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The Journal of Immunology, 1998, 161: 5555-5563.
Copyright © 1998 by The American Association of Immunologists

Differential Interaction of Crkl with Cbl or C3G, Hef-1, and {gamma} Subunit Immunoreceptor Tyrosine-Based Activation Motif in Signaling of Myeloid High Affinity Fc Receptor for IgG (Fc{gamma}RI)1

Wade T. Kyono*, Ron de Jong{dagger}, Rae Kil Park{ddagger}, Yenbou Liu*, Nora Heisterkamp{dagger}, John Groffen{dagger} and Donald L. Durden2,*

* Neil Bogart Memorial Laboratories, Division of Hematology-Oncology, and {dagger} Section of Molecular Carcinogenesis, Department of Pathology, Childrens Hospital Los Angeles Research Institute and University of Southern California School of Medicine, 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
 
Cbl-Crkl and Crkl-C3G interactions have been implicated in T cell and B cell receptor signaling and in the regulation of the small GTPase, Rap1. Recent evidence suggests that Rap1 plays a prominent role in the regulation of immunoreceptor tyrosine-based activation motif (ITAM) signaling. To gain insight into the role of Crkl in myeloid ITAM signaling, we investigated Cbl-Crkl and Crkl-C3G interactions following Fc{gamma}RI aggregation in U937IF cells. Fc{gamma}RI cross-linking of U937IF cells results in the tyrosine phosphorylation of Cbl, Crkl, and Hef-1, an increase in the association of Crkl with Cbl via direct SH2 domain interaction and increased Crkl-Hef-1 binding. Crkl constitutively binds to the guanine nucleotide-releasing protein, C3G, via direct SH3 domain binding. Our data show that distinct Cbl-Crkl and Crkl-C3G complexes exist in myeloid cells, suggesting that these complexes may modulate distinct signaling events. Anti-Crkl immunoprecipitations demonstrate that the ITAM-containing {gamma} subunit of Fc{gamma}RI is induced to form a complex with the Crkl protein, and Crkl binds to the cytoskeletal protein, Hef-1. The induced association of Crkl with Cbl, Hef-1, and Fc{gamma}RI{gamma} after Fc{gamma}RI activation and the constitutive association between C3G and Crkl provide the first evidence that a Fc{gamma}RI{gamma}-Crkl-C3G complex may link ITAM receptors to the activation of Rap1 in myeloid cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FcR signaling evokes a variety of cellular responses in reaction to activation by Ag-Ab complexes, Ab-dependent cytotoxicity, phagocytosis, and mast cell degranulation leading to immune complex-mediated inflammation. FcRs also regulate proliferation and Ab production in lymphoid cells (1). The family of FcRs for IgG includes a high affinity receptor (Fc{gamma}RI)3 and low affinity receptors (Fc{gamma}RII and Fc{gamma}RIII). Fc{gamma}RI is a membrane glycoprotein comprised of a ligand-binding {alpha} subunit containing C2 class Ig binding domains (2) along with a transmembrane and cytoplasmic domain consisting of a homodimeric {gamma} subunit that contains an immunoreceptor tyrosine-based activation motif (ITAM) (3). Fc{gamma}RI and Fc{gamma}RII are expressed on monocytic cells such as the human histiocytic lymphoma cell line U937, which has been used as a model to examine FcR-mediated signal transduction (4, 5, 6, 7). In monocytes and monocytic cell lines such as U937, IFN-{gamma} increases Fc{gamma}RI expression by as much as 20-fold and facilitates the examination of Fc{gamma}RI signaling events (8, 9) leading to activation of the respiratory burst (oxidant signaling) (4, 5, 6, 7, 10). The mechanisms linking FcRs to activation of small GTPases and oxidant production are poorly understood but probably involve adapter proteins and nucleotide exchange proteins. Since Rap1 is involved in the regulation of the respiratory burst response in myeloid cells and since the adapter protein Crkl via its binding to the nucleotide exchange protein, C3G, is involved in the regulation of Rap1, we hypothesized that the Fc{gamma}RI signal may involve the Crkl adapter protein. Using U937 cells differentiated in IFN-{gamma} (U937IF cells) we have proceeded to characterize the involvement of the Cbl-Crkl, Crkl-Hef-1, and Crkl-C3G interactions in Fc{gamma}RI signaling in the current study.

Crkl, or Crk-like protein, is a 38-kDa adapter protein with an N-terminal SH2 domain, two C-terminal SH3 domains, no catalytic function, and a 60% homology to the Crk adapter protein (11). The Crkl SH2 domain shows specificity for YXXP sequences present on the proto-oncoprotein Cbl and the cytoskeleton-associated proteins paxillin, p130 Cas, and Hef-1. The N-terminal Crkl SH3 domain has been found associated with the guanine nucleotide-releasing proteins, C3G and SOS; the proto-oncoprotein Abl; and the Bcr-Abl fusion protein implicated in CML and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia. Crkl is tyrosine phosphorylated in Bcr-Abl-transformed cells, suggesting a role in malignant transformation. Crkl is present in a variety of nontransformed cell types, predominantly hemopoietic cells, and interacts with Cbl in T cell (12, 13), B cell (14, 15), and EGF (16) receptor signaling. The Crkl protein has been observed to undergo tyrosine phosphorylation upon cell surface receptor activation, but the physiologic significance of this phosphorylation event is unclear.

The 120-kDa Cbl is a complex adapter protein expressed in hemopoietic cells and is the cellular homologue of the transforming protein of the murine Cas NS-1 retrovirus that causes pro-B, pre-B, and myeloid leukemias in mice (17). It is the mammalian homologue of the sli-1 gene described in Caenorhabditis elegans as a negative regulator of Ras in the EGF receptor signaling pathway. Cbl is known to interact with adapter proteins (e.g., Grb2, Crk, Crkl, and Nck) that regulate guanine nucleotide exchange factors in mammalian cells, and Cbl has been implicated in Fc{gamma} receptor signaling (18, 19, 20, 21). In this report we demonstrate the first evidence implicating Crkl and the Cbl-Crkl, Crkl-Hef-1, and Crkl-C3G interactions in Fc{gamma}RI signaling. Upon Fc{gamma}RI stimulation, we observe the tyrosine phosphorylation of Crkl, Cbl, and Hef-1. We observe the inductive direct association of Crkl with Cbl and the constitutive direct binding of C3G with Crkl. The Fc{gamma}RI-induced association of Crkl with the Fc{gamma}RI {gamma} subunit and Hef-1 provides the first evidence for Crkl and Hef-1 in Fc{gamma}RI signal relay and the first direct evidence for Crkl, Hef-1, and Abl in {gamma}ITAM receptor signaling. The data suggest a model for regulation of Rap1 in myeloid oxidant signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Abs and GST fusion proteins

The Fc{gamma}RI-specific Abs were provided by Medarex (West Lebanon, NH). mAb 32.2 was a F(ab')2 IgG specific for Fc{gamma}RI, while mAb 197 was a whole Ab specific for the receptor. The cross-linking Ab was a rabbit anti-mouse (R{alpha}M) F(ab')2 purchased from Organon Teknika (Durham, NC). Polyclonal anti-Cbl, anti-Crkl, anti-Grb2, anti-C3G, and anti-SOS Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The mAb that recognizes both p130 CAS and p110 Hef-1 and anti-Grb2 mAb were obtained from Transduction Laboratories (Lexington, KY). Anti-peptide serum against the {gamma} subunit of Fc{gamma}RI (Ab 5927.3) specific for the extreme C-terminal sequence of the {gamma} subunit (i.e., NQETYETLKHEKPPQ) was used in {gamma} immunoblots (4). 4D8 anti-{gamma} subunit mAb was provided by J. Kochan of Hoffmann-La Roche (Nutley, NJ). GST-Crkl (residues 1–303), GST-Crkl SH2 (residues 7–128), and GST-Crkl SH33 (both SH3 domains in tandem, residues 115–303) as previously described by ten Hoeve et al. (22) were used in precipitations and Far Western blotting.

Cells

The U937 histiocytic lymphoma cell line was obtained from the American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 with 10% FBS. IFN-{gamma}-differentiated U937 (U937IF) cells were prepared by culturing U937 cells in RPMI 1640 with 10% FBS and 250 U/ml human rIFN-{gamma} for 5 or 6 days (Genentech, San Francisco, CA). U937IF cells were maintained at a concentration of 5 x 105 cells/ml, and the medium was replaced with fresh medium containing IFN-{gamma} every 2 or 3 days as previously described (4, 6).

Fc{gamma}RI cross-linking of U937IF cells

U937IF cells were collected and washed in cold HBSS and adjusted to a concentration of 4 x 107/ml. mAbs against Fc{gamma}RI were used to activate the cells. Cells (2 x 107) in 0.5 ml of RPMI were incubated on ice for 30 min with 0.25 µg/sample of the anti-Fc{gamma}RI (F(ab')2) Ab, mAb 32.2, or whole Ab, mAb 197. We then added the secondary R{alpha}M Ab at a concentration of 10 µg/ml and incubated the cells at 37°C for different times. The addition of the secondary Ab at 37°C was considered the start of stimulation with rapid cooling by the addition of an equal volume of cold HBSS at the stop time. For PMA (Sigma, St. Louis, MO) stimulation of cells we added PMA at 0.01 µmol to 2 x 107 cells for 5 min followed by rapid cooling by addition of cold HBSS. Samples were then centrifuged at 500 x g in a refrigerated centrifuge for 5 min, and the supernatant was quickly aspirated. Cells were lysed in 800 µl of Triton X-100 extraction buffer on ice for 30 min followed by centrifugation at 15,000 x g for 30 min. Immunoprecipitations or GST fusion protein pull-downs were performed as described below.

Immunoprecipitation

Cell lysates were prepared in 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, 0.1% 2-ME, 5 µM phenylarsine oxide, and 100 µM sodium orthovanadate. Lysates were cleared by centrifugation at 15,000 x g at 4°C for 30 min. To precipitate Cbl, Crkl, Grb2, C3G, and SOS, we added 1 µg of the polyclonal anti-Cbl, Crkl, Grb2, C3G, or SOS antisera to these lysates. After a 2-h incubation on ice, 30 µl of a 10% suspension of formalin-fixed Staphylococcus aureus (Pansorbin, Calbiochem, La Jolla, CA) was added to the immunoprecipitates and incubated for another hour on ice. The adsorbed immune complexes were washed three times with extraction buffer. We resuspended the samples in 25 µl of sample buffer, heated these samples at 98°C for 5 min, and resolved proteins using SDS-PAGE.

Electrophoresis and immunoblotting

Immunoprecipitates and whole cell lysates were resolved on 15% acrylamide-0.193% bisacrylamide gels by SDS-PAGE (6). Proteins were transferred to nitrocellulose filters (1 mA-h/cm2) using a dry transfer system (Ellard, Seattle, WA) (4). The blot was incubated with a blocking solution (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5% powdered milk, and Tween-20) at room temperature for 1 h and incubated with specific Ab at room temperature for 2 h with continuous agitation. After three washes in rinse solution (10 mM Tris-HCl (pH 7.5) and 150 mM NaCl), the membranes were incubated at room temperature for 1 h with secondary anti-mouse Ab conjugated with horseradish peroxidase for enhanced chemiluminescence (ECL kit, Amersham, Arlington Heights, IL) or conjugated with alkaline phosphatase for colorimetric development. Immunoblotting with polyclonal anti-C3G, anti-SOS, anti-Cbl, and anti-Crkl antisera was performed on sectioned portions of the monoclonal anti-Tyr(p) blot with detection using the ECL system.

In vitro GST fusion protein precipitations

Cell lysates were prepared as described above followed by precipitation with GST fusion proteins. Ten micrograms of GST fusion protein was preincubated with 50 µl of extraction buffer washed glutathione-Sepharose beads (glutathione-Sepharose 4B, Pharmacia Biotech, Piscataway, NJ) for 1 h and added to each lysate for 2 h. Beads were washed three times with ice-cold extraction buffer. Samples were resuspended in 25 µl of sample buffer and heated at 98°C for 5 min before proteins were separated using SDS-PAGE. Western blots were performed as described above.

Far Western assays

Immunoprecipitations with anti-Crkl Ab were performed as described above. Far Western assays were performed as follows. Nitrocellulose membranes were blocked with 25 mM sodium phosphate, 150 mM NaCl, 0.1% Tween-20, 2.5 mM EDTA, 20 mM NaF, and 1 mM DTT (Far Western binding buffer) in 2% nonfat milk at 4°C for 1 h. Membranes were washed with 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, and 1 mM DTT (PBS-T). Membranes were incubated with 2 µg/ml of GST fusion proteins in Far Western binding buffer and 2% nonfat milk overnight at 4°C, washed with PBS-T, then incubated with anti-GST Ab (1/1000) at room temperature for 2 h. Membranes were washed with PBS-T, incubated with horseradish peroxidase-conjugated rabbit anti-mouse Ab in Far Western binding buffer and 2% nonfat milk at room temperature for 2 h, and developed using the ECL system.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cbl is inducibly phosphorylated in Fc{gamma}RI-stimulated U937IF cells

To determine whether Cbl is involved in Fc{gamma}RI signaling, we compared the phosphotyrosine pattern of Cbl immunoprecipitated from resting and Fc{gamma}RI-stimulated U937IF cells. We stimulated U937IF cells with anti-Fc{gamma}RI mAb (32.2, F(ab')2). The 120-kDa Cbl displays a basal level of tyrosine phosphorylation in resting cells (Fig. 1Go, lane 2), and the intensity of phosphorylation increased within 1 min of receptor aggregation (lane 5), peaked in intensity by 5–10 min (lanes 6 and 7), and began to disappear by 30 min (lane 9) of Fc{gamma}RI stimulation. An additional, slower migrating, less intense Tyr(p) band appeared upon receptor activation and followed a similar pattern of inducible phosphorylation as the 120-kDa band. Both Tyr(p) bands were immunoreactive with anti-Cbl Ab, consistent with the presence of Cbl and its slower migrating isoform (18, 19, 20). It was interesting to note that the mobility-shifted Cbl isoform present after receptor activation was also seen in PMA-stimulated cells (Fig. 1Go, anti-Cbl blot, lane 3). Under these stimulation conditions, Cbl is dephosphorylated (Fig. 1Go, anti-Tyr(p) blot, compare lane 2 with lane 3).



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  		FIGURE 1. Crkl coprecipitates with Cbl following Fc{gamma}RI stimulation. Precipitation of U937IF cell lysates was performed with anti-Cbl Ab after Fc{gamma}RI stimulation. Anti-Tyr(p), anti-Cbl, and anti-Crkl immunoblots were performed. Lane 1, Precipitation performed with preimmune rabbit antisera. Lane 2 represents resting cells, while lane 3 contains a lysate of cells incubated with PMA for 5 min. Lanes 4–9 represent cells stimulated by Fc{gamma}RI cross-linking using the mAb 32.2 F(ab')2 (anti-Fc{gamma}RI) for 10 s and 1, 5, 10, 20, and 30 min, respectively. A whole cell lysate (1 x 106 cell equivalents) of stimulated U937IF cells is shown in lane 10. Blotting Abs are indicated on the left. Locations of Cbl, Crkl, and the m.w. markers are indicated on the right.

 
The Cbl-Crkl interaction is induced by Fc{gamma}RI stimulation

The results in Fig. 1Go, upper panel, demonstrate that Cbl undergoes increased tyrosine phosphorylation upon Fc{gamma}RI aggregation. Cbl immunoprecipitates probed with anti-Crkl antiserum revealed that a small amount of Crkl was associated with Cbl in resting cells, with a marked increased Crkl-Cbl binding noted within the first 10 s to 1 min of Fc{gamma}RI stimulation (five- to sixfold increase; Fig. 1Go, lanes 4 and 5). Crkl-Cbl binding peaked by 5–10 min of receptor stimulation (Fig. 1Go, lanes 6 and 7) and appeared to be kinetically related to the phosphorylation of Cbl (Fig. 1Go, lanes 4–7). By 20–30 min after Fc{gamma}RI stimulation the Crkl-Cbl association began to decrease, paralleling the dephosphorylation of Cbl (Fig. 1Go, lanes 8 and 9). PMA stimulation resulted in a slight decrease in the resting association of Crkl with Cbl and correlated with the dephosphorylation of Cbl (Fig. 1Go, lane 3). We interpret these data to suggest that the Cbl-Crkl interaction is dependent upon the tyrosine phosphorylation of Cbl.

To provide additional support for the presence of a Cbl-Crkl interaction in myeloid cells and to implicate this adapter complex in Fc{gamma}RI signaling, we performed reciprocal immunoprecipitations with Crkl antisera in resting and Fc{gamma}RI-stimulated U937IF cells (Fig. 2Go, A and B). Similar to the data shown in Fig. 1Go, we observed a basal level of tyrosine-phosphorylated Cbl in the Crkl immunoprecipitated from resting cells (Fig. 2Go, A and B, lane 2). Within 10 s of Fc{gamma}RI stimulation a marked increase was seen in amount of tyrosine-phosphorylated proteins bound to Crkl (Fig. 2GoA, compare lane 2 with lane 4). Cbl immunoblotting confirmed the identity of one of these proteins as p120 Cbl (Fig. 2GoB, anti-Cbl blot). Coprecipitation of Cbl with Crkl peaked by 5 min (Fig. 2GoB, anti-Cbl blot, lane 6) and gradually lessened by 20 min of receptor activation, correlating with the tyrosine phosphorylation of Cbl (lane 9). PMA stimulation resulted in the decreased tyrosine phosphorylation of Cbl and a decreased amount of Cbl associated with Crkl compared with Crkl precipitations performed on resting cells (Fig. 2Go, A and B, lane 3). The induced tyrosine phosphorylation of Cbl and Crkl and the augmented Cbl-Crkl interaction that follows Fc{gamma}RI stimulation implicate Cbl and Crkl in Fc{gamma}RI signaling.



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  		FIGURE 2. Cbl and C3G coprecipitate with Crkl. Anti-Crkl antisera was used to immunoprecipitate Crkl from lysates of U937IF (A and B) cells. Anti-Tyr(p), anti-Cbl, and anti-Crkl immunoblots were performed. A, Anti-Tyr(p) immunoblot. Lane 1 represents precipitation with preimmune sera. Lane 2 consists of resting cell lysates, while lane 3 represents a lysate of cells stimulated with PMA for 5 min. Lanes 4–9 represent cells stimulated with anti-Fc{gamma}RI 32.2 Ab for 10 s and 1, 5, 10, 20, and 30 min, respectively. B, Anti-C3G, Cbl, and Crkl immunoblots. Blotting Abs are indicated on the left of the membranes, while the locations of Cbl, Crkl, and C3G are indicated on the right. The m.w. markers are on the right.

 
Fc{gamma}RI stimulation induces Crkl tyrosine phosphorylation

Previous reports demonstrate Crkl tyrosine phosphorylation in Bcr/Abl-transformed cells (23). Anti-Tyr(p) immunoblots of the Crkl precipitations demonstrated an immunoreactive band migrating at 38 kDa, of which the intensity increased with Fc{gamma}RI activation (Fig. 2GoA, anti-Tyr(p) blot). Immunoblot analysis with polyclonal anti-Crkl Ab demonstrated that the Tyr(p) band directly superimposed with a slower migrating form of Crkl (compare anti-Tyr(p) blot of Fig. 2GoA with anti-Crkl blots of Fig. 2GoB). Previous experiments have shown that the slower migrating form of Crkl represents the tyrosine-phosphorylated protein, whereas the more rapidly migrating form consists of nonphosphorylated Crkl (23). Crkl immunoprecipitates demonstrate increasing tyrosine phosphorylation that peaked 5 min after Fc{gamma}RI stimulation (Fig. 2GoA, lane 6), which corresponded to the induction of the slower migrating Crkl isoforms. Interestingly, the mobility-shifted Crkl isoform described above in Crkl immunoprecipitates was not observed to coimmunoprecipitate with Cbl or C3G (Fig. 1Go, lanes 2–9; Fig. 3Go, compare lanes 8and 9 to lanes 2, 3, 10, and 11, anti-Crkl blots).



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  		FIGURE 3. Crkl-Cbl and Crkl-C3G complexes are distinct. Separate Cbl, Grb2, SOS, Crkl, and C3G immunoprecipitates were performed using U937IF cells under resting conditions or stimulation for 1 min after cross-linking with anti-Fc{gamma}RI mAb 197 Ab. Immunoblotting was performed with anti-C3G, Tyr(P), Cbl, Crkl, and Grb2 Abs. Lane 1 represents an immunoprecipitate performed with preimmune rabbit antisera. Abs used for precipitations for all other lanes are indicated at the top of the figures. A, Cbl, Grb2, SOS, Crkl, and C3G immunoprecipitations. B, To probe for SOS proteins that were near C3G in m.w., we performed a duplicate precipitation to that in A and performed an anti-SOS immunoblot. Blotting Abs are indicated on the left, while the positions of C3G, Cbl, and Crkl are indicated on the right. The m.w. markers are indicated on the right.

 
Crkl-Cbl and Crkl-C3G interactions are direct, and the signaling complexes are distinct

It has been suggested that the Crkl-C3G interaction serves to activate Rap1 during receptor engagement. In contrast, the function of the Crkl-Cbl interaction is less clear. Our Crkl immunoprecipitates demonstrated the coprecipitation of Crkl with both Cbl and C3G (Fig. 2GoB, lanes 2 and 4–9; Fig. 4Go, lanes 6 and 7). We designed a series of experiments to determine whether the Cbl-Crkl and Crkl-C3G protein complexes were distinct. Although Crkl was detected in Cbl precipitations, we were unable to detect C3G in the same immunoprecipitates (Fig. 3Go, lanes 2 and 3). Similarly, C3G immunoprecipitates demonstrated Crkl, but no Cbl could be detected (Fig. 3GoA, lanes 10 and 11); however, we could show trimolecular complexes consisting of Crkl, Cbl, and Grb2 using the same methods (Fig. 3GoA, lanes 8 and 9). Crkl precipitation following the immunodepletion of Cbl demonstrated that the immunodepletion of Cbl occurs with no decrease in the amount of C3G complexed with Crkl, providing additional support for the argument that Cbl-Crkl-C3G complexes are not present to a significant extent in vivo (data not shown). GST fusion protein pull-down experiments demonstrate that the full-length Crkl protein binds both Cbl and C3G (Fig. 4GoA, lanes 3 and 4). The Crkl-SH2 domain binds to Cbl, and the Crkl-SH3 domain binds C3G in U937IF cell lysates (Fig. 4GoA, lanes 5–8). Far Western immunoblotting with full-length Crkl-GST fusion proteins demonstrated direct Crkl binding to Cbl and C3G (Fig. 4GoB, lanes 1 and 2), that the Crkl-SH2 domain binds directly to Cbl (Fig. 4BGo, lanes 3 and 4), and that the Crkl-SH3 domain binds directly to C3G in myeloid cell lysates (Fig. 4GoB, lanes 5 and 6). Consistent with our other data, Fc{gamma}RI stimulation is noted to induce more Cbl-Crkl-SH2 binding in Far Western (Fig. 4GoB) and coimmunoprecipitation experiments (Fig. 3Go, compare lanes 8 and 9). The data above provide evidence for a direct interaction between Cbl and the Crkl-SH2 domain and between C3G and the Crkl-SH3 motif, and support a model for the mutually exclusive interaction between Cbl and Crkl vs Crkl and C3G in myeloid cells.



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  		FIGURE 4. GST-Crkl fusion protein experiments. A, GST-Crkl, GST-Crkl SH2, and GST-Crkl SH33 fusion proteins were used to precipitate associated proteins in resting and stimulated cell lysates. Lanes 1 and 2 contain resting and stimulated cell lysates (5 min with 32.2 anti-Fc{gamma}RI) that were precipitated with GST alone. Lanes 3 and 4 show resting and stimulated cell lysates precipitated with GST-Crkl. Lanes 5 and 6 are similar samples precipitated with GST-Crkl SH2, while lanes 7 and 8 are precipitated with GST-Crkl SH33. The blots were reacted with Abs against C3G and Cbl as indicated on the left. The locations of Cbl and C3G along with the m.w. markers are indicated on the right. B, Far Western analysis of Cbl-Crkl and Crkl-C3G interactions. Resting and stimulated cells were immunoprecipitated with anti-Crkl Ab and then Far Western blotted with GST-Crkl, GST-Crkl SH2, and GST-Crkl SH33. Lanes 1, 3, and 5 represent resting cell lysates, and lanes 2, 4, and 6 are Fc{gamma}RI-stimulated cell lysates. The blotting fusion proteins are indicated at the top of the figure, while the locations of C3G and Cbl are noted on the right. The m.w. markers are shown on the right.

 
Crkl association with Hef-1 and Abl proteins in myeloid cells

Crkl immunoprecipitates probed with antiphosphotyrosine Ab revealed a prominent phosphoprotein migrating at 110–120 kDa (Fig. 2GoA, lanes 7 and 8) identified as Cbl (Fig. 2GoB). We probed anti-Crkl immunoprecipitates with Abs against Cbl, Hef-1, and c-Abl kinase (Fig. 5Go). These data demonstrate that Crkl binds to Cbl, Hef-1, and Abl in myeloid cells (Fig. 5Go, lanes 2–9). The Crkl-Abl interaction is constitutive, with slightly increased binding occurring after 20- to 30-min stimulation (Fig. 5Go, lanes 8 and 9). The Cbl-Crkl and Crkl-Hef-1 interactions are constitutive but inducible upon Fc{gamma}RI stimulation. The induced associations between Cbl-Crkl and Crkl-Hef-1 correlate with the augmented tyrosine phosphorylation of Cbl and Hef-1, respectively. In the Crkl immunoprecipitate it is clear that the kinetics of Cbl tyrosine phosphorylation differ from those of the Fc{gamma}RI-induced phosphorylation of Hef-1, in that Hef-1 tyrosine phosphorylation and Hef-1-Crkl binding are later events occurring 20–30 min following Fc{gamma}RI cross-linking (Fig. 5Go, lanes 7–9). The tyrosine phosphorylation of Cbl and the induced Cbl-Crkl interaction is an earlier event, peaking 1–5 min after receptor engagement (Figs. 1Go and 5Go, lanes 2–6). The data provide the first evidence for a role for Hef-1 and Abl in Fc{gamma}RI signaling.



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  		FIGURE 5. Crkl associates with Hef-1 and Abl in myeloid cells. Anti-Crkl antiserum was used to immunoprecipitate Crkl from U937IF cells. Anti-Tyr(p), anti-Hef-1, and anti-Abl immunoblots were performed on these Crkl immunoprecipitates. Lane 1 represents precipitation performed with preimmune Ab. Lane 2 consists of resting cell lysates, while lane 3 is lysate prepared from PMA-stimulated cells (1 µg/ml). Lanes 4–9 are lysates prepared from Fc{gamma}RI cross-linked cells (using the 32.2, F(ab')2). A whole cell lysate is loaded in lane 10.

 
The {gamma} subunit of Fc{gamma}RI is complexed with Crkl after receptor aggregation

We previously reported that Fc{gamma}RI signals through a homodimeric {gamma} subunit containing an ITAM (4). It is possible that this {gamma} subunit signaling complex may bind to downstream signaling molecules such as Crkl to activate the small GTPase, Rap1. To test this hypothesis we immunoprecipitated Crkl and probed these immunoprecipitates for the presence of {gamma} subunit protein. The {gamma} subunit of the Fc{gamma}RI (Fc{gamma}RI{gamma}) coprecipitated with Crkl (Fig. 6GoA, {gamma} blot) in an inducible and transient manner after receptor activation. There was some constitutive binding of the {gamma} subunit to Crkl-containing complexes in cells at rest (Fig. 6GoA, lane 2), followed by an inducible increase with receptor stimulation that peaked by 1 min (three- to fivefold increase; Fig. 6GoA, lane 5). PMA stimulation resulted in a decrease in the amount of the {gamma} subunit coprecipitated with Crkl (Fig. 6GoA, lane 3), with no change in the amount of Crkl immunoprecipitated (Fig. 6GoA, lane 3, Crkl blot). Preimmune antisera did not immunoprecipitate Crkl, Cbl, or the {gamma} subunit in resting cell lysates. Peptide blocking experiments were performed on identical parallel anti-Crkl immunoprecipitates with a peptide (NQETYETLKHEKPPQ; Fig. 6GoB) specific for the 5927.3 anti-{gamma} subunit Ab (4). The {gamma} subunit-specific bands shown to coimmunoprecipitate with Crkl antisera (Fig. 6GoB, lanes 1–4) were completely blocked by preincubation of blotting Ab with the {gamma} subunit-specific peptide (Fig. 6GoB, lanes 6–9) and confirmed the identity and the specificity of the coprecipitating receptor complexes. From these data we conclude that Crkl binds to the Fc{gamma}RI{gamma} subunit in aggregated Fc{gamma}RI complexes.



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  		FIGURE 6. The {gamma} subunit of Fc{gamma}RI is coimmunoprecipitated with Crkl. A, U937IF cells were stimulated with anti-Fc{gamma}RI mAb 32.2 and then precipitated with anti-Crkl Ab. Immunoblotting was performed with anti-Crkl and anti-{gamma} Abs (5927.3) as indicated on the left. Lane 1 contains U937IF cells immunoprecipitated with preimmune antisera. Lane 2 contains U937IF cell lysates at rest, while lane 3 contains cell lysates activated with PMA for 5 min. Lanes 4–9 contain U937IF cell lysates stimulated for 10 s and 1, 5, 10, 20, and 30 min, respectively. Lane 10 contains a whole cell lysate of Fc{gamma}RI-stimulated U927IF cells (1 x 106 cell equivalents). The positions of Crkl and the {gamma} subunit along with the m.w. markers are indicated on the right. B, After immunoprecipitation using anti-Crkl Ab, divided samples were immunoblotted with 5927.3 anti-{gamma} subunit Ab alone or 5927.3 Ab preincubated with 5927.2 {gamma} subunit peptide. Cells were incubated with anti-Fc{gamma}RI mAb 32.2, then stimulated for 0 s, 10 s, 1 min, and 5 min (lanes 1–4 and lanes 6–9). Lanes 5 and 10 contain Fc{gamma}RI-stimulated U927IF cell lysates (1 x 106 cell equivalents). Lanes 1–5 were immunoblotted with anti-{gamma} subunit (5927.3) Ab, while lanes 6–10 were immunoblotted with anti-{gamma} subunit Ab preincubated with 5927.3 {gamma} subunit peptide. Blotting Abs are indicated on the bottom, while the position of the {gamma} subunit is indicated on the right. The m.w. markers are indicated on the right.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The study of signal transduction is fundamental to the understanding of macrophage and neutrophil activation. Previous work from our laboratory has established the use of U937 cells differentiated in {gamma}-IFN (U937IF) as a model system to study oxidant signaling (4, 5, 6, 7). The Crkl-C3G interaction has been implicated in the control of Rap1 (15), and the conversion of Rap1 to its GTP-bound state has been shown to regulate the activity of the NADPH oxidase complex in myeloid cells (24). We reported that the detection of superoxide anion production following Fc{gamma}RI stimulation of U937IF cells with 32.2 anti-Fc{gamma}RI cross-linking begins 1 min after receptor cross-linking is initiated (4, 5, 6, 7). The goal of the experiments reported herein was to characterize the upstream signaling events associated with the activation of Fc{gamma}RI that may lead to the activation of Rap1. We hypothesized roles for Crkl, Cbl, and C3G in Fc{gamma}RI oxidant signaling. Herein we present evidence that the Fc{gamma}RI {gamma} subunit associates with Crkl and that Crkl-Cbl and Crkl-C3G interactions are involved in Fc{gamma}RI signal transduction in myeloid cells. The demonstration of Crkl-Cbl (13, 25, 26, 27) and Crkl-C3G (13, 28) interactions in other signaling pathways suggests a potential role for Crkl in linking Cbl with C3G in myeloid cells. The association of C3G with Rap1 (15, 29) and that of Rap1 with the NADPH oxidase complex (24, 30) suggest that Crkl may link Fc{gamma}RI stimulation with the generation of the respiratory burst (4, 5). Herein, we present data directly implicating Crkl in the Fc{gamma}RI signaling pathway through its association with Cbl or C3G, Hef-1, and the ITAM-containing Fc{gamma}RI{gamma} subunit.

Cell surface receptor aggregation activates intracytoplasmic signals through assembly of adapter protein complexes at the plasma membrane, which serves to localize nucleotide exchange proteins, SOS, C3G, etc. Recent data from our laboratory suggest that Grb2 binds to the complex adapter protein, Cbl, and that this interaction may participate in Fc{gamma}RI signaling and the activation of Ras (21). Our data and those of Rellahan et al. and Buday et al. are consistent with an "adapter shield" exchange function for Cbl as it relates to the control of Grb2-SOS interaction (21, 31, 32). Unlike the Cbl-Grb2 interaction that occurs without Fc{gamma}RI stimulation, the Cbl-Crkl interaction is primarily induced (Figs. 1Go, 2Go, and 5Go, A and B). Reciprocal precipitations with Crkl- and Cbl-specific antisera confirm that Crkl inducibly associates with phosphorylated Cbl after Fc{gamma}RI cross-linking in U937IF cells (Fig. 1Go and 2Go, A and B), an interaction we also reproducibly found in the THP1 myeloid cell line (data not shown). Our data demonstrate increasing amounts of Crkl that associate with Cbl through SH2 domain interactions during the first 30 min of receptor activation, paralleling Cbl phosphorylation. The baseline phosphorylation of Cbl in resting U937IF cells also occurs in THP1 cells and primary macrophages derived from human bone marrow and is probably not due to transformation of our cell lines or an effect of IFN-{gamma} (our unpublished observations). Inducible phosphorylation of Cbl is also seen following activation of other receptors such as TCR (13, 33, 34), BCR (14, 35), EGF receptor (16, 36, 37, 38), erythropoietin receptor (39), granulocyte-macrophage CSF receptor (39), and thrombopoietin receptor (40). Crkl associates with Cbl in TCR, BCR, and EGF receptor signaling (14, 15, 16, 41), interactions that appear to be dependent on Cbl phosphorylation and possible SH2 interactions. Crkl SH2 association with Cbl is consistent with the findings of others in Bcr-Abl- and v-Abl-transformed cells (13, 25, 26, 27). Boussiotis et al. have implicated Cbl tyrosine phosphorylation and Cbl-Crkl interaction as activators of Rap1 in T cells (42). Their data suggest that the Cbl-Crkl-C3G-Rap1 suppresses TCR activation in T cells and may be responsible for T cell anergy.

In addition to Crkl SH2-Cbl domain interactions, evidence from several laboratories suggests the presence of SH3 domain-mediated Crkl-C3G and Crk-C3G interactions that potentially modulate the exchange of Rap1-GDP to Rap1-GTP (12, 13, 15, 28, 29, 43, 44, 45, 46, 47). Constitutive Crkl SH3-C3G complexes are observed in U937 cells, consistent with Crkl performing a role in linking SH2 binding proteins such as Cbl with downstream SH3 domain binding nucleotide exchange proteins. Cbl-Crkl and Crkl-C3G complexes appear prominently in our immunoprecipitations, which also demonstrate Crkl-Cbl-Grb2 ternary complexes but no appreciable Cbl-Crkl-C3G complexes, arguing for a lower stability or an absence of ternary complexes consisting of Crkl, Cbl, and C3G. In activated Jurkat T cells small amounts of C3G coimmunoprecipitate with Cbl (13), suggesting that Cbl-Crkl-C3G complexes may potentially form after TCR activation, while in Jurkat T cells overexpressing Cbl more C3G coimmunoprecipitates with Cbl (13). Our Cbl immunodepletion experiments support the argument that the Cbl-Crkl interaction is distinct from the Crkl-C3G interaction (data not shown). We propose that Cbl-Crkl and Crkl-C3G complexes may differentially form after Fc{gamma}RI activation to sequentially link receptor activation with downstream activation of Rap1. These observations are similar to our results (21) and the data reported by Rellahan et al. supporting an exchange function for Cbl in regulation of Grb2-SOS interaction (32). The ability of Cbl to bind to Crkl or Grb2 with potential downstream signaling via SOS-Ras or C3G-Rap1 interactions suggests potential cooperative signaling between the Ras and Rap1 pathways after Fc{gamma}RI stimulation and that Cbl may coordinately regulate Ras and Rap1 pathways through interactions with Grb2 and Crkl, respectively, in the cell.

The tyrosine phosphorylation of Crkl has previously been observed in Bcr/Abl-transformed leukemia cells (23) and under conditions of BCR and ß1 integrin stimulation (48). We observed the tyrosine phosphorylation of Crkl after Fc{gamma}RI activation, similar to that seen when Crkl is phosphorylated on tyrosine by the Bcr-Abl and Abl kinases (23). The phosphorylated fraction of Crkl in our experiments and in Bcr/Abl-transformed cells is mobility shifted (Figs. 2GoB and 4), and the slower migrating phosphorylated Crkl isoform is not detected in our Cbl and C3G immunoprecipitations (Fig. 3Go, compare lanes 8 and 9 to lanes 2, 3, 10, and 11). The physiologic significance of Crkl tyrosine phosphorylation is unknown. The phosphorylation of Crkl after Fc{gamma}RI cross-linking may serve to negatively regulate its interaction with Cbl and/or C3G via an intramolecular interaction similar to what has been suggested for Crk, since this isoform is not associated with these molecules in vivo. In contrast, we (23) recently demonstrated that mutating Y207, such that it cannot be phosphorylated by Bcr-Abl, does not enhance or decrease the association of Crkl with C3G, SOS, or Cbl. The further analysis of regulated Crkl phosphorylation following Fc{gamma}RI stimulation in the non-Bcr/Abl-transformed U937IF cell line or normal macrophages may provide insight into the significance of Crkl phosphorylation and dephosphorylation in a nontransformed setting.

We sought direct evidence that Crkl is involved in Fc{gamma}RI signaling. If Crkl participates in Fc{gamma}RI signaling, we postulated that the Crkl protein may be recruited to this receptor complex. Crkl immunoprecipitates probed with anti-Fc{gamma}RI{gamma}-specific antisera revealed coassociation between Crkl and the {gamma} subunit, a result that was confirmed in our peptide inhibition studies (Fig. 6Go, A and B). Unlike the direct binding of full-length Crkl to Cbl and C3G seen in Fig. 4GoB, experiments performed with identical Crkl fusion proteins on {gamma} subunit immunoprecipitates failed to demonstrate direct binding of Crkl to Fc{gamma}RI{gamma} (data not shown). Possible candidates for binding to the {gamma} subunit and Crkl include phosphorylated Cbl and Syk, both upstream effectors of the Fc{gamma}R signal transduction pathway. Our laboratory has previously reported that the nonreceptor protein tyrosine kinase, Syk, is tyrosine phosphorylated with increased kinase activity and associates with the Fc{gamma}RI {gamma} subunit after receptor cross-linking (5). Ota and Samelson reported that Cbl is a potential negative regulator of Syk kinase in Fc{epsilon}RI signaling in mast cells (49). The coprecipitation of the {gamma} subunit with Crkl suggests that Cbl may serve to link Crkl to the ITAM-containing {gamma} subunit, possibly after its phosphorylation by the Syk kinase. This would then provide a mechanism for the suppression of Syk kinase by Cbl. The inductive association of Crkl with the Fc{gamma}RI {gamma} subunit provides the first direct evidence that this adapter protein may play a role in ITAM-mediated receptor signaling in myeloid cells.

The recruitment of the nucleotide exchange factors, SOS and C3G, to adapter proteins and the localization of SOS and C3G to the plasma membrane where they can contact and activate the small GTPases Ras and Rap1 lead to the propagation of downstream signal transduction including the generation of superoxide anions in myeloid cells (24). Other data from our laboratory support a role for Crk and Crkl in integrin signaling pathways involving adhesion and motility responses (50). Our results demonstrate that Crkl interacts in myeloid cells with Hef-1 and Abl kinase, suggesting that other functions for Crkl probably exist in myeloid cells. The Crkl-Abl interaction we observed is consistent with other data from our laboratory that it occurs in a constitutive manner through the Crkl-SH3 domain, whereas the Crkl-Hef-1 interaction is induced by Fc{gamma}RI stimulation (Fig. 5Go, lanes 7–9) driven through the Crkl-SH2 domain (unpublished observation) (50). Interestingly, the Crkl-Hef-1 interaction is induced as a late event following Fc{gamma}RI cross-linking compared with the rapid on-loading of Crkl to Cbl that occurs within 1 min of stimulation (Fig. 1Go, lane 5, and Fig. 2Go, lanes 5 and 6). The binding of Crkl to Hef-1 occurs at a time when the Crkl-Cbl interaction is decreasing and correlates with the augmented tyrosine phosphorylation of Hef-1 (Fig. 5Go, lanes 7–9). In addition to increased Crkl-Hef-1 interaction, we observed a small increase in Abl binding to Crkl under conditions of Fc{gamma}RI stimulation (Fig. 5Go, lanes 7–9). These data constitute the first evidence that Hef-1 and Abl are involved in ITAM signal relay and suggest alternative functions for Crkl in Fc{gamma}RI signaling. Moreover, the data support the concept that Fc{gamma}RI activation leads to a cascade of signaling events involving Cbl, Crkl, Hef-1, and C3G that potentially leads to the activation of Rap1 and the generation of the respiratory burst in myeloid cells. Alternatively, the activation of Rap1 may suppress Ras and Rac and negatively regulate oxidant signaling and other phenotypic responses in myeloid cells. Recent evidence suggests that Ras can activate the Rac pathway, suggesting a potential link between Ras and the NADPH oxidase system (24, 51, 52). This suggests a potential role for SOS-Ras-Rac interactions in the generation of the respiratory burst in addition to C3G-Rap1 interactions. We propose a model in which initially Src family kinases phosphorylate the ITAM of the {gamma} subunit (6, 53) that recruits and activates Syk kinase (3, 5, 54, 55), which, in turn, binds to and phosphorylates a number of substrates, including Cbl, Shc, Crkl, and Hef-1 (10, 14, 20, 21). Other data from our laboratory have confirmed that the Fc{gamma}RI signals through these adapter proteins to activate Ras (10, 56) (unpublished observation). We postulate that the phosphorylation of Cbl and Hef-1 is followed by increased Cbl-Crkl and Hef-1-Crkl interactions in receptor aggregates that sequentially lead to the downstream activation of Rap1 via Crkl-C3G binding. The Hef-1-Crkl interaction may provide an as yet unknown connection between Rap1 and the cytoskeletal compartment participating in the orchestration of oxidant signaling events in adherent macrophages. Studies are ongoing with mutant forms of Crkl, Cbl, and Rap1 to test this two-step model for the functional involvement of distinct Cbl-Crkl and Crkl-C3G-Rap1 interactions in Fc{gamma}RI-induced signaling.


   Acknowledgments
 
We thank Dr. Anat Erdreich-Epstein for careful reading of the manuscript before submission.


   Footnotes
 
1 This work was supported in part by National Institutes of Health Grants CA75637 (to D.L.D.), CA47456 (to J.G.), and CA50248 (to N.H.). The work was performed in the Neil Bogart Memorial Laboratories supported by the T. J. Martell Foundation for Leukemia, Cancer, and AIDS Research; a grant from the American Cancer Society (RPG-98-244-01-LBC); a Career Development Award from the Children’s Hospital Los Angeles Research Institute; and the STOP Cancer Foundation. R.d.J. and R.K.P. are both recipients of the Childrens Hospital Los Angeles Career Development Fellowship. R.K.P. was supported by the Wonkwang University School of Medicine. W.T.K. is supported by National Cancer Institute Research Training Grant T32CA09659 from the National Institutes of Health. R.d.J. is a recipient of the American Cancer Society Postdoctoral Fellowship (PF-q8-130-01-LBC). Back

2 Address correspondence and reprint requests to Dr. Donald L. Durden, Department of Pediatrics, Division of Hematology-Oncology, M/S #57, Childrens Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027. E-mail address: Back

3 Abbreviations used in this paper: Fc{gamma}RI, high affinity Fc receptor for IgG; Fc{gamma}RII, low affinity Fc receptor for IgG; Fc{gamma}RIII, low affinity Fc receptor for IgG; ITAM, immunoreceptor tyrosine-based activation motif; SH2, Src-homology 2 domain; SH3, Src-homology 3 domain; EGF, epidermal growth factor; GST, glutathione S-transferase; R{alpha}M, rabbit anti-mouse Ab; ECL, enhanced chemiluminescence; Tyr(p), phosphotyrosine; PBS-T, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, and 1 mM dithithreitol; NADPH, nicotinamide adenine dinucleotide phosphate oxidase system; BCR, B cell receptor. Back

Received for publication April 23, 1998. Accepted for publication July 13, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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