Fc of Myeloid High Affinity Fc Receptor for Igg Tyrosine-Based Activation

Differential Interaction of Crkl with Cbl or C3G, Hef-1, and γ Subunit Immunoreceptor Tyrosine-Based Activation Motif in Signaling of Myeloid High Affinity Fc Receptor for IgG This information is current as (Fc γRI) of September 27, 2021. Wade T. Kyono, Ron de Jong, Rae Kil Park, Yenbou Liu, Nora Heisterkamp, John Groffen and Donald L. Durden J Immunol 1998; 161:5555-5563; ; http://www.jimmunol.org/content/161/10/5555 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Differential Interaction of Crkl with Cbl or C3G, Hef-1, and ␥ Subunit Immunoreceptor Tyrosine-Based Activation Motif in Signaling of Myeloid High Afﬁnity Fc Receptor for IgG (Fc␥RI)1

Wade T. Kyono,* Ron de Jong,† Rae Kil Park,‡ Yenbou Liu,* Nora Heisterkamp,† John Groffen,† and Donald L. Durden2*

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 Downloaded from Crkl-C3G interactions following Fc␥RI aggregation in U937IF cells. Fc␥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 ␥ subunit of Fc␥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 http://www.jimmunol.org/ with Cbl, Hef-1, and Fc␥RI␥ after Fc␥RI activation and the constitutive association between C3G and Crkl provide the ﬁrst evidence that a Fc␥RI␥-Crkl-C3G complex may link ITAM receptors to the activation of Rap1 in myeloid cells. The Journal of Immunology, 1998, 161: 5555–5563.

cR signaling evokes a variety of cellular responses in re- and low affinity receptors (Fc␥RII and Fc␥RIII). Fc␥RI is a mem- action to activation by Ag-Ab complexes, Ab-dependent brane glycoprotein comprised of a ligand-binding ␣ subunit con- F cytotoxicity, phagocytosis, and mast cell degranulation taining C2 class Ig binding domains (2) along with a transmem- leading to immune complex-mediated inflammation. FcRs also brane and cytoplasmic domain consisting of a homodimeric ␥ regulate proliferation and Ab production in lymphoid cells (1). The subunit that contains an immunoreceptor tyrosine-based activation by guest on September 27, 2021 family of FcRs for IgG includes a high affinity receptor (Fc␥RI)3 motif (ITAM) (3). Fc␥RI and Fc␥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 † *Neil Bogart Memorial Laboratories, Division of Hematology-Oncology, and Sec- transduction (4–7). In monocytes and monocytic cell lines such as tion of Molecular Carcinogenesis, Department of Pathology, Childrens Hospital Los Angeles Research Institute and University of Southern California School of Medicine, U937, IFN-␥ increases Fc␥RI expression by as much as 20-fold Los Angeles, CA 90027; and ‡Department of Microbiology and Immunology, and facilitates the examination of Fc␥RI signaling events (8, 9) Wonkwang University School of Medicine, Iksan Jeonbuk, Korea leading to activation of the respiratory burst (oxidant signaling) Received for publication April 23, 1998. Accepted for publication July 13, 1998. (4–7, 10). The mechanisms linking FcRs to activation of small The costs of publication of this article were defrayed in part by the payment of page GTPases and oxidant production are poorly understood but prob- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ably involve adapter proteins and nucleotide exchange proteins. 1 This work was supported in part by National Institutes of Health Grants CA75637 Since Rap1 is involved in the regulation of the respiratory burst (to D.L.D.), CA47456 (to J.G.), and CA50248 (to N.H.). The work was performed in response in myeloid cells and since the adapter protein Crkl via its the Neil Bogart Memorial Laboratories supported by the T. J. Martell Foundation for binding to the nucleotide exchange protein, C3G, is involved in the 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 regulation of Rap1, we hypothesized that the Fc RI signal may Los Angeles Research Institute; and the STOP Cancer Foundation. R.d.J. and R.K.P. involve the Crkl adapter protein. Using U937 cells differentiated in are both recipients of the Childrens Hospital Los Angeles Career Development Fel- IFN-␥ (U937IF cells) we have proceeded to characterize the in- lowship. R.K.P. was supported by the Wonkwang University School of Medicine. W.T.K. is supported by National Cancer Institute Research Training Grant volvement of the Cbl-Crkl, Crkl-Hef-1, and Crkl-C3G interactions T32CA09659 from the National Institutes of Health. R.d.J. is a recipient of the Amer- in Fc␥RI signaling in the current study. ican Cancer Society Postdoctoral Fellowship (PF-q8-130-01-LBC). Crkl, or Crk-like protein, is a 38-kDa adapter protein with an 2 Address correspondence and reprint requests to Dr. Donald L. Durden, Department N-terminal SH2 domain, two C-terminal SH3 domains, no cata- of Pediatrics, Division of Hematology-Oncology, M/S #57, Childrens Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027. E-mail address: lytic function, and a 60% homology to the Crk adapter protein [email protected] (11). The Crkl SH2 domain shows specificity for YXXP sequences 3 Abbreviations used in this paper: Fc␥RI, high affinity Fc receptor for IgG; Fc␥RII, present on the proto-oncoprotein Cbl and the cytoskeleton-associ- ␥ low affinity Fc receptor for IgG; Fc RIII, low affinity Fc receptor for IgG; ITAM, ated proteins paxillin, p130 Cas, and Hef-1. The N-terminal Crkl immunoreceptor tyrosine-based activation motif; SH2, Src-homology 2 domain; SH3, Src-homology 3 domain; EGF, epidermal growth factor; GST, glutathione S-trans- SH3 domain has been found associated with the guanine nucleoti- ferase; R␣M, rabbit anti-mouse Ab; ECL, enhanced chemiluminescence; Tyr(p), de-releasing proteins, C3G and SOS; the proto-oncoprotein Abl; phosphotyrosine; PBS-T, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM and the Bcr-Abl fusion protein implicated in CML and Philadel- KH2PO4, and 1 mM dithithreitol; NADPH, nicotinamide adenine dinucleotide phos- ϩ phate oxidase system; BCR, B cell receptor. phia chromosome-positive (Ph ) acute lymphoblastic leukemia.

Crkl is tyrosine phosphorylated in Bcr-Abl-transformed cells, sug- rated. Cells were lysed in 800 ␮l of Triton X-100 extraction buffer on ice gesting a role in malignant transformation. Crkl is present in a for 30 min followed by centrifugation at 15,000 ϫ g for 30 min. Immu- variety of nontransformed cell types, predominantly hemopoietic noprecipitations or GST fusion protein pull-downs were performed as described below. 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 Immunoprecipitation to undergo tyrosine phosphorylation upon cell surface receptor ac- Cell lysates were prepared in extraction buffer containing 1% Triton tivation, but the physiologic significance of this phosphorylation X-100, 10 mM Tris (pH 7.6), 50 mM NaCl, 0.1% BSA, 1 mM PMSF, 1% event is unclear. aprotinin, 5 mM EDTA, 50 mM NaF, 0.1% 2-ME, 5 ␮M phenylarsine The 120-kDa Cbl is a complex adapter protein expressed in oxide, and 100 ␮M sodium orthovanadate. Lysates were cleared by cen- ϫ hemopoietic cells and is the cellular homologue of the transform- trifugation at 15,000 g at 4°C for 30 min. To precipitate Cbl, Crkl, Grb2, C3G, and SOS, we added 1 ␮g of the polyclonal anti-Cbl, Crkl, Grb2, ing protein of the murine Cas NS-1 retrovirus that causes pro-B, C3G, or SOS antisera to these lysates. After a 2-h incubation on ice, 30 ␮l pre-B, and myeloid leukemias in mice (17). It is the mammalian of a 10% suspension of formalin-fixed Staphylococcus aureus (Pansorbin, homologue of the sli-1 gene described in Caenorhabditis elegans Calbiochem, La Jolla, CA) was added to the immunoprecipitates and in- as a negative regulator of Ras in the EGF receptor signaling path- cubated for another hour on ice. The adsorbed immune complexes were washed three times with extraction buffer. We resuspended the samples in way. Cbl is known to interact with adapter proteins (e.g., Grb2, 25 ␮l of sample buffer, heated these samples at 98°C for 5 min, and re- Crk, Crkl, and Nck) that regulate guanine nucleotide exchange solved proteins using SDS-PAGE. factors in mammalian cells, and Cbl has been implicated in Fc␥ receptor signaling (18–21). In this report we demonstrate the first Electrophoresis and immunoblotting evidence implicating Crkl and the Cbl-Crkl, Crkl-Hef-1, and Crkl- Immunoprecipitates and whole cell lysates were resolved on 15% acryl- Downloaded from C3G interactions in Fc␥RI signaling. Upon Fc␥RI stimulation, we amide-0.193% bisacrylamide gels by SDS-PAGE (6). Proteins were trans- 2 observe the tyrosine phosphorylation of Crkl, Cbl, and Hef-1. We ferred to nitrocellulose filters (1 mA-h/cm ) using a dry transfer system (Ellard, Seattle, WA) (4). The blot was incubated with a blocking solution observe the inductive direct association of Crkl with Cbl and the (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5% powdered milk, and constitutive direct binding of C3G with Crkl. The Fc␥RI-induced Tween-20) at room temperature for 1 h and incubated with specific Ab at association of Crkl with the Fc␥RI ␥ subunit and Hef-1 provides room temperature for 2 h with continuous agitation. After three washes in the first evidence for Crkl and Hef-1 in Fc␥RI signal relay and the rinse solution (10 mM Tris-HCl (pH 7.5) and 150 mM NaCl), the mem- http://www.jimmunol.org/ first direct evidence for Crkl, Hef-1, and Abl in ␥ITAM receptor branes were incubated at room temperature for 1 h with secondary anti- mouse Ab conjugated with horseradish peroxidase for enhanced chemilu- signaling. The data suggest a model for regulation of Rap1 in minescence (ECL kit, Amersham, Arlington Heights, IL) or conjugated myeloid oxidant signaling. with alkaline phosphatase for colorimetric development. Immunoblotting with polyclonal anti-C3G, anti-SOS, anti-Cbl, and anti-Crkl antisera was Materials and Methods performed on sectioned portions of the monoclonal anti-Tyr(p) blot with detection using the ECL system. Abs and GST fusion proteins The Fc␥RI-specific Abs were provided by Medarex (West Lebanon, NH). In vitro GST fusion protein precipitations Ј ␥ mAb 32.2 was a F(ab )2 IgG specific for Fc RI, while mAb 197 was a

Cell lysates were prepared as described above followed by precipitation by guest on September 27, 2021 whole Ab speciﬁc for the receptor. The cross-linking Ab was a rabbit with GST fusion proteins. Ten micrograms of GST fusion protein was ␣ Ј anti-mouse (R M) F(ab )2 purchased from Organon Teknika (Durham, preincubated with 50 ␮l of extraction buffer washed glutathione-Sepharose NC). Polyclonal anti-Cbl, anti-Crkl, anti-Grb2, anti-C3G, and anti-SOS beads (glutathione-Sepharose 4B, Pharmacia Biotech, Piscataway, NJ) for Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). 1 h and added to each lysate for 2 h. Beads were washed three times with The mAb that recognizes both p130 CAS and p110 Hef-1 and anti-Grb2 ice-cold extraction buffer. Samples were resuspended in 25 ␮l of sample mAb were obtained from Transduction Laboratories (Lexington, KY). An- buffer and heated at 98°C for 5 min before proteins were separated using ␥ ␥ ti-peptide serum against the subunit of Fc RI (Ab 5927.3) speciﬁc for the SDS-PAGE. Western blots were performed as described above. extreme C-terminal sequence of the ␥ subunit (i.e., NQETYETLKHEK PPQ) was used in ␥ immunoblots (4). 4D8 anti-␥ subunit mAb was pro- Far Western assays vided 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 Immunoprecipitations with anti-Crkl Ab were performed as described SH3 domains in tandem, residues 115–303) as previously described by ten above. Far Western assays were performed as follows. Nitrocellulose Hoeve et al. (22) were used in precipitations and Far Western blotting. 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 West- Cells ern 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 Na HPO , 1.8 mM The U937 histiocytic lymphoma cell line was obtained from the American 2 4 KH2PO4, and 1 mM DTT (PBS-T). Membranes were incubated with 2 Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 with ␮ ␥ g/ml of GST fusion proteins in Far Western binding buffer and 2% nonfat 10% FBS. IFN- -differentiated U937 (U937IF) cells were prepared by cul- milk overnight at 4°C, washed with PBS-T, then incubated with anti-GST turing U937 cells in RPMI 1640 with 10% FBS and 250 U/ml human ␥ Ab (1/1000) at room temperature for 2 h. Membranes were washed with rIFN- for 5 or 6 days (Genentech, San Francisco, CA). U937IF cells were PBS-T, incubated with horseradish peroxidase-conjugated rabbit anti- maintained at a concentration of 5 ϫ 105 cells/ml, and the medium was ␥ mouse Ab in Far Western binding buffer and 2% nonfat milk at room replaced with fresh medium containing IFN- every 2 or 3 days as previ- temperature for 2 h, and developed using the ECL system. ously described (4, 6). Fc␥RI cross-linking of U937IF cells Results ␥ U937IF cells were collected and washed in cold HBSS and adjusted to a Cbl is inducibly phosphorylated in Fc RI-stimulated U937IF concentration of 4 ϫ 107/ml. mAbs against Fc␥RI were used to activate the cells cells. Cells (2 ϫ 107) in 0.5 ml of RPMI were incubated on ice for 30 min ␥ ␮ ␥ Ј To determine whether Cbl is involved in Fc RI signaling, we com- with 0.25 g/sample of the anti-Fc RI (F(ab )2) Ab, mAb 32.2, or whole Ab, mAb 197. We then added the secondary R␣M Ab at a concentration of pared the phosphotyrosine pattern of Cbl immunoprecipitated from 10 ␮g/ml and incubated the cells at 37°C for different times. The addition resting and Fc␥RI-stimulated U937IF cells. We stimulated U937IF ␥ Ј of the secondary Ab at 37°C was considered the start of stimulation with cells with anti-Fc RI mAb (32.2, F(ab )2). The 120-kDa Cbl dis- rapid cooling by the addition of an equal volume of cold HBSS at the stop plays a basal level of tyrosine phosphorylation in resting cells (Fig. time. For PMA (Sigma, St. Louis, MO) stimulation of cells we added PMA at 0.01 ␮mol to 2 ϫ 107 cells for 5 min followed by rapid cooling by 1, lane 2), and the intensity of phosphorylation increased within 1 addition of cold HBSS. Samples were then centrifuged at 500 ϫ g in a min of receptor aggregation (lane 5), peaked in intensity by 5–10 refrigerated centrifuge for 5 min, and the supernatant was quickly aspi- min (lanes 6 and 7), and began to disappear by 30 min (lane 9)of The Journal of Immunology 5557

FIGURE 1. Crkl coprecipitates with Cbl following Fc␥RI stimulation. Precipitation of U937IF cell lysates was performed with anti-Cbl Ab after Fc␥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 Downloaded from incubated with PMA for 5 min. Lanes 4–9 represent cells stimulated by ␥ Ј ␥ Fc RI cross-linking using the mAb 32.2 F(ab )2 (anti-Fc RI) for 10 s and 1, 5, 10, 20, and 30 min, respectively. A whole cell lysate (1 ϫ 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. http://www.jimmunol.org/

Fc␥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 (18–20). It was interesting to note that the mobility-shifted Cbl isoform present after receptor activation was also seen in PMA- by guest on September 27, 2021 stimulated cells (Fig. 1, anti-Cbl blot, lane 3). Under these stimulation conditions, Cbl is dephosphorylated (Fig. 1, anti-Tyr(p) blot, compare lane 2 with lane 3).

The Cbl-Crkl interaction is induced by Fc␥RI stimulation The results in Fig. 1, upper panel, demonstrate that Cbl undergoes increased tyrosine phosphorylation upon Fc␥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␥RI stimulation (five- to sixfold increase; Fig. 1, lanes 4 and 5). Crkl-Cbl binding peaked by 5–10 min of receptor stimulation (Fig. 1, lanes 6 and 7) and appeared to be kinetically related to the phosphorylation of Cbl (Fig. 1, lanes 4–7). By FIGURE 2. Cbl and C3G coprecipitate with Crkl. Anti-Crkl antisera was used to immunoprecipitate Crkl from lysates of U937IF (A and B) 20–30 min after Fc␥RI stimulation the Crkl-Cbl association began cells. Anti-Tyr(p), anti-Cbl, and anti-Crkl immunoblots were performed. A, to decrease, paralleling the dephosphorylation of Cbl (Fig. 1, lanes Anti-Tyr(p) immunoblot. Lane 1 represents precipitation with preimmune 8 and 9). PMA stimulation resulted in a slight decrease in the sera. Lane 2 consists of resting cell lysates, while lane 3 represents a lysate resting association of Crkl with Cbl and correlated with the de- of cells stimulated with PMA for 5 min. Lanes 4–9 represent cells stim- phosphorylation of Cbl (Fig. 1, lane 3). We interpret these data to ulated with anti-Fc␥RI 32.2 Ab for 10 s and 1, 5, 10, 20, and 30 min, suggest that the Cbl-Crkl interaction is dependent upon the ty- respectively. B, Anti-C3G, Cbl, and Crkl immunoblots. Blotting Abs are rosine phosphorylation of Cbl. indicated on the left of the membranes, while the locations of Cbl, Crkl, and To provide additional support for the presence of a Cbl-Crkl C3G are indicated on the right. The m.w. markers are on the right. interaction in myeloid cells and to implicate this adapter complex in Fc␥RI signaling, we performed reciprocal immunoprecipitations with Crkl antisera in resting and Fc␥RI-stimulated U937IF cells lane 2 with lane 4). Cbl immunoblotting confirmed the identity of (Fig. 2, A and B). Similar to the data shown in Fig. 1, we observed one of these proteins as p120 Cbl (Fig. 2B, anti-Cbl blot). Copre- a basal level of tyrosine-phosphorylated Cbl in the Crkl immuno- cipitation of Cbl with Crkl peaked by 5 min (Fig. 2B, anti-Cbl blot, precipitated from resting cells (Fig. 2, A and B, lane 2). Within 10 s lane 6) and gradually lessened by 20 min of receptor activation, of Fc␥RI stimulation a marked increase was seen in amount of correlating with the tyrosine phosphorylation of Cbl (lane 9). PMA tyrosine-phosphorylated proteins bound to Crkl (Fig. 2A, compare stimulation resulted in the decreased tyrosine phosphorylation of 5558 Crkl, Cbl, AND Hef-1 IN Fc␥RI SIGNALING Downloaded from http://www.jimmunol.org/ 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␥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 FIGURE 3. Crkl-Cbl and Crkl-C3G complexes are distinct. Separate by guest on September 27, 2021 Cbl, Grb2, SOS, Crkl, and C3G immunoprecipitates were performed using the m.w. markers are indicated on the right. B, Far Western analysis of U937IF cells under resting conditions or stimulation for 1 min after cross- Cbl-Crkl and Crkl-C3G interactions. Resting and stimulated cells were linking with anti-Fc␥RI mAb 197 Ab. Immunoblotting was performed with immunoprecipitated with anti-Crkl Ab and then Far Western blotted with anti-C3G, Tyr(P), Cbl, Crkl, and Grb2 Abs. Lane 1 represents an immu- GST-Crkl, GST-Crkl SH2, and GST-Crkl SH33. Lanes 1, 3, and 5 repre- ␥ noprecipitate performed with preimmune rabbit antisera. Abs used for present resting cell lysates, and lanes 2, 4, and 6 are Fc RI-stimulated cell cipitations for all other lanes are indicated at the top of the figures. A, Cbl, lysates. The blotting fusion proteins are indicated at the top of the figure, Grb2, SOS, Crkl, and C3G immunoprecipitations. B, To probe for SOS while the locations of C3G and Cbl are noted on the right. The m.w. proteins that were near C3G in m.w., we performed a duplicate precipita- markers are shown on the right. tion 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 indi- Crkl immunoprecipitates demonstrate increasing tyrosine phos- cated on the right. The m.w. markers are indicated on the right. phorylation that peaked 5 min after Fc␥RI stimulation (Fig. 2A, lane 6), which corresponded to the induction of the slower migrating Crkl isoforms. Interestingly, the mobility-shifted Crkl isoform Cbl and a decreased amount of Cbl associated with Crkl compared described above in Crkl immunoprecipitates was not observed to with Crkl precipitations performed on resting cells (Fig. 2, A and coimmunoprecipitate with Cbl or C3G (Fig. 1, lanes 2–9; Fig. 3, B, lane 3). The induced tyrosine phosphorylation of Cbl and Crkl compare lanes 8 and 9 to lanes 2, 3, 10, and 11, anti-Crkl blots). and the augmented Cbl-Crkl interaction that follows Fc␥RI stimulation implicate Cbl and Crkl in Fc␥RI signaling. Crkl-Cbl and Crkl-C3G interactions are direct, and the signaling complexes are distinct ␥ Fc RI stimulation induces Crkl tyrosine phosphorylation It has been suggested that the Crkl-C3G interaction serves to ac- Previous reports demonstrate Crkl tyrosine phosphorylation in tivate Rap1 during receptor engagement. In contrast, the function Bcr/Abl-transformed cells (23). Anti-Tyr(p) immunoblots of the of the Crkl-Cbl interaction is less clear. Our Crkl immunoprecipi- Crkl precipitations demonstrated an immunoreactive band migrat- tates demonstrated the coprecipitation of Crkl with both Cbl and ing at 38 kDa, of which the intensity increased with Fc␥RI acti- C3G (Fig. 2B, lanes 2 and 4–9; Fig. 4, lanes 6 and 7). We designed vation (Fig. 2A, anti-Tyr(p) blot). Immunoblot analysis with poly- a series of experiments to determine whether the Cbl-Crkl and clonal anti-Crkl Ab demonstrated that the Tyr(p) band directly Crkl-C3G protein complexes were distinct. Although Crkl was de- superimposed with a slower migrating form of Crkl (compare anti- tected in Cbl precipitations, we were unable to detect C3G in the Tyr(p) blot of Fig. 2A with anti-Crkl blots of Fig. 2B). Previous same immunoprecipitates (Fig. 3, lanes 2 and 3). Similarly, C3G experiments have shown that the slower migrating form of Crkl immunoprecipitates demonstrated Crkl, but no Cbl could be de- represents the tyrosine-phosphorylated protein, whereas the more tected (Fig. 3A, lanes 10 and 11); however, we could show trimo- rapidly migrating form consists of nonphosphorylated Crkl (23). lecular complexes consisting of Crkl, Cbl, and Grb2 using the The Journal of Immunology 5559

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 ␮g/ml). Lanes 4–9 are lysates prepared from Downloaded from ␥ Ј Fc RI cross-linked cells (using the 32.2, F(ab )2). A whole cell lysate is loaded in lane 10. same methods (Fig. 3A, lanes 8 and 9). Crkl precipitation follow- event, peaking 1–5 min after receptor engagement (Figs. 1 and 5, ing the immunodepletion of Cbl demonstrated that the immu- lanes 2–6). The data provide the first evidence for a role for Hef-1 nodepletion of Cbl occurs with no decrease in the amount of C3G and Abl in Fc␥RI signaling. http://www.jimmunol.org/ complexed with Crkl, providing additional support for the argu- The ␥ subunit of Fc␥RI is complexed with Crkl after receptor ment that Cbl-Crkl-C3G complexes are not present to a significant aggregation extent in vivo (data not shown). GST fusion protein pull-down experiments demonstrate that the full-length Crkl protein binds We previously reported that Fc␥RI signals through a homodimeric both Cbl and C3G (Fig. 4A, lanes 3 and 4). The Crkl-SH2 domain ␥ subunit containing an ITAM (4). It is possible that this ␥ subunit binds to Cbl, and the Crkl-SH3 domain binds C3G in U937IF cell signaling complex may bind to downstream signaling molecules lysates (Fig. 4A, lanes 5–8). Far Western immunoblotting with such as Crkl to activate the small GTPase, Rap1. To test this hy- full-length Crkl-GST fusion proteins demonstrated direct Crkl pothesis we immunoprecipitated Crkl and probed these immuno- binding to Cbl and C3G (Fig. 4B, lanes 1 and 2), that the Crkl-SH2 precipitates for the presence of ␥ subunit protein. The ␥ subunit of by guest on September 27, 2021 domain binds directly to Cbl (Fig. 4B, lanes 3 and 4), and that the the Fc␥RI (Fc␥RI␥) coprecipitated with Crkl (Fig. 6A, ␥ blot) in an Crkl-SH3 domain binds directly to C3G in myeloid cell lysates inducible and transient manner after receptor activation. There was (Fig. 4B, lanes 5 and 6). Consistent with our other data, Fc␥RI some constitutive binding of the ␥ subunit to Crkl-containing com- stimulation is noted to induce more Cbl-Crkl-SH2 binding in Far plexes in cells at rest (Fig. 6A, lane 2), followed by an inducible Western (Fig. 4B) and coimmunoprecipitation experiments (Fig. 3, increase with receptor stimulation that peaked by 1 min (three- to compare lanes 8 and 9). The data above provide evidence for a fivefold increase; Fig. 6A, lane 5). PMA stimulation resulted in a direct interaction between Cbl and the Crkl-SH2 domain and be- decrease in the amount of the ␥ subunit coprecipitated with Crkl tween C3G and the Crkl-SH3 motif, and support a model for the (Fig. 6A, lane 3), with no change in the amount of Crkl immuno- mutually exclusive interaction between Cbl and Crkl vs Crkl and precipitated (Fig. 6A, lane 3, Crkl blot). Preimmune antisera did C3G in myeloid cells. not immunoprecipitate Crkl, Cbl, or the ␥ subunit in resting cell lysates. Peptide blocking experiments were performed on identical Crkl association with Hef-1 and Abl proteins in myeloid cells parallel anti-Crkl immunoprecipitates with a peptide (NQETY Crkl immunoprecipitates probed with antiphosphotyrosine Ab re- ETLKHEKPPQ; Fig. 6B) specific for the 5927.3 anti-␥ subunit Ab vealed a prominent phosphoprotein migrating at 110–120 kDa (4). The ␥ subunit-specific bands shown to coimmunoprecipitate (Fig. 2A, lanes 7 and 8) identified as Cbl (Fig. 2B). We probed with Crkl antisera (Fig. 6B, lanes 1–4) were completely blocked anti-Crkl immunoprecipitates with Abs against Cbl, Hef-1, and by preincubation of blotting Ab with the ␥ subunit-specific peptide c-Abl kinase (Fig. 5). These data demonstrate that Crkl binds to (Fig. 6B, lanes 6–9) and confirmed the identity and the specificity Cbl, Hef-1, and Abl in myeloid cells (Fig. 5, lanes 2–9). The Crkl- of the coprecipitating receptor complexes. From these data we Abl interaction is constitutive, with slightly increased binding oc- conclude that Crkl binds to the Fc␥RI␥ subunit in aggregated curring after 20- to 30-min stimulation (Fig. 5, lanes 8 and 9). The Fc␥RI complexes. Cbl-Crkl and Crkl-Hef-1 interactions are constitutive but inducible upon Fc␥RI stimulation. The induced associations between Cbl- Discussion Crkl and Crkl-Hef-1 correlate with the augmented tyrosine phos- The study of signal transduction is fundamental to the understand- phorylation of Cbl and Hef-1, respectively. In the Crkl immuno- ing of macrophage and neutrophil activation. Previous work from precipitate it is clear that the kinetics of Cbl tyrosine our laboratory has established the use of U937 cells differentiated phosphorylation differ from those of the Fc␥RI-induced phosphor- in ␥-IFN (U937IF) as a model system to study oxidant signaling ylation of Hef-1, in that Hef-1 tyrosine phosphorylation and Hef- (4–7). The Crkl-C3G interaction has been implicated in the control 1-Crkl binding are later events occurring 20–30 min following of Rap1 (15), and the conversion of Rap1 to its GTP-bound state Fc␥RI cross-linking (Fig. 5, lanes 7–9). The tyrosine phosphory- has been shown to regulate the activity of the NADPH oxidase lation of Cbl and the induced Cbl-Crkl interaction is an earlier complex in myeloid cells (24). We reported that the detection of 5560 Crkl, Cbl, AND Hef-1 IN Fc␥RI SIGNALING Downloaded from http://www.jimmunol.org/

FIGURE 6. The ␥ subunit of Fc␥RI is coimmunoprecipitated with Crkl. A, U937IF cells were stimulated with anti-Fc␥RI mAb 32.2 and then precipitated with anti-Crkl Ab. Immunoblotting was performed with anti-Crkl and anti-␥ 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␥RI-stimulated U927IF cells (1 ϫ 106 cell equivalents). The positions of Crkl and the ␥ 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-␥ subunit Ab alone or 5927.3 Ab preincubated with 5927.2 ␥ subunit peptide. Cells were incubated with anti-Fc␥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 ␥ ϫ 6 ␥ and 10 contain Fc RI-stimulated U927IF cell lysates (1 10 cell equivalents). Lanes 1–5 were immunoblotted with anti- subunit (5927.3) Ab, while by guest on September 27, 2021 lanes 6–10 were immunoblotted with anti-␥ subunit Ab preincubated with 5927.3 ␥ subunit peptide. Blotting Abs are indicated on the bottom, while the position of the ␥ subunit is indicated on the right. The m.w. markers are indicated on the right. superoxide anion production following Fc␥RI stimulation of Cbl-Grb2 interaction that occurs without Fc␥RI stimulation, the U937IF cells with 32.2 anti-Fc␥RI cross-linking begins 1 min after Cbl-Crkl interaction is primarily induced (Figs. 1, 2, and 5, A and receptor cross-linking is initiated (4–7). The goal of the experi- B). Reciprocal precipitations with Crkl- and Cbl-specific antisera ments reported herein was to characterize the upstream signaling confirm that Crkl inducibly associates with phosphorylated Cbl events associated with the activation of Fc␥RI that may lead to the after Fc␥RI cross-linking in U937IF cells (Fig. 1 and 2, A and B), activation of Rap1. We hypothesized roles for Crkl, Cbl, and C3G an interaction we also reproducibly found in the THP1 myeloid ␥ in Fc RI oxidant signaling. Herein we present evidence that the cell line (data not shown). Our data demonstrate increasing ␥ ␥ Fc RI subunit associates with Crkl and that Crkl-Cbl and Crkl- amounts of Crkl that associate with Cbl through SH2 domain in- ␥ C3G interactions are involved in Fc RI signal transduction in my- teractions during the first 30 min of receptor activation, paralleling eloid cells. The demonstration of Crkl-Cbl (13, 25–27) and Crkl- Cbl phosphorylation. The baseline phosphorylation of Cbl in rest- C3G (13, 28) interactions in other signaling pathways suggests a ing U937IF cells also occurs in THP1 cells and primary macro- potential role for Crkl in linking Cbl with C3G in myeloid cells. phages derived from human bone marrow and is probably not due The association of C3G with Rap1 (15, 29) and that of Rap1 with to transformation of our cell lines or an effect of IFN-␥ (our un- the NADPH oxidase complex (24, 30) suggest that Crkl may link published observations). Inducible phosphorylation of Cbl is also Fc␥RI stimulation with the generation of the respiratory burst (4, seen following activation of other receptors such as TCR (13, 33, 5). Herein, we present data directly implicating Crkl in the Fc␥RI signaling pathway through its association with Cbl or C3G, Hef-1, 34), BCR (14, 35), EGF receptor (16, 36–38), erythropoietin re- and the ITAM-containing Fc␥RI␥ subunit. ceptor (39), granulocyte-macrophage CSF receptor (39), and Cell surface receptor aggregation activates intracytoplasmic sig- thrombopoietin receptor (40). Crkl associates with Cbl in TCR, nals through assembly of adapter protein complexes at the plasma BCR, and EGF receptor signaling (14–16, 41), interactions that membrane, which serves to localize nucleotide exchange proteins, appear to be dependent on Cbl phosphorylation and possible SH2 SOS, C3G, etc. Recent data from our laboratory suggest that Grb2 interactions. Crkl SH2 association with Cbl is consistent with the binds to the complex adapter protein, Cbl, and that this interaction findings of others in Bcr-Abl- and v-Abl-transformed cells (13, may participate in Fc␥RI signaling and the activation of Ras (21). 25–27). Boussiotis et al. have implicated Cbl tyrosine phosphor- Our data and those of Rellahan et al. and Buday et al. are consis- ylation and Cbl-Crkl interaction as activators of Rap1 in T cells tent with an “adapter shield” exchange function for Cbl as it relates (42). Their data suggest that the Cbl-Crkl-C3G-Rap1 suppresses to the control of Grb2-SOS interaction (21, 31, 32). Unlike the TCR activation in T cells and may be responsible for T cell anergy. The Journal of Immunology 5561

In addition to Crkl SH2-Cbl domain interactions, evidence for binding to the ␥ subunit and Crkl include phosphorylated Cbl from several laboratories suggests the presence of SH3 domain- and Syk, both upstream effectors of the Fc␥R signal transduction mediated Crkl-C3G and Crk-C3G interactions that potentially pathway. Our laboratory has previously reported that the nonre- modulate the exchange of Rap1-GDP to Rap1-GTP (12, 13, 15, ceptor protein tyrosine kinase, Syk, is tyrosine phosphorylated 28, 29, 43–47). Constitutive Crkl SH3-C3G complexes are ob- with increased kinase activity and associates with the Fc␥RI ␥ served in U937 cells, consistent with Crkl performing a role in subunit after receptor cross-linking (5). Ota and Samelson reported linking SH2 binding proteins such as Cbl with downstream SH3 that Cbl is a potential negative regulator of Syk kinase in Fc⑀RI domain binding nucleotide exchange proteins. Cbl-Crkl and signaling in mast cells (49). The coprecipitation of the ␥ subunit Crkl-C3G complexes appear prominently in our immunopre- with Crkl suggests that Cbl may serve to link Crkl to the ITAM- cipitations, which also demonstrate Crkl-Cbl-Grb2 ternary containing ␥ subunit, possibly after its phosphorylation by the Syk complexes but no appreciable Cbl-Crkl-C3G complexes, argu- kinase. This would then provide a mechanism for the suppression ing for a lower stability or an absence of ternary complexes of Syk kinase by Cbl. The inductive association of Crkl with the consisting of Crkl, Cbl, and C3G. In activated Jurkat T cells Fc␥RI ␥ subunit provides the ﬁrst direct evidence that this adapter small amounts of C3G coimmunoprecipitate with Cbl (13), sug- protein may play a role in ITAM-mediated receptor signaling in gesting that Cbl-Crkl-C3G complexes may potentially form af- myeloid cells. ter TCR activation, while in Jurkat T cells overexpressing Cbl The recruitment of the nucleotide exchange factors, SOS and more C3G coimmunoprecipitates with Cbl (13). Our Cbl im- C3G, to adapter proteins and the localization of SOS and C3G munodepletion experiments support the argument that the Cbl- to the plasma membrane where they can contact and activate the Downloaded from Crkl interaction is distinct from the Crkl-C3G interaction (data small GTPases Ras and Rap1 lead to the propagation of down- not shown). We propose that Cbl-Crkl and Crkl-C3G complexes stream signal transduction including the generation of superox- ␥ may differentially form after Fc RI activation to sequentially ide anions in myeloid cells (24). Other data from our laboratory link receptor activation with downstream activation of Rap1. support a role for Crk and Crkl in integrin signaling pathways These observations are similar to our results (21) and the data involving adhesion and motility responses (50). Our results reported by Rellahan et al. supporting an exchange function for demonstrate that Crkl interacts in myeloid cells with Hef-1 and Cbl in regulation of Grb2-SOS interaction (32). The ability of Abl kinase, suggesting that other functions for Crkl probably http://www.jimmunol.org/ Cbl to bind to Crkl or Grb2 with potential downstream signaling exist in myeloid cells. The Crkl-Abl interaction we observed is via SOS-Ras or C3G-Rap1 interactions suggests potential co- consistent with other data from our laboratory that it occurs in operative signaling between the Ras and Rap1 pathways after a constitutive manner through the Crkl-SH3 domain, whereas Fc␥RI stimulation and that Cbl may coordinately regulate Ras the Crkl-Hef-1 interaction is induced by Fc␥RI stimulation and Rap1 pathways through interactions with Grb2 and Crkl, (Fig. 5, lanes 7–9) driven through the Crkl-SH2 domain (un- respectively, in the cell. published observation) (50). Interestingly, the Crkl-Hef-1 inter- The tyrosine phosphorylation of Crkl has previously been ob- action is induced as a late event following Fc␥RI cross-linking served in Bcr/Abl-transformed leukemia cells (23) and under con-

compared with the rapid on-loading of Crkl to Cbl that occurs by guest on September 27, 2021 ditions of BCR and ␤ integrin stimulation (48). We observed the 1 within 1 min of stimulation (Fig. 1, lane 5, and Fig. 2, lanes 5 tyrosine phosphorylation of Crkl after Fc␥RI activation, similar to and 6). The binding of Crkl to Hef-1 occurs at a time when the that seen when Crkl is phosphorylated on tyrosine by the Bcr-Abl Crkl-Cbl interaction is decreasing and correlates with the aug- and Abl kinases (23). The phosphorylated fraction of Crkl in our experiments and in Bcr/Abl-transformed cells is mobility shifted mented tyrosine phosphorylation of Hef-1 (Fig. 5, lanes 7–9). (Figs. 2B and 4), and the slower migrating phosphorylated Crkl In addition to increased Crkl-Hef-1 interaction, we observed a ␥ isoform is not detected in our Cbl and C3G immunoprecipitations small increase in Abl binding to Crkl under conditions of Fc RI (Fig. 3, compare lanes 8 and 9 to lanes 2, 3, 10, and 11). The stimulation (Fig. 5, lanes 7–9). These data constitute the first physiologic significance of Crkl tyrosine phosphorylation is un- evidence that Hef-1 and Abl are involved in ITAM signal relay ␥ known. The phosphorylation of Crkl after Fc␥RI cross-linking and suggest alternative functions for Crkl in Fc RI signaling. ␥ may serve to negatively regulate its interaction with Cbl and/or Moreover, the data support the concept that Fc RI activation C3G via an intramolecular interaction similar to what has been leads to a cascade of signaling events involving Cbl, Crkl, suggested for Crk, since this isoform is not associated with these Hef-1, and C3G that potentially leads to the activation of Rap1 molecules in vivo. In contrast, we (23) recently demonstrated that and the generation of the respiratory burst in myeloid cells. mutating Y207, such that it cannot be phosphorylated by Bcr-Abl, Alternatively, the activation of Rap1 may suppress Ras and Rac does not enhance or decrease the association of Crkl with C3G, and negatively regulate oxidant signaling and other phenotypic SOS, or Cbl. The further analysis of regulated Crkl phosphoryla- responses in myeloid cells. Recent evidence suggests that Ras tion following Fc␥RI stimulation in the non-Bcr/Abl-transformed can activate the Rac pathway, suggesting a potential link be- U937IF cell line or normal macrophages may provide insight into tween Ras and the NADPH oxidase system (24, 51, 52). This the significance of Crkl phosphorylation and dephosphorylation in suggests a potential role for SOS-Ras-Rac interactions in the a nontransformed setting. generation of the respiratory burst in addition to C3G-Rap1 We sought direct evidence that Crkl is involved in Fc␥RI sig- interactions. We propose a model in which initially Src family naling. If Crkl participates in Fc␥RI signaling, we postulated that kinases phosphorylate the ITAM of the ␥ subunit (6, 53) that the Crkl protein may be recruited to this receptor complex. Crkl recruits and activates Syk kinase (3, 5, 54, 55), which, in turn, immunoprecipitates probed with anti-Fc␥RI␥-specific antisera re- binds to and phosphorylates a number of substrates, including vealed coassociation between Crkl and the ␥ subunit, a result that Cbl, Shc, Crkl, and Hef-1 (10, 14, 20, 21). Other data from our was confirmed in our peptide inhibition studies (Fig. 6, A and B). laboratory have confirmed that the Fc␥RI signals through these Unlike the direct binding of full-length Crkl to Cbl and C3G seen adapter proteins to activate Ras (10, 56) (unpublished observa- in Fig. 4B, experiments performed with identical Crkl fusion pro- tion). We postulate that the phosphorylation of Cbl and Hef-1 is teins on ␥ subunit immunoprecipitates failed to demonstrate direct followed by increased Cbl-Crkl and Hef-1-Crkl interactions in binding of Crkl to Fc␥RI␥ (data not shown). Possible candidates receptor aggregates that sequentially lead to the downstream 5562 Crkl, Cbl, AND Hef-1 IN Fc␥RI SIGNALING activation of Rap1 via Crkl-C3G binding. The Hef-1-Crkl in- 24. Gabig, T. G., C. D. Crean, P. L. Mantel, and R. Rosli. 1995. 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