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Connexins are chordate gap junction channel proteins that, by enabling direct communication between the cytosols of adjacent cells, create a unique cell signalling network. Gap junctional intercellular communication (GJIC) has important roles in controlling cell growth and differentiation and in tissue development and homeostasis. Moreover, several non-canonical connexin functions unrelated to GJIC have been discovered. Of the 21 members of the human connexin family, connexin 43 (Cx43) is the most widely expressed and studied. The long cytosolic C-terminus (CT) of Cx43 is subject to extensive post-translational modifications that modulate its intracellular trafficking and gap junction channel gating. Moreover, the Cx43 CT contains multiple domains involved in protein interactions that permit crosstalk between Cx43 and cytoskeletal and regulatory proteins. These domains endow Cx43 with the capacity to affect cell growth and differentiation independently of GJIC. Here, we review the current understanding of the regulation and unique functions of the Cx43 CT, both as an essential component of full-length Cx43 and as an independent signalling hub. We highlight the complex regulatory and signalling networks controlled by the Cx43 CT, including the extensive protein interactome that underlies both gap junction channel-dependent and -independent functions. We discuss these data in relation to the recent discovery of the direct translation of specific truncated forms of Cx43. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

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 comprise arrays of intercellular channels formed by connexin proteins and provide for the direct communication between adjacent cells. This type of intercellular communication permits the coordination of cellular activities and plays key roles in the control of cell growth and differentiation and in the maintenance of tissue homoeostasis. After more than 50 years, deciphering the links among connexins, gap junctions and cancer, researchers are now beginning to translate this knowledge to the clinic. The emergence of new strategies for connexin targeting, combined with an improved understanding of the molecular bases underlying the dysregulation of connexins during cancer development, offers novel opportunities for clinical applications. However, different connexin isoforms have diverse channel-dependent and-independent functions that are tissue and stage specific. This can elicit both pro-and anti-tumorigenic effects that engender significant challenges in the path towards personalised medicine. Here, we review the current understanding of the role of connexins and gap junctions in cancer, with particular focus on the recent progress made in determining their prognostic and therapeutic potential.