Knockout of c‐Cbl slows EGFR endocytic trafficking and enhances EGFR signaling despite incompletely blocking receptor ubiquitylation

Abstract

Epidermal growth factor receptor (EGFR) activity is necessary and sufficient for corneal epithelial homeostasis. However, the addition of exogenous Epidermal Growth Factor (EGF) does not reliably restore the corneal epithelium when wounded. This is likely due to high levels of endogenous EGF in tear fluid as well as desensitization of the EGFR following ligand stimulation. We hypothesize that preventing receptor downregulation is an alternative mechanism to enhance EGFR signaling and promote the restoration of compromised corneas. Ligand‐dependent EGFR ubiquitylation is associated with the targeted degradation of the receptor. In this manuscript, we determine whether knockout of c‐Cbl, an E3 ubiquitin ligase that ubiquitylates the EGFR, is sufficient to prolong EGFR phosphorylation and sustain signaling. Using CRISPR/Cas9 gene editing, we generated immortalized human corneal epithelial (hTCEpi) cells lacking c‐Cbl. Knockout (KO) cells expressed the other E3 ligases at the same levels as the control cells, indicating other E3 ligases were not up‐regulated. As compared to the control cells, EGF‐stimulated EGFR ubiquitylation was reduced in KO cells, but not completely abolished. Similarly, EGF:EGFR trafficking was slowed, with a 35% decrease in the rate of endocytosis and a twofold increase in the receptor half‐life. This resulted in a twofold increase in the magnitude of EGFR phosphorylation, with no change in duration. Conversely, Mitogen Activating Protein Kinase (MAPK) phosphorylation did not increase in magnitude but was sustained for 2–3 h as compared to control cells. We propose antagonizing c‐Cbl will partially alter receptor ubiquitylation and endocytic trafficking but this is sufficient to enhance downstream signaling.

Epidermal Growth Factor Receptor Expression in the Corneal Epithelium

“…The E3 ubiquitin ligase, c-Cbl, has emerged as a viable target for prolonging EGFR signaling. c-Cbl ubiquitylates the EGFR in response to ligand treatment and regulates the rate of EGF:EGFR complex internalization and degradation [120]. Further, the loss of c-Cbl enhances EGFR-dependent cell migration and in vivo wound healing [69].…”

c-Cbl Regulates Murine Subventricular Zone-Derived Neural Progenitor Cells in Dependence of the Epidermal Growth Factor Receptor

“…However, c-Cbl's role in regulating endogenous EGFRs that contribute to tissue homeostasis is less well studied. All members of the Cbl family (c-Cbl, Cbl-b, and Cbl-3) have been reported to be E3 ligases for the EGFR [44].…”

Modulating Growth Factor Receptor Signaling to Promote Corneal Epithelial Homeostasis

“…Evidence to support ubiquitylation as a target comes from studies with the EGFR. The knockdown and/or knockout of c-Cbl decreased EGFR ubiquitylation and increased the rate of corneal epithelial in vitro wound healing [58,65]. Indirect pharmacologic inhibition of ubiquitylation via PP1 (Src inhibitor) resulted in faster in vitro and in vivo corneal reepithelialization [58].…”