Akt Phosphorylates Connexin43 on Ser373, a “Mode-1” Binding Site for 14-3-3

Natural Products & Cancer Biology Program, Cancer Research Center, University of Hawaii at Manoa, Honolulu, Hawaii 96813, USA.
Cell Communication & Adhesion (Impact Factor: 1.52). 09/2007; 14(5):211-26. DOI: 10.1080/15419060701755958
Source: PubMed


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 (52.
Park D J, Freitas T A, Wallick C J, Guyette C V, Warn-Cramer B J. Molecular dynamics and in vitro analysis of Connexin43: A new 14-3-3 mode-1 interacting protein. Protein Sci 2006; 15: 2344–2355View all references). 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.

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    • "More recently, we showed that proteasomal inhibition increased gap junction stability through activation of Akt (Dunn et al., 2012). Because S373 has been described as a possible Akt kinase substrate (Park et al., 2007), we decided to create a phosphospecific antibody that reacts with Cx43 when it is phosphorylated at S373 (pS373). To examine Cx43 phosphorylation in cells, we used an MDCK cell line that was null for Cx43 in the parental line (Jordan et al., 1999), which upon stable transfection expresses Cx43 at nominal levels (MDCK43) and routinely forms functional gap junctions (Dunn et al., 2012). "
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    ABSTRACT: The proteins that form vertebrate gap junctions, the connexins, are highly regulated and have short (< 2 h) half-lives. Phosphorylation of connexin43 (Cx43) is generally known to affect gap junction assembly, channel gating and turnover. After finding dramatic effects on gap junctions with Akt inhibitors, we created an antibody specific for Cx43 phosphorylated on S373, a potential Akt substrate. We found S373 phosphorylation in cells and skin or heart almost exclusively in larger gap junctional structures that increased dramatically after wounding or hypoxia. We were able to mechanistically show that Akt-dependent S373 phosphorylation increases gap junction size and communication by completely eliminating interaction between Cx43 and ZO-1. Thus, phosphorylation on S373 acts as a molecular "switch" to rapidly increase gap junctional communication potentially leading to initiation of activation and migration of keratinocytes or ischemic injury response in skin and heart, respectively.
    Preview · Article · Nov 2013 · Journal of Cell Science
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    • "In addition, a recent study found that Akt activation is crucial to disrupt dye transfer in v-Src transformed cells, but inhibition of Akt only recovered the metabolic coupling to 60% (Ito et al., 2010). Since Akt may cause phosphorylation of Ser369 and Ser373 in Cx43 (Park et al., 2007; for details see section on Ser364, Ser365, Ser368, Ser369, Ser372, and Ser373), this indicates that Src kinases controls several intracellular signaling pathways, and thereby affects the phosphorylation of both serine and tyrosine residues in the C-terminal tail of Cx43 with different effects on electrical and metabolic coupling. Together, these studies outlines that the control of gap junction coupling by Src kinases is extremely complex; It depends of a combination of direct phosphorylation of both Tyr247 and Tyr265, as well as interplay with several intracellular signaling pathways controlling Cx43 serine phosphorylation. "
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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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    • "src, via SH2 and 3 domains (Warn- Cramer et al. 1996)], adapter [e.g. ZO-1via PDZ 1(Giepmans and Moolenaar 1998; Toyofuku et al. 1998); 14-3-3 protein (Park et al., 2007)], and cytoskeletal elements [e.g. tubulin (Giepmans et al. 2001)] may also be relevant to longer term regulation of coupling by growth factors. "
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    ABSTRACT: Attenuation in gap junctional coupling has consistently been associated with induction of rapid or synchronous cell division in normal and pathological conditions. In the case of the v-src oncogene, gating of Cx43 gap junction channels has been linked to both direct phosphorylation of tyrosines (Y247 and 265) and phosphorylation of the serine targets of Erk1/2 (S255, 279 and 282) on the cytoplasmic C-terminal domain of Cx43. However, only the latter has been associated with acute, rather than chronic, gating of the channels immediately after v-src expression, a process that is mediated through a "ball-and-chain" mechanism. In this study we show that, while ERK1/2 is necessary for acute closure of gap junction channels, it is not sufficient. Rather, multiple pathways converge to regulate Cx43 coupling in response to expression of v-src, including parallel signaling through PKC and MEK1/2, with additional positive and negative regulatory effects mediated by PI3 kinase, distinguished by the involvement of Akt.
    Full-text · Article · Sep 2012 · Journal of Membrane Biology
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