CCN3 (NOV) interacts with connexin43 in C6 glioma cells: possible mechanism of connexin-mediated growth suppression

Many tumor cells exhibit aberrant gap junctional intercellular communication, which can be restored by transfection with connexin genes. We have previously discovered that overexpression of connexin43 (Cx43) in C6 glioma cells not only reduces proliferation but also leads to production of soluble growth-inhibitory factors. We identified that several members of the CCN (Cyr61/connective tissue growth factor/nephroblastoma-overexpressed) family are up-regulated following Cx43 expression, including CCN3 (NOV). We now report evidence for an association between CCN3 and Cx43. Western blot analysis demonstrated that the 48-kDa full-length CCN3 protein was present in the lysate and conditioned medium of growth-suppressed C6-Cx43 cells, as well as primary astrocytes, but not in C6 parental and human glioma cells. Immunocytochemical examination of CCN3 revealed diffuse localization in parental C6 cells, whereas transfection of C6 cells with Cx43 (C6-Cx43) or with a modified Cx43 tagged to green fluorescent protein on its C terminus (Cx43-GFP) resulted in punctate staining, suggesting that CCN3 co-localizes with Cx43 in plaques at the plasma membrane. In cells expressing a C-terminal truncation of Cx43 (Cx43Delta244-382), this co-localization was lost. Glutathione S-transferase pull-down assay and co-immunoprecipitation demonstrated that CCN3 was able to physically interact with Cx43. In contrast, CCN3 was not found to associate with Cx43Delta244-382. Similar experiments revealed that CCN3 did not co-localize or associate with other connexins, including Cx40 or Cx32. Taken together, these data support an interaction of CCN3 with the C terminus of Cx43, which could play an important role in mediating growth control induced by specific gap junction proteins.     

Dose-dependent differential upregulation of CCN1/Cyr61 and CCN3/NOV by the gap junction protein Connexin43 in glioma cells

Gap junctions form channels that allow exchange of materials between cells and are composed of transmembrane protein subunits called connexins. While connexins are believed to mediate cellular signaling by permitting intercellular communication to occur, there is also increasing evidence that suggest connexins may mediate growth control via a junction-independent mechanism. Connexin43 (Cx43) is the most abundant gap junction protein found in astrocytes, and gliomas exhibit reduced Cx43 expression. We have previously observed that restoration of Cx43 levels in glioma cells led to increased expression of CCN3 (NOV) proteins. We now report that overexpression of Cx43 in C6-glioma cells (C6-Cx43) also upregulates the expression of CCN1 (Cyr61). Both CCN1 and CCN3 belong to the Cyr61/Connective tissue growth factor/Nephroblastoma-overexpressed (CCN) family of secretory proteins. The CCN proteins are tightly associated with the extracellular matrix and have important roles in cell proliferation and migration. CCN1 promotes growth in glioma cells, as shown by the increased proliferation rate of CCN1-overexpressing C6 cells. In addition to its effect on cell growth, CCN1 also increased the motility of glioma cells in the presence of extracellular substrates such as fibronectin. Gliomas expressing high levels of Cx43 preferentially upregulated CCN3 which resulted in reduced growth rate. CCN3 could also be observed in Cx43 gap junction plaques in confluent C6-Cx43H culture at the stationary phase of their growth. Our results suggest that the dissimilar growth characteristics between high and low Cx43 expressors may be due to differential regulation of CCN3 by varying levels of Cx43.     

A 14‐3‐3 Mode‐1 Binding Motif Initiates Gap Junction Internalization During Acute Cardiac Ischemia

Altered phosphorylation and trafficking of connexin 43 (Cx43) during acute ischemia contributes to arrhythmogenic gap junction remodeling, yet the critical sequence and accessory proteins necessary for Cx43 internalization remain unresolved. 14‐3‐3 proteins can regulate protein trafficking, and a 14‐3‐3 mode‐1 binding motif is activated upon phosphorylation of Ser373 of the Cx43 C‐terminus. We hypothesized that Cx43^Ser373 phosphorylation is important to pathological gap junction remodeling. Immunofluorescence in human heart reveals the enrichment of 14‐3‐3 proteins at intercalated discs, suggesting interaction with gap junctions. Knockdown of 14‐3‐3τ in cell lines increases gap junction plaque size at cell–cell borders. Cx43^S373A mutation prevents Cx43/14‐3‐3 complexing and stabilizes Cx43 at the cell surface, indicating avoidance of degradation. Using Langendorff‐perfused mouse hearts, we detect phosphorylation of newly internalized Cx43 at Ser373 and Ser368 within 30 min of no‐flow ischemia. Phosphorylation of Cx43 at Ser368 by protein kinase C and Ser255 by mitogen‐activated protein kinase has previously been implicated in Cx43 internalization. The Cx43^S373A mutant is resistant to phosphorylation at both these residues and does not undergo ubiquitination, revealing Ser373 phosphorylation as an upstream gatekeeper of a posttranslational modification cascade necessary for Cx43 internalization. Cx43^Ser373 phosphorylation is a potent target for therapeutic interventions to preserve gap junction coupling in the stressed myocardium.      

Fusion of GFP to the Carboxyl Terminus of Connexin43 Increases Gap Junction Size in HeLa Cells

The pattern of gap junctional coupling between cells is thought to be important for the proper function of many types of tissues. At present, little is known about the molecular mechanisms that control the size and distribution of gap junctions. We addressed this issue by expressing connexin43 (Cx43) constructs in HeLa cells, a connexin-deficient cell line. HeLa cells expressing exogenously introduced wild-type Cx43 formed small, punctate gap junctions. By contrast, cells expressing Cx43-GFP formed large, sheet-like gap junctions. These results suggest that the GFP tag, which is fused to the carboxyl terminus of Cx43, alters gap junction size by masking the carboxyl terminal amino acids of Cx43 that comprise a zonula occludins-1 (ZO-1) binding site. We are currently testing this hypothesis using deletion and dominant-negative constructs that directly target the interaction between Cx43 and ZO-1.     

Fusion of GFP to the Carboxyl Terminus of Connexin43 Increases Gap Junction Size in HeLa Cells

The pattern of gap junctional coupling between cells is thought to be important for the proper function of many types of tissues. At present, little is known about the molecular mechanisms that control the size and distribution of gap junctions. We addressed this issue by expressing connexin43 (Cx43) constructs in HeLa cells, a connexin-deficient cell line. HeLa cells expressing exogenously introduced wild-type Cx43 formed small, punctate gap junctions. By contrast, cells expressing Cx43-GFP formed large, sheet-like gap junctions. These results suggest that the GFP tag, which is fused to the carboxyl terminus of Cx43, alters gap junction size by masking the carboxyl terminal amino acids of Cx43 that comprise a zonula occludins-1 (ZO-1) binding site. We are currently testing this hypothesis using deletion and dominant-negative constructs that directly target the interaction between Cx43 and ZO-1.     

Fusion of GFP to the carboxyl terminus of connexin43 increases gap junction size in HeLa cells

The pattern of gap junctional coupling between cells is thought to be important for the proper function of many types of tissues. At present, little is known about the molecular mechanisms that control the size and distribution of gap junctions. We addressed this issue by expressing connexin43 (Cx43) constructs in HeLa cells, a connexin-deficient cell line. HeLa cells expressing exogenously introduced wild-type Cx43 formed small, punctate gap junctions. By contrast, cells expressing Cx43-GFP formed large, sheet-like gap junctions. These results suggest that the GFP tag, which is fused to the carboxyl terminus of Cx43, alters gap junction size by masking the carboxyl terminal amino acids of Cx43 that comprise a zonula occludins-1 (ZO-1) binding site. We are currently testing this hypothesis using deletion and dominant-negative constructs that directly target the interaction between Cx43 and ZO-1.     

Disruption of the Cx43/miR21 pathway leads to osteocyte apoptosis and increased osteoclastogenesis with aging

Skeletal aging results in apoptosis of osteocytes, cells embedded in bone that control the generation/function of bone forming and resorbing cells. Aging also decreases connexin43 (Cx43) expression in bone; and osteocytic Cx43 deletion partially mimics the skeletal phenotype of old mice. Particularly, aging and Cx43 deletion increase osteocyte apoptosis, and osteoclast number and bone resorption on endocortical bone surfaces. We examined herein the molecular signaling events responsible for osteocyte apoptosis and osteoclast recruitment triggered by aging and Cx43 deficiency. Cx43‐silenced MLO‐Y4 osteocytic (Cx43^def) cells undergo spontaneous cell death in culture through caspase‐3 activation and exhibit increased levels of apoptosis‐related genes, and only transfection of Cx43 constructs able to form gap junction channels reverses Cx43^def cell death. Cx43^def cells and bones from old mice exhibit reduced levels of the pro‐survival microRNA miR21 and, consistently, increased levels of the miR21 target phosphatase and tensin homolog (PTEN) and reduced phosphorylated Akt, whereas PTEN inhibition reduces Cx43^def cell apoptosis. miR21 reduction is sufficient to induce apoptosis of Cx43‐expressing cells and miR21 deletion in miR21^fl/fl bones increases apoptosis‐related gene expression, whereas a miR21 mimic prevents Cx43^def cell apoptosis, demonstrating that miR21 lies downstream of Cx43. Cx43^def cells release more osteoclastogenic cytokines [receptor activator of NFκB ligand (RANKL)/high‐mobility group box‐1 (HMGB1)], and caspase‐3 inhibition prevents RANKL/HMGB1 release and the increased osteoclastogenesis induced by conditioned media from Cx43^def cells, which is blocked by antagonizing HMGB1‐RAGE interaction. These findings identify a novel Cx43/miR21/HMGB1/RANKL pathway involved in preventing osteocyte apoptosis that also controls osteoclast formation/recruitment and is impaired with aging.     

Connexin43 interacts with βarrestin: a pre-requisite for osteoblast survival induced by parathyroid hormone

Parathyroid hormone (PTH) promotes osteoblast survival through a mechanism that depends on cAMP-mediated signaling downstream of the G protein-coupled receptor PTHR1. We present evidence herein that PTH-induced survival signaling is impaired in cells lacking connexin43 (Cx43). Thus, expression of functional Cx43 dominant negative proteins or Cx43 knock-down abolished the expression of cAMP-target genes and anti-apoptosis induced by PTH in osteoblastic cells. In contrast, cells lacking Cx43 were still responsive to the stable cAMP analog dibutyril-cAMP. PTH survival signaling was rescued by transfecting wild type Cx43 or a truncated dominant negative mutant of βarrestin, a PTHR1-interacting molecule that limits cAMP signaling. On the other hand, Cx43 mutants lacking the cytoplasmic domain (Cx43(Δ245)) or unable to be phosphorylated at serine 368 (Cx43(S368A)), a residue crucial for Cx43 trafficking and function, failed to restore the anti-apoptotic effect of PTH in Cx43-deficient cells. In addition, overexpression of wild type βarrestin abrogated PTH survival signaling in Cx43-expressing cells. Moreover, βarrestin physically associated in vivo to wild type Cx43 and to a lesser extent to Cx43(S368A) ; and this association and the phosphorylation of Cx43 in serine 368 were reduced by PTH. Furthermore, induction of Cx43(S368) phosphorylation or overexpression of wild type Cx43, but not Cx43(Δ245) or Cx43(S368A) , reduced the interaction between βarrestin and the PTHR1. These studies demonstrate that βarrestin is a novel Cx43-interacting protein and suggest that, by sequestering βarrestin, Cx43 facilitates cAMP signaling, thereby exerting a permissive role on osteoblast survival induced by PTH.     

New protein–protein interactions of mitochondrial connexin 43 in mouse heart

Connexin 43 (Cx43), the gap junction protein involved in cell‐to‐cell coupling in the heart, is also present in the subsarcolemmal fraction of cardiomyocyte mitochondria. It has been described to regulate mitochondrial potassium influx and respiration and to be important for ischaemic preconditioning protection, although the molecular effectors involved are not fully characterized. In this study, we looked for potential partners of mitochondrial Cx43 in an attempt to identify new molecular pathways for cardioprotection. Mass spectrometry analysis of native immunoprecipitated mitochondrial extracts showed that Cx43 interacts with several proteins related with mitochondrial function and metabolism. Among them, we selected for further analysis only those present in the subsarcolemmal mitochondrial fraction and known to be related with the respiratory chain. Apoptosis‐inducing factor (AIF) and the beta‐subunit of the electron‐transfer protein (ETFB), two proteins unrelated to date with Cx43, fulfilled these conditions, and their interaction with Cx43 was proven by direct and reverse co‐immunoprecipitation. Furthermore, a previously unknown molecular interaction between AIF and ETFB was established, and protein content and sub‐cellular localization appeared to be independent from the presence of Cx43. Our results identify new protein–protein interactions between AIF‐Cx43, ETFB‐Cx43 and AIF‐ETFB as possible players in the regulation of the mitochondrial redox state.     

Matricellular Protein CCN3 (NOV) Regulates Actin Cytoskeleton Reorganization

CCN3 (NOV), a putative ligand for integrin receptors, is tightly associated with the extracellular matrix and mediates diverse cellular functions, including cell adhesion and proliferation. CCN3 has been shown to negatively regulate growth although it promotes migration in a cell type-specific manner. In this study, overexpression of CCN3 reduces growth and increases intercellular adhesion of breast cancer cells. Interestingly, CCN3 overexpression also led to the formation of multiple pseudopodia that are enriched in actin, CCN3, and vinculin. Breast cancer cells preincubated with exogenous CCN3 protein also induced the same phenotype, indicating that secreted CCN3 is sufficient to induce changes in cell morphology. Surprisingly, extracellular CCN3 is internalized to the early endosomes but not to the membrane protrusions, suggesting pseudopodia-enriched CCN3 may derive from a different source. The presence of an intracellular variant of CCN3 will be consistent with our finding that the cytoplasmic tail of the gap junction protein connexin43 (Cx43) associates with CCN3. Cx43 is a channel protein permitting intercellular communication to occur. However, neither the channel properties nor the protein levels of Cx43 are affected by the CCN3 protein. In contrast, CCN3 proteins are down-regulated in the absence of Cx43. Finally, we showed that overexpression of CCN3 increases the activity of the small GTPase Rac1, thereby revealing a pathway that links Cx43 directly to actin reorganization.The CCN (CYR61/Connective Tissue Growth Factor/Nephroblastoma Overexpressed) family of multimodular proteins mediates diverse cellular functions, including cell adhesion, migration, and proliferation (1–3). Overexpression of CCN3, one of the founding members of the family, inhibits proliferation in most types of tumors such as glioblastoma and Ewing sarcoma (4, 5). Similarly, down-regulation of CCN3 has been suggested to promote melanoma progression (6). On the other hand, CCN3 can also promote migration in sarcoma and glioblastoma (4, 7), although a separate study shows that it decreases the invasion of melanoma (6). Therefore, in contrast to its role in growth suppression, the role of CCN3 signaling in cell motility is less clear.Most evidence suggests CCN3 mediates its effects by binding to the integrin proteins, such as the αVβ3 receptors (8, 9), and that CCN3 alters cell adhesion in an integrin-dependent fashion (4, 10). In melanocytes, the discoidin domain receptor 1 mediates CCN3-dependent adhesion (11). CCN3 has also been observed to associate with Notch1 (12), fibulin 1C (13), S100A4 (14), and the gap junction protein Cx43³ (15, 16), suggesting that CCN3 may also modulate cell growth via non-integrin signaling pathways.Gap junction proteins are best known for forming channels between cells, contributing to intercellular communication by allowing the exchange of small ions and molecules (17, 18). Consequently, attenuated intercellular communication has been implicated in promoting carcinogenesis (19, 20). Recent evidence has indicated that connexins can mediate channel-independent growth control through interaction of their C-terminal cytoplasmic tail with various intracellular signaling molecules (21–23). In addition, many Cx43-interacting proteins, including ZO-1 (zonula occludens-1) (24), Drebrin (25), and N-cadherin (26) associate with F-actin, thus placing Cx43 in close proximity to the actin cytoskeleton.In this study, we show for the first time that CCN3 reorganizes the actin cytoskeleton of the breast cancer cells MDA-MB-231 with the formation of multiple cell protrusions, possibly by activating the small GTPase Rac1. Our results also suggest an alternative route by which Cx43 may be functionally linked to actin cytoskeletal signaling via CCN3.     

Structural changes in the carboxyl terminus of the gap junction protein connexin 40 caused by the interaction with c-Src and zonula occludens-1

c-Src can disrupt the connexin 43 (Cx43) and zonula occludens-1 (ZO-1) interaction, leading to down-regulation of gap junction intercellular communication. Previously, the authors characterized the interaction of domains from these proteins with the carboxyl terminus of Cx43 (Cx43CT) and found that binding of the c-Src SH3 domain to Cx43CT disrupted the Cx43CT/ZO-1 PDZ-2 domain complex. Because Cx43 and Cx40 form heteromeric connexons and display similar mechanisms of pH regulation, the authors addressed whether Cx40CT interacts with these domains in a similar manner as Cx43CT. Nuclear magnetic resonance (NMR) data indicate that Cx40CT is an intrinsically disordered protein. NMR titrations determined that PDZ-2 affected the last 28 Cx40CT residues and SH3 shifted numerous amino-terminal Cx40CT residues. Finally, the Cx40CT/PDZ-2 complex was unaffected by SH3 and both domains interacted simultaneously with Cx40CT. This result differs from when the same experiment was performed with Cx43CT, suggesting different mechanisms of regulation exist between connexin isoforms, even when involving the same molecular partners.     

Role of connexin 43 in the mechanism of action of alendronate: dissociation of anti-apoptotic and proliferative signaling pathways

Bisphosphonates (BPs) inhibit osteocyte and osteoblast apoptosis via opening of connexin (Cx) 43 hemichannels and activating the extracellular signal regulated kinases ERKs. Previously, we hypothesized that intracellular survival signaling is initiated by interaction of BPs with Cx43. However, using whole cell binding assays with [(3)H]-alendronate, herein we demonstrated the presence of saturable, specific and high affinity binding sites in the Cx43-expressing ROS 17/2.8 osteoblastic cells, authentic osteoblasts and MLO-Y4 cells expressing Cx43 or not, as well as in HeLa cells lacking Cx43 expression and ROS 17/2.8 cells pretreated with agents that disassemble Cx channels. In addition, both BPs and the PTP inhibitor Na(3)VO(4) increased proliferation of cells expressing Cx43 or not. Furthermore, although BPs are internalized and inhibit intracellular enzymes in osteoclasts, whether the drugs penetrate non-resorptive bone cells is not known. To clarify this, we evaluated the osteoblastic uptake of AF-ALN, a fluorescently labeled analog of alendronate. AF-ALN was rapidly internalized in cells expressing Cx43 or not indicating that this process is not mediated via Cx43 hemichannels. Altogether, these findings suggest that although required for triggering intracellular survival signaling by BPs, Cx43 is dispensable for cellular BP binding, its uptake, as well as the proliferative effects of these agents.     

Connexin43 PDZ2 binding domain mutants create functional gap junctions and exhibit altered phosphorylation

Connexin43 (Cx43) is the most abundantly expressed gap junction protein. The C-terminal tail of Cx43 is important for regulation of gap junctions via phosphorylation of specific tyrosine and serine residues and through interactions with cellular proteins. The C-terminus of Cx43 has been shown to interact with the PDZ2 domain of the tight and adherens junction associated zona occludens 1 (ZO-1) protein. Analysis of the PDZ2 binding domain of Cx43 indicated that positions -3 and -2, and the final hydrophobic amino acid at the C-terminus, are critical for ZO-1 binding. In addition, the C-termini of connexins 40 and 45, but not Cx32, interacted with ZO-1. To evaluate the functional significance of the Cx43-ZO-1 interaction, Cx43 wild type (Cx43wt) and mutants lacking either the C-terminal hydrophobic isoleucine (Cx43deltaI382) or the last five amino acids (Cx43delta378-382), required for ZO-1 binding in vitro, were introduced into a Cx43-deficient MDCK cell line. In vitro binding studies and coimmunoprecipitation assays indicated that these Cx43 mutants failed to interact with ZO-1. Confocal and deconvolution microscopy revealed that a fraction of Cx43wt colocalized with ZO-1 at the plasma membrane. A similar colocalization pattern was observed for the Cx43deltaI382 and Cx43 delta378-382 mutants, which were translocated to the plasma membrane and formed functional gap junction channels. The wt and mutant Cx43 appeared to have similar turnover rates. However, the P2 and P3 phosphoisoforms of the Cx43 mutants were significantly reduced compared to Cx43wt. These studies indicated that the interaction of Cx43 with ZO-1 may contribute to the regulation of Cx43 phosphorylation.     

The carboxy‐terminal tail of connexin43 gap junction protein is sufficient to mediate cytoskeleton changes in human glioma cells

Connexin43 (Cx43) is a ubiquitously expressed member of the gap junction protein family that mediates gap junction intercellular communication (GJIC) by allowing exchange of cytosolic materials. Previous studies have used Cx43 truncated at the cytoplasmic tail (C‐tail) to demonstrate that the C‐tail is essential to regulate cell growth and motility. Therefore, the aim of our study was to delineate the respective role of the truncated Cx43 and the C‐tail in mediating Cx43‐dependent signaling. A truncated Cx43 expressing the channel part of the protein (TrCx43, amino acid 1–242) and a construct encompassing only the C‐tail from amino acid 243 (243Cx43) were transduced into LN18 human glioma cells. Our results showed that the ability of Cx43 to suppress growth was independent of GJIC as assessed by dye transfer, but was dependent on the presence of a rigid extracellular matrix. We further demonstrated that the C‐tail alone is sufficient to promote motility. Surprisingly, Cx43 is also able to increase migration in the absence of the C‐tail, suggesting the presence of at least two distinct signaling mechanisms utilized by Cx43 to affect motility. Finally, we used time‐lapse imaging to examine the behavior of migrating cells and it was apparent that the C‐tail was associated with a lamellipodia‐based migration not observed in either mock or TrCx43 expressing LN18 cells. Our study shows for the first time that a free C‐tail is sufficient to induce Cx43‐dependent changes in cell morphology and that Cx43 signaling is linked to the regulation of the actin cytoskeleton. J. Cell. Biochem. 110: 589–597, 2010. © 2010 Wiley‐Liss, Inc.     

RACK1 identified as the PCBP1‐interacting protein with a novel functional role on the regulation of human MOR gene expression

Poly C binding protein 1 (PCBP1) is an expressional regulator of the mu‐opioid receptor (MOR) gene. We hypothesized the existence of a PCBP1 co‐regulator modifying human MOR gene expression by protein–protein interaction with PCBP1. A human brain cDNA library was screened using the two‐hybrid system with PCBP1 as the bait. Receptor for activated protein kinase C (RACK1) protein, containing seven WD domains, was identified. PCBP1‐RACK1 interaction was confirmed via in vivo validation using the two‐hybrid system, and by co‐immunoprecipitation with anti‐PCBP1 antibody and human neuronal NMB cell lysate, endogenously expressing PCBP1 and RACK1. Further co‐immunoprecipitation suggested that RACK1‐PCBP1 interaction occurred in cytosol alone. Single and serial WD domain deletion analyses demonstrated that WD7 of RACK1 is the key domain interacting with PCBP1. RACK1 over‐expression resulted in a dose‐dependent decrease of MOR promoter activity using p357 plasmid containing human MOR promoter and luciferase reporter gene. Knock‐down analysis showed that RACK1 siRNA decreased the endogenous RACK1 mRNA level in NMB, and elevated MOR mRNA level as indicated by RT‐PCR. Likewise, a decrease of RACK1 resulted in an increase of MOR proteins, verified by ³H‐diprenorphine binding assay. Collectively, this study reports a novel role of RACK1, physically interacting with PCBP1 and participating in the regulation of human MOR gene expression in neuronal NMB cells.     

Connexin43 PDZ2 Binding Domain Mutants Create Functional Gap Junctions and Exhibit Altered Phosphorylation

Connexin43 (Cx43) is the most abundantly expressed gap junction protein. The C-terminal tail of Cx43 is important for regulation of gap junctions via phosphorylation of specific tyrosine and serine residues and through interactions with cellular proteins. The C-terminus of Cx43 has been shown to interact with the PDZ2 domain of the tight and adherens junction associated zona occludens 1 (ZO-1) protein. Analysis of the PDZ2 binding domain of Cx43 indicated that positions -3 and -2, and the final hydrophobic amino acid at the C-terminus, are critical for ZO-1 binding. In addition, the C-termini of connexins 40 and 45, but not Cx32, interacted with ZO-1. To evaluate the functional significance of the Cx43-ZO-1 interaction, Cx43 wild type (Cx43wt) and mutants lacking either the C-terminal hydrophobic isoleucine (Cx43ΔI382) or the last five amino acids (Cx43Δ378-382), required for ZO-1 binding in vitro, were introduced into a Cx43-deficient MDCK cell line. In vitro binding studies and coimmunoprecipitation assays indicated that these Cx43 mutants failed to interact with ZO-1. Confocal and deconvolution microscopy revealed that a fraction of Cx43wt colocalized with ZO-1 at the plasma membrane. A similar colocalization pattern was observed for the Cx43ΔI382 and Cx43Δ378-382 mutants, which were translocated to the plasma membrane and formed functional gap junction channels. The wt and mutant Cx43 appeared to have similar turnover rates. However, the P2 and P3 phosphoisoforms of the Cx43 mutants were significantly reduced compared to Cx43wt. These studies indicated that the interaction of Cx43 with ZO-1 may contribute to the regulation of Cx43 phosphorylation.     

Protein Kinase Cδ-mediated Phosphorylation of Connexin43 Gap Junction Channels Causes Movement within Gap Junctions followed by Vesicle Internalization and Protein Degradation

Phosphorylation of gap junction proteins, connexins, plays a role in global signaling events involving kinases. Connexin43 (Cx43), a ubiquitous and important connexin, has several phosphorylation sites for specific kinases. We appended an imaging reporter tag for the activity of the δ isoform of protein kinase C (PKCδ) to the carboxyl terminus of Cx43. The FRET signal of this reporter is inversely related to the phosphorylation of serine 368 of Cx43. By activating PKC with the phorbol ester phorbol 12,13-dibutyrate (PDBu) or a natural stimulant, UTP, time lapse live cell imaging movies indicated phosphorylated Ser-368 Cx43 separated into discrete domains within gap junctions and was internalized in small vesicles, after which it was degraded by lysosomes and proteasomes. Mutation of Ser-368 to an Ala eliminated the response to PDBu and changes in phosphorylation of the reporter. A phosphatase inhibitor, calyculin A, does not change this pattern, indicating PKC phosphorylation causes degradation of Cx43 without dephosphorylation, which is in accordance with current hypotheses that cells control their intercellular communication by a fast and constant turnover of connexins, using phosphorylation as part of this mechanism.     

Focus on lens connexins

The lens is an avascular organ composed of an anterior epithelial cell layer and fiber cells that form the bulk of the organ. The lens expresses connexin43 (Cx43), connexin46 (Cx46) and connexin50 (Cx50). Epithelial Cx50 has critical roles in cell proliferation and differentiation, likely involving growth factor-dependent signaling pathways. Both Cx46 and Cx50 are crucial for lens transparency; mutations in their genes have been linked to congenital and age-related cataracts. Congenital cataract-associated connexin mutants can affect protein trafficking, stability and/or function, and the functional effects may differ between gap junction channels and hemichannels. Dominantly inherited cataracts may result from effects of the connexin mutant on its wild type isotype, the other co-expressed wild type connexin and/or its interaction with other cellular components.