How the cell converts graded signals into threshold-activated responses is a question of great biological relevance. Here, we uncover a nonlinear modality of epidermal growth factor receptor (EGFR)-activated signal transduction, by demonstrating that the ubiquitination of the EGFR at the PM is threshold controlled. The ubiquitination threshold is mechanistically determined by the cooperative recruitment of the E3 ligase Cbl, in complex with Grb2, to the EGFR. This, in turn, is dependent on the simultaneous presence of two phosphotyrosines, pY1045 and either one of pY1068 or pY1086, on the same EGFR moiety. The dose–response curve of EGFR ubiquitination correlate precisely with the non-clathrin endocytosis (NCE) mode of EGFR internalization. Finally, EGFR-NCE mechanistically depends on EGFR ubiquitination, as the two events can be simultaneously re-engineered on a phosphorylation/ubiquitination-incompetent EGFR backbone. Since NCE controls the degradation of the EGFR, our findings have implications for how the cell responds to increasing levels of EGFR signalling, by varying the balance of receptor signalling and degradation/attenuation.

Following activation by its cognate ligand(s), the epidermal growth factor receptor (EGFR) is rapidly routed to the lysosome for degradation in a ubiquitination-dependent fashion. This pathway represents the major mechanism of long-term attenuation of EGFR signaling, and its deregulation is a significant feature in different types of cancers. Here we demonstrate, through a systematic RNAi-based approach, that several deubiquitinating (DUB) enzymes extend or decrease EGFR half-life upon EGF stimulation. We focus on USP9X, whose depletion severely affects EGFR turnover, interfering with its internalization and trafficking. We identify the endocytic protein Eps15 as one of the critical substrates of USP9X, and we map the Eps15 ubiquitination sites. We found that Eps15 monoubiquitination occurs already at minimal dose of EGF stimulation and is essential for EGFR internalization. Overall, our findings identify USP9X as a novel regulator of EGFR endocytosis and suggest a model whereby cycles of ubiquitination and deubiquitination events on endocytic accessory proteins may regulate the internalization and trafficking of the EGFR toward the lysosomes.

Summary Adaptor protein 2 (AP2) is a major constituent of clathrin-coated pits (CCPs). Whether it is essential for all forms of clathrin-mediated endocytosis (CME) in mammalian cells is an open issue. Here, we demonstrate, by live TIRF microscopy, the existence of a subclass of relatively short-lived CCPs lacking AP2 under physiological, unperturbed conditions. This subclass is retained in AP2-knockout cells and is able to support the internalization of epidermal growth factor receptor (EGFR) but not of transferrin receptor (TfR). The AP2-independent internalization mechanism relies on the endocytic adaptors eps15, eps15L1, and epsin1. The absence of AP2 impairs the recycling of the EGFR to the cell surface, thereby augmenting its degradation. Accordingly, under conditions of AP2 ablation, we detected dampening of EGFR-dependent AKT signaling and cell migration, arguing that distinct classes of CCPs could provide specialized functions in regulating EGFR recycling and signaling.

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