Eps15 Is Recruited to the Plasma Membrane upon Epidermal Growth Factor Receptor Activation and Localizes to Components of the Endocytic Pathway during Receptor Internalization

Abstract

Eps15 is a substrate for the tyrosine kinase of the epidermal growth factor receptor (EGFR) and is characterized by the presence of a novel protein:protein interaction domain, the EH domain. Eps15 also stably binds the clathrin adaptor protein complex AP-2. Previous work demonstrated an essential role for eps15 in receptor-mediated endocytosis. In this study we show that, upon activation of the EGFR kinase, eps15 undergoes dramatic relocalization consisting of 1) initial relocalization to the plasma membrane and 2) subsequent colocalization with the EGFR in various intracellular compartments of the endocytic pathway, with the notable exclusion of coated vesicles. Relocalization of eps15 is independent of its binding to the EGFR or of binding of the receptor to AP-2. Furthermore, eps15 appears to undergo tyrosine phosphorylation both at the plasma membrane and in a nocodazole-sensitive compartment, suggesting sustained phosphorylation in endocytic compartments. Our results are consistent with a model in which eps15 undergoes cycles of association:dissociation with membranes and suggest multiple roles for this protein in the endocytic pathway.

Cbl-dependent Ubiquitination Is Required for Progression of EGF Receptors into Clathrin-coated Pits

“…However, our current data do not establish that cargo itself needs to be ubiquitinated. Grb2 and Cbl were interestingly found to specifically localize to the rim of EGFR-positive clathrin coats in the same manner as previously reported for Eps15 (Torrisi et al, 1999;Stang et al, 2000). We have further demonstrated that also hSpry2 localizes to the rim of coated pits in transfected cells.…”

“…It was reported that Grb2 colocalized with the ␤2 subunit of AP-2 and EGF upon incubation of PAE cells with EGF for 1 h at 4°C . We have previously demonstrated that the EGFR colocalizes with Cbl at the plasma membrane under similar conditions (Longva et al, 2002), and it has also been shown that Eps15 is recruited to the plasma membrane when cells are incubated with EGF on ice (Torrisi et al, 1999). As expected, we found by immunofluorescence and confocal microscopy that incubation of Hep2 cells with EGF on ice for 60 min recruited Grb2, Cbl, and Eps15 to the plasma membrane ( Figure 4A).…”

Large‐scale proteomic analysis of tyrosine‐phosphorylation induced by T‐cell receptor or B‐cell receptor activation reveals new signaling pathways

“…The clathrin-dependent pathway has been shown to be the primary pathway for ligand-induced BCR endocytosis, and raft structures appear to be required for efficient phosphorylation of clathrin heavy chain [21,33]. Tyrosine-phosphorylation of clathrin heavy chain is induced by an SRC-type protein kinase in response to stimulation of various receptor types, and the level of phosphorylation correlates with the rate of receptor internalization [21,[34][35][36][37]. Our data support a requirement for phosphorylation of clathrin heavy chain as well as for that of other endocytosis-related factors in BCR internalization.…”

PHD3 regulates EGFR internalization and signalling in tumours

“…EGFR pathway substrate (Eps) 15 and epsin1 are members of a family of ubiquitin-interacting motif (UIM) containing proteins that govern the early steps of EGFR endocytosis [30][31][32] . Therefore, we investigated whether the loss of PHD3 affects the recruitment of Eps15 and epsin1 to EGFR.…”