Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and -deficient cell lines.

Abstract

Abstract. Connexin43 is a member of the highly homologous connexin family of gap junction proteins. We have studied how connexin monomers are assembled into functional gap junction plaques by examining the biosynthesis of connexin43 in cell types that differ greatly in their ability to form functional gap junctions. Using a combination of metabolic radiolabeling and immunoprecipitation, we have shown that connexin43 is synthesized in gap junctional communication-competent cells as a 42-kD protein that is efficiently converted to a ,'-,46-kD species (connexin43-P2) by the posttranslational addition of phosphate. Surprisingly, certain cell lines severely deficient in gap junctional communication and known cell-cell adhesion molecules (S180 and L929 cells) also expressed 42-kD connexin43. Connexin43 in these communication-deficient cell lines was not, however, phosphorylated to the P2 form. Conversion of S180 cells to a communication-competent phenotype by transfection with a cDNA encoding the cell-cell adhesion molecule L-CAM induced phosphorylation of connexin43 to the P2 form; conversely, blocking junctional communication in ordinarily communicationcompetent cells inhibited connexin43-P2 formation. Immunohistochemical localization studies indicated that only communication-competent cells accumulated connexin43 in visible gap junction plaques. Together, these results establish a strong correlation between the ability of cells to process connexin43 to the P2 form and to produce functional gap junctions. Connexin43 phosphorylation may therefore play a functional role in gap junction assembly and/or activity.

Actions of HSVtk and connexin43 gene delivery on gap junctional communication and drug sensitization in hepatocellular carcinoma

“…26,27 Hepa1-6 cells express Cx43, but, in general, at low levels as reported by Wilgembus et al 25 and confirmed by the present study. Immunoblot assay was carried out to examine if the HSVtk could induce some posttranslational changes of Cx43 protein.…”

“…The decrease in gap junctional communication has been suggested as a common characteristic of cancer cells. 40,41 However, it was demonstrated that some transformed cells such as the mouse cell lines S180 and L929 express high gap junctional intercellular communication (GJIC), 26 indicating that the loss of GJIC is not necessarily associated with neoplastic transformation. However, even if the GJIC is present in these cells, they remain without functionality.…”

“…22,[26][27][28] This phosphorylation has been widely suggested to be linked to both gap junction assembly and gating events, and can be the consequence of a protein kinase. 29,30 To determine if the elevated level of the HSVtk can act on the phosphorylated/ dephosphorylated status of connexin43 protein, we have transfected Hepa1-6 cells with different plasmids carrying the HSVtk gene.…”

Key Connexin 43 Phosphorylation Events Regulate the Gap Junction Life Cycle

“…Cx43 electrophoreses as multiple isoforms when analyzed by SDS-PAGE, including a faster migrating form that includes non-phosphorylated (P0 or NP) Cx43, and at least two slower migrating forms, commonly termed P1 and P2 (Crow et al, 1990;Musil et al, 1990). Pulse chase analysis indicated that the Cx43 isoforms progress from P0 to P1 to P2 and the P2 isoform is associated with gap junctional structures (Musil & Goodenough, 1991).…”

The role of cellular- and prodrug-associated factors in the bystander effect induced by the Varicella zoster and Herpes simplex viral thymidine kinases in suicide gene therapy

“…The 46 -kDa band corresponded to the serine -diphosphorylated form, which is known to be the most active species of the functional connexon. 43 To check if the Cx43 expressed in the transduced cells adopted the proper surface topology, immunolocalization studies were performed ( Fig 5 ) using wild -type 9L cells and MDA -MB -435 cells as positive and negative controls, respectively (Fig 5A and B ). Spots of Cx43 were observed at the periphery of the breast -transduced cells ( Fig 5C and D ) .…”