Dataset Information

Phosphorylation-Dependent Intra-Domain Interaction of the Cx37 Carboxyl-Terminus Controls Cell Survival.

ABSTRACT: Differential phosphorylation of the carboxyl-terminus of connexin 37 (Cx37-CT) regulates phenotypic switching between cell growth phenotypes (cell death, cell cycle arrest, proliferation). The specific phosphorylation events in the Cx37-CT that are necessary for these growth regulatory effects are currently unknown. Through the combined use of deletion and site specific (de)phospho-mimetic Cx37-CT mutants, our data suggest a phosphorylation-dependent interaction between the mid-tail (aa 273?317) and end-tail (aa 318?333) portions of the Cx37-CT that regulates cell survival. As detected by mass spectrometry, Cx37 was phosphorylated at serines 275, 321, and 328; phosphomimetic mutations of these sites resulted in cell death when expressed in rat insulinoma cells. Alanine substitution at S328, but not at S275 or S321, also triggered cell death. Cx37-S275D uniquely induced the death of only low density, non-contact forming cells, but neither hemichannel open probability nor channel conductance distinguished death-inducing mutants. As channel function is necessary for cell death, together the data suggest that the phosphorylation state of the Cx37-CT controls an intra-domain interaction within the CT that modifies channel function and induces cell death.

SUBMITTER: Jacobsen NL

PROVIDER: S-EPMC6406260 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Phosphorylation-Dependent Intra-Domain Interaction of the Cx37 Carboxyl-Terminus Controls Cell Survival.

Jacobsen Nicole L NL Pontifex Tasha K TK Langlais Paul R PR Burt Janis M JM

Cancers 20190206 2

Differential phosphorylation of the carboxyl-terminus of connexin 37 (Cx37-CT) regulates phenotypic switching between cell growth phenotypes (cell death, cell cycle arrest, proliferation). The specific phosphorylation events in the Cx37-CT that are necessary for these growth regulatory effects are currently unknown. Through the combined use of deletion and site specific (de)phospho-mimetic Cx37-CT mutants, our data suggest a phosphorylation-dependent interaction between the mid-tail (aa 273⁻317) ...[more]

PMID: 30736283

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Project description:Background ? Carboxyl terminus 1 (?CT1) is a 25-amino acid therapeutic peptide incorporating the zonula occludens-1 (ZO-1)-binding domain of connexin 43 (Cx43) that is currently in phase 3 clinical testing on chronic wounds. In mice, we reported that ?CT1 reduced arrhythmias after cardiac injury, accompanied by increases in protein kinase C? phosphorylation of Cx43 at serine 368. Herein, we characterize detailed molecular mode of action of ?CT1 in mitigating cardiac ischemia-reperfusion injury. Methods and Results To study ?CT1-mediated increases in phosphorylation of Cx43 at serine 368, we undertook mass spectrometry of protein kinase C? phosphorylation assay reactants. This indicated potential interaction between negatively charged residues in the ?CT1 Asp-Asp-Leu-Glu-Iso sequence and lysines (Lys345, Lys346) in an ?-helical sequence (helix 2) within the Cx43-CT. In silico modeling provided further support for this interaction, indicating that ?CT1 may interact with both Cx43 and ZO-1. Using surface plasmon resonance, thermal shift, and phosphorylation assays, we characterized a series of ?CT1 variants, identifying peptides that interacted with either ZO-1-postsynaptic density-95/disks large/zonula occludens-1 2 or Cx43-CT, but with limited or no ability to bind both molecules. Only peptides competent to interact with Cx43-CT, but not ZO-1-postsynaptic density-95/disks large/zonula occludens-1 2 alone, prompted increased pS368 phosphorylation. Moreover, in an ex vivo mouse model of ischemia-reperfusion injury, preischemic infusion only with those peptides competent to bind Cx43 preserved ventricular function after ischemia-reperfusion. Interestingly, a short 9-amino acid variant of ?CT1 (?CT11) demonstrated potent cardioprotective effects when infused either before or after ischemic injury. Conclusions Interaction of ?CT1 with the Cx43, but not ZO-1, is correlated with cardioprotection. Pharmacophores targeting Cx43-CT could provide a translational approach to preserving heart function after ischemic injury.

Dataset Information

Phosphorylation-Dependent Intra-Domain Interaction of the Cx37 Carboxyl-Terminus Controls Cell Survival.

Publications

Phosphorylation-Dependent Intra-Domain Interaction of the Cx37 Carboxyl-Terminus Controls Cell Survival.

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