In vitro and in vivo assays of monoubiquitination of receptor tyrosine kinases.
Growth factor receptors, such as the epidermal growth factor receptor (EGFR), stimulate a variety of signal transduction pathways upon binding a ligand molecule at the cell surface. Desensitization of signaling initiates when active receptors are recruited to clathrin-coated regions of the plasma membrane and subsequently sorted to intracellular degradation in lysosomes. Sorting for lysosomal degradation entails receptor conjugation with ubiquitin molecules, which are recognized by the endocytic machinery. Unlike degradation in the 26S proteasome, which requires a chain of four or more units of ubiquitin (polyubiquitination), covalent addition of a monomeric ubiquitin (monoubiquitination) appears sufficient for receptor sorting to lysosomal degradation. In this chapter we describe two methods that contrast polyubiquitination with monoubiquitination of EGFR. Because monoubiquitination enables evasion from proteasomal degradation, the methods we describe may be useful for the analysis of other monoubiquitination events.
Katz
Menachem
M
Department of Biological Regulation, The Weizmann Institute of Science, Rehovot, Israel.
Mosesson
Yaron
Y
Yarden
Yosef
Y
D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*
D025801:Ubiquitin; D066246:ErbB Receptors; D020794:Receptor Protein-Tyrosine Kinases