Alan F. Lau

Connexins: Mechanisms regulating protein levels and intercellular communication

Intercellular communication can occur through gap junction channels, which are comprised of connexin proteins. Therefore, levels of connexins can directly correlate with gap junctional intercellular communication. Because gap junctions have a critical role in maintaining cellular homeostasis, the regulation of connexin protein levels is important. In the connexin life cycle, connexin protein levels can be modified through differential gene transcription or altered through trafficking and degradation mechanisms. More recently, significant attention has been directed to the pathways that cells utilize to increase or decrease connexin levels and thus indirectly, gap junctional communication. Here, we review the studies revealing the mechanisms that affect connexin protein levels and gap junctional intercellular communication.

Regulation of Connexin-43 Gap Junctional Intercellular Communication by Mitogen-activated Protein Kinase

Activation of the Ras/Raf/mitogen-activated protein kinase kinase/mitogen-activated protein (MAP) kinase signaling cascade is initiated by activation of growth factor receptors and regulates many cellular events, including cell cycle control. Our previous studies suggested that the connexin-43 gap junction protein may be a target of activated MAP kinase and that MAP kinase may regulate connexin-43 function. We identified the sites of MAP kinase phosphorylation in in vitro studies as the consensus MAP kinase recognition sites in the cytoplasmic carboxyl tail of connexin-43, Ser255, Ser279, and Ser282. In this study, we demonstrate that activation of MAP kinase by ligand-induced activation of the epidermal growth factor (EGF) or lysophosphatidic acid receptors or by pervanadate-induced inhibition of tyrosine phosphatases results in increased phosphorylation on connexin-43. EGF and lysophosphatidic acid-induced phosphorylation on connexin-43 and the down-regulation of gap junctional communication in EGF-treated cells were blocked by a specific mitogen-activated protein kinase kinase inhibitor (PD98059) that prevented activation of MAP kinase. These studies confirm that connexin-43 is a MAP kinase substrate in vivo and that phosphorylation on Ser255, Ser279, and/or Ser282 initiates the down-regulation of gap junctional communication. Studies with connexin-43 mutants suggest that MAP kinase phosphorylation at one or more of the tandem Ser279/Ser282 sites is sufficient to disrupt gap junctional intercellular communication.

v-Src phosphorylation of connexin 43 on Tyr247 and Tyr265 disrupts gap junctional communication

The mechanism by which v-Src disrupts connexin (Cx)43 intercellular gap junctional communication (GJC) is not clear. In this study, we determined that Tyr247 (Y247) and the previously identified Tyr265 (Y265) site of Cx43 were the primary phosphorylation targets for activated Src in vitro. We established an in vivo experimental system by stably expressing v-Src and wild-type (wt) Cx43, or Y247F, Y265F, or Y247F/Y265F Cx43 mutants in a Cx43 knockout mouse cell line. Wt and mutant Cx43 localized to the plasma membrane in the absence or presence of v-Src. When coexpressed with v-Src, the Y247F, Y265F, and Y247F/Y265F Cx43 mutants exhibited significantly reduced levels of tyrosine phosphorylation compared with wt Cx43, indicating that Y247 and Y265 were phosphorylation targets of v-Src in vivo. Most importantly, GJC established by the Y247F, Y265F, and Y247F/Y265F Cx43 mutants was resistant to disruption by v-Src. Furthermore, we did not find evidence for a role for mitogen-activated protein kinase in mediating the disruption of GJC by v-Src. We conclude that phosphorylation on Y247 and Y265 of Cx43 is responsible for disrupting GJC in these mammalian cells expressing v-Src.

Regulation of connexin43 function by activated tyrosine protein kinases

Gap junctions are specialized membrane structures that are involved in the normal functioning of numerous mammalian tissues and implicated in several human disease processes. This mini-review focuses on the regulation of gap junctions through phosphorylation of connexin43 induced by the v-Src or epidermal growth factor receptor tyrosine kinases. These tyrosine kinases markedly disrupt gap junctional communication in mammalian cells. Here, we describe work correlating the alteration of connexin43 function with the ability of the v-Src tyrosine kinase to phosphorylate connexin43 directly on two distinct tyrosine sites in mammalian cells (Y247 and Y265). We also present evidence that proline-rich regions and phosphotyrosine sites of connexin43 may mediate interactions with the SH3 and SH2 domains of v-Src. In contrast to v-Src, the activated epidermal growth factor receptor acts indirectly through activated MAP kinase which may stimulate phosphorylation of connexin43 exclusively on serine. This phosphorylation event is complex because MAP kinase phosphorylates three serine sites in connexin43 (S255, S279, and S282). These findings suggest novel interactions between connexin43, the v-Src tyrosine kinase, and activated MAP kinase that set the stage for future investigations into the regulation of gap junctions by protein phosphorylation.

Activation of Akt, Not Connexin 43 Protein Ubiquitination, Regulates Gap Junction Stability

Background: The gap junctional protein connexin 43 (Cx43) has a 1–3 h half-life, and both the proteasome and lysosome have been implicated in Cx43 turnover. Results: Use of lysine to arginine mutants and kinase inhibitors showed that Akt activation stabilizes gap junctions. Conclusion: Akt phosphorylation of Cx43 stabilizes gap junctions. Significance: Linkage of Akt to gap junction stability may mechanistically explain their cell survival/growth roles. The pore-forming gap junctional protein connexin 43 (Cx43) has a short (1–3 h) half-life in cells in tissue culture and in whole tissues. Although critical for cellular function in all tissues, the process of gap junction turnover is not well understood because treatment of cells with a proteasomal inhibitor results in larger gap junctions but little change in total Cx43 protein whereas lysosomal inhibitors increase total, mostly nonjunctional Cx43. To better understand turnover and identify potential sites of Cx43 ubiquitination, we prepared constructs of Cx43 with different lysines converted to arginines. However, when transfected into cells, a mutant version of Cx43 with all lysines converted to arginines behaved similarly to wild type in the presence of proteasomal and lysosomal inhibitors, indicating that ubiquitination of Cx43 did not appear to be playing a role in gap junction stability. Through the use of inhibitors and dominant negative constructs, we found that Akt (protein kinase B) activity controlled gap junction stability and was necessary to form larger stable gap junctions. Akt activation was increased upon proteasomal inhibition and resulted in phosphorylation of Cx43 at Akt phosphorylation consensus sites. Thus, we conclude that Cx43 ubiquitination is not necessary for the regulation of Cx43 turnover; rather, Akt activity, probably through direct phosphorylation of Cx43, controls gap junction stability. This linkage of a kinase involved in controlling cell survival and growth to gap junction stability may mechanistically explain how gap junctions and Akt play similar regulatory roles.

Akt phosphorylates Connexin43 on Ser373, a "mode-1" binding site for 14-3-3.

Connexin43 (Cx43) is a membrane-spanning protein that forms channels that bridge the gap between adjacent cells and this allows for the intercellular exchange of information. Cx43 is regulated by phosphorylation and by interacting proteins. “Mode-1” interaction with 14-3-3 requires phosphorylation of Ser373 on Cx43 (). Akt phosphorylates and targets a number of proteins to interactions with 14-3-3. Here we demonstrate that Akt phosphorylates Cx43 on Ser373 and Ser369; antibodies recognizing Akt-phosphorylated sites or phospho-Ser “mode-1” 14-3-3-binding sites recognize a protein from EGF-treated cells that migrates as Cx43, and GST-14-3-3 binds to Cx43 phosphorylated endogenously in EGF-treated cells. Confocal microscopy supports the co-localization of Cx43 with Akt and with 14-3-3 at the outer edges of gap junctional plaques. These data suggest that Akt could target Cx43 to an interaction with 14-3-3 that may play a role in the forward trafficking of Cx43 multimers and/or their incorporation into existing gap junctional plaques.

In vivo association of pp60v-src and the gap-junction protein connexin 43 in v-src–transformed fibroblasts

v‐src–transformed fibroblasts have significantly reduced levels of gap junction–mediated intercellular communication. This observed downregulation of cellular communication has been associated with tyrosine phosphorylation of the gap‐junction protein connexin 43 (Cx43). Previously, we demonstrated that purified, kinase‐active pp60src phosphorylates Cx43 in vitro (J Biol Chem 1995;270:12751–12761). More recently, we reported that this association is mediated by the SH2 and SH3 domains of pp60v‐src (J Biol Chem 1997;272:22824–22831). In this report, we present in vivo evidence supporting the hypothesis that Cx43 is an endogenous substrate of pp60v‐src in v‐src–transformed fibroblasts. Cytological localization studies with confocal microscopy demonstrated that pp60v‐src and Cx43 were partially co‐localized in regions of the plasma membrane. Cx43 and pp60v‐src co‐immunoprecipitated from v‐src–transformed fibroblasts, indicating that the two proteins were associated, and form a stable complex. Furthermore, pp60v‐src could phosphorylate co‐immunoprecipitated Cx43 in an immune‐complex kinase assay. Two‐dimensional phosphopeptide mapping of the immune‐complexed Cx43 phosphorylated in vitro demonstrated that the sites of tyrosine phosphorylation were consistent with previously identified sites of pp60v‐src phosphorylation. These results provide additional in vivo evidence that Cx43 is a direct substrate of pp60v‐src in v‐src–transformed fibroblasts. The ability of pp60v‐src to alter gap junction–mediated cellular communication may serve as one mechanism by which pp60v‐src initiates and/or maintains aspects of cellular transformation. Mol. Carcinog. 25:187–195, 1999.

A Novel Connexin43-interacting Protein, CIP75, Which Belongs to the UbL-UBA Protein Family, Regulates the Turnover of Connexin43

The degradation of connexin43 (Cx43) has been reported to involve both lysosomal and proteasomal degradation pathways; however, very little is known about the mechanisms regulating these Cx43 degradation pathways. Using yeast two-hybrid, glutathione S-transferase pull-down, and co-immunoprecipitation approaches, we have identified a novel Cx43-interacting protein of ∼75 kDa, CIP75. Laser confocal microscopy showed that CIP75 is located primarily at the endoplasmic reticulum, as indicated by the calnexin marker, with Cx43 co-localization in this perinuclear region. CIP75 belongs to the UbL (ubiquitin-like)-UBA (ubiquitin-associated) domain-containing protein family with a N-terminal UbL domain and a C-terminal UBA domain. The UBA domain of CIP75 is the main element mediating the interaction with Cx43, whereas the CIP75-interacting region in Cx43 resides in the PY motif and multiphosphorylation sites located between Lys264 and Asn302. Interestingly, the UbL domain interacts with the S2/RPN1 and S5a/RPN10 protein subunits of the regulatory 19 S proteasome cap subunit of the 26 S proteasome complex. Overexpression experiments suggested that CIP75 is involved in the turnover of Cx43 as measured by a significant stimulation of Cx43 degradation and reduction in its half-life with the opposite effects on Cx43 degradation observed in small interference RNA knockdown experiments.

A 17mer Peptide Interferes With Acidification-Induced Uncoupling of Connexin43

Structure/function analysis shows that the carboxyl terminal (CT) domain of connexin43 (Cx43) is essential for the chemical regulation of cell-cell communication. Of particular interest is the region between amino acids 260 and 300. Structural preservation of this region is essential for acidification-induced uncoupling (ie, pH gating). In this study, we report data showing that a 17mer peptide of the same sequence as amino acids 271 to 287 of Cx43 (CSSPTAPLSPMSPPGYK) can prevent pH gating of Cx43-expressing oocytes. Experiments were carried out in pairs of Xenopus oocytes previously injected with connexin38 antisense and expressing wild-type Cx43. Junctional conductance was measured electrophysiologically. pHi was determined from the light emission of the proton-sensitive dye dextran-seminaphthorhodafluor. Intracellular acidification was induced by superfusion with a bicarbonate-buffered solution gassed with a progressively increasing concentration of CO2. Injection of water alone into both oocytes of a Cx43-expressing pair or injection of a peptide from region 321 to 337 of Cx43 did not modify pH sensitivity. However, injection of a polypeptide corresponding to amino acids 241 to 382 of Cx43 interfered with the ability of gap junctions to close on acidification. Similar results were obtained when a 17mer peptide (region 271 to 287) was injected into both oocytes of the pair. Normal Cx43 pH gating was observed if (1) the amino acid sequence of the 17mer peptide was scrambled or (2) the N and the C ends of the 17mer peptide were not included in the sequence. This is the first demonstration of a molecule that can interfere with the chemical regulation of connexin channels in a cell pair. The data may lead to the development of small molecules that can be used in Cx43-expressing multicellular preparations to study the role of gap junction regulation in normal as well as diseased states.

Ubiquitin-independent Proteasomal Degradation of Endoplasmic Reticulum-localized Connexin43 Mediated by CIP75

Connexin43 (Cx43) is a transmembrane protein that forms gap junction channels. Regulation of Cx43 turnover is one mechanism to control the level of intercellular communication that occurs through gap junction channels. Proteasomal degradation of Cx43 is regulated in part through CIP75, a ubiquitin-like and ubiquitin-associated domain containing protein. CIP75 interacts with endoplasmic reticulum-localized Cx43, as demonstrated through co-immunoprecipitation and immunofluorescence microscopy experiments. CIP75 also binds to free monoubiquitin and lysine 48-linked tetraubiquitin chains in vitro and binds to ubiquitinated proteins in cellular lysates. However, analysis of Cx43 that immunoprecipitated with CIP75 demonstrated that the Cx43 associated with CIP75 was not ubiquitinated, and a mutant form of Cx43 that lacked lysines capable of ubiquitination retained the capacity to interact with CIP75. These results suggest that although CIP75 can interact with ubiquitinated cellular proteins, its interaction with Cx43 and stimulation of Cx43 proteasomal degradation does not require the ubiquitination of Cx43.