src- and v-Fps-expressed cells (14). Shc is tyrosine phosphorylated on catalytic activation of receptor or nonreceptor protein tyrosine kinases, inducing its binding to the Grb2-SH2 domain (10, 12, 14).

Recently, our attention has focused on the role of the complex adaptor protein, Cbl, in the regulation of Ras. p120c-Cbl is tyrosine phosphorylated after stimulation of growth factor receptors, cytokine receptors, and immunoreceptors (TCR, BCR, or Fc receptors) (15, 16, 17). The cbl protooncogene is the mammalian cellular counterpart of the transforming component v-cbl in Cas NS-1 murine retrovirus. Both members of the Cbl-family, c-Cbl and Cbl-b, are expressed in myeloid cells where they bind to SH3-containing proteins, including Fyn, Grb2, Lck, Fgr, Nck, Crk, PLC{gamma}1, and p85 of PI-3K (17, 18, 19, 20). Also, tyrosine-phosphorylated Cbl can interact with the SH2 domain of Fyn, Lck, or Blk (18, 21). Recent results from Cbl knockout mice suggest a negative regulatory role for Cbl in T lymphocyte development (22). The specific role of Cbl-adaptor protein complex the regulation of Ras has not yet been clearly elucidated in mammalian cells.

Previously, we reported that Shc is markedly tyrosine phosphorylated at Fc{gamma}RI activation and that tyrosine-phosphorylated Shc forms a Shc-Grb2 complex in a phosphotyrosine-dependent manner (23). Data from our laboratory have implicated Cbl and the Raf-1 and MAP kinases in the Fc{gamma}RI signaling pathway (24, 25, 26). These observations prompted us to examine the interaction of the adaptor protein complex, Shc-Grb2-Sos, and Cbl in Fc{gamma}RI-mediated activation of Ras and the respiratory burst. Here we demonstrate that activation of Fc{gamma}RI induced a mobility shift of Sos followed by a transient increase of GTP-Ras. Grb2-Sos complexes were localized to the membrane in resting U937IF cells and were augmented after Fc{gamma}RI cross-linking. Sos coprecipitated Shc and Grb2, and Shc-Grb2-Sos complexes were significantly increased (6- to 8-fold) on Fc{gamma}RI stimulation. Grb2 inducibly associated with tyrosine-phosphorylated Shc, p120–145, and p35 proteins. Cbl was heavily phosphorylated on Fc{gamma}RI stimulation and interacted with Grb2 constitutively and with Shc inducibly via its carboxyl terminus. A selective Src family kinase inhibitor, PP1, was observed to completely abrogate the Shc tyrosine phosphorylation, the Shc-Grb2 interaction, and the myeloid respiratory burst induced by Fc{gamma}R aggregation. Our data provide the first evidence that Src regulates the superoxide response. These data combined with other results from our laboratory suggest that the mutually exclusive Cbl-Grb2-Shc and Shc-Grb2-Sos complexes exist (26) and support a model whereby Src kinases regulate the Grb2-Shc interaction critical for the activation of Ras and the myeloid superoxide response following immunoreceptor stimulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies

The Fc{gamma}RI-specific Abs were kindly provided by Medarex (Annandale, NJ). The mAb 197, 32.2, and 22 are specific for the Fc{gamma}RI{alpha} subunit, and mAb 32.2 and 22 are F(ab')2 fragments of IgG. The cross-linking Ab was a rabbit anti-mouse F(ab')2 fragment (R{alpha}M) obtained from Organon Teknika (West Chester, PA). Anti-Phosphotyrosine and anti-Shc were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-Grb2, anti-Cbl, and anti-Sos were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). anti-Ras (Y13-259) was purchased from Oncogene Science (Uniondale, NY). The PP1 Src family selective kinase inhibitor was purchased from Calbiochem (La Jolla, CA).

Differentiation and cross-linking of U937 cells

U937 cells were maintained in RPMI 1640 with 10% FBS and differentiated with 250 U/ml human recombinant IFN-{gamma} for 4 days (U937IF cells). U937IF cells were cultured at a concentration of 5 x 105 cells/ml, and the medium was replenished with fresh IFN-{gamma} every 2 days, as described (27). For cross-linking of Fc{gamma}RI receptors of U937IF cells, the cells were washed twice in cold HBSS and adjusted to a concentration of 2 x 107 cells/0.5 ml. Cells in 0.5 ml volume were incubated on ice for 30 min with anti-Fc{gamma}RI (0.50 µg/sample). We then added R{alpha}M (5 µg/sample) at 37°C for different periods. Stimulated cells were rapidly cooled with 0.8 ml of cold HBSS and spun down at 500 x g at 4°C for 5 min. The cell pellet was lysed with 0.8 ml Triton X-100 extraction buffer on ice for 30 min.

Immunoprecipitation

Cell lysates were prepared in a Triton X-100 extraction buffer containing 1% Triton X-100, 10 mM Tris, pH 7.6, 50 mM NaCl, 0.1% BSA, 1 mM PMSF, 1% aprotinin, 5 mM EDTA, 50 mM NaF, 10 µM phenylarsine oxide, and 2 mM sodium o-vanadate. Lysates were cleared by centrifugation at 15,000 x g at 4°C for 30 min. For immunoprecipitation of protein, we added 1 µg of anti-Cbl, anti-Shc, anti-Grb2, and anti-Sos to clarified cell lysates. After incubation on ice for 1 h, 50 µl of a 10% solution of formalin-fixed Staphylococcus aureus were added to immunoprecipitates and incubated on ice for 1 h. The adsorbed immune complexes were washed three times in Triton X-100 extraction buffer and resuspended with 25 µl of 1x sample buffer. After boiling at 98°C for 5 min, immune complexes were resolved by SDS-PAGE.

GST fusion protein production

Escherichia coli organisms expressing the GST-Shc SH2 domain (GST-ShcSH2) were kindly provided by Dr. Larry Rohrschneider, Fred Hutchinson Cancer Center, Seattle, WA. A fragment containing the C-terminal Cbl, aa 462–906, was amplified by PCR from full length human Cbl cDNA (American Type Culture Collection, Manassas, VA) and ligated into the BamHI and EcoRI sites of pGEX2 (Pharmacia Biotech, Uppsala, Sweden). Fusion proteins were produced by isopropyl-ß-D-thiogalactopyranoside induction and purified on glutathione-Sepharose 4B beads (Pharmacia Biotech) (28). Approximately 5–10 µg of protein were used in vitro pull-down experiments.

Electrophoresis and immunoblotting

Immunoprecipitates or GST fusion protein-associated precipitates were resolved on 7.5–12.5% acrylamide and 0.193% bisacrylamide gels by SDS-PAGE. Proteins were transferred onto a nitrocellulose membrane (11 milli-amp hours (mA-h)/cm2) with the use of a semidry blotting transfer system (Ellard, Seattle, WA), as described (29). The membrane was incubated with blocking solution (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5% powdered milk) at room temperature for 1 h and then incubated with specific anti-Tyr(p), anti-Shc, anti-Grb2, or anti-Sos with continuous agitation. After three washes in rinse solution (10 mM Tris-HCl (pH 7.5), 150 mM NaCl), the membrane was incubated at room temperature for 1 h with anti-mouse or anti-rabbit Ab conjugated with HRP for enhanced chemiluminescence detection (Amersham, Arlington Heights, IL) or conjugated with alkaline phosphatase for colorimetric development. For reprobing, the membrane was stripped with 0.1 M glycine, pH 2.5, at room temperature for 30 min and then reblotted with primary Ab.

Exchange assay of Ras-GDP to Ras-GTP

U937IF cells (2 x 107 per experimental group) were cultured in phosphate-free RPMI 1640 for 12 h in a 5% CO2 chamber and labeled with 0.3 mCi [32P]orthophosphate/ml at 37°C for 4 h. After labeling and washing free isotope out, U937IF cells were stimulated with 1 µg anti-Fc{gamma}RI, mAb 197, on ice for 30 min and subsequently cross-linked with R{alpha}M for various periods. Cells were lysed with 0.8 ml Triton X-100 extraction buffer. The lysates were cleared by centrifugation and then immunoprecipitated with anti-Ras on ice for 1 h. Immune complexes were adsorbed with fixed S. aureus and eluted in 25 µl elution buffer containing 20 mM Tris-HCl (pH 7.5), 20 mM EDTA, 2% SDS, 0.5 mM GTP, and 0.5 mM GDP by heating at 68°C for 5 min. TLC was performed on polyethylenediamine cellulose in 0.75 M KH2PO4, pH 3.4. A TLC plate was exposed for the autoradiogram, and the ratio of GTP-Ras to (GDP + GTP)-Ras was quantitated using a PhosphorImager (Bio-Rad, Hercules, CA) as described (30).

Cell fractionations

Cell fractionation was performed as described (31) with some modifications. Briefly, U937IF cells (30 x 106 cells in 0.5 ml RPMI) were cross-linked (32.2 F(ab')2) as described above, and the cell pellet was osmotically lysed on ice for 10 min in 1.5 ml buffer A (10 mM Tris-HCl (pH 7.5), 0.5 mM MgCl2, 1 mM sodium o-vanadate, 10 µM phenylarsine oxide, 1 mM PMSF, 0.1 U/ml aprotinin, 10 µM leupeptin, 4 µg/ml pepstatin A). The cell lysate was subsequently homogenized on ice (Dounce homogenizer, tight pestle, 30 strokes) and adjusted to contain 150 mM NaCl, 5 mM EDTA, and 0.1% BSA. Unbroken cells and nuclei were removed by centrifugation at 500 x g for 5 min. The lysate then underwent centrifugation at 100,000 x g (SW 50.1) for 45 min at 4°C, and the supernatant (cytosolic fraction) was adjusted to contain 300 mM NaCl and 1% Triton X-100 in a final volume of 1.6 ml/sample. The pellet (membranes and insoluble fraction) was resuspended in 1.6 ml of buffer B (10 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% BSA, 300 mM NaCl, 1% Triton X-100, 1 mM sodium o-vanadate, 10 µM phenylarsine oxide, 1 mM PMSF, 0.1 U/ml aprotinin, 10 µM leupeptin, 4 µg/ml pepstatin A), and the solubilized membranes (supernatant) were separated from the insoluble fraction (pellet) at 10,000 x g for 15 min. The insoluble fraction (pellet) was resuspended in 1.6 ml buffer B. All fractions were then adjusted to contain 0.1% SDS in an equal final volume (1.6 ml) and incubated on ice for 10 min. An equal volume (16–50 µl) from each sample was removed for assessment of purity of the fractions and SDS-PAGE, and the remainder was subjected to immunoprecipitation, which was performed as for other experiments in this paper, except that all the incubations and washes were performed in buffer B. This subcellular fractionation method assures that the immunoprecipitations are performed under identical SDS concentration, ionic, and pH conditions for comparison of induced protein-protein interactions. The purity of the fractions was evaluated by measurement of lactate dehydrogenase activity (cytosolic marker) and assessment of the content of the {gamma} subunit of Fc{gamma}RI (membrane marker) (32) by 20% SDS-PAGE of 1% of each of the fractionated samples (0.3 x 106 cell equivalents/sample) as described.

Respiratory burst assay

U937IF cells were pretreated with the Src-specific protein tyrosine kinase inhibitor, PP1 (Calbiochem) (concentrations included 1, 5, and 10 µM), or DMSO as control for 45 min at 37°C. Cross-linking Ab or immune complexes (BSA anti-BSA insoluble immune complexes) were then added to the prewarmed cells followed by biochemical analysis of Shc phosphorylation, Shc-Grb2 interaction, and measurement of respiratory burst as described previously (25). Briefly, this involves the quantitation of superoxide anions as measured by the reduction in ferricytochrome c as determined by optical density at 550 nm.. Data are expressed in nanomols of superoxide liberated from 2 x 106 cells during 30 min.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mobility shift of Sos on Fc{gamma}RI activation

Previously, we demonstrated that activation of Fc{gamma}RI results in tyrosine phosphorylation of Raf-1 and a mobility shift of MAP kinase which are downstream of Ras (25). Growth factor-induced phosphorylation of Sos is modulated by the Raf-1/mitogen-activated protein/extracellular signal-related kinase kinase/MAP kinases (6, 9, 10, 11). These concepts led us to explore whether the Fc{gamma}RI-mediated signaling might induce the activation of Ras through Sos. To determine whether Sos was involved in signaling through the Fc{gamma}RI receptor, U937IF cells were stimulated with anti-Fc{gamma}RI followed by R{alpha}M cross-linking. Previous data from our laboratory demonstrated that U937IF cells stimulated with mAb 197 displayed a more rapid and more dramatic activation of Raf-1, MAP kinase, and Shc than did cells stimulated with mAb 32.2 or 22 (25, 33). On the basis of these experiments, we evaluated a point in the kinetics of each Fc{gamma}RI-specific mAb at which we observe maximal activation of downstream signals (Fig. 1GoA, lanes 3–5). Anti-Sos blot revealed that Sos underwent a significant retardation in electrophoretic mobility in U937IF cells stimulated with all three anti-Fc{gamma}RI Abs (Fig. 1AGo, top, lanes 3–5). The mobility shift of Sos was reversed by potato acid phosphatase treatment of cell lysates (unpublished observation). From these data, we conclude that Fc{gamma}RI stimulation induces the phosphorylation of Sos in myeloid cells.



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  		FIGURE 1. Mobility shift of Sos and augmented Grb2-Sos interaction after Fc{gamma}RI activation. U937IF cells were stimulated with anti-Fc{gamma}RI Abs as described in Materials and Methods and immunoprecipitated with anti-Sos. A, anti-Sos immunoprecipitates (IP) of 2 x 107 U937IF cells were immunoblotted for Sos (top), Grb2 (middle), and Shc (bottom). Lane 1 represents a preimmune immunoprecipitation. Lane 2 corresponds to resting U937IF cells. Other lanes include cells stimulated with mAb 197 for 1 min (lane 3), with mAb 32.2 for 5 min (lane 4), and with mAb 22 for 10 min (lane 5). Lane 6 is a whole cell lysate of U937IF cells stimulated with mAb 197. B, Kinetics of Sos mobility shift and Grb2-Sos recruitment after mAb 197 stimulation. Anti-Sos immunoprecipitates were probed for Sos (top), Grb2 (middle), and Shc (bottom). Lane 1 represents preimmune immunoprecipitates, and lane 8 represents whole cell lysates of Fc{gamma}RI-stimulated U937IF cells. Other lanes correspond to resting U937IF cells (lane 2) and those stimulated for 1 min (lane 3), 5 min (lane 4), 10 min (lane 5), 30 min (lane 6), 60 min (lane 7), respectively.

 
Recruitment of Sos to Grb2 after Fc{gamma}RI stimulation

Shuttling of Sos to the plasma membrane, where Sos exchanges Ras-GDP to Ras-GTP, depends on a molecular interaction with Grb2 (10, 11, 12). Immunoprecipitation studies of Grb2 and Sos demonstrated that the Grb2-Sos complex existed constitutively in resting state but that this complex was markedly increased after Fc{gamma}RI activation (6-fold increase with mAb 197) (Fig. 1GoA, middle, lanes 3–5, mAb 197, and Fig. 2GoA, lanes 6–10, 32.2 F(ab')2). Fc{gamma}RI stimulation leads to interaction of Grb2 with Shc (25). Hence we examined whether the Grb2-Sos complex contained Shc. Anti-Sos precipitates contained the p52 isoform of Shc only following Fc{gamma}RI-activation (Fig. 1GoA, bottom, lanes 3–5), and there was no detectable Shc band in resting U937IF cells. Additionally, anti-Tyr(p) blot revealed that tyrosine-phosphorylated Shc was associated with Sos only in Fc{gamma}RI-stimulated U937IF cells (data not shown). The most dramatic association between Shc and Sos was observed with mAb anti-Fc{gamma}RI Ab cross-linking with less Shc coimmunoprecipitating with Sos following 32.2 and 22 mAb stimulation (Fig. 1AGo, compare lane 3 with lanes 4 and 5). These data are consistent with our previous observations that mAb 197 is a more potent stimulator of the Fc{gamma}RI receptor in our system. These results indicated that cross-linking of Fc{gamma}RI induced the mobility shift of Sos and the recruitment of the Sos-Grb2 complex which contains the tyrosine-phosphorylated p52 isoform of Shc, thus forming a Shc-Grb2-Sos multimolecular complex in Fc{gamma}RI-stimulated myeloid cells.



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  		FIGURE 2. Grb2-Sos complexes are recruited to the membrane by Fc{gamma}RI stimulation. U937IF cells (30 x 106 cells/sample) were cross-linked for 0, 1, 3, 10, or 30 min with 32.2 (Fab')2 and then lysed and fractionated as described in Materials and Methods. One percent of the fractionated lysates (0.3 x 106 cell equivalents) was analyzed by 10% SDS-PAGE (D–F), and the remainder of the fractionated lysates were immunoprecipitated (IP) with anti-Grb2 Ab in 0.15 mM NaCl, 0.05 mM Tris-HCl (pH 7.2), 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS buffer (RIPA buffer) and resolved by a separate 10% SDS-PAGE (A–C). A–C: lanes 1–5 and 11, cytosolic fractions (cyto); lanes 6–10 and 12, Triton X-100-soluble membrane fractions (mem); lanes 1–10, anti-Grb2 Ips; lanes 11 and 12, anti-rabbit IgG IP. D–F: lanes 1–5, cytosolic fractions; lanes 6–10, Triton X-100-soluble membrane fractions; lane 11, unfractionated whole cell lysate (0.3 x 106 cell equivalents) stimulated for 1 min with mAb 197 cross-linking.

 
We assessed the kinetics of the Shc-Grb2-Sos complex to determine whether the formation of this complex was transient or propagated after Fc{gamma}RI activation. U937IF lysates from resting or Fc{gamma}RI-stimulated cells (using mAb 197) were immunoprecipitated with ant-Sos antisera and subjected to Western blot analysis. Fc{gamma}RI stimulation of U937IF cells resulted in a mobility shift of Sos detectable as early as 1 min after stimulation (Fig. 1GoB, top, lanes 3–7, and Fig. 2GoA, lanes 6–10). The Sos mobility shift was very rapid, reaching a maximum at 5–10 min and decreasing by 30 min after Fc{gamma}RI stimulation. Anti-Grb2 blot of anti-Sos precipitates revealed that a Grb2-Sos complex was present in a resting state of U937IF cells and that on stimulation with mAb 197 this complex rapidly increased (6- to 8-fold) followed by gradual decline (Fig. 1Go, A, middle, lanes 2–5, and B, middle, lanes 2–7). Although Sos returned to its baseline mobility on SDS-PAGE, the Grb2-Sos complexes were relatively stable and only partially dissociated by 60 min after mAb 197 stimulation. Sos precipitated from U937IF cells stimulated with mAb 197 bound less Shc for a shorter interval of time than did amount of Shc binding to Grb2 performed under similar conditions (compare Fig. 1GoB, bottom, lanes 3–7; Figs. 4GoB and 5B). Anti-Tyr(p) blot for Shc in anti-Sos precipitates revealed predominantly the p52 Shc band shown in Fig. 1GoB (bottom) (data not shown). Consistent with the result in Fig. 1GoA (bottom), the association of Sos with Shc was an inducible event (Fig. 1GoB, bottom). The Sos-Shc interaction paralleled the Sos-Grb2 interaction, decreasing over time. There was no detectable association of Sos with Shc in resting U937IF cells (Fig. 1Go, bottom panels, lane 2). These results indicate that Fc{gamma}RI stimulation induced a rapid and transient mobility shift of Sos, which is associated with the increased binding of Sos to Grb2 and Shc and the tyrosine-phosphorylated p52 Shc. Interestingly, when Sos returned to its basal position on SDS PAGE 30 min after stimulation, it continued to complex with Grb2 but not with Shc (3-fold decrease in Sos-Grb2 interaction). From these data, we conclude that the formation of a Shc-Grb2-Sos complex in vivo is an extremely short-lived biochemical event in the cell.



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  		FIGURE 4. Interaction of Shc and Grb2 is induced by Fc{gamma}RI activation. Tyrosine phosphorylation of Shc and its association with Grb2 were examined in U937IF cells under conditions of Fc{gamma}RI stimulation. A, Anti-Shc precipitation performed on 2 x 107 U937IF cells after stimulation with mAb 197, 32.2, or 22. The membrane was blotted with anti-Tyr(p). B, Membranes were probed with anti-Shc (top) and anti-Grb2 (bottom). Lane 1 represents preimmune (Pre-im) immunoprecipitates (IP). Other lanes correspond to resting U937IF cells (lane 2) and cells stimulated with mAb 197 for 1 min (lane 3), mAb 32.2 for 5 min (lane 4), and mAb 22 for 10 min (lane 5). Lane 6 is a whole lysate of Fc{gamma}RI-stimulated U937IF cells.

 
Sos-Grb2-Shc complexes are localized to the membrane and are augmented after Fc{gamma}RI stimulation

To further examine the highly induced association of the Sos-Grb2-Shc complex, we fractionated U937IF cells stimulated with Fc{gamma}RI for various lengths of time into cytosolic fraction, Triton X-100-solubilized membrane fraction, and Triton X-100 insoluble fraction and performed Grb2 immunoprecipitations on the cytosolic and the Triton-soluble membrane fractions. As can be seen from Fig. 2Go, D and F, Sos and Grb2 were present in both the cytosolic and the Triton X-100-soluble membrane fractions of U937IF cell lysates in similar amounts in resting cells and throughout stimulation, (each lane represents the cytosolic fraction from 0.3 x 106 cell equivalents). Lysates comprising the Triton X-100 insoluble membrane fractions contained undetectable or only minimally detectable Sos and Grb2 (not shown). Grb2 immunoprecipitations of the fractionated samples from resting U937IF cells (30 x 106 cells/immunoprecipitation) revealed that a significant amount of Sos was associated with Grb2 in the Triton X-100-soluble membrane fraction (Fig. 2GoA, lane 6), whereas no Grb2-Sos complex was detected in the cytosolic fraction in the resting cells (Fig. 2GoA, lane 1). The amount of Sos bound to Grb2 in the membrane fraction rapidly increased above baseline after Fc{gamma}RI stimulation, reaching a maximum 1–3 min after stimulation. The electrophoretic mobility of the Grb2-bound membrane-associated Sos was retarded after stimulation, with maximal retardation 10 min after stimulation, and returning to baseline after >30 min from the time of stimulation (Fig. 2GoA, lanes 6–10). This mobility shift is similar to that seen in Fig. 1Go in unfractionated U937IF cells, most likely reflecting serine/threonine phosphorylation of Sos (9, 10, 11). Either none or only minimal amounts of cytosolic Sos were associated with Grb2 in the Grb2 immunoprecipitations of the cytosolic fractions, either at rest or after stimulation (Fig. 2GoA, lanes 1–5, and data not shown). Fig. 2GoC confirms that similar amounts of Grb2 were immunoprecipitated in the cytosolic and membrane fractions at the various time points.

Examination of Shc in the same experiments reveals that in the fractionated cell lysates the majority of Shc was in the cytosolic fraction, the remainder being found in the Triton X-100-soluble membrane fraction (Fig. 2GoE). Only minimal amounts of Shc were associated with Grb2 in Grb2 immunoprecipitations of fractionated resting U937IF cells (Fig. 2GoB, lanes 1 and 6). After Fc{gamma}RI stimulation, Shc rapidly bound to Grb2 in both the cytosol and the soluble membrane fractions with similar kinetics, and with more Shc bound to Grb2 in the cytosol, and a smaller amount in the Triton X-100-soluble membrane fraction (Fig. 2GoB). The kinetics of Shc binding to Grb2 was very similar to the kinetics of the mobility shift of Grb2-bound Sos in the membrane fraction, beginning as early as 1 min after stimulation, reaching a maximum at 10 min, and declining by 30 min (Fig. 2Go, A and B). The inducible transient increase in the tyrosine-phosphorylated Shc band coprecipitating with Grb2 on the anti-Tyr(p) blot (not shown) followed kinetics similar to that of the Shc-Grb2 complex formation as seen on the Shc-specific blot (Fig. 2GoB), indicating that the increase in the tyrosine-phosphorylated band probably reflected the increase and subsequent decrease in Shc onloading to Grb2. Shc and Sos were only minimally detected (or not at all) in the Triton X-100-insoluble membrane fraction on blots of both the fractionated cell lysates and the Grb2 immunoprecipitations (not shown). The cytosolic fraction and both membrane fractions (Triton X-100 soluble and insoluble) were at least 90% pure, as determined by assessment of lactate dehydrogenase activity (cytosolic marker) and the {gamma} subunit of the Fc{gamma}RI receptor (soluble membrane marker). To verify that the Grb2-Sos complex was preformed in the membrane in resting cells, we performed Sos immunoprecipitations on similarly fractionated U937IF cells at rest and found that in resting U937IF Grb2 coprecipitates with Sos exclusively in the Triton X-100-soluble membrane fraction despite the presence of similar amounts of Grb2 in the cytosolic and soluble membrane fractions (not shown). From these data, we conclude that following ITAM stimulation, the formation of Shc-Grb2-Sos complex is a highly induced event occurring in the plasma membrane compartment of the cell.

Fc{gamma}RI Stimulation induces the conversion of GDP-Ras to GTP-Ras

It has been shown that mobility shift of Sos is due to serine/threonine phosphorylation and reflects the activation of GNRF to exchange Ras-bound GDP to GTP (10). Therefore, we determined the amount of GTP-Ras in U937IF cells after Fc{gamma}RI activation. p21Ras activation by Fc{gamma}RI in U937IF cells was rapid and transient (Fig. 3GoA). The Ras-GTP was increased after 1 min of Fc{gamma}RI stimulation, reached a maximum by 5 min, and gradually declined there after. Using a PhosphorImager, we quantitated that the ratio of GTP-Ras to (GTP-Ras + GDP-Ras) was 12% in a resting state, reached a maximum of 32% by 5 min of stimulation, and returned to 16% by 30 min after Fc{gamma}RI stimulation (Fig. 3GoB). These results demonstrate that a baseline level of GTP-Ras existed in resting myeloid cells and was markedly increased for a short period upon Fc{gamma}RI activation.



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  		FIGURE 3. Fc{gamma}RI stimulation induced the conversion of GDP-Ras to GTP-Ras. U937IF cells were radiolabeled with 32PO4 and stimulated with mAb 197 followed by R{alpha}M Ab. After stimulation, p21ras of U937IF cells was immunoprecipitated with anti-Ras (Y13–259), and guanine nucleotides were separated by TLC as described in Materials and Methods. Lane 1 represents preimmune (Pre-im) immunoprecipitates (IP) of U937IF cells stimulated for 1 min. Other lanes correspond to resting U937IF cells (lane 2) and cells stimulated for 1 min (lane 3), 5 min (lane 4), 10 min (lane 5), and 30 min (lane 6), respectively. The autoradiogram and PhosphorImager quantitation are shown in A and B, respectively. Data are representative of three separate experiments.

 
Interaction of Shc with Grb2 was inducible on Fc{gamma}RI activation

We next examined the molecular interaction of the adaptor molecules Shc and Grb2, the complexes of which shuttle Sos to Ras at the plasma membrane (11). Tyrosine phosphorylation of Shc is critically required for the activation of Ras in many systems (6). Thus, we tested the tyrosine phosphorylation of Shc and its association with Grb2 after stimulation of U937IF cells with mAb 197, 32.2, or 22. Anti-Tyr(p) blot of anti-Shc precipitates showed that Fc{gamma}RI activation markedly induced the tyrosine phosphorylation of both p46 and p52 isoforms of Shc and other phosphoproteins including p145 and p35 (Fig. 4GoA, lanes 2–5). To confirm the identity of the p46 and p52 phosphoproteins, the membrane was stripped and reprobed with a polyclonal anti-Shc Ab. An equivalent amount of p46 and p52 Shc was detected in all lanes except in immunoprecipitation with normal rabbit serum (preimmune) (Fig. 4GoB, top). Both the p46 and p52 bands immunoreactive with anti-Shc in Fig. 4GoB (top) could be accurately superimposed with the phosphotyrosine bands in the anti-Shc precipitates (Fig. 4AGo). To assess the recruitment of Grb2 with Shc after Fc{gamma}RI activation, the same membrane was probed for Grb2. Anti-Grb2 blot revealed that stimulation of U937IF cells with all three anti-Fc{gamma}RI Abs (mAb 197, 32.2, or 22) significantly increased the amount of Grb2 bound to Shc (Fig. 4GoB, bottom, lanes 3–5). As shown previously (25), recruitment of Grb2 by Shc was dependent on the tyrosine phosphorylation of Shc. To confirm the association of Shc with Grb2 in Fc{gamma}RI signaling, we performed a reciprocal in vivo immunoprecipitation with anti-Grb2 Ab. We observed that the Grb2 IP coprecipitated phosphoproteins including p145, p110–120, p46, and p52 of Shc and p35 in U937IF cells on Fc{gamma}RI activation (Fig. 5GoA, lanes 3–5). We confirmed that both p46 and p52 phosphoproteins were Shc by probing with rabbit anti-Shc (Fig. 5GoB, upper panel). Grb2 was not associated with Shc in the resting state and was markedly induced after Fc{gamma}RI stimulation (Fig. 5GoB, top, lanes 3–5). Anti-Grb2 blot confirmed that an equal amount of Grb2 was precipitated in all lanes, except in the preimmune precipitate (Fig. 5GoB, middle, lanes 2–5). These results suggest that Shc and Grb2 are recruited by each other and inducibly formed Shc-Grb2 complexes in the Fc{gamma}RI-mediated signaling pathway in U937IF cells.



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  		FIGURE 5. Grb2 associates with Shc. A, Anti-Grb2 precipitates were probed for Tyr(p) Ab. Lane 1 represents preimmune (Pre-im) immunoprecipitate (IP). Other lanes of the membrane correspond to resting U937IF cells (lane 2) and cells stimulated with mAb 197 for 1 min (lane 3), mAb 32.2 for 5 min (lane 4), and mAb 22 for 10 min (lane 5). Lane 6 is a whole lysate of Fc{gamma}RI-stimulated U937IF cells. B: top, anti-Shc immunoblot; middle, anti-Grb2 blot; bottom, anti-Sos blot. Lanes are identical with those in A.

 
Tyrosine phosphorylation of Cbl and its association with Grb2 and Shc

Recently, several lines of evidence have shown p120c-Cbl to be a major substrate for protein tyrosine kinases as well as an adaptor-binding protein in the signaling pathway of Ig gene superfamily receptors such as TCR, BCR and Fc receptors (15, 16, 17). To determine whether Cbl is involved in signaling of Fc{gamma}RI in U937IF cells, lysates were immunoprecipitated with anti-Cbl and analyzed by Western blot. Fc{gamma}RI stimulation induced a marked tyrosine phosphorylation of 120-kDa proteins with mobility retardation (Fig. 6Go, A and B, top panel, lanes 3–5). p120 was also tyrosine phosphorylated in the resting state without any change in mobility but the level of tyrosine phosphorylation was stoichiometrically less than that in Fc{gamma}RI-activated cells (~60%). To confirm the identity of the 120-kDa protein, the same membrane was stripped and reprobed for Cbl. The p120 immunoreactive bands of the anti-Tyr(p) blot superimposed with the Cbl bands in both anti-Cbl precipitates and the positive control U937IF lysates (Fig. 6Go, A, second panel, lanes 2–6, and B, second panel, lanes 2–5). Preimmune precipitates did not bring down any specific band in this m.w. range (Fig. 6Go, A and B, second panel, lane 1).



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  		FIGURE 6. Tyrosine phosphorylation of Cbl and its association with Grb2 and Shc. A, Tyrosine phosphorylation of Cbl and its association with Grb2 and Shc were examined in U937IF cells stimulated with anti-Fc{gamma}RI Abs. Anti-Cbl precipitates from 2 x 107 U937IF cells were probed for Tyr(p) (top panel), Cbl (second panel), Grb2 (third panel), and Shc (bottom panel). Lane 1 represents preimmune (Pre-im) immunoprecipitates (IP). Other lanes correspond to resting U937IF cells (lane 2) and cells stimulated with mAb 197 for 1 min (lane 3), mAb 32.2 for 5 min (lane 4), and mAb 22 for 10 min (lane 5). Lane 6 is a whole lysate of Fc{gamma}RI-stimulated U937IF cells. B, Kinetics of tyrosine phosphorylation of Cbl and Shc on Fc{gamma}RI activation. U937IF cells stimulated with anti-Fc{gamma}RI mAb 197 followed by rabbit anti-mouse cross-linking (F(ab')2). Cell lysates were immunoprecipitated with anti-Cbl and immunoblotted for Tyr(p) (top panel), Cbl (second panel), and Grb2 (third panel), Shc (bottom panel). Lane 1 represents preimmune immunoprecipitates, and lane 8 is a whole lysate of Fc{gamma}RI-stimulated U937IF cells. Other lanes correspond to resting U937IF cells (lane 2) and cells stimulated for 1 min (lane 3), 5 min (lane 4), 10 min (lane 5), 30 min (lane 6), and 60 min (lane 7), respectively. C, Shc-SH2 binds to Cbl. GST fusion protein pull-down experiments were performed on U937IF lysates of resting and Fc{gamma}RI-stimulated cells with 5 µg GST alone (lanes 1–4) or GST-ShcSH2 fusion proteins (lanes 5–8). The membrane was immunoblotted for Cbl. Stimulation with anti-Fc{gamma}RI Abs was performed as described in Materials and Methods with three different Fc{gamma}RI specific Abs (lanes 6–8).

 
Cbl is associated with adaptor proteins including Grb2, p85 subunit of PI-3 kinase, Nck, Crk, and CrkL (15, 16, 17, 18, 19, 21). To examine whether Cbl was associated with Grb2 on Fc{gamma}RI activation, the same membrane of anti-Cbl precipitates (Fig. 6Go) was immunoblotted for Grb2. Both resting and U937IF cells maximally stimulated with anti-Fc{gamma}RI brought down a similar amount of Grb2, except in preimmune precipitates (Fig. 6GoA, third panel, lanes 2–5). The upper band represents the immunoreactive light chain of IgG. We then tested whether Cbl could coprecipitate Shc after Fc{gamma}RI activation. Shc was not detectable in anti-Cbl precipitates of resting U937IF cells; however, Cbl-Shc associations were induced after Fc{gamma}RI stimulation (Fig. 6GoA, bottom panel, compare lane 2 with lanes 3–5; Fig. 6GoB, bottom panel, compare lane 2 with lanes 3–7). Anti-Tyr(p) blot showed that Cbl-bound Shc underwent tyrosine phosphorylation after Fc{gamma}RI activation (data not shown). To examine whether the Cbl and Shc interaction is mediated by the SH2 domain of Shc, 5 µg GST or GST-ShcSH2 fusion protein were incubated with U937IF lysates. p120Cbl was detected in the precipitates of GST-ShcSH2 fusion protein in both resting and Fc{gamma}RI-stimulated cell lysates (Fig. 6GoC). Cbl precipitated with GST-ShcSH2 fusion protein from Fc{gamma}RI-activated U937IF cells was a doublet, displaying prominent mobility retardation, whereas in the resting state it was represented by a single band (Fig. 6GoC, lanes 5–8). As we expected, {alpha}-Tyr(p) blot showed the same pattern of Cbl as represented in Fig. 6GoB (data not shown). Interestingly, the intensity of Cbl bands from U937IF lysate stimulated with mAb 197 was much weaker than that of resting state, and the in vitro interaction observed by the analysis of resting cell lysates (Fig. 6GoC) is not observed when we immunoprecipitate Shc or Cbl in vivo (Fig. 6Go, A and B). The reason for this disparity between in vivo and in vitro analysis is not clear and will require further analysis with mutants of Shc in vivo. An equal amount of GST or GSTShcSH2 fusion protein was loaded and confirmed by Coomassie blue stain (data not shown).

Kinetics of tyrosine phosphorylation of Cbl and Shc on Fc{gamma}RI activation

We were interested in the kinetics of tyrosine phosphorylation as well as the molecular interaction among Cbl, Shc, and Grb2. U937IF lysates were precipitated with anti-Cbl and subjected to Western blot analysis (Fig. 6GoB). The phosphotyrosine content of the Cbl IP dramatically increased at 1 min and then declined sharply by 30 min after mAb 197 stimulation (Fig. 6GoB, top panel, lanes 2–7). The tyrosine-phosphorylated p120 bands superimposed with Cbl bands in anti-Cbl blot (Fig. 6GoB, second panel, lanes 2–8). We found that tyrosine phosphorylation of Cbl is not always coincident with mobility retardation of the protein. Both 30- and 60-min stimulated U937IF lysates continued to show Cbl bands with mobility shift in anti-Cbl blot but there were no or very faint bands of phosphoprotein in the anti-Tyr(p) blot. The anti-Cbl precipitates were then blotted for Shc and Grb2. Cbl IPs precipitated an equivalent amount of Grb2 in resting U937IF cells as well as those stimulated with mAb 197 for 1 min (Fig. 6GoB, third panel, lanes 2–3). However, interaction of Cbl with Grb2 was beginning to dissociate 5 min after stimulation (Fig. 6GoB, third panel, lanes 4–7). The association of Cbl with p52 Shc was detectable in U937IF cells stimulated with mAb 197 against Fc{gamma}RI (Fig. 6GoB, bottom panel, lanes 3–7). In the resting state, there was no detectable Cbl-Shc interaction (Fig. 6GoB, lane 2). The amount of Shc in anti-Cbl precipitates increased rapidly by 1 min after stimulation and then decreased during 5–60 min. The middle band migrating between p52 and p46 Shc is the heavy chain of IgG. Anti-Tyr(p) blot for Shc showed a similar pattern to other anti-Shc blots (not shown). The results indicated that Cbl was significantly phosphorylated on tyrosine in a kinetic manner after Fc{gamma}RI activation, constitutively maintaining a stable complex with Grb2 and inducibly recruiting tyrosine-phosphorylated p52 Shc on Fc{gamma}RI activation.

C terminus of Cbl was associated with Shc and Grb2

Our observations that Shc binds to Grb2 in an inducible manner after Fc{gamma}RI stimulation and that Cbl is bound to Grb2 in a constitutive manner prompted us to investigate the molecular basis for the Cbl-Shc interaction in myeloid cells. To further determine whether a specific domain of Cbl mediates the interaction between Cbl and the adaptor molecules, Shc and Grb2, we generated a GST fusion protein encoding C terminus of Cbl (containing the Grb2 binding site) as described in Materials and Methods. We conducted an in vitro binding assay using the GST-Cbl C terminus (GST-Cbl-CT) fusion protein. GST or GST-Cbl-CT fusion protein, 5 µg, was incubated with lysates from resting cells or U937IF cells stimulated with mAb 197. As shown in Fig. 7Go, Cbl-CT coprecipitated both Shc and Grb2 in a significantly different manner. Cbl-CT recruited a considerable amount of Shc by 5 min after stimulation of U937IF cells with mAb 197 (Fig. 7GoA, lanes 4–6). Shc was not detectable in resting cells and reached a maximum level around 1 min after stimulation, then gradually disappeared by 30 min after stimulation. Anti-Tyr(p) blot of Shc in GST-Cbl-CT precipitates showed the of an inducible association of Shc with Cbl (data not shown). However, in contrast to the Cbl-Shc interaction, GST-Cbl-CT brought down an equivalent amount of Grb2 in both resting and stimulated U937IF lysates with mAb 197 (Fig. 7GoB). We confirmed that equivalent amounts of GST or GST-Cbl-CT proteins were loaded by Coomassie blue staining of the gel (data not shown). These results indicate that the Cbl-CT mediated a constitutive interaction with Grb2 and inducibly binds to Shc via the Shc-Grb2 interaction. Cbl might have at least two ways to modulate the Shc-Grb2-Sos complexes through either its inducible direct interaction with Shc (through the YxxL, Y at position 92) or its relatively stable association with Grb2-SH3.



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  		FIGURE 7. C terminus of Cbl associates with Shc and Grb2. U937IF lysates from resting or cells stimulated with mAb 197 were precipitated with 5 µg GST alone (lanes 1 and 2) or GST-Cbl CT fusion proteins (lanes 3–8). A, The membrane was immunoblotted for Shc. Lanes correspond to resting U937IF cells (lanes 1 and 3) and cells stimulated for 1 min (lanes 2 and 4), 5 min (lane 5), 10 min (lane 6), 30 min (lane 7), 60 min (lane 8), respectively. Lane 9 represents a whole cell lysate of Fc{gamma}RI-stimulated U937IF cells. B, The membrane shown in A was probed for Grb2. Lanes are identical with those in. A.

 
Src kinase inhibitor, PP1, abrogates Shc phosphorylation, Shc-Grb2 interaction, and superoxide response

Previous data from our laboratory demonstrated that Fc{gamma}RI stimulation results in the activation of the Src family kinase, Hck, and the tyrosine phosphorylation of the Shc and Cbl adaptor proteins (25, 26, 29). Hanke et al. (34) recently described a protein tyrosine kinase inhibitor which at concentrations between 1 and 10 µM selectively inhibited the Src family protein tyrosine kinases and recent crystallographic data of Schindler et al. (35) confirms this selective interaction between PP1 and Hck kinase. To study the role of Src kinases in Fc{gamma}RI signaling, we incubated U937IF cells with the tyrosine kinase inhibitor, PP1, followed by Fc{gamma}R cross-linking (Fig. 8Go). PP1 completely abrogated the tyrosine phosphorylation of Shc (Fig. 8GoA, lanes 5–6) which correlated with an inhibition of in vivo formation of Shc-Grb2 complexes (Fig. 8Go, B and C, lanes 5–6). This block in Shc-Grb2 complex formation was associated with decreased recruitment of Grb2-Sos complex formation in U937IF cells (data not shown), and a complete abrogation of the Fc{gamma}R-induced respiratory burst response. An identical effect of PP1 was observed when the Fc{gamma}RI-specific mAb, 32.2 (F(ab')2 fragment), was used to induce NADPH oxidase activity (data not shown). Moreover, the phorbol ester (PMA)-induced activation of the superoxide production was not inhibited by pretreatment of cells with PP1 (Fig. 8GoD), suggesting that the effect of PP1 was specific for Fc{gamma}RI-induced respiratory burst. From these data, we conclude that Src kinase activation is essential for the downstream tyrosine phosphorylation of adaptor complexes that control Fc{gamma}RI-induced adaptor-nucleotide exchange interactions and the myeloid oxidant response.



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  		FIGURE 8. Src family kinase inhibitor, PP1, abrogates the Fc{gamma}RI-induced Shc phosphorylation, Shc-Grb2 interaction, and the NADPH oxidase response. Shc was precipitated from resting (lane 1) or Fc{gamma}R-stimulated (lanes 2–7) U937IF cells preincubated at 37°C with 1 µM PP1 (lane 4), 5 µM PP1 (lane 5), 10 µM PP1 (lane 6) or DMSO (lane 3). Lane 7 represents a whole cell lysate of Fc{gamma}R-stimulated U937IF cells. A, Membranes were probed with anti-Tyr(p) ({alpha}PY) Ab. B, Anti-Shc blots. C, anti-Grb2 immunoblots. D, Effects of PP1 on myeloid Fc{gamma}R- and PMA-induced respiratory burst response. The NADPH oxidase activity of U937IF cells preincubated at 37°C for 45 min. with 1 µM, 5 µM, or 10 µM PP1 or DMSO was quantitated by measuring superoxide production as superoxide dismutase-inhibitable reduction of ferricytochrome c as described in Materials and Methods. Data are the means and SD of triplicate samples in each experimental group (see legend for experimental groups) IP, immunoprecipitate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We previously reported that Fc{gamma}R induces the activation of Src, and we implicated the Shc-Grb2 complex and Raf-1/MAP kinases in Fc{gamma}RI signaling (25, 29). Recent results from our laboratory (26)and the data of Rellahan et al. (36)and Buday et al. (37)have suggested that the Cbl protein may serve as a complex adapter protein binding Grb2 and controlling the formation of the Grb2-Sos complex in response to ITAM stimulation. In this report, we designed experiments to determine whether cross-linking of Fc{gamma}RI induces Ras activation and to further define the role of Src kinases, the Shc-Grb2-Sos complex and Cbl in Fc{gamma}RI signaling pathways that lead to the activation of Ras and the superoxide response.

Our data demonstrate that the electrophoretic mobility of Sos was retarded by Fc{gamma}RI stimulation (Fig. 1Go, top). This may reflect serine/threonine phosphorylation as demonstrated in growth factor signaling. Potato acid phosphatase treatment reversed the mobility shift of Sos after Fc{gamma}RI stimulation in U937IF cells (our unpublished data). Previously, it has been reported that the mobility shift in Sos is associated with serine/threonine phosphorylation of Sos, which then mediates the activation of the Ras/Raf-1/MEK/ERK (mitogen-activated protein/extracellular signal-related kinase kinase) pathway after stimulation with insulin or EGF (38). However, the biological significance of Sos phosphorylation has not been clearly elucidated. One possibility is that phosphorylation of Sos directly stimulates its catalytic activity toward Ras. This notion is supported by the report that phosphatase inhibitors are required for the purification of GNRFs (39). The other possibility is that phosphorylation may induce the conformational change of Sos, affecting its binding to Grb2 or its cytoplasmic localization leading to an increase in the activity of GNRF (40). The other possibility is that Sos phosphorylation is not important for Sos activity. The molecular interaction of Sos with Grb2 is important for the activation of Ras (6, 41, 42, 43). Our coimmunoprecipitation studies demonstrated that Sos constitutively forms a complex with Grb2 in the resting state and was recruited to bind more Grb2 after Fc{gamma}RI activation (6-fold increase) (Fig. 1GoA, middle, lanes 2–5). This Grb2-Sos complex binds to tyrosine phosphorylated Shc only in U937IF cells stimulated with anti-Fc{gamma}RI. These results suggest that the Grb2/Sos complex is induced in myeloid cells after Fc{gamma}RI activation. Moreover, the phosphorylation-dependent association of Shc with the Sos-Grb2 complex observed in Ramos cells stimulated with anti-IgM further supports this contention (44).

In mammalian cells, Shc and Grb2 functionally couple upstream protein tyrosine kinases to Ras (9, 12, 45). In addition, Tyr317 within the consensus Tyr-Val-Asn-Val Shc CH domain interacts with the Grb2 SH2 domain after stimulation with growth factors, TCR, BCR, and many cytokines (12, 13, 14). In our studies, Shc was tyrosine phosphorylated and displayed kinetic changes in binding to Grb2 and Sos on Fc{gamma}RI activation (Figs. 4Go and 5Go). Furthermore, Shc coprecipitated with other phosphoproteins, including p115–120, p145, and p35, after Fc{gamma}RI activation. Recently, Damen et al. (46) reported that the Shc-associated p140–145 exhibited both phosphatidylinositol 3,4,5-triphosphatase and inositol 1,3,4,5-tetraphosphate 5-phosphatase activity. p140–145, now identified as the SH2-containing inositol phosphatase, is thought to be a bridge molecule connecting Ras and inositol signaling pathways. We have confirmed that the p145 protein present in our {alpha}-Shc and {alpha}-Grb2 IPs is SH2-containing inositol phosphatase (Figs. 4GoA and 5A) (data not shown). A tyrosine-phosphorylated protein of 35–38 kDa has been described previously as a substrate of TCR-activated protein tyrosine kinases. It has been shown to interact with numerous SH2-containing proteins including Grb2, PLC{gamma}1, GAP, and Src family kinases (47). The p35–38 protein, now known as Lnk, may play a critical role in coupling tyrosine kinases signals through Grb2 to the phosphatidylinositol pathways. Hence, we conclude that Shc by virtue of binding to these phosphoproteins may modulate specific protein-protein interactions after Fc{gamma}RI stimulation.

Cbl is markedly tyrosine phosphorylated in response to Fc{gamma}R and Fc{gamma}RI engagement (16, 26). Yoon et al. (48) have shown that Cbl functions as a negative signaling modulator in Caenorhabditis elegans vulval development. In C. elegans, Cbl functions at the level of Sem5/Grb2 to control Ras (48). Our data demonstrated that tyrosine phosphorylation of Cbl occurred rapidly and disappeared by 30 min after stimulation, although Cbl continued to display mobility retardation which has been suggested to reflect ubiquitination of Cbl (49). Previously, it has been demonstrated that Cbl interacts with Grb2 and other adapter proteins via both SH2 and SH3 domains (16, 17, 18, 19, 21, 26). Herein, we demonstrate that the Cbl-Grb2 complex contains tyrosine-phosphorylated Shc and that these Cbl-Shc-Grb2 complexes are induced rapidly and are short-lived in U937IF cells stimulated with anti-Fc{gamma}RI (Fig. 6GoB). In contrast, it has been reported that Shc binds to Cbl for up to 60 min after CSF-1 stimulation of BAC1.2F5 macrophages (49). Our data indicate that Cbl, Shc, Grb2, and Sos form a transient induced multimolecular adaptor complex. This complex may modulate the activation of Ras and other small GTPases. The C terminus of Cbl (Cbl-CT) has 11 proline-rich regions as well as a NXXY motif (consensus binding site for Shc PTB domain). In GST-Cbl CT fusion protein precipitation studies, the Cbl and Grb2 interaction was constitutive whereas the Cbl and Shc association was remarkably inducible (Fig. 7Go). GST-ShcSH2 fusion protein precipitated Cbl in both resting and activated U937IF cells (Fig. 6GoC). In other fusion protein experiments, we observed decreased Shc-SH2 binding to Cbl at 1 min after Fc{gamma}RI stimulation, a time when anti-Cbl IP show an increased binding of Cbl to Shc (Fig. 6Go, A and B) (data not shown). The decreased Cbl that precipitated with GST-ShcSH2 fusion protein by 1 min of Fc{gamma}RI stimulation compared with that detected in the resting state, which correlates with a maximum level of tyrosine phosphorylation of Cbl at 1 min of stimulation. The kinetics of GST-ShcSH2 fusion protein precipitation showed that Cbl was detected as a single band in the basal status, declined in intensity by 1 min after stimulation, and by 5 min it returned to a doublet with similar intensity as Cbl in the resting state (unpublished data). However, the amount of Shc present in anti-Cbl precipitates from cell lysates between 1 and 5 min of stimulation was not decreased. Our in vivo and in vitro precipitation studies regarding the Cbl-Grb2 complex demonstrated that the amount of Cbl-bound Grb2 in the basal state was similar to that seen in U937IF activated with anti-Fc{gamma}RI for 1 min. By 1 min of stimulation, Cbl in GST-Grb2SH2 precipitates was still detected as a doublet, whereas GST-Grb2CSH3 did not precipitate Cbl (26). These results suggest that the Cbl-Shc interaction may be dependent on at least two phosphotyrosine-binding sites of Shc, the Shc-SH2 (via YxxL, Y at position 92 of Cbl) and the Shc-PTB domain (via NxxY, Y at position 674 of Cbl) (12, 13). Our potato acid phosphatase experiments, in which the Cbl-Shc interaction was eliminated with phosphatase treatment, confirm that Cbl interacts with Shc in a phosphorylation-dependent manner (unpublished data). A possible role for the tyrosine phosphorylation of Cbl may be to induce a conformational change leading to an alteration in Cbl-Shc and/or Cbl-Grb2 binding. We propose a model that Cbl may alter its interaction with Shc (i.e., initially Cbl is bound to Shc-SH2; then on stimulation Shc binds to CBL via the Shc-PTB domain). Work is in progress on the construction of a full length Cbl mutant lacking the NXXY motif (the putative Shc-PTB binding site) and Shc-PTB constructs to test this hypothesis. We argue that Shc or Cbl tyrosine phosphorylation qualitatively modulates their interaction with Grb2 and that this Shc-Grb2 interaction may indirectly modulate the Grb2-Sos complex after ITAM stimulation to control Ras. This model is supported by data shown in Fig. 2Go, in which the Grb2-Sos interaction is induced within the membrane fraction. It is formally possible that Shc binding to Cbl modulated the binding of Grb2 to Cbl and hence controls the exchange of Cbl for Sos in receptor aggregates.

Considerable evidence suggests a role for the small GTPase, Rac2, in regulation of NADPH oxidase activity in myeloid cells (50, 51). Despite significant progress, the mechanisms by which upstream receptors including ITAM-based receptors activate the myeloid respiratory burst are unclear. One possible link between ITAM and GTPase would be the phosphorylation and subsequent aggregation of adapter protein complexes, which would then recruit nucleotide exchange factors to stimulate the conversion of GDP-Ras to GTP-Ras. Nimnual et al. (52) have now linked Sos exchange protein to the coordinate regulation of Ras and Rac in fibroblasts. These data and other studies support a paradigm by which Sos acts as nucleotide exchanger for Ras, resulting in activation of PI-3 kinase. PI-3 phosphate then binds to the Sos pleckstrin homology domain, resulting in activation of the Sos Dbl homology domain and exchange activity toward Rac (53). Alternatively, PI-3 phosphate can also activate another exchange factor for Rac2, the Vav protein. Unpublished data from our laboratory have demonstrated that the PI-3 kinase inhibitor, wortmannin, completely abrogates the Fc{gamma}R-induced activation of the respiratory burst. Hence, it is possible that Sos and/or Vav, both regulated by PI-3 kinase, subsequently control NADPH oxidase and superoxide generation in myeloid cells.

Using the Src family selective protein tyrosine kinase inhibitor (34, 35), PP1, we report the first evidence that Src kinases are essential for the upstream activation of Fc{gamma}R-induced oxidant signaling. Wang et al. (4) demonstrated that the Fc{gamma}RI receptor physically associates with Hck and Lyn kinases, and we (29) previously reported evidence implicating Hck in the Fc{gamma}RI signaling pathway. These combined data suggest that a potential downstream target for Hck and Lyn activation may be the phosphorylation of Shc which induces the formation of a Shc-Grb2 complex leading to recruitment of nucleotide exchange activity to the myeloid Fc{gamma}RI receptor. We suggest that the induced binding of Shc to the Grb2-Cbl complex may alter the dynamic balance between Grb2-Cbl in favor of formation of the Grb2-Sos complex. This Grb2-Sos complex is important in the control of Ras and potentially in control of effectors downstream of Ras (PI-3 kinase, Raf-1, etc.). This model is further supported by data recently reported from our laboratory (26, 54) and work of Rellahan et al. (36), that Cbl-Grb2 complexes are distinct from Grb2-Sos interaction and that Cbl is exchanged for Sos on ITAM stimulation. We observed that, consistent with this model, PP1 completely blocks the Fc{gamma}RI-induced activation of Erk2 in U937IF cells (data not shown). Alternatively, the effect of PP1 on the Shc-Grb2-Sos complex may not play a causative role in the control of all Ras signals or NADPH oxidase. Cbl binds the p85 subunit of PI-3 kinase and hence could serve as a scaffolding protein to increase the local concentration of PI-3 kinase after Fc{gamma}RI stimulation. Therefore, we propose that phosphorylation of Shc by myeloid-specific Src kinases modulates the formation of the membrane-localized Grb2-Sos complex (Fig. 2Go) after receptor aggregation to control Ras, PI-3 kinase, and Rac2 activation and oxidant signaling in myeloid cells. Proof of this concept will require further investigation with specific mutants of Shc and Cbl, experiments that are currently ongoing in our laboratory.


   Acknowledgments
 
We thank Dr. Norbert Berndt for the use of his metabolic labeling facility.


   Footnotes
 
1 This work was supported by Grant RO1 CA7563701 from the National Cancer Institute, Grant RPG-98-244-01-LBC from the American Cancer Society, and a Career Development Award from the STOP Cancer Foundation to D.L.D. A.E.-E. is funded by the Concern II Foundation and the Childrens Cancer Research Fund. R.K.P. was supported by Wonkwang University School of Medicine in 1999. Back

2 Address correspondence and reprint requests to Dr. Donald L. Durden, Herman B. Wells Center for Pediatric Research, Cancer Reseach Institute, Indiana University School of Medicine, 1044 West Walnut Street, Indianapolis, IN 46202. E-mail address: Back

3 Abbreviations used in this paper: Fc{gamma}RI, high affinity Fc receptor for IgG Fc portion; ITAM, immunoreceptor tyrosine-based activation motif; U937IF cells, IFN-{gamma}-differentiated U937 cells for 4 days; SH2, Src homology domain 2; SH3, Src homology domain 3; GAP, Ras GTPase-activating protein; EGF, epidermal growth factor; Fc{gamma}RI{alpha}, {alpha} subunit of Fc{gamma}RI; BCR, B cell receptor; Fc{epsilon}RI, high affinity Fc receptor for IgE; R{alpha}M, rabbit anti-mouse F(ab')2 fragment; MAP kinase, mitogen-activated protein kinase; PI-3 kinase, phosphoinositol 3-kinase; GNRF, Ras guanine nucleotide-releasing factor; CH, collagen homology; PTB, phosphotyrosine binding; PLC{gamma}1, phospholipase C{gamma}1; Tyr(p), phosphotyrosine; GST-ShcSH2, GST-Shc SH2 domain; GST-Cbl-CT, GST-Cbl C terminus. Back

Received for publication February 8, 1999. Accepted for publication September 8, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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