Regulation of EGFR Endocytosis by CBL During Mitosis

Abstract

The overactivation of epidermal growth factor (EGF) receptor (EGFR) is implicated in various cancers. Endocytosis plays an important role in EGFR-mediated cell signaling. We previously found that EGFR endocytosis during mitosis is mediated differently from interphase. While the regulation of EGFR endocytosis in interphase is well understood, little is known regarding the regulation of EGFR endocytosis during mitosis. Here, we found that contrary to interphase cells, mitotic EGFR endocytosis is more reliant on the activation of the E3 ligase CBL. By transfecting HeLa, MCF-7, and 293T cells with CBL siRNA or dominant-negative 70z-CBL, we found that at high EGF doses, CBL is required for EGFR endocytosis in mitotic cells, but not in interphase cells. In addition, the endocytosis of mutant EGFR Y1045F-YFP (mutation at the direct CBL binding site) is strongly delayed. The endocytosis of truncated EGFR Δ1044-YFP that does not bind to CBL is completely inhibited in mitosis. Moreover, EGF induces stronger ubiquitination of mitotic EGFR than interphase EGFR, and mitotic EGFR is trafficked to lysosomes for degradation. Furthermore, we showed that, different from interphase, low doses of EGF still stimulate EGFR endocytosis by non-clathrin mediated endocytosis (NCE) in mitosis. Contrary to interphase, CBL and the CBL-binding regions of EGFR are required for mitotic EGFR endocytosis at low doses. This is due to the mitotic ubiquitination of the EGFR even at low EGF doses. We conclude that mitotic EGFR endocytosis exclusively proceeds through CBL-mediated NCE.

Clathrin Heavy Chain Knockdown Impacts CXCR4 Signaling and Post-translational Modification

“…Redundancy of endocytic pathways is not a new idea and work in this area has been pioneered with EGFR (Sigismund et al, 2005). Interestingly, we found that while clathrin knockdown increased CXCR4 PTM, this was not the case for EGFR as the antibody used to measure total EGFR has been previously shown to not be sensitive to EGFR PTMs (Wee and Wang, 2018) and previous research suggests that EGFR PTMs are unaffected by clathrin inhibition (Garay et al, 2015). Additionally, while EGFR is preferentially internalized by CME at increasing high ligand levels, at times it is also internalized by clathrin-independent mechanisms (Sigismund et al, 2005).…”

“…Cycloheximide chase experiments in RPE cells overexpressing c-terminal myc tagged CXCR4 further confirmed that clathrin knockdown did not lead to a change in CXCR4 degradation kinetics (Supplementary Figures 3C,D) and additionally total CXCR4 labeling using the myc antibody confirmed that CXCR4 total protein levels were unchanged in the overexpression CXCR4 model as well (Supplementary Figure 3E). To test whether this phenomenon was specific to CXCR4 or more broadly applicable to other receptors, we measured EGFR protein levels using an antibody (A-10 clone) not expected to be sensitive to receptor PTM (Wee and Wang, 2018). Interestingly, clathrin knockdown significantly reduced EGFR detection in both HeLa and RPE cells as well.…”

The crosstalk between microtubules, actin and membranes shapes cell division

“…Finally, other NCE pathways remain active during mitosis, such as the one responsible for the uptake of the EGFR [130,131], as well as some macropinocytic events [132].…”

G protein pathway suppressor 2 suppresses gastric cancer by destabilizing epidermal growth factor receptor

“…Non-clathrin-mediated endocytosis plays a major role in the regulation of EGFR fate by targeting it to lysosomes for degradation. 9 Wang et al also reported that EGFR is degraded through the proteasome pathway. 40 We used As mentioned above, the ubiquitination regulation of EGFR by Cbl is either directly, through binding to its pY1045 site, or indirectly, through Grb2 binding to the pY1068 or pY1086 site.…”

“…EGFR‐mediated cell signaling and its endocytosis are strictly regulated. 9 The endocytosis of EGFR can lead to two different fates of this receptor: circulation back to the plasma membrane or degradation in lysosomes. 10 Promoting EGFR degradation could be a potential strategy for tumor treatment.…”