Gap junctions are specialized plasma membrane domains enriched in connexin proteins that form channels between adjacent cells. Gap junctions are highly dynamic, and modulation of the connexin turnover rate is considered to play an important role in the regulation of gap junctional intercellular communication. In the present study, we show that the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) induces ubiquitination of connexin-43 (Cx43) in IAR20 rat liver epithelial cells. The accelerated ubiquitination of Cx43 in response to TPA occurred concomitantly with Cx43 hyperphosphorylation and inhibition of cell-cell communication via gap junctions. The TPA-induced ubiquitination of Cx43 was mediated via protein kinase C and partly involved the mitogen-activated protein kinase pathway. Following ubiquitination, Cx43 was internalized and degraded. The loss of Cx43 protein was counteracted by ammonium chloride, indicating that acidification of internalized Cx43 gap junctions is a prerequisite for its degradation. Furthermore, the Cx43 degradation was partly counteracted by leupeptin, an inhibitor of cathepsin B, H, and L. Cx43 internalization and subsequent degradation were blocked by inhibitors of the proteasome. Evidence is provided that Cx43 is modified by multiple monoubiquitins rather than a polyubiquitin chain in response to TPA. Moreover, the TPA-induced ubiquitination of Cx43 was blocked by proteasomal inhibitors. Taken together, the data indicate that Cx43 ubiquitination is a highly regulated process. Moreover, the results suggest that the proteasome might play an indirect role in Cx43 degradation by affecting the level of monoubiquitin conjugation and trafficking of Cx43 to endosomal compartments.

factor regulates ubiquitination, internalization and proteasome-dependent degradation of connexin43. J. Cell Sci. 117, 1211-1220.In the online version of this article the bottom line of Fig. 8B was missing. The correct figure is shown below. We apologise for any inconvenience caused. Erratum IntroductionGap junctions are specialized domains in the plasma membrane containing intercellular channels between neighbouring cells. Gap junction channels are formed by the docking of two hemichannels of connexin proteins contributed by each adjacent cell. These channels are found in most animal tissues and enable cells to exchange cytoplasmic components (<1 kDa) directly, including second messengers, nucleotides and ions (Goodenough et al., 1996). Gap junctions are thought to be important in embryonic development, cellular growth control and differentiation (Guthrie and Gilula, 1989;Loewenstein, 1979;Mehta et al., 1986). The most widely expressed member of the connexin family in tissues and cell lines, connexin43 (Cx43), has been reported to behave as a classical tumour suppressor gene both in cell culture and animal tests, and restores the growth regulatory and differentiation properties of carcinoma cells (Hirschi et al., 1996;Huang et al., 1998;Omori and Yamasaki, 1998;Qin et al., 2002;Rose et al., 1993). Connexins have four hydrophobic membrane-spanning domains, and phosphorylation of the cytoplasmic C-terminal region is an important way to modulate the function of gap junctions . Phosphorylation of connexins might directly affect gap junction channel gating or modulate connexin intracellular trafficking, channel assembly and connexin turnover (Crow et al., 1990;Lampe, 1994;Lau et al., 1991;Musil and Goodenough, 1991;Oh et al., 1991;Puranam et al., 1993;TenBroek et al., 2001). Thus, an important step towards understanding the molecular mechanisms underlying the regulation of gap junctional intercellular communication (GJIC) is to identify the signalling pathways involved in the diverse aspects of the life cycle of connexins.Gap junction endocytosis is a unique process in which the entire gap junction or a fragment of it is internalized into one of the apposing cells. The internalized gap junction, termed an annular gap junction, is then degraded or possibly reused (Gaietta et al., 2002;Jordan et al., 2001;Larsen et al., 1979;Naus et al., 1993). Degradation of Cx43 involves both the lysosome and the ubiquitin-proteasome system (Laing and Beyer, 1995;Laing et al., 1997;Larsen and Hai, 1978;Musil et al., 2000;Qin et al., 2003;Thomas et al., 2003). In the ubiquitin-proteasome system, proteins are marked for

Gap junctions are dynamic plasma membrane domains, and their protein constituents, the connexins, have a high turnover rate in most tissue types. However, the molecular mechanisms involved in degradation of gap junctions have remained largely unknown. Here, we show that ubiquitin is strongly relocalized to connexin-43 (Cx43; also known as Gja1) gap junction plaques in response to activation of protein kinase C. Cx43 remained ubiquitylated during its transition to a Triton X-100-soluble state and along its trafficking to early endosomes. Following internalization, Cx43 partly colocalized with the ubiquitin-binding proteins Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate; also known as Hgs) and Tsg101 (tumor susceptibility gene 101). Depletion of Hrs or Tsg101 by small interfering RNA abrogated trafficking of Cx43 from early endosomes to lysosomes. Under these conditions, Cx43 was able to undergo dephosphorylation and deubiquitylation, locate to the plasma membrane and form functional gap junctions. Simultaneous depletion of Hrs and Tsg101 caused accumulation of a phosphorylated and ubiquitylated subpopulation of Cx43 in early endosomes and in hybrid organelles between partly degraded annular gap junctions and endosomes. Collectively, these data reveal a central role of early endosomes in sorting of ubiquitylated Cx43, and identify Hrs and Tsg101 as crucial regulators of trafficking of Cx43 to lysosomes.

This article is the first to show that loss of connexin43 (Cx43) expression in colorectal tumors is correlated with significantly shorter relapse-free and overall survival. Cx43 was further found to negatively regulate growth of colon cancer cells, in part by enhancing apoptosis. In addition, Cx43 was found to colocalize with b-catenin and reduce Wnt signaling. The study represents the first evidence that Cx43 acts as a colorectal cancer tumor suppressor and that loss of Cx43 expression during colorectal cancer development is associated with reduced patient survival. The study has important implications for the assessment of Cx43 as a prognostic marker and target in colorectal cancer prevention and therapy. Gap junctions consist of intercellular channels that permit direct transfer of ions and small molecules between adjacent cells. The gap junction channel protein Cx43 plays important roles in cell growth control and differentiation and is frequently dysregulated in human cancers. However, the functional importance and clinical relevance of Cx43 in cancer development has remained elusive. Here, we show that Cx43 is downregulated or aberrantly localized in colon cancer cell lines and colorectal carcinomas, which is associated with loss of gap junction intercellular communication. The in situ protein expression of Cx43 was analyzed in colorectal tumors in a cohort of 674 patients and related to established clinicopathological variables and survival. A subgroup of the patients had weak or no expression of Cx43 in tumors. Loss of Cx43 expression was significantly correlated with shorter relapse-free and overall survival. Loss of Cx43 further identified a high-risk subgroup among stage I and stage II patients with reduced relapse-free and overall survival. Ectopic expression of Cx43 in the colon cancer cell line HT29 was associated with reduced growth in monolayer and soft agar cultures and in tumor xenografts. Cx43 was found to colocalize with b-catenin and negatively regulate the Wnt signaling pathway, and expression of Cx43 was associated with increased levels of apoptosis. Altogether, these data indicate that Cx43 is a colorectal cancer tumor suppressor protein that predicts clinical outcome.Colorectal cancer is among the most common types of cancer worldwide.