The E3 ubiquitin ligase NEDD4 induces endocytosis and lysosomal sorting of connexin 43 to promote loss of gap junctions

Abstract

Intercellular communication via gap junctions has an important role in controlling cell growth and in maintaining tissue homeostasis. Connexin 43 (Cx43; also known as GJA1) is the most abundantly expressed gap junction channel protein in humans and acts as a tumor suppressor in multiple tissue types. Cx43 is often dysregulated at the post-translational level during cancer development, resulting in loss of gap junctions. However, the molecular basis underlying the aberrant regulation of Cx43 in cancer cells has remained elusive. Here, we demonstrate that the oncogenic E3 ubiquitin ligase NEDD4 regulates the Cx43 protein level in HeLa cells, both under basal conditions and in response to protein kinase C activation. Furthermore, overexpression of NEDD4, but not a catalytically inactive form of NEDD4, was found to result in nearly complete loss of gap junctions and increased lysosomal degradation of Cx43 in both HeLa and C33A cervical carcinoma cells. Collectively, the data provide new insights into the molecular basis underlying the regulation of gap junction size and represent the first evidence that an oncogenic E3 ubiquitin ligase promotes loss of gap junctions and Cx43 degradation in human carcinoma cells.

Role of connexin 43 in different forms of intercellular communication – gap junctions, extracellular vesicles and tunnelling nanotubes

“…1B). In accordance, impairing Nedd4-mediated ubiquitylation of Cx43 results in the accumulation of gap junctions at the plasma membrane with a concomitant increase of intercellular communication (Leykauf et al, 2006;Girão et al, 2009, Totland et al, 2017. Ubiquitylation of Cx43 has also been linked to proteasome-dependent quality control mechanisms in the context of endoplasmic reticulum (ER)-associated protein degradation (ERAD) (Kopanic et al, 2015;VanSlyke and Musil, 2002).…”

The ubiquitin-specific protease USP8 deubiquitinates and stabilizes Cx43

“…Moreover, both Lys 63 -and Lys 48 -polyubiquitin linkages can be recognized by the autophagy adaptor p62/ SQSTM1, via its UBA-domain, to trigger autophagy-mediated degradation. It is well-established that endocytosed Cx43-containing GJs are degraded through the endolysosomal and autophagosomal pathways (19,20). Therefore, it is expected that USP8 can prevent endocytosed Cx43-containing GJs from entering these two degradation pathways via removing both Lys 63 -and Lys 48 -polyubiquitin chains.…”

“…The endocytosed GJs form a double-membrane vacuole called annular gap junction (AGJ) or connexosome (17,18). The AGJ may be recycled to the cell surface, or destined for degradation through the endolysosomal and/or autophagosomal pathways (19,20). Ubiquitination of Cx43 may occur at several subcellular locations and is involved in multiple stages of degradation.…”

“…The latter is eventually fused with lysosomes where the contents are degraded. Cx43 ubiquitination was reported to be regulated by several E3 ubiquitin ligases including NEDD4 (20), WWP1 (23), and TRIM21 (24).…”

“…They may act at different stages/levels in the regulation of the intracellular trafficking and degradation of Cx43-containing GJs. For example, NEDD4 promotes endocytosis of Cx43, and controls its degradation via either the endolysosomal or autophagosomal pathway (20,21).…”