c-Cbl Is Involved in Met Signaling in B Cells and Mediates Hepatocyte Growth Factor-Induced Receptor Ubiquitination This information is current as Taher E. I. Taher, Esther P. M. Tjin, Esther A. Beuling, of October 2, 2021. Jannie Borst, Marcel Spaargaren and Steven T. Pals J Immunol 2002; 169:3793-3800; ; doi: 10.4049/jimmunol.169.7.3793 http://www.jimmunol.org/content/169/7/3793 Downloaded from References This article cites 66 articles, 46 of which you can access for free at: http://www.jimmunol.org/content/169/7/3793.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 *average by guest on October 2, 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 © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology c-Cbl Is Involved in Met Signaling in B Cells and Mediates Hepatocyte Growth Factor-Induced Receptor Ubiquitination Taher E. I. Taher,1* Esther P. M. Tjin,1* Esther A. Beuling,* Jannie Borst,† Marcel Spaargaren,* and Steven T. Pals2* Hepatocyte growth factor/scatter factor (HGF) and its receptor tyrosine kinase Met are key regulators of epithelial motility and morphogenesis. Recent studies indicate that the HGF/Met pathway also plays a role in B cell differentiation, whereas uncontrolled Met signaling may lead to B cell neoplasia. These observations prompted us to explore HGF/Met signaling in B cells. In this study, we demonstrate that HGF induces strong tyrosine phosphorylation of the proto-oncogene product c-Cbl in B cells and increases Cbl association with the Src family tyrosine kinases Fyn and Lyn, as well as with phosphatidylinositol-3 kinase and CrkL. In addition, we demonstrate that c-Cbl mediates HGF-induced ubiquitination of Met. This requires the juxtamembrane tyrosine Y1001 (Y2) of Met, but not the multifunctional docking site (Y14/15) or any additional C-terminal tyrosine residues (Y13–16). In Downloaded from contrast to wild-type c-Cbl, the transforming mutants v-Cbl and 70Z/3 Cbl, which lack the ubiquitin ligase RING finger domain, suppress Met ubiquitination. Our findings identify c-Cbl as a negative regulator of HGF/Met signaling in B cells, mediating ubiquitination and, consequently, proteosomal degradation of Met, and suggest a role for Cbl in Met-mediated tumorigenesis. The Journal of Immunology, 2002, 169: 3793–3800. epatocyte growth factor/scatter factor (HGF)3 is a mul- tion from apoptosis (20–24). In B cell neoplasia, by contrast, auto- http://www.jimmunol.org/ tifunctional cytokine with a domain structure and pro- and/or paracrine stimulation of Met, rather than receptor mutation, H teolytic mechanism of activation similar to that of the appears to be the most important mechanism for transformation. It serine protease plasminogen. By binding to the receptor tyrosine was recently demonstrated that HGF is a potent growth and sur- kinase Met, the product of the proto-oncogene c-met, HGF triggers vival factor for plasma cell myelomas (25). These tumors fre- intracellular signals regulating cell proliferation, migration, and quently coexpress HGF and Met, suggesting the presence of an survival (1–9). In mice, HGF or Met deficiency results in embry- autocrine loop (26). For HGF-negative myelomas, bone marrow onic death due to severe developmental defects in the placenta and stromal cells may present an alternative, paracrine, source of HGF liver, and disrupts the migration of myogenic precursors into the (27). Similarly, HGF produced by follicular dendritic cells and limb buds (6, 9). Other studies have provided evidence for an stromal cells in lymphoid tissues may stimulate the growth and by guest on October 2, 2021 important role of HGF in angiogenesis and in the three-dimen- survival of Met-positive non-Hodgkins lymphomas (10, 11). Consis- sional organization of epithelial tissues, including kidney tubules tent with a role for HGF/Met in myeloma progression, patients with and mammary glands (3–5). More recently, the HGF/Met pathway high serum levels of HGF have an unfavorable prognosis (28). has also been implicated in B cell differentiation. Specifically, HGF/Met signaling has been extensively studied in epithelial HGF produced by follicular dendritic cells or stromal cells has cells. These studies revealed a prominent role for the multifunc- been shown to regulate integrin-mediated adhesion and migration tional docking site, consisting of tyrosine residues Y1349 (Y14) of germinal center B cells and plasma cells (10, 11). and Y1356 (Y15) (3, 29). Upon phosphorylation, this docking site Apart from these physiologic functions, uncontrolled activation mediates the interaction with Grb2, resulting in activation of the of Met is oncogenic and can promote tumor growth, invasion, and Ras/mitogen-activated protein kinase pathway. In addition, the metastasis via several distinct mechanisms (3, 12–19). In heredi- docking protein Gab1 plays an important role in HGF/Met signal- tary papillary renal carcinoma, Met mutations cause hyperactiva- ing as it is also able to interact directly with the docking site of tion of the receptor in response to HGF stimulation. These receptor Met, as well as with several signal-transducing proteins, including mutants can mediate transformation, invasive growth, and protec- phosphatidylinositol-3 kinase (PI-3K), CrkL, and SHP-2 (30–32). Despite the role of HGF/Met signaling in normal B cell differen- *Department of Pathology, Academic Medical Center, University of Amsterdam, tiation and malignancy (3, 10, 11, 25, 26), hardly anything is Amsterdam, The Netherlands; and †Department of Cellular Biochemistry, The Neth- known about the underlying signal transduction mechanism in B erlands Cancer Institute, Amsterdam, The Netherlands cells. Recently, we have reported the presence of two prominent Received for publication February 6, 2002. Accepted for publication July 29, 2002. phosphoproteins of 110 and 120 kDa after HGF stimulation of B The costs of publication of this article were defrayed in part by the payment of page cells (33), of which the 110-kDa phosphoprotein was identified as charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Gab1 (34). In the present study, we identify the other major phos- 1 T.E.I.T. and E.P.M.T. contributed equally to this manuscript. phoprotein as c-Cbl, which is a prominent target for B cell Ag receptor signaling as well (35–38). HGF induces a strong and tran- 2 Address correspondence and reprint requests to Dr. Steven T. Pals, Department of Pathology, Academic Medical Center, University Of Amsterdam, Meibergdreef 9, sient tyrosine phosphorylation of c-Cbl, resulting in an increased 1105 AZ Amsterdam, The Netherlands. E-mail address: [email protected] association with Fyn, Lyn, PI-3K, and CrkL. In addition, we dem- 3 Abbreviations used in this paper: HGF, hepatocyte growth factor; EGF, epidermal onstrate that c-Cbl, but not its oncogenic forms v-Cbl or 70Z/3 growth factor; ERK, extracellular signal-regulated kinase; HA, hemagglutinin; KD, kinase dead; NGF, nerve growth factor; PI-3K, phosphatidylinositol-3 kinase; WT, Cbl, negatively regulates Met by inducing ubiquitination of its wild type. cytoplasmic domain. Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00 3794 Cbl IN Met SIGNALING Materials and Methods 1 (Gab1), an adaptor protein that can associate with the cytoplas- Antibodies mic docking site of Met (34). By performing immunodepletion experiments, we now identified the larger prominent phosphopro- mAbs used were anti-phosphotyrosine, PY20 (Affiniti, Nottingham, U.K.), and anti-hemagglutinin tag, 12CA5 (anti-HA) (C. de Vries, Department of tein in the lysates of HGF-stimulated cells as c-Cbl (Fig. 1A). Biochemistry, AMC, Amsterdam, The Netherlands). The rabbit polyclonal Immunoblotting of Cbl immunoprecipitates with Abs against Abs used were: anti-ubiquitin (DAKO, Glostrup, Denmark); anti-human phosphotyrosine confirmed that HGF stimulation leads to a rapid Met, C-12; anti-mouse Met, SP260; anti-Fyn, FYN3; anti-Lyn, 44; anti- and transient phosphorylation of Cbl on tyrosine residues, peaking CrkL, C-20; anti-Cbl, C-15 (all: Santa Cruz Biotechnology, Santa Cruz, at 1 min and decreasing after 5 min (Fig. 1B). CA); and anti-PI-3K p85 (Upstate Biotechnology, Lake Placid, NY). Plasmids HGF stimulation leads to enhanced association of c-Cbl with Fyn, Lyn, PI-3K, and CrkL The c-Cbl cDNA was a kind gift from W. Y. Langdon (University of Western Australia, Nedlands, Australia). pMT2-encoding HA-tagged hu- The above observations prompted us to explore the function of Cbl man c-Cbl, v-Cbl, and 70Z/3 Cbl were generated from this cDNA by PCR. in Met signaling. Although Cbl itself lacks kinase activity, its char- The constructs encoding Trk-Met (a chimeric receptor that consists of the acteristic modular structure enables it to act as a scaffold for var- extracellular domain of the nerve growth factor (NGF) receptor, Trk A, and the cytoplasmic domain of c-Met), either wild type (WT) or mutants of ious signaling molecules, including cytoplasmic tyrosine kinases either tyrosine residue 1001 (Y2), 1232 and 1233 (kinase dead (KD)), 1347 (40). To determine whether HGF stimulation leads to changes in (Y14), 1354 (Y15), 1347 and 1354 (Y14/15), or 1311, 1347, 1354, and the kinase activity associated with Cbl, we conducted in vitro ki- 1363 (Y13–16), were a kind gift from W.