GAP JUNCTION TURNOVER, INTRACELLULAR TRAFFICKING, AND PHOSPHORYLATIONOF CONNEXIN43 IN BREFELDIN-A-TREATED RAT MAMMARY-TUMOR CELLS

Intercellular gap junction channels are thought to form when oligomers of connexins from one cell (connexons) register and pair with connexo ns from a neighboring cell en route to forming tightly packed arrays ( plaques). In the current study we used the rat mammary BICR-M1R(k) tum or cell line to examine the trafficking, maturation, and kinetics of c onnexin43 (Cx43). Cx43 was conclusively shown to reside in the Golgi a pparatus in addition to sites of cell-cell apposition in these cells a nd in normal rat kidney cells. Brefeldin A (BFA) blocked Cx43 traffick ing to the surface of the mammary cells and also prevented phosphoryla tion of the 42-kD form of Cx43 to 44- and 46-kD species. However, phos phorylation of Cx43 occurred in the presence of BFA while it was still a resident of the ER or Golgi apparatus yielding a 43-kD form of Cx43 . Moreover, the 42- and 43-kD forms of Cx43 trapped in the ER/Golgi co mpartment were available for gap junction assembly upon the removal of BFA. Mammary cells treated with BFA for 6 h lost preexisting gap junc tion ''plaques,'' as well as the 44- and 46-kD forms of Cx43 and funct ional coupling. These events were reversible 1 h after the removal of BFA and not dependent on protein synthesis. In summary, we provide str ong evidence that in BICR-M1R(k) tumor cells: (a) Cx43 is transiently phosphorylated in the ER/Golgi apparatus, (b) Cx43 trapped in the ER/G olgi compartment is not subject to rapid degradation and is available for the assembly of new gap junction channels upon the removal of BFA, (c) the rapid turnover of gap junction plaques is correlated with the loss of the 44- and 46-kD forms of Cx43.