Abstract
Ubiquitination of the epidermal growth factor receptor (EGFR) is an important intracellular signal that occurs upon EGF stimulation and controls EGFR trafficking at multiple steps, finally destining the receptor to lysosomal degradation. In this chapter, we give an overview of the biochemical methods to investigate EGFR ubiquitination.
Firstly, we describe the in vitro ubiquitination assay, a method where, in the presence of the minimal ubiquitination machinery, the biological milieu for EGFR ubiquitination is reproduced in a test tube. In the second protocol, we explain how to immunoprecipitate the EGFR from total lysate and reveal its ubiquitinated form by western blot analysis. Then, with an ELISA-derived assay, we illustrate a robust and reliable method to assess EGFR ubiquitination from low amount of sample; lastly, we illustrate an immunofluorescence protocol to visualize ubiquitinated species (including the EGFR itself) within the EGFR-positive endocytic compartments upon EGF stimulation.
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References
Citri A, Yarden Y (2006) EGF-ERBB signalling: towards the systems level. Nat Rev Mol Cell Biol 7(7):505–516. doi:10.1038/nrm1962
Zwick E, Bange J, Ullrich A (2001) Receptor tyrosine kinase signalling as a target for cancer intervention strategies. Endocr Relat Cancer 8(3):161–173
Kovacs E, Zorn JA, Huang Y, Barros T, Kuriyan J (2015) A structural perspective on the regulation of the epidermal growth factor receptor. Annu Rev Biochem 84:739–764. doi:10.1146/annurev-biochem-060614-034402
Levkowitz G, Waterman H, Zamir E, Kam Z, Oved S, Langdon WY, Beguinot L, Geiger B, Yarden Y (1998) C-Cbl/Sli-1 regulates endocytic sorting and ubiquitination of the epidermal growth factor receptor. Genes Dev 12(23):3663–3674
Sigismund S, Algisi V, Nappo G, Conte A, Pascolutti R, Cuomo A, Bonaldi T, Argenzio E, Verhoef LG, Maspero E, Bianchi F, Capuani F, Ciliberto A, Polo S, Di Fiore PP (2013) Threshold-controlled ubiquitination of the EGFR directs receptor fate. EMBO J 32(15):2140–2157. doi:10.1038/emboj.2013.149
Sigismund S, Argenzio E, Tosoni D, Cavallaro E, Polo S, Di Fiore PP (2008) Clathrin-mediated internalization is essential for sustained EGFR signaling but dispensable for degradation. Dev Cell 15(2):209–219. doi:10.1016/j.devcel.2008.06.012
Goh LK, Sorkin A (2013) Endocytosis of receptor tyrosine kinases. Cold Spring Harb Perspect Biol 5(5):a017459. doi:10.1101/cshperspect.a017459
Waterman H, Levkowitz G, Alroy I, Yarden Y (1999) The RING finger of c-Cbl mediates desensitization of the epidermal growth factor receptor. J Biol Chem 274(32):22151–22154
Waterman H, Katz M, Rubin C, Shtiegman K, Lavi S, Elson A, Jovin T, Yarden Y (2002) A mutant EGF-receptor defective in ubiquitylation and endocytosis unveils a role for Grb2 in negative signaling. EMBO J 21(3):303–313. doi:10.1093/emboj/21.3.303
Jiang X, Huang F, Marusyk A, Sorkin A (2003) Grb2 regulates internalization of EGF receptors through clathrin-coated pits. Mol Biol Cell 14(3):858–870. doi:10.1091/mbc.E02-08-0532
Capuani F, Conte A, Argenzio E, Marchetti L, Priami C, Polo S, Di Fiore PP, Sigismund S, Ciliberto A (2015) Quantitative analysis reveals how EGFR activation and downregulation are coupled in normal but not in cancer cells. Nat Commun 6:7999. doi:10.1038/ncomms8999
Huang F, Kirkpatrick D, Jiang X, Gygi S, Sorkin A (2006) Differential regulation of EGF receptor internalization and degradation by multiubiquitination within the kinase domain. Mol Cell 21(6):737–748. doi:10.1016/j.molcel.2006.02.018
Conte A, Sigismund S (2016) Chapter six – the ubiquitin network in the control of EGFR endocytosis and signaling. Prog Mol Biol Transl Sci 141:225–276. doi:10.1016/bs.pmbts.2016.03.002
Goh LK, Huang F, Kim W, Gygi S, Sorkin A (2010) Multiple mechanisms collectively regulate clathrin-mediated endocytosis of the epidermal growth factor receptor. J Cell Biol 189(5):871–883. doi:10.1083/jcb.201001008
Sigismund S, Woelk T, Puri C, Maspero E, Tacchetti C, Transidico P, Di Fiore PP, Polo S (2005) Clathrin-independent endocytosis of ubiquitinated cargos. Proc Natl Acad Sci U S A 102(8):2760–2765. doi:10.1073/pnas.0409817102
Haglund K, Shimokawa N, Szymkiewicz I, Dikic I (2002) Cbl-directed monoubiquitination of CIN85 is involved in regulation of ligand-induced degradation of EGF receptors. Proc Natl Acad Sci U S A 99(19):12191–12196. doi:10.1073/pnas.192462299
Tomas A, Futter CE, Eden ER (2014) EGF receptor trafficking: consequences for signaling and cancer. Trends Cell Biol 24(1):26–34. doi:10.1016/j.tcb.2013.11.002
Berndsen CE, Wolberger C (2011) A spectrophotometric assay for conjugation of ubiquitin and ubiquitin-like proteins. Anal Biochem 418(1):102–110. doi:10.1016/j.ab.2011.06.034
Maspero E, Polo S (2016) In vitro ubiquitination: self-ubiquitination, chain formation, and substrate ubiquitination assays. Methods Mol Biol 1449:153–160. doi:10.1007/978-1-4939-3756-1_7
Acknowledgments
The authors gratefully acknowledge Rosalind Gunby for critically reviewing this manuscript and Roberta Pascolutti for the EGFR in vitro ubiquitination experiment. SS was supported by a grant from Worldwide Cancer Research (16-1245). AC was supported by FIRC Fellowship 14909.
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Conte, A., Sigismund, S. (2017). Methods to Investigate EGFR Ubiquitination. In: Wang, Z. (eds) ErbB Receptor Signaling. Methods in Molecular Biology, vol 1652. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7219-7_5
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DOI: https://doi.org/10.1007/978-1-4939-7219-7_5
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