EGF receptor ubiquitination is not necessary for its internalization

Abstract

Ubiquitination of the EGF receptor (EGFR) has been implicated in EGF-induced receptor internalization, lysosomal degradation, and down-regulation. Mutation of EGFR ubiquitination sites identified by mass spectrometry yielded receptor mutants that are weakly ubiquitinated and not down-regulated by EGF. However, these EGFR mutants were normally internalized. To examine whether this internalization was mediated by the residual ubiquitination, systematic mutagenesis of lysine residues in the kinase domain of the EGFR was performed to generate a receptor mutant that is not ubiquitinated. Mutations of a number of lysines inhibited kinase activity of the EGFR, thus leading to the inhibition of receptor internalization. However, a mutant lacking 15 lysine residues (15KR), which was negligibly ubiquitinated and normally phosphorylated, was internalized at a rate similar to that of the wild-type EGFR. As in the case of the wild-type EGFR, internalization of the 15KR mutant depended on the presence of clathrin, Grb2 adaptor, and Cbl ubiquitin ligase. These data imply that EGFR ubiquitination is not necessary for its internalization by clathrincoated pits. Interestingly, the reconstitution of two major ubiquitination sites in the 16KR receptor mutant, which had impaired kinase activity and slow internalization kinetics, resulted in a partial rescue of ubiquitination and a complete rescue of receptor internalization. This result suggests that ubiquitination of the kinase-impaired receptor can mediate its internalization by the clathrin pathway. Altogether these data emphasize the robustness of the EGFR internalization process, which can be controlled by multiple kinase-and ubiquitination-dependent and -independent mechanisms. endocytosis ͉ clathrin ͉ ubiquitin A ctivation of the EGF receptor (EGFR) by EGF or other ligands at the cell surface triggers several signaling cascades leading to a cell-specific biological response (1). Activated EGFR also is rapidly endocytosed by clathrin-coated pits (2). After internalization, ligand-receptor complexes traffic through a series of endosomal compartments and are either returned back to the plasma membrane or degraded in lysosomes. The accelerated internalization and efficient sorting of activated EGFR to lysosomes result in the dramatic down-regulation of the receptor and serve as a negative-feedback regulation of receptor signaling (3). At the same time, internalized EGFR continues to signal from endosomes, and this endosomal signaling is thought to play an important role in determining the duration, intensity, and specificity of signaling processes (2).Endocytosis and intracellular sorting of EGFR have been the most popular experimental models to study pathways and mechanisms of endocytic trafficking that are specific to signaling receptors. However, despite extensive research for more than two decades, the mechanisms of internalization and degradation of EGFR, as well as other receptor tyrosine kinases, are not well understood. Our understanding of the molecular interactions lead...

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“…Thus, several mechanisms that impair Cbl targeting to the EGFR and inhibit its downregulation could promote transformation by generating an increase in total levels and surface levels of the EGFR. New data by Sorkin and coworkers show that EGFR ubiquitination is not required for internalization but plays a role in the targeting of the EGFR to late endosomes and multivesicular bodies for degradation, providing additional mechanistic detail on the role of Cbl in EGFR downregulation (Huang et al, 2007). Interestingly, activated Cdc42 sequesters Cbl away from the EGFR and inhibits its downregulation (Wu et al, 2003).…”

Tyrosine 1045 Codon Mutations in Exon 27 of EGFR are Infrequent in Oral Squamous Cell Carcinomas

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“…Many yeast surface proteins have been demonstrated to require ubiquitination for their internalization (15), but the situation is less clear in mammals. In the case of the EGF receptor, mutation of multiple intracellular lysines within the kinase domain of the protein leads to an almost complete loss of EGF receptor ubiquitination but did not result in the decrease of its internalization rate (16). Regarding Notch ligands, it has been shown that the E3 ligase Mind Bomb 1 promotes ubiquitination and internalization of mammalian Dll1 (17).…”