Article

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

Journal of Cell Science (Impact Factor: 5.43). 05/2012; 125(17). DOI: 10.1242/jcs.093500
Source: PubMed

ABSTRACT

Gap 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 (siRNA) 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.

    • "For example, it has been shown that Cx43 phosphorylation by EGF (epidermal growth factor) and TPA (12-O-tetradecanoylphorbol-13-acetate) promotes interaction between Cx43 and the E3 ubiquitin ligase Nedd4 (neuronal precursor cell-expressed developmentally down-regulated 4) leading to the subsequent ubiquitination of Cx43[89,90]. 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[91], Smurf2[92], and Nedd48990919293. Since these studies were done in cell culture systems, it still remains largely unclear how Cx43 ubiquitination is regulated in the cardiomyocytes. "
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    ABSTRACT: With each heartbeat, Connexin43 (Cx43) cell-cell communication gap junctions are needed to rapidly spread and coordinate excitation signals for an effective heart contraction. The correct formation and delivery of channels to their respective membrane subdomain is referred to as protein trafficking. Altered Cx43 trafficking is a dangerous complication of diseased myocardium which contributes to the arrhythmias of sudden cardiac death. Cx43 has also been found to regulate many other cellular processes that cannot be explained by cell-cell communication. We recently identified the existence of up to six endogenous internally translated Cx43 N-terminal truncated isoforms from the same full-length mRNA molecule. This is the first evidence that alternative translation is possible for human ion channels and in human heart. Interestingly, we found that these internally translated isoforms, more specifically the 20kDa isoform (GJA1-20k), is important for delivery of Cx43 to its respective membrane subdomain. This review covers recent advances in Cx43 trafficking and potential importance of alternatively translated Cx43 truncated isoforms.
    No preview · Article · Nov 2015 · Biochimica et Biophysica Acta
    • "As such, it would appear that the still developing postnatal heart is a dynamic entity with much more plasticity of gene expression, allowing it to cope with alterations in electroconduction that accompany a 69–82% decrease of Cx43 protein in the myocardium arising from either conditional loss of Cx43 or from overexpression of Wwp1. Wwp1 belongs to the same subfamily of HECT E3s as Nedd4 and Smurf2, ubiquitin ligases that have previously been shown to contribute to the downregulation of Cx43 in cell culture systems either directly or indirectly141516. All members of this subfamily are widely expressed with numerous other substrates and alternative splice isoforms reported for many of them[17], prompting the questions of whether these ligases serve redundant roles in targeting Cx43 and/or associated proteins, or, if not, how is specificity in various cellular, tissue, and biological contexts achieved? "
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    ABSTRACT: Gap junctions (GJ) are intercellular channels composed of connexin subunits that play a critical role in a diverse number of cellular processes in all tissue types. In the heart, GJs mediate electrical coupling between cardiomyocytes and display mislocalization and/or downregulation in cardiac disease (a process known as GJ remodeling), producing an arrhythmogenic substrate. The main constituent of GJs in the ventricular myocardium is connexin 43 (Cx43), an integral membrane protein that is rapidly turned over and shows decreased expression or function with age. We hypothesized that Wwp1, an ubiquitin ligase whose expression in known to increase in aging-related pathologies, may regulate Cx43 in vivo by targeting it for ubiquitylation and degradation and yield tissue-specific Cx43 loss of function phenotypes. When Wwp1 was globally overexpressed in mice under the control of a β-actin promoter, the highest induction of Wwp1 expression was observed in the heart which was associated with a 90% reduction in cardiac Cx43 protein levels, left ventricular hypertrophy (LVH), and the development of lethal ventricular arrhythmias around 8weeks of age. This phenotype was completely penetrant in two independent founder lines. Cardiomyocyte-specific overexpression of Wwp1 confirmed that this phenotype was cell autonomous and delineated Cx43-dependent and -independent roles for Wwp1 in arrhythmogenesis and LVH, respectively. Using a cell-based system, it was determined that Wwp1 co-immunoprecipitates with and ubiquitylates Cx43, causing a decrease in the steady state levels of Cx43 protein. These findings offer new mechanistic insights into the regulation of Cx43 which may be exploitable in various gap junctionopathies.
    No preview · Article · Sep 2015 · Journal of Molecular and Cellular Cardiology
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    • "Only the WW2 domain binds to the PY motif (Leykauf et al., 2006). Other groups have demonstrated interaction of Cx43 with additional E3 ligases, including the other members of the HECT family smad ubiquitination regulatory factor-2 (Smurf2; Fykerud et al., 2012) and WWP1 as well as the RING E3 ligase Tripartite motif-containing protein 2 (TRIM21) as reviewed by Su and Lau (2012). "
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    ABSTRACT: Gap junctions are comprised of connexins that form cell-to-cell channels which couple neighboring cells to accommodate the exchange of information. The need for communication does, however, change over time and therefore must be tightly controlled. Although the regulation of connexin protein expression by transcription and translation is of great importance, the trafficking, channel activity and degradation are also under tight control. The function of connexins can be regulated by several post translational modifications, which affect numerous parameters; including number of channels, open probability, single channel conductance or selectivity. The most extensively investigated post translational modifications are phosphorylations, which have been documented in all mammalian connexins. Besides phosphorylations, some connexins are known to be ubiquitinated, SUMOylated, nitrosylated, hydroxylated, acetylated, methylated, and γ-carboxyglutamated. The aim of the present review is to summarize our current knowledge of post translational regulation of the connexin family of proteins.
    Full-text · Article · Oct 2013 · Frontiers in Pharmacology
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