Regulation of ubiquitination and degradation of the gap junction channel protein Connexin43 involves the concerted action of multiple members of the NEDD4 family of E3 ubiquitin ligases

Intercellular communication via gap junctions is often lost during cancer development, which may contribute to increased tumor growth and affect how cancer cells respond to radio- and chemotherapy. Gap junction channels comprise transmembrane proteins belonging to the connexin family, of which connexin43 (Cx43) is the most ubiquitously expressed in humans. SMAD ubiquitination regulatory factor 2 (SMURF2), a member of the neural precursor cell expressed developmentally down-regulated protein 4 (NEDD4) family of E3 ubiquitin ligases, often accumulates in the cytoplasm in cancer cells, where it may display oncogenic properties. Here, we demonstrate that SMURF2 interacts with Cx43 and promotes its ubiquitination in HeLa cells, which is associated with loss of Cx43-based gap junctions and reduced levels of Cx43. Moreover, SMURF2 was found to cooperate with two other NEDD4 family members, NEDD4 and ITCH, to regulate Cx43 ubiquitination and degradation. Simultaneous depletion of these three E3 ubiquitin ligases significantly reduced the Cx43 ubiquitination and degradation rate compared with their single depletion. Their combined knockdown was also found to reduce the ubiquitination and degradation of Cx43 following exposure to the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA). Collectively, these data identify SMURF2 as a negative regulator of gap junctional intercellular communication in cancer cells by inducing the loss of Cx43-based gap junctions. The study also establishes that Cx43 ubiquitination and degradation are controlled by the concurrent participation of multiple E3 ubiquitin ligases, both under basal conditions and in response to TPA exposure.