Epidermal growth factor regulates ubiquitination, internalization and proteasome-dependent degradation of connexin43

Connexins are membrane-spanning proteins that form gap junction channels between adjacent cells. Connexin43 (Cx43), the most widely expressed member of the connexin family in tissues and cell lines, has a rapid turnover rate and its degradation involves both the lysosomal and ubiquitin-proteasome pathway. It was previously shown that the proteasome is involved in regulating the number of functional gap junctions at the plasma membrane. However, little is known about how proteasome-dependent turnover of Cx43 is controlled. Epidermal growth factor (EGF) induces hyperphosphorylation of CA3 and a rapid, transient decrease in gap junctional intercellular communication. In this study, we show that, along with inhibition of gap junctional intercellular communication, EGF induces disorganization, internalization and degradation of CA3 gap junction plaques in IAR20 rat liver epithelial cells. These EGF-induced modifications of Cx43 were counteracted by the MEK1 inhibitor PD98059, indicating that the effects were mediated by the mitogen-activated protein kinase pathway. The EGF-induced destruction of CA3 was proteasome-dependent, because the loss of CA3 protein was counteracted by the proteasome inhibitor MG132 but not the lysosomal inhibitor leupeptin. Furthermore, EGF induced ubiquitination of CA3, which was associated with the CA3 hyperphosphorylation. The EGF-induced Cx43 ubiquitination was counteracted by PD98059. The EGF-induced internalization of CA3 was blocked by hypertonic sucrose treatment, indicating that EGF mediates internalization of CA3 via a clathrin-dependent mechanism. Our results indicate that ubiquitination of CA3 occurs at the plasma membrane before Cx43 internalization. Taken together, these data provide the first evidence that EGF-induced phosphorylation of CA3 induces binding of ubiquitin and targets Cx43 for internalization and degradation in a proteasome-dependent manner.