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

TLDR

The Ras/Raf/MAPK signaling cascade, activated by growth factor receptors, regulates cellular events such as cell cycle control and has been implicated as a regulator of the connexin‑43 gap junction protein. MAP kinase phosphorylates connexin‑43 at Ser255, Ser279, and Ser282, and ligand‑induced MAPK activation increases this phosphorylation, which down‑regulates gap‑junction communication—a process blocked by the MEK inhibitor PD98059 and shown to be sufficient when Ser279/Ser282 are phosphorylated.

Abstract

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 <i>in vitro</i> 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 <i>in vivo</i> 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.