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Signaling Immunoreceptor Tyrosine Activation Motif CBL-GRB2

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Signaling Immunoreceptor Tyrosine Activation Motif CBL-GRB2 CBL-GRB2 Interaction in Myeloid Immunoreceptor Tyrosine Activation Motif Signaling This information is current as Rae Kil Park, Wade T. Kyono, Yenbou Liu and Donald L. Durden of September 26, 2021. J Immunol 1998; 160:5018-5027; ; http://www.jimmunol.org/content/160/10/5018 Downloaded from References This article cites 42 articles, 31 of which you can access for free at: http://www.jimmunol.org/content/160/10/5018.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • 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 *average by guest on September 26, 2021 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. CBL-GRB2 Interaction in Myeloid Immunoreceptor Tyrosine Activation Motif Signaling1 Rae Kil Park,*† Wade T. Kyono,* Yenbou Liu,* and Donald L. Durden2* In this study, we provide the first evidence for role of the CBL adapter protein interaction in FcgRI receptor signal transduction. We study the FcgRI receptor, an immunoreceptor tyrosine activation motif (ITAM)-linked signaling pathway, using IFN-g- differentiated U937 myeloid cells, termed U937IF cells. CBL is constitutively associated with both GRB2 and the ITAM-containing receptor subunit, FcgRIg of FcgRI, providing direct evidence that CBL functions in myeloid ITAM signaling. FcgRI cross-linking of U937IF cells induces the tyrosine phosphorylation of CBL that is associated with an altered CBL-GRB2 interaction. Both GRB2-SH3 and SH2 domains bind CBL in resting cell lysates; upon FcgRI stimulation, phosphorylated CBL binds exclusively to the GRB2-SH2 domain. Glutathione-S-transferase fusion protein data demonstrate that the constitutive interaction of CBL with GRB2 and CRKL is mediated via two discrete regions of the CBL C terminus. The proximal C terminus (residues 461–670) binds Downloaded from to GRB2 constitutively, and under conditions of receptor activation binds to the tyrosine-phosphorylated SHC adapter molecule. The distal C terminus of CBL (residues 671–906) binds the CRKL adapter protein. The data demonstrate that the CBL-GRB2 and GRB2-SOS protein complexes are distinct and mutually exclusive in U937IF cells, supporting a model by which the CBL- GRB2 and GRB2-SOS complexes function in separate pathways for myeloid FcgRI signaling. The Journal of Immunology, 1998, 160: 5018–5027. http://www.jimmunol.org/ he cbl gene, originally described as the transforming gene with adapter proteins (e.g., GRB2, CRK, CRKL, NCK) that reg- of the Cas NS-1 murine retrovirus, induces pre-B cell ulate the guanine nucleotide exchange factors, “son of sevenless” T lymphomas and myeloid leukemias in mice (1, 2). Inter- (SOS) and C3G in mammalian cells following the activation of the estingly, the viral oncoprotein has lost a portion of its C terminus TCR (12–14). Marcilla et al. reported that stimulation of multiple that encodes the GRB2-SH3 binding site. The N terminus of the FcgR classes (FcgRI, FcgRII, and FcgRIII) in HL-60 cells with CBL protein is closely related to the SLI-1 gene product recently IgG/anti-IgG complexes induces the tyrosine phosphorylation of cloned in Caenorhabditis elegans, which is a putative negative CBL (9). Stimulation with these immune complexes results in the regulator of RAS in the LET-23 pathway (epidermal growth factor g g g activation of Fc RI, Fc RIIA, and Fc RIII receptors, making it by guest on September 26, 2021 related) for vulval development (3). Several reports suggest that more difficult to interpret these results. Matsuo et al. and Tanaka et cbl p120 is involved in the regulation of small GTPases that are al. subsequently implicated CBL in FcgRII/III signaling in mac- activated by receptor protein tyrosine kinases (4–7). rophages and THP-1 cells, respectively (14, 15). The function of CBL is tyrosine phosphorylated following the activation of re- CBL tyrosine phosphorylation and/or the interaction of CBL with ceptors belonging to the Ig gene superfamily (TCR, B cell recep- adapter proteins as it relates to specific signaling through the g tor, and Fc receptors). These multisubunit receptors signal FcgRI receptor in myeloid cells and the regulation of RAS have 3 through an immunoreceptor tyrosine activation motif (ITAM) not been thoroughly studied. (YXXLX6–8YXXL, consensus) (8–11). CBL is known to interact We investigated the role of the CBL adapter protein interaction following specific cross-linking of the FcgRI receptor in myeloid g g g *Neil Bogart Memorial Laboratories, Division of Hematology-Oncology, Children’s signaling. CBL is bound to the Fc RI subunit of the Fc RI re- Hospital Los Angeles Research Institute and University of Southern California School ceptor in myeloid cells, providing direct evidence that CBL is in- of Medicine, Norris Cancer Center, Los Angeles, CA 90027; and †Department of Microbiology and Immunology, Wonkwang University School of Medicine, Iksan, volved in ITAM signaling. Our data demonstrate that CBL binds Korea in vitro to GRB2 and CRKL molecules via different domains of the Received for publication September 2, 1997. Accepted for publication January CBL C terminus. CBL is tyrosine phosphorylated after FcgRI 21, 1998. cross-linking, and this phosphorylation is associated with an al- The costs of publication of this article were defrayed in part by the payment of page tered CBL-GRB2 interaction. At the same time, the GRB2-SH2 charges. This article must therefore be hereby marked advertisement in accordance g with 18 U.S.C. Section 1734 solely to indicate this fact. domain inducibly binds to SHC after Fc RI stimulation. These events are associated with the conversion of GDPras to GTPras 1 This work was partially supported by a grant from National Institutes of Health, RO1 CA 37256-01 to D.L.D. Work was performed in Neil Bogart Memorial Labo- (unpublished observation). Taken together, our data support a ratories, as supported by T. J. Martell Foundation for Leukemia, Cancer, and AIDS model by which the CBL-GRB2 interaction may modulate the in- Research. D.L.D. is supported by a Career Development Award from Children’s g Hospital Los Angeles Research Institute and STOP Cancer Foundation and a grant teraction between GRB2 and SOS in Fc RI signaling in myeloid from Robert E. and May R. Wright Foundation through USC School of Medicine. cells. R.K.P. was supported by Wonkwang University in 1996. 2 Address correspondence and reprint requests to Dr. Donald L. Durden, Department of Pediatrics, Division of Hematology-Oncology (M/S #57), Children’s Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027. E-mail address: Materials and Methods ddurden%[email protected] Antibodies 3 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine activation motif; ECL, enhanced chemiluminescence; GST, glutathione-S-transferase; IP, immunopre- The FcgRIa-specific cross-linking Abs were generously provided by Me- cipitate; MAP, mitogen-activated protein; SOS, son of sevenless. darex (West Lebanon, NH). The mAb 197 and mAb 32.2 are specific for Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 The Journal of Immunology 5019 g a 9 the Fc RI subunit; mAb 32.2 is a F(ab )2 fragment of IgG. The cross- 9 linking Ab was a rabbit anti-mouse F(ab )2 fragment purchased from Or- ganon Teknika (West Chester, PA). Anti-CBL Ab was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphotyrosine, anti- SHC Abs, and anti-CRKL antisera were purchased from Upstate Biotech- nology (Lake Placid, NY), and the anti-GRB2 mAb (G16720) was ob- tained from Transduction Laboratories (Lexington, KY). GRB2 immunoprecipitations were performed with polyclonal anti-GRB2 (C-231) against residues 195–217 of human GRB2 molecule from Santa Cruz Bio- technology. The anti-g subunit (FcgRIg) antisera 5927 was prepared in our laboratory, as described (16, 17), and the 4D8 anti-g mAb was generously provided by J. Kochan (Hoffman-La Roche, Nutley, NJ) (18). Preimmune immunoprecipitations were performed with an equal amount of purified rabbit IgG. Differentiation and stimulation of U937 cells U937 cells were maintained in RPMI 1640 with 10% FCS and differenti- ated with 250 U/ml human rIFN-g (obtained from Genentech, San Fran- cisco, CA) for 4 days (termed U937IF cells). U937IF cells were cultured at a concentration of 5 3 105 cells/ml, and the medium was replenished with fresh IFN-g (250 U/ml) every 2 days, as described (16, 19). At the time of performing cross-linking experiments, the U937IF cells are 48 h from the Downloaded from addition of fresh IFN-g. Flow-cytometric analysis of U937IF cells dem- onstrated the expression of the FcgRI and FcgRII receptors on these cells (data not shown). For stimulation of FcgRI receptors on U937IF cells, cells were washed twice in cold HBSS and adjusted to a concentration of 4 3 107 cells/ml; 0.5-ml aliquots were incubated on ice for 30 min with anti- FcgRI Abs (0.25 mg/sample). Cross-linking Abs used in Figure 1 were the 32.2 (F(ab9) fragment) and 197 (whole IgG) anti-FcgRI mAbs. Experi- 2 http://www.jimmunol.org/ ments shown in Figures 2, 3, 6, and 7 were conducted with the 32.2 9 (F(ab )2 fragment), and Figures 4 and 5 were performed with 197 Ab cross-linking, as described (19).
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