Threshold-controlled ubiquitination of the EGFR directs receptor fate

Abstract

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.

PHD3 regulates EGFR internalization and signalling in tumours

“…To obtain additional mechanistic insight into the function of PHD3 in EGFR internalization and to confirm that PHD3 acts as a scaffolding adaptor that regulates the early steps of EGFR endocytosis, we next analysed the role of PHD3 in the recruitment of known EGFR interactors that play a central part in EGFR internalization and signalling. Binding of the E3 ligase Cbl, in complex with Grb2, to phosphorylated EGFR has been shown to act as a switch that tightly regulates the ubiquitination of the receptor and can trigger its internalization 29 . Therefore, we first investigated whether the recruitment of Cbl and Grb2, as well as the ubiquitination levels of EGFR, were altered following PHD3 loss.…”

Analytical reduction of combinatorial complexity arising from multiple protein modification sites

“…To accomplish this, we created a new site that takes into account the simultaneous phosphorylation states of Y1045 and Y1068 while still using our model reduction assumptions (see the electronic supplementary material). Using this extended model, we performed simulations to explore whether these assumptions could reproduce the cooperative behaviour in cCbl binding to EGFR observed [33]. Surprisingly, we found that there was no predicted difference in recruitment of Grb2 or cCbl as a function of EGF dose (figure 3c), despite the higher affinity of the Grb2-cCbl complex and the reliance on two sites to be simultaneously phosphorylated.…”

“…Although there are certainly some potential deviations from the assumptions in a strict sense, in many respects, we conclude that they may reasonably apply to the EGFR system. One significant deviation is the cooperative recruitment of the E3 ubiquitin ligase c-Cbl to the EGFR, depending on the presence of Grb2 [33]. The theory as developed above does not allow for such a possibility, but we derive a special case here that allows us to introduce this feature without considering the full array combinatorial complexity.…”

“…A recent study has shown that cCbl is cooperatively recruited to EGFR as EGF dose is increased, and that this cooperativity is dependent on Grb2 and the presence of the recruitment sites for both Grb2 (Y1068) and cCbl (Y1045) [33]. It was suggested that this effect may be due to cooperativity on the level of the phosphorylation sites themselves, implying that a Grb2-cCbl complex may bind cooperativity to EGFR, because Y1045 and Y1068 are more likely to be phosphorylated together.…”

The regulation and mechanisms of EGFR-mediated apoptosis in MDA-MB-468 cells.

“…concentrations, where as with higher concentrations of ligand, (10-100 ng/mL) the receptor undergoes Clathrin-independent endocytosis via caveolae [25,26].…”