Edward Leithe

Epidermal growth factor regulates ubiquitination, internalization and proteasome-dependent degradation of connexin43

Connexins are membrane-spanning proteins that form gap junction channels between adjacent cells. Connexin43 (Cx43), the most widely expressed member of the connexin family in tissues and cell lines, has a rapid turnover rate and its degradation involves both the lysosomal and ubiquitin-proteasome pathway. It was previously shown that the proteasome is involved in regulating the number of functional gap junctions at the plasma membrane. However, little is known about how proteasome-dependent turnover of Cx43 is controlled. Epidermal growth factor (EGF) induces hyperphosphorylation of Cx43 and a rapid, transient decrease in gap junctional intercellular communication. In this study, we show that, along with inhibition of gap junctional intercellular communication, EGF induces disorganization, internalization and degradation of Cx43 gap junction plaques in IAR20 rat liver epithelial cells. These EGF-induced modifications of Cx43 were counteracted by the MEK1 inhibitor PD98059, indicating that the effects were mediated by the mitogen-activated protein kinase pathway. The EGF-induced destruction of Cx43 was proteasome-dependent, because the loss of Cx43 protein was counteracted by the proteasome inhibitor MG132 but not the lysosomal inhibitor leupeptin. Furthermore, EGF induced ubiquitination of Cx43, which was associated with the Cx43 hyperphosphorylation. The EGF-induced Cx43 ubiquitination was counteracted by PD98059. The EGF-induced internalization of Cx43 was blocked by hypertonic sucrose treatment, indicating that EGF mediates internalization of Cx43 via a clathrin-dependent mechanism. Our results indicate that ubiquitination of Cx43 occurs at the plasma membrane before Cx43 internalization. Taken together, these data provide the first evidence that EGF-induced phosphorylation of Cx43 induces binding of ubiquitin and targets Cx43 for internalization and degradation in a proteasome-dependent manner.

Ubiquitination and Down-regulation of Gap Junction Protein Connexin-43 in Response to 12-O-Tetradecanoylphorbol 13-Acetate Treatment

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.

Ubiquitylation of the gap junction protein connexin-43 signals its trafficking from early endosomes to lysosomes in a process mediated by Hrs and Tsg101

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.

Portrait of the PI3K/AKT pathway in colorectal cancer ☆

PI3K/AKT signaling leads to reduced apoptosis, stimulates cell growth and increases proliferation. Under normal conditions, PI3K/AKT activation is tightly controlled and dependent on both extracellular growth signals and the availability of amino acids and glucose. Genetic aberrations leading to PI3K/AKT hyper-activation are observed at considerable frequency in all major nodes in most tumors. In colorectal cancer the most commonly observed pathway changes are IGF2 overexpression, PIK3CA mutations and PTEN mutations and deletions. Combined, these alterations are found in about 40% of large bowel tumors. In addition, but not mutually exclusive to these, KRAS mutations are observed at a similar frequency. There are however additional, less frequent and more poorly understood events that may also push the PI3K/AKT pathway into overdrive and thus promote malignant growth. Here we discuss aberrations of components at the genetic, epigenetic, transcriptional, post-transcriptional, translational and post-translational level where perturbations may drive excessive PI3K/AKT signaling. Integrating multiple molecular levels will advance our understanding of this cancer critical circuit and more importantly, improve our ability to pharmacologically target the pathway in view of clonal development, tumor heterogeneity and drug resistance mechanisms. In this review, we revisit the PI3K/AKT pathway cancer susceptibility syndromes, summarize the known aberrations at the different regulatory levels and the prognostic and predictive values of these alterations in colorectal cancer.

Endocytic processing of connexin43 gap junctions: a morphological study

Gap junctions are plasma membrane areas enriched in channels that provide direct intercellular communication. Gap junctions have a high turnover rate; however, the mechanisms by which gap junctions are degraded are incompletely understood. In the present study, we show that in response to phorbol ester treatment, the gap junction channel protein Cx43 (connexin43) is redistributed from the plasma membrane to intracellular vesicles positive for markers for early and late endosomes and for the endolysosomal protease cathepsin D. Immunoelectron microscopy studies indicate that the double membranes of internalized gap junctions undergo separation and cutting, resulting in multivesicular endosomes enriched in Cx43 protein. Using preloading of BSA-gold conjugates to mark lysosomes, we provide evidence suggesting that the degradation process of the double-membrane structure of annular gap junctions occurs prior to transport of Cx43 to the lysosome. The results further suggest that bafilomycin A1, an inhibitor of vacuolar H+-ATPases, causes accumulation of Cx43 in early endosomes. Taken together, these findings indicate that internalized gap junctions undergo a maturation process from tightly sealed double-membrane vacuoles to connexin-enriched multivesicular endosomes with a single limiting membrane. The results further suggest that along with the processing of the double-membrane structure of annular gap junctions, connexins are trafficked via early and late endosomes, finally resulting in their endolysosomal degradation.

Interplay between PKC and the MAP kinase pathway in Connexin43 phosphorylation and inhibition of gap junction intercellular communication

Gap junction channels are made of a family proteins called connexins. The best-studied type of connexin, Connexin43 (Cx43), is phosphorylated at several sites in its C-terminus. The tumor-promoting phorbol ester TPA strongly inhibits Cx43 gap junction channels. In this study we have investigated mechanisms involved in TPA-induced phosphorylation of Cx43 and inhibition of gap junction channels. The data show that TPA-induced inhibition of gap junction intercellular communication (GJIC) is dependent on both PKC and the MAP kinase pathway. The data suggest that PKC-induced activation of MAP kinase partly involves Src-independent trans-activation of the EGF receptor, and that TPA-induced shift in SDS-PAGE gel mobility of Cx43 is caused by MAP kinase phosphorylation, whereas phosphorylation of S368 by PKC does not alter gel migration of Cx43. We also show that TPA, in addition to phosphorylation of S368, also induces phosphorylation of S255 and S262, in a MAP kinase-dependent manner. The data add to our understanding of the molecular mechanisms involved in the interplay between signaling pathways in regulation of GJIC.

Regulation of gap junction intercellular communication by the ubiquitin system

Intercellular communication via gap junctions plays a critical role in numerous cellular processes, including the control of cell growth and differentiation, maintenance of tissue homeostasis and embryonic development. Gap junctions are aggregates of intercellular channels that enable adjacent cells in solid tissues to directly exchange ions and small molecules. These channels are formed by a family of integral membrane proteins called connexins, of which the best studied is connexin43. Connexins have a high turnover rate in most tissue types, and degradation of connexins is considered to be a tightly regulated process. Post-translational modification of connexins by ubiquitin is emerging as an important event in the regulation of connexin degradation. Ubiquitination is involved in endoplasmic reticulum-associated degradation of connexins as well as in trafficking of connexins to lysosomes. At both the endoplasmic reticulum and the plasma membrane, ubiquitination of connexins is strongly affected by changes in the extracellular environment. There is increasing evidence that the regulation of connexin ubiquitination might be an important mechanism for rapidly modifying the level of functional gap junctions at the plasma membrane, under both normal and pathological conditions. This review discusses the current knowledge about the regulation of intercellular communication via gap junctions by ubiquitination of connexins.

Ubiquitination of gap junction proteins.

Gap junctions are plasma membrane domains containing arrays of channels that exchange ions and small molecules between neighboring cells. Gap junctional intercellular communication enables cells to directly cooperate both electrically and metabolically. Several lines of evidence indicate that gap junctions are important in regulating cell growth and differentiation and for maintaining tissue homeostasis. Gap junction channels consist of a family of transmembrane proteins called connexins. Gap junctions are dynamic structures, and connexins have a high turnover rate in most tissues. Connexin43 (Cx43), the best-studied connexin isoform, has a half-life of 1.5–5 h; and its degradation involves both the lysosomal and proteasomal systems. Increasing evidence suggests that ubiquitin is important in the regulation of Cx43 endocytosis. Ubiquitination of Cx43 is thought to occur at the plasma membrane and has been shown to be regulated by protein kinase C and the mitogen-activated protein kinase pathway. Cx43 binds to the E3 ubiquitin ligase Nedd4, in a process modulated by Cx43 phosphorylation. The interaction between Nedd4 and Cx43 is mediated by the WW domains of Nedd4 and involves a proline-rich sequence conforming to a PY (XPPXY) consensus motif in the C terminus of Cx43. In addition to the PY motif, an overlapping tyrosine-based sorting signal conforming to the consensus of an YXXϕ motif is involved in Cx43 endocytosis, indicating that endocytosis of gap junctions involves both ubiquitin-dependent and -independent pathways. Here, we discuss current knowledge on the ubiquitination of connexins.

Connexin43 acts as a colorectal cancer tumor suppressor and predicts disease outcome

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 β‐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 β‐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.

The Gap Junction Channel Protein Connexin 43 Is Covalently Modified and Regulated by SUMOylation

Background: Gap junctions are intercellular plasma membrane domains enriched in channels that provide direct communication between adjacent cells. Results: The gap junction channel protein connexin 43 is posttranslationally modified by SUMOylation. Conclusion: SUMOylation of connexin 43 is a novel mechanism for regulating gap junctions. Significance: The study has important implications for understanding how gap junctions are regulated under normal and pathological conditions. SUMOylation is a posttranslational modification in which a member of the small ubiquitin-like modifier (SUMO) family of proteins is conjugated to lysine residues in specific target proteins. Most known SUMOylation target proteins are located in the nucleus, but there is increasing evidence that SUMO may also be a key determinant of many extranuclear processes. Gap junctions consist of arrays of intercellular channels that provide direct transfer of ions and small molecules between adjacent cells. Gap junction channels are formed by integral membrane proteins called connexins, of which the best-studied isoform is connexin 43 (Cx43). Here we show that Cx43 is posttranslationally modified by SUMOylation. The data suggest that the SUMO system regulates the Cx43 protein level and the level of functional Cx43 gap junctions at the plasma membrane. Cx43 was found to be modified by SUMO-1, -2, and -3. Evidence is provided that the membrane-proximal lysines at positions 144 and 237, located in the Cx43 intracellular loop and C-terminal tail, respectively, act as SUMO conjugation sites. Mutations of lysine 144 or lysine 237 resulted in reduced Cx43 SUMOylation and reduced Cx43 protein and gap junction levels. Altogether, these data identify Cx43 as a SUMOylation target protein and represent the first evidence that gap junctions are regulated by the SUMO system.