Gap junctions consist of arrays of intercellular channels that enable adjacent cells to communicate both electrically and metabolically. Gap junctions have a wide diversity of physiological functions, playing critical roles in both excitable and non-excitable tissues. Gap junction channels are formed by integral membrane proteins called connexins. Inherited or acquired alterations in connexins are associated with numerous diseases, including heart failure, neuropathologies, deafness, skin disorders, cataracts and cancer. Gap junctions are highly dynamic structures and by modulating the turnover rate of connexins, cells can rapidly alter the number of gap junction channels at the plasma membrane in response to extracellular or intracellular cues. Increasing evidence suggests that ubiquitination has important roles in the regulation of endoplasmic reticulum-associated degradation of connexins as well as in the modulation of gap junction endocytosis and post-endocytic sorting of connexins to lysosomes. In recent years, researchers have also started to provide insights into the physiological roles of connexin ubiquitination in specific tissue types. This review provides an overview of the advances made in understanding the roles of connexin ubiquitination in the regulation of gap junction intercellular communication and discusses the emerging physiological and pathophysiological implications of these processes.

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.

In order to achieve accurate chromosome segregation, eukaryotic cells undergo a dramatic change in morphology to obtain a spherical shape during mitosis. Interphase cells communicate directly with each other by exchanging ions and small molecules via gap junctions, which have important roles in controlling cell growth and differentiation. As cells round up during mitosis, the gap junctional communication between mitotic cells and adjacent interphase cells ceases. Whether mitotic cells use alternative mechanisms for mediating direct cell-cell communication during rounding is currently unknown. Here, we have studied the mechanisms involved in the remodeling of gap junctions during mitosis. We further demonstrate that mitotic cells are able to form actin-based plasma membrane bridges with adjacent cells during rounding. These structures, termed “mitotic nanotubes,” were found to be involved in mediating the transport of cytoplasm, including Rab11-positive vesicles, between mitotic cells and adjacent cells. Moreover, a subpool of the gap-junction channel protein connexin43 localized in these intercellular bridges during mitosis. Collectively, the data provide new insights into the mechanisms involved in the remodeling of gap junctions during mitosis and identify actin-based plasma membrane bridges as a novel means of communication between mitotic cells and adjacent cells during rounding.

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clear. Here, we have analysed the effect of FAT1 on SWH pathway in glioma. Material and methods Glioma cell lines (U87MG, U373, A172, GOS3 and SW1088) were transfected with FAT1 specific siRNA/control siRNA and analysed the expression of SWH pathway molecules by qPCR/Western blot. Protein-protein interactions were analysed by Co-immunoprecipitation (Co-IP) after overexpression of YAP1 (wild-type and mutated) and TEAD1 with and without FAT1 knockdown. Sub-cellular localization of proteins was analysed by Confocal microscopy. Results and discussions The mRNA expression of FAT1 and SWH pathway molecules (MST1, LATS1, LATS2, YAP1 and TEAD1) was highest in U87MG cells followed by A172, U373MG and GOS3. After FAT1 knockdown, the mRNA expression of MST1 and BIRC2 were significantly decreased with no change in the levels of LATS1, LATS2, YAP1, TEAD1 and BIRC5. At protein level, increased YAP1 and phospho-YAP1 was observed after FAT1 knockdown with increased total as well as phospho-YAP1 in the cytoplasmic extract as compared to the nuclear extract. There was significant reduction in the interaction between YAP1 and TEAD1 in siFAT1 treated cells as compared to siControl treated cells. Conclusion Knockdown of FAT1 (i) increases the YAP1 protein level, could be by increasing the protein stability as no change was observed at the mRNA level (ii) it relieves the inhibitory effect on YAP1 phosphorylation, thereby increasing the phospho-YAP1 level (iii) it affects the sub-cellular localization of YAP1 by retaining YAP1 in the cytosol and thereby (iv) decrease in the YAP1:TEAD1 interaction with decreased expression of their target gene Birc2.This finding of the effect of FAT1 on YAP1 in GBM is novel with features pointing towards the oncogenic role of FAT1 by regulating YAP1 sub-cellular localization and co-transcriptional activity independent of SWH pathway.

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Introduction: NEDD4 (neural precursor cell-expressed developmentally down-regulated 4), a member of the HECT (homologous to E6AP C-terminus) family of E3 ubiquitin ligases, has been shown to be an important regulator of multiple proteins involved in cancer development, including the tumor suppressor PTEN and the proto-oncogene MDM2. Furthermore, NEDD4 has been shown to be overexpressed and act as an oncogene in multiple cancer types. We have previously shown that NEDD4 is overexpressed in colorectal cancer (CRC) and that it can promote growth of colon cancer cells independently of PTEN and PI3K/AKT signaling. In the present study, we investigated the role of NEDD4 in regulating the PTEN/PI3K pathway and the MDM2/p53 axis in CRC. Materials and Methods: The CRISPR/Cas9 system was applied to generate a Caco2 NEDD4 knockout cell line, and NEDD4 knockdown was performed in LS174T and SW480 cells. Gene expression profiles of 412 primary CRCs, 51 normal mucosa samples, 38 CRC cell lines, as well as the Caco2 parental and NEDD4 knockout cell lines, were generated using exon-resolution Affymetrix Human Exon Arrays or Human Transcriptome Arrays. The NEDD4 expression level was correlated with the mutation status of KRAS, PTEN, TP53, PIK3CA, BRAF and NRAS. Western blotting was used to detect and quantify NEDD4, PTEN and MDM2 protein levels. Results and Discussions: Gene expression analysis of the patient material confirmed our previous study that NEDD4 is significantly upregulated in CRC as compared to normal colonic mucosa. There was no correlation between NEDD4 expression and mutations in KRAS, BRAF, NRAS, PTEN, PIK3CA and TP53. CRISPR/Cas9-mediated NEDD4 knockout in Caco2 cells resulted in a reduction (P <0.01) in both the PTEN and MDM2 protein levels as compared to control cells. Gene set analysis showed that PI3K/AKT/MTOR signaling was upregulated in the NEDD4 knockout cells as compared to control cells. siRNA-mediated depletion of NEDD4 in LS174T cells was associated with reduced levels of MDM2, but did not affect the PTEN protein level. In SW480 cells, depletion of NEDD4 affected neither the MDM2 nor the PTEN protein level. By analyzing the expression of NEDD4, PTEN and MDM2 in 38 CRC cell lines by Western blotting, a positive correlation (P<0.01) was observed between NEDD4 and PTEN protein, while there was no significant correlation between NEDD4 and MDM2. Conclusion: The data show that NEDD4 is significantly upregulated in CRC, and that NEDD4 expression correlates with PTEN expression. The data further suggest that NEDD4 has the ability to regulate the PTEN and MDM2 protein levels in colon cancer cells in a cell line-specific manner. Citation Format: Lars M. Knudsen, Anita Sveen, Christer A. Andreassen, Christian H. Bergsland, Ina A. Eilertsen, Nikoline L. Rasmussen, Max Z. Totland, Peter W. Eide, Jarle Bruun, Ragnhild A. Lothe, Edward Leithe. Role of the E3 ubiquitin ligase NEDD4 in the regulation of PTEN and MDM2 in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1433.