Smad ubiquitination regulatory factor-2 controls gap junction intercellular communication by modulating endocytosis and degradation of connexin43

Abstract

SummaryGap junctions consist of arrays of intercellular channels that enable adjacent cells to communicate both electrically and metabolically. Gap junction channels are made of a family of integral membrane proteins called connexins, of which the best-studied member is connexin43. Gap junctions are dynamic plasma membrane domains, and connexin43 has a high turnover rate in most tissue types. However, the mechanisms involved in the regulation of connexin43 endocytosis and transport to lysosomes are still poorly understood. Here, we demonstrate by live-cell imaging analysis that treatment of cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) induces endocytosis of subdomains of connexin43 gap junctions. The internalized, connexin43-enriched vesicles were found to fuse with early endosomes, which was followed by transport of connexin43 to the lumen of early endosomes. The HECT E3 ubiquitin ligase smad ubiquitination regulatory factor-2 (Smurf2) was found to be recruited to connexin43 gap junctions in response to TPA treatment. Depletion of Smurf2 by small interfering RNA resulted in enhanced levels of connexin43 gap junctions between adjacent cells and increased gap junction intercellular communication. Smurf2 depletion also counteracted the TPA-induced endocytosis and degradation of connexin43. Collectively, these data identify Smurf2 as a novel regulator of connexin43 gap junctions.

Mitotic cells form actin-based bridges with adjacent cells to provide intercellular communication during rounding

“…Depletion of SMURF2 resulted in an overall increase in Cx43 protein levels, in accordance with our previous study (Fig. 2B and Fykerud et al 35 …”

“…We have previously demonstrated that the E3 ubiquitin ligase SMURF2 regulates the endocytosis of Cx43 gap junctions in IAR20 cells under basal conditions and in response to PKC activation 35 . In the present study, we found that SMURF2 has a major role in regulating the endocytosis of Cx43 gap junctions during mitosis in IAR20 cells.…”

“…In these experiments, the SMURF2 protein level was reduced by approximately 70% compared with cells transfected with a control siRNA sequence (Fig. 2B and Fykerud et al 35 …”

“…Moreover, whether the increased endocytosis of Cx43 during mitosis is a prerequisite for the remodeling of gap junctions during mitosis is currently unknown. We have previously demonstrated that the E3 ubiquitin ligase SMAD ubiquitination regulatory factor-2 (SMURF2) controls the endocytosis of Cx43 gap junctions under basal conditions and in response to activation of protein kinase C (PKC) 35 . In mitotic IAR20 cells, SMURF2 was found to partly colocalize with Cx43 gap junctions at the plasma membrane and in intracellular vesicles (Fig.…”

Connexin 43 and CaV1.2 Ion Channel Trafficking in Healthy and Diseased Myocardium

“…There is increasing evidence that Cx43 ubiquitination plays an important role in regulating gap junction internalization and degradation and several E3 ubiquitin ligases have been shown to regulate Cx43 internalization from the plasma membrane including TRIM21 69 , Smurf2 70 , and Nedd4 66, 68 . Since these studies were done in cell culture systems, it still remains largely unclear how Cx43 ubiquitination is regulated in the cardiomyocytes.…”