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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 References This article cites 55 articles, 39 of which you can access for free at: http://www.jimmunol.org/content/161/10/5555.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 27, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 g Subunit Immunoreceptor Tyrosine-Based Activation Motif in Signaling of Myeloid High Affinity Fc Receptor for IgG (FcgRI)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 FcgRI aggregation in U937IF cells. FcgRI 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 g subunit of FcgRI 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 FcgRIg after FcgRI activation and the constitutive association between C3G and Crkl provide the first evidence that a FcgRIg-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 (FcgRII and FcgRIII). FcgRI is a mem- action to activation by Ag-Ab complexes, Ab-dependent brane glycoprotein comprised of a ligand-binding a 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 g 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 (FcgRI)3 motif (ITAM) (3). FcgRI and FcgRII 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-g increases FcgRI expression by as much as 20-fold Los Angeles, CA 90027; and ‡Department of Microbiology and Immunology, and facilitates the examination of FcgRI 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 g (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-g (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 FcgRI 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: FcgRI, high affinity Fc receptor for IgG; FcgRII, present on the proto-oncoprotein Cbl and the cytoskeleton-associ- g 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; RaM, 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- 1 phate oxidase system; BCR, B cell receptor. phia chromosome-positive (Ph ) acute lymphoblastic leukemia. Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 5556 Crkl, Cbl, AND Hef-1 IN FcgRI SIGNALING Crkl is tyrosine phosphorylated in Bcr-Abl-transformed cells, sug- rated. Cells were lysed in 800 ml 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 3 g for 30 min. Immu- variety of nontransformed cell types, predominantly hemopoietic noprecipitations or GST fusion protein pull-downs were performed as de- scribed 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 mM phenylarsine The 120-kDa Cbl is a complex adapter protein expressed in oxide, and 100 mM sodium orthovanadate. Lysates were cleared by cen- 3 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 mg 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 ml 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.
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