Casein Kinase 1-delta Dynamically Modulates Connexin-43 and Gap Junction Intercellular Communication in Homeostatic and Stressed Corneal Endothelium

Abstract

Purpose: : Corneal endothelial (CE) cells communicate extensively with each other through connexin-composed gap junctions. In response to various stress exposures, many cells rapidly alter gap junction intercellular communication (GJIC) primarily through kinase-mediated phosphorylation of connexin. Casein kinase 1-delta (CK1Δ), a proposed Cx43 kinase, modifies the stability and function of its substrates through phosphorylation. We sought to determine how CK1Δ regulates CE Cx43 and GJIC in both homeostatic and stressed conditions.

Methods: : Primary bovine CE cells were used to examine the relationship between CK1Δ and Cx43. CK1Δ expression was evaluated by PCR, Western, and immunofluorescent staining (IF). CK1Δ:Cx43 interaction was assessed by co-immunoprecipitation (Co-IP) and IF. In vitro kinase assays with GST-Cx43 C-terminus were performed with recombinant CK1Δ ( rCK1Δ ). An antibody specific for Cx43 phosphorylated at amino acids 325, 328 & 330 (anti-pCx43) was generated and validated. The effect of CK1Δ inhibition on Cx43 and GJIC was tested with inhibitor PF670462. Genotoxic stressors were used to determine changes in CK1Δ and its role in modifying Cx43 and GJIC.

Results: : Bovine CE cells expressed CK1Δ, which was localized in nuclear, peri-nuclear, and membrane compartments. Co-IP and IF demonstrated CK1Δ interaction with Cx43. rCK1Δ phosphorylated the C-terminus of Cx43 which created an epitope recognized by anti-pCx43 antibody. Chemical inhibition of CK1Δ affected the stability of Cx43, prevented its accumulation into gap junction plaques, and altered GJIC. Genotoxic stress increased levels of CK1Δ and pCx43_325/328/330, events which were affected by inhibition of CK1Δ.

Conclusions: : CK1Δ plays a role in regulation of CE Cx43 and GJIC in both homeostasis and stressed states. Alterations in CK1Δ-mediated phosphorylation of Cx43 affect Cx43 stability, localization, and function. Stressed CE cells appear to enhance CK1Δ activity to phosphorylate Cx43 which may promote cell survival and protection against genotoxic insults.

Keywords: cornea: endothelium • gap junctions/coupling • stress response