Phosphorylation of connexin43 gap junction protein in uninfected and Rous sarcoma virus-transformed mammalian fibroblasts.
Gap junctions are membrane channels that permit the interchange of ions and other low-molecular-weight molecules between adjacent cells. Rous sarcoma virus (RSV)-induced transformation is marked by an early and profound disruption of gap-junctional communication, suggesting that these membrane structures may serve as sites of pp60v-src action. We have begun an investigation of this possibility by identifying and characterizing putative proteins involved in junctional communication in fibroblasts, the major cell type currently used to study RSV-induced transformation. We found that uninfected mammalian fibroblasts do not appear to contain RNA or protein related to connexin32, the major rat liver gap junction protein. In contrast, vole and mouse fibroblasts contained a homologous 3.0-kilobase RNA similar in size to the heart tissue RNA encoding the gap junction protein, connexin43. Anti-connexin43 peptide antisera specifically reacted with three proteins of approximately 43, 45 and 47 kilodaltons (kDa) from communicating fibroblasts. Gap junctions of heart cells contained predominantly 45- and 47-kDa species similar to those found in fibroblasts. Uninfected fibroblast 45- and 47-kDa proteins were phosphorylated on serine residues. Phosphatase digestions of 45- and 47-kDa proteins and pulse-chase labeling studies indicated that these proteins represented phosphorylated forms of the 43-kDa protein. Phosphorylation of connexin protein appeared to occur shortly after synthesis, followed by an equally rapid dephosphorylation. In comparison with these results, connexin43 protein in RSV-transformed fibroblasts contained both phosphotyrosine and phosphoserine. Thus, the presence of phosphotyrosine in connexin43 correlates with the loss of gap-junctional communication observed in RSV-transformed fibroblasts.
Crow
D S
DS
Cancer Research Center, University of Hawaii, Honolulu 96813.
Beyer
E C
EC
Paul
D L
DL
Kobe
S S
SS
Lau
A F
AF
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003411:Arvicolinae; D001358:Avian Sarcoma Viruses / Q000235:genetics*; D015152:Blotting, Northern; D002450:Cell Communication; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D017630:Connexins; D015342:DNA Probes; D005347:Fibroblasts / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D007106:Immune Sera; D007365:Intercellular Junctions / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009206:Myocardium / Q000378:metabolism; D010766:Phosphorylation; D012313:RNA / Q000235:genetics
D000596:Amino Acids; D017630:Connexins; D015342:DNA Probes; D007106:Immune Sera; D008565:Membrane Proteins; D012313:RNA