A protein kinase A-ezrin complex regulates connexin 43 gap junction communication in liver epithelial cells
Introduction
Gap junctions (GJs) are channels composed of integral membrane proteins termed connexins (Cxs), which form hexameric pores that transverse the cell membrane of two adjacent cells and allow the passage of fluid, ions and molecules smaller than approximately 1 kDa between cells [1]. The Cx family includes 21 members in humans, and are numbered according to their molecular weight in kilodaltons [2]. Cx43 is the most widely expressed and predominant Cx in many primary cell types as well as in a number of cell lines. GJ intercellular communication (GJIC) plays important roles in development, cell growth control, as well as metabolic homeostasis and synchronized physiological functions of cells [3]. Aberrant GJIC has been linked to several human diseases, including cancer [4], [5].
Phosphorylation plays an important role in the acute regulation of GJ channels. The C-terminus of Cx43 contains several putative phosphorylation sites that are prone to phosphorylation by many kinases, including cAMP-dependent protein kinase (PKA) [6]. Spatial organization brings specificity to cAMP signaling via A-kinase anchoring proteins (AKAPs) that place PKA close to their substrates, also ensuring appropriate temporal control [7], [8]. Besides the A-kinase binding domain (AKB), all AKAPs have specific targeting domains, which enable PKA-AKAP complexes to be positioned in a membrane, organelle or other subcellular organization. In addition, these complexes may contain binding domains for other cAMP effectors and signaling molecules [9], [10].
Ezrin belongs to the ERM (ezrin-radixin-moesin) family of proteins [11]. In addition to the role of ERM proteins in organizing the cortical cytoskeleton by connecting actin filaments to the plasma membrane, ERM proteins also serve as scaffolds to directly organize signaling components, for example in T cell receptor signaling where Ezrin serves as an AKAP for PKA, in the insulin secretory pathway and during apoptosis [12], [13], [14], [15], [16]. In a soluble state the N-terminal, transmembrane receptor binding domain of ERM proteins interacts with the c-terminal, actin-binding domain, leaving ERM proteins in a folded and inactive state [17]. The association with PKA is hindered when Ezrin is in the folded configuration, but the Ezrin AKB, located in the middle alpha-helical region, becomes accessible for PKA binding when Ezrin is activated by phosphorylation of the C-terminal threonine (T567) and unfolds [14], [18].
We recently showed that in placental cytotrophoblasts, Ezrin functions as an AKAP to confer human chorion gonadotropin (hCG)-driven cAMP regulation of cell fusion [19]. In the placenta, GJ opening is accomplished by Cx43 phosphorylation by an anchored pool of PKA, bound to Ezrin in complex with Cx43 [19]. In order to assess whether Ezrin regulates GJ opening also in other nonfusogenic cell types, we examined an epithelial liver cell line with Cx43 as the predominant GJ protein and found that Ezrin coordinates a supramolecular complex with PKA and Cx43 that facilitates regulation of GJ communication by cAMP.
Section snippets
Cells
The rat liver epithelial cell line IAR20 was cultured at 37 °C 5% CO2 in DMEM high glucose GlutaMAX medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% PenStrep (Gibco). All the experiments were done with 90–100% confluent cells (grown in different dishes depending on the experiment). The cells were routinely examined for infections, tested for mycoplasma and were free of contamination.
Reagents
8-CPT-cAMP (Biolog), [32P]γ-ATP (Pierce), ATP (Sigma), cAMP (Sigma).
Peptides
All peptides used were
The GJ protein Cx43 forms a supramolecular complex with ezrin and PKA at the cell membrane of IAR20 cells
In order to examine the presence and localization of the AKAP Ezrin and PKA in epithelial cells, the liver cell line IAR20, which forms abundant GJs, was subjected to confocal immunofluorescence analysis. Immunostaining showed that Ezrin is present in IAR20 cell membranes and that it co-distributed with Cx43, which is known to be the predominant Cx isoform expressed in this cell line [23], [24] (Fig. 1). The main region of co-distribution between Cx43 and Ezrin appeared to be in GJ plaques [24]
Discussion
The discovery that Ezrin is a dual-specific AKAP in placental trophoblasts responsible for regulation of GJIC, raised the question of whether Ezrin also in other cell systems serves as an AKAP that confers PKA phosphorylation of Cx43 and channel opening. In the placenta, cytotrophoblasts fuse and form multinucleated syncytium, which is a hormone driven process that is known to involve the cAMP signaling pathway [37]. Fusogenic signal transmission through Cx43 GJs is needed for the fusion
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
This work was supported by the Norwegian Cancer Society [grant number 419544], the Research Council of Norway [grant number 187615], the K.G. Jebsen Foundation [grant number 2012/21 and 2012/23] and the Novo Nordic Foundation [grant number NFF14OC001090]. The authors acknowledge the use of the RCN supported NorMIC Oslo Imaging platform.
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