Abstract
The human proto-oncogene product c-Cbl and a similar protein in Caenorhabditis elegans (Sli-1) contain a proline-rich COOH-terminal region that binds Src homology 3 (SH3) domains of proteins such as the adapter Grb2. Cb1-Grb2 complexes can be recruited to tyrosine-phosphorylated epidermal growth factor (EGF) receptors through the SH2 domain of Grb2. Here we identify by molecular cloning a Drosophila cDNA encoding a protein (Drosophila Cbl [D-Cbl]) that shows high sequence similarity to the N-terminal region of human c-Cbl but lacks proline-rich sequences and fails to bind Grb2. Nonetheless, in COS-1 cells, expression of hemagglutinin epitope-tagged D-Cbl results in its coimmunoprecipitation with EGF receptors in response to EGF. EGF also caused tyrosine phosphorylation of D-Cbl in such cells, but no association of phosphatidylinositol 3-kinase was detected in assays using anti-p85 antibody. A point mutation in D-Cbl (G305E) that suppresses the negative regulation of LET-23 by the Cbl homolog Sli-1 in C. elegans prevented tyrosine phosphorylation of D-Cbl as well as binding to the liganded EGF receptor in COS-1 cells. Colocalization of EGF receptors with both endogenous c-Cbl or expressed D-Cbl in endosomes of EGF-treated COS-1 cells is also demonstrated by immunofluorescence microscopy. In lysates of adult transgenic Drosophila melanogaster, GST-DCbl binds to the tyrosine-phosphorylated 150-kDa torso-DER chimeric receptor. Expression of D-Cbl directed by the sevenless enhancer in intact Drosophila compromises severely the development of the R7 photoreceptor neuron. These data suggest that despite the lack of Grb2 binding sites, D-Cbl functions as a negative regulator of receptor tyrosine kinase signaling in the Drosophila eye by a mechanism that involves its association with EGF receptors or other tyrosine kinases.
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- Andoniou C. E., Thien C. B., Langdon W. Y. Tumour induction by activated abl involves tyrosine phosphorylation of the product of the cbl oncogene. EMBO J. 1994 Oct 3;13(19):4515–4523. doi: 10.1002/j.1460-2075.1994.tb06773.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Basler K., Christen B., Hafen E. Ligand-independent activation of the sevenless receptor tyrosine kinase changes the fate of cells in the developing Drosophila eye. Cell. 1991 Mar 22;64(6):1069–1081. doi: 10.1016/0092-8674(91)90262-w. [DOI] [PubMed] [Google Scholar]
- Blake T. J., Heath K. G., Langdon W. Y. The truncation that generated the v-cbl oncogene reveals an ability for nuclear transport, DNA binding and acute transformation. EMBO J. 1993 May;12(5):2017–2026. doi: 10.1002/j.1460-2075.1993.tb05851.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Blake T. J., Shapiro M., Morse H. C., 3rd, Langdon W. Y. The sequences of the human and mouse c-cbl proto-oncogenes show v-cbl was generated by a large truncation encompassing a proline-rich domain and a leucine zipper-like motif. Oncogene. 1991 Apr;6(4):653–657. [PubMed] [Google Scholar]
- Bowtell D. D., Langdon W. Y. The protein product of the c-cbl oncogene rapidly complexes with the EGF receptor and is tyrosine phosphorylated following EGF stimulation. Oncogene. 1995 Oct 19;11(8):1561–1567. [PubMed] [Google Scholar]
- Buday L., Khwaja A., Sipeki S., Faragó A., Downward J. Interactions of Cbl with two adapter proteins, Grb2 and Crk, upon T cell activation. J Biol Chem. 1996 Mar 15;271(11):6159–6163. doi: 10.1074/jbc.271.11.6159. [DOI] [PubMed] [Google Scholar]
- Daga A., Banerjee U. Resolving the sevenless pathway using sensitized genetic backgrounds. Cell Mol Biol Res. 1994;40(3):245–251. [PubMed] [Google Scholar]
- Donovan J. A., Wange R. L., Langdon W. Y., Samelson L. E. The protein product of the c-cbl protooncogene is the 120-kDa tyrosine-phosphorylated protein in Jurkat cells activated via the T cell antigen receptor. J Biol Chem. 1994 Sep 16;269(37):22921–22924. [PubMed] [Google Scholar]
- Freeman M. Reiterative use of the EGF receptor triggers differentiation of all cell types in the Drosophila eye. Cell. 1996 Nov 15;87(4):651–660. doi: 10.1016/s0092-8674(00)81385-9. [DOI] [PubMed] [Google Scholar]
- Fukazawa T., Miyake S., Band V., Band H. Tyrosine phosphorylation of Cbl upon epidermal growth factor (EGF) stimulation and its association with EGF receptor and downstream signaling proteins. J Biol Chem. 1996 Jun 14;271(24):14554–14559. doi: 10.1074/jbc.271.24.14554. [DOI] [PubMed] [Google Scholar]
- Fukazawa T., Reedquist K. A., Trub T., Soltoff S., Panchamoorthy G., Druker B., Cantley L., Shoelson S. E., Band H. The SH3 domain-binding T cell tyrosyl phosphoprotein p120. Demonstration of its identity with the c-cbl protooncogene product and in vivo complexes with Fyn, Grb2, and phosphatidylinositol 3-kinase. J Biol Chem. 1995 Aug 11;270(32):19141–19150. doi: 10.1074/jbc.270.32.19141. [DOI] [PubMed] [Google Scholar]
- Galisteo M. L., Dikic I., Batzer A. G., Langdon W. Y., Schlessinger J. Tyrosine phosphorylation of the c-cbl proto-oncogene protein product and association with epidermal growth factor (EGF) receptor upon EGF stimulation. J Biol Chem. 1995 Sep 1;270(35):20242–20245. doi: 10.1074/jbc.270.35.20242. [DOI] [PubMed] [Google Scholar]
- Hartley D., Meisner H., Corvera S. Specific association of the beta isoform of the p85 subunit of phosphatidylinositol-3 kinase with the proto-oncogene c-cbl. J Biol Chem. 1995 Aug 4;270(31):18260–18263. doi: 10.1074/jbc.270.31.18260. [DOI] [PubMed] [Google Scholar]
- Honegger A. M., Dull T. J., Felder S., Van Obberghen E., Bellot F., Szapary D., Schmidt A., Ullrich A., Schlessinger J. Point mutation at the ATP binding site of EGF receptor abolishes protein-tyrosine kinase activity and alters cellular routing. Cell. 1987 Oct 23;51(2):199–209. doi: 10.1016/0092-8674(87)90147-4. [DOI] [PubMed] [Google Scholar]
- Lai W. H., Cameron P. H., Doherty J. J., 2nd, Posner B. I., Bergeron J. J. Ligand-mediated autophosphorylation activity of the epidermal growth factor receptor during internalization. J Cell Biol. 1989 Dec;109(6 Pt 1):2751–2760. doi: 10.1083/jcb.109.6.2751. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lee J., Jongeward G. D., Sternberg P. W. unc-101, a gene required for many aspects of Caenorhabditis elegans development and behavior, encodes a clathrin-associated protein. Genes Dev. 1994 Jan;8(1):60–73. doi: 10.1101/gad.8.1.60. [DOI] [PubMed] [Google Scholar]
- Levkowitz G., Klapper L. N., Tzahar E., Freywald A., Sela M., Yarden Y. Coupling of the c-Cbl protooncogene product to ErbB-1/EGF-receptor but not to other ErbB proteins. Oncogene. 1996 Mar 7;12(5):1117–1125. [PubMed] [Google Scholar]
- Lupher M. L., Jr, Reedquist K. A., Miyake S., Langdon W. Y., Band H. A novel phosphotyrosine-binding domain in the N-terminal transforming region of Cbl interacts directly and selectively with ZAP-70 in T cells. J Biol Chem. 1996 Sep 27;271(39):24063–24068. doi: 10.1074/jbc.271.39.24063. [DOI] [PubMed] [Google Scholar]
- Meisner H., Conway B. R., Hartley D., Czech M. P. Interactions of Cbl with Grb2 and phosphatidylinositol 3'-kinase in activated Jurkat cells. Mol Cell Biol. 1995 Jul;15(7):3571–3578. doi: 10.1128/mcb.15.7.3571. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Meisner H., Czech M. P. Coupling of the proto-oncogene product c-Cbl to the epidermal growth factor receptor. J Biol Chem. 1995 Oct 27;270(43):25332–25335. doi: 10.1074/jbc.270.43.25332. [DOI] [PubMed] [Google Scholar]
- Odai H., Sasaki K., Iwamatsu A., Hanazono Y., Tanaka T., Mitani K., Yazaki Y., Hirai H. The proto-oncogene product c-Cbl becomes tyrosine phosphorylated by stimulation with GM-CSF or Epo and constitutively binds to the SH3 domain of Grb2/Ash in human hematopoietic cells. J Biol Chem. 1995 May 5;270(18):10800–10805. doi: 10.1074/jbc.270.18.10800. [DOI] [PubMed] [Google Scholar]
- Olivier J. P., Raabe T., Henkemeyer M., Dickson B., Mbamalu G., Margolis B., Schlessinger J., Hafen E., Pawson T. A Drosophila SH2-SH3 adaptor protein implicated in coupling the sevenless tyrosine kinase to an activator of Ras guanine nucleotide exchange, Sos. Cell. 1993 Apr 9;73(1):179–191. doi: 10.1016/0092-8674(93)90170-u. [DOI] [PubMed] [Google Scholar]
- Panchamoorthy G., Fukazawa T., Miyake S., Soltoff S., Reedquist K., Druker B., Shoelson S., Cantley L., Band H. p120cbl is a major substrate of tyrosine phosphorylation upon B cell antigen receptor stimulation and interacts in vivo with Fyn and Syk tyrosine kinases, Grb2 and Shc adaptors, and the p85 subunit of phosphatidylinositol 3-kinase. J Biol Chem. 1996 Feb 9;271(6):3187–3194. doi: 10.1074/jbc.271.6.3187. [DOI] [PubMed] [Google Scholar]
- Reedquist K. A., Fukazawa T., Druker B., Panchamoorthy G., Shoelson S. E., Band H. Rapid T-cell receptor-mediated tyrosine phosphorylation of p120, an Fyn/Lck Src homology 3 domain-binding protein. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4135–4139. doi: 10.1073/pnas.91.10.4135. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Reichman-Fried M., Dickson B., Hafen E., Shilo B. Z. Elucidation of the role of breathless, a Drosophila FGF receptor homolog, in tracheal cell migration. Genes Dev. 1994 Feb 15;8(4):428–439. doi: 10.1101/gad.8.4.428. [DOI] [PubMed] [Google Scholar]
- Rivero-Lezcano O. M., Sameshima J. H., Marcilla A., Robbins K. C. Physical association between Src homology 3 elements and the protein product of the c-cbl proto-oncogene. J Biol Chem. 1994 Jul 1;269(26):17363–17366. [PubMed] [Google Scholar]
- Rogge R., Cagan R., Majumdar A., Dulaney T., Banerjee U. Neuronal development in the Drosophila retina: the sextra gene defines an inhibitory component in the developmental pathway of R7 photoreceptor cells. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5271–5275. doi: 10.1073/pnas.89.12.5271. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Soltoff S. P., Cantley L. C. p120cbl is a cytosolic adapter protein that associates with phosphoinositide 3-kinase in response to epidermal growth factor in PC12 and other cells. J Biol Chem. 1996 Jan 5;271(1):563–567. doi: 10.1074/jbc.271.1.563. [DOI] [PubMed] [Google Scholar]
- Sorkin A., Waters C. M. Endocytosis of growth factor receptors. Bioessays. 1993 Jun;15(6):375–382. doi: 10.1002/bies.950150603. [DOI] [PubMed] [Google Scholar]
- Tanaka S., Neff L., Baron R., Levy J. B. Tyrosine phosphorylation and translocation of the c-cbl protein after activation of tyrosine kinase signaling pathways. J Biol Chem. 1995 Jun 16;270(24):14347–14351. doi: 10.1074/jbc.270.24.14347. [DOI] [PubMed] [Google Scholar]
- Tsygankov A. Y., Mahajan S., Fincke J. E., Bolen J. B. Specific association of tyrosine-phosphorylated c-Cbl with Fyn tyrosine kinase in T cells. J Biol Chem. 1996 Oct 25;271(43):27130–27137. doi: 10.1074/jbc.271.43.27130. [DOI] [PubMed] [Google Scholar]
- Tyers M., Tokiwa G., Nash R., Futcher B. The Cln3-Cdc28 kinase complex of S. cerevisiae is regulated by proteolysis and phosphorylation. EMBO J. 1992 May;11(5):1773–1784. doi: 10.1002/j.1460-2075.1992.tb05229.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yoon C. H., Lee J., Jongeward G. D., Sternberg P. W. Similarity of sli-1, a regulator of vulval development in C. elegans, to the mammalian proto-oncogene c-cbl. Science. 1995 Aug 25;269(5227):1102–1105. doi: 10.1126/science.7652556. [DOI] [PubMed] [Google Scholar]
- Zipursky S. L., Rubin G. M. Determination of neuronal cell fate: lessons from the R7 neuron of Drosophila. Annu Rev Neurosci. 1994;17:373–397. doi: 10.1146/annurev.ne.17.030194.002105. [DOI] [PubMed] [Google Scholar]
- de Jong R., ten Hoeve J., Heisterkamp N., Groffen J. Crkl is complexed with tyrosine-phosphorylated Cbl in Ph-positive leukemia. J Biol Chem. 1995 Sep 15;270(37):21468–21471. doi: 10.1074/jbc.270.37.21468. [DOI] [PubMed] [Google Scholar]