Cbl is a multi-adaptor protein involved in ligand-induced downregulation of receptor tyrosine kinases. It is thought that Cbl-mediated ubiquitination of active receptors is essential for receptor degradation and cessation of receptor-induced signal transduction1,2,3,4,5. Here we demonstrate that Cbl additionally regulates epidermal growth factor (EGF) receptor endocytosis. Cbl rapidly recruits CIN85 (Cbl-interacting protein of 85K; ref. 6) and endophilins (regulatory components of clathrin-coated vesicles7,8,9,10) to form a complex with activated EGF receptors, thus controlling receptor internalization. CIN85 was constitutively associated with endophilins, whereas CIN85 binding to the distal carboxy terminus of Cbl was increased on EGF stimulation. Inhibition of these interactions was sufficient to block EGF receptor internalization, delay receptor degradation and enhance EGF-induced gene transcription, without perturbing Cbl-directed receptor ubiquitination. Thus, the evolutionary divergent C terminus of Cbl uses a mechanism that is functionally separable from the ubiquitin ligase activity of Cbl to mediate ligand-dependent downregulation of receptor tyrosine kinases.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Thien, C. B. & Langdon, W. Y. Cbl: many adaptations to regulate protein tyrosine kinases. Nature Rev. Mol. Cell Biol. 2, 294–307 (2001).
Joazeiro, C. A. et al. The tyrosine kinase negative regulator c-Cbl as a RING-type, E2-dependent ubiquitin-protein ligase. Science 286, 309–312 (1999).
Levkowitz, G. et al. Ubiquitin ligase activity and tyrosine phosphorylation underlie suppression of growth factor signaling by c-Cbl/Sli-1. Mol. Cell 4, 1029–1040 (1999).
Waterman, H. & Yarden, Y. Molecular mechanisms underlying endocytosis and sorting of ErbB receptor tyrosine kinases. FEBS Lett. 490, 142–152 (2001).
Di Fiore, P. P. & De Camilli, P. Endocytosis and signaling. An inseparable partnership. Cell 106, 1–4 (2001).
Take, H. et al. Cloning and characterization of a novel adaptor protein, CIN85, that interacts with c-Cbl. Biochem. Biophys. Res. Commun. 268, 321–328 (2000).
Schmidt, A. et al. Endophilin I mediates synaptic vesicle formation by transfer of arachidonate to lysophosphatidic acid. Nature 401, 133–141 (1999).
Ringstad, N. et al. Endophilin/SH3p4 is required for the transition from early to late stages in clathrin-mediated synaptic vesicle endocytosis. Neuron 24, 143–154 (1999).
Simpson, F. et al. SH3-domain-containing proteins function at distinct steps in clathrin-coated vesicle formation. Nature Cell Biol. 1, 119–124 (1999).
Gad, H. et al. Fission and uncoating of synaptic clathrin-coated vesicles are perturbed by disruption of interactions with the SH3 domain of endophilin. Neuron 27, 301–312 (2000).
Yoon, C. H., Chang, C., Hopper, N. A., Lesa, G. M. & Sternberg, P. W. Requirements of multiple domains of SLI-1, a Caenorhabditis elegans homologue of c-Cbl, and an inhibitory tyrosine in LET-23 in regulating vulval differentiation. Mol. Biol. Cell 11, 4019–4031 (2000).
Gout, I. et al. Negative regulation of PI 3-kinase by Ruk, a novel adaptor protein. EMBO J. 19, 4015–4025 (2000).
Bogler, O. et al. SETA: a novel SH3 domain-containing adapter molecule associated with malignancy in astrocytes. Neuro-oncology. 2, 6–15 (2000).
Kirsch, K. H. et al. The adapter type protein CMS/CD2AP binds to the proto-oncogenic protein c-Cbl through a tyrosine phosphorylation-regulated Src homology 3 domain interaction. J. Biol. Chem. 276, 4957–4963 (2001).
Watanabe, S. et al. Characterization of the CIN85 adaptor protein and identification of components involved in CIN85 complexes. Biochem. Biophys. Res. Commun. 278, 167–174 (2000).
Borinstein, S. C. et al. SETA is a multifunctional adapter protein with three SH3 domains that binds Grb2, Cbl, and the novel SB1 proteins. Cell Signal 12, 769–779 (2000).
Ringstad, N., Nemoto, Y. & De Camilli, P. The SH3p4/Sh3p8/SH3p13 protein family: binding partners for synaptojanin and dynamin via a Grb2-like Src homology 3 domain. Proc. Natl Acad. Sci. USA 94, 8569–8574 (1997).
Micheva, K. D., Ramjaun, A. R., Kay, B. K. & McPherson, P. S. SH3 domain-dependent interactions of endophilin with amphiphysin. FEBS Lett. 414, 308–312 (1997).
Farsad, K. et al. Generation of high curvature membranes mediated by direct endophilin bilayer interactions. J. Cell Biol. 155, 193–200 (2001).
de Melker, A. A., van Der Horst, G., Calafat, J., Jansen, H. & Borst, J. c-Cbl ubiquitinates the EGF receptor at the plasma membrane and remains receptor associated throughout the endocytic route. J. Cell Sci. 114, 2167–2178 (2001).
Levkowitz, G. et al. c-Cbl/Sli-1 regulates endocytic sorting and ubiquitination of the epidermal growth factor receptor. Genes Dev. 12, 3663–3674 (1998).
Petrelli, A. et al. The endophilin–CIN85–Cbl complex mediates ligand-dependent downregulation of c-Met. Nature 416, 187–190 (2001).
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. 270, 20242–20245 (1995).
Scaife, R. M. & Langdon, W. Y. c-Cbl localizes to actin lamellae and regulates lamellipodia formation and cell morphology. J. Cell Sci. 113(2), 215–226 (2000).
Dikic, I., Tokiwa, G., Lev, S., Courtneidge, S. A. & Schlessinger, J. A role for Pyk2 and Src in linking G-protein-coupled receptors with MAP kinase activation. Nature 383, 547–550 (1996).
Ivankovic-Dikic, I., Gronroos, E., Blaukat, A., Barth, B. U. & Dikic, I. Pyk2 and FAK regulate neurite outgrowth induced by growth factors and integrins. Nature Cell Biol. 2, 574–581 (2000).
Sorkin, A., Mazzotti, M., Sorkina, T., Scotto, L. & Beguinot, L. Epidermal growth factor receptor interaction with clathrin adaptors is mediated by the Tyr974-containing internalization motif. J. Biol. Chem. 271, 13377–13384 (1996).
We thank S. Giordano and Y. Yarden for communication of unpublished results, and L. Claesson-Welsh and C.-H. Heldin for critical reading of the manuscript. We also thank other members of the Dikic laboratory for valuable comments and help with various reagents. This work was supported by a Marie Curie Fellowship of the European Community programme to P.S., and the Boehringer Ingelheim Fonds and the Swedish Strategic Funds to I.D.
The authors declare no competing financial interests.
About this article
Cite this article
Soubeyran, P., Kowanetz, K., Szymkiewicz, I. et al. Cbl–CIN85–endophilin complex mediates ligand-induced downregulation of EGF receptors. Nature 416, 183–187 (2002). https://doi.org/10.1038/416183a
CD5 signalosome coordinates antagonist TCR signals to control the generation of Treg cells induced by foreign antigens
Proceedings of the National Academy of Sciences (2020)
Biochemical Journal (2020)
<i>Cindr</i>, the <i>Drosophila</i> Homolog of the <i>CD2AP</i> Alzheimer's Disease Susceptibility Gene, is Required for Synaptic Transmission and Proteostasis
SSRN Electronic Journal (2019)