ON THE MECHANISMS OF CELL UNCOUPLING INDUCED BY A TUMOR PROMOTER PHORBOL ESTER IN CLONE-9 CELLS, A RAT-LIVER EPITHELIAL-CELL LINE

It is known that in Clone 9 (C9) cells, intercellular gap junctional c ommunication (IGJC) is rapidly blocked by the tumor promoter phorbol e ster, 12-O-tetradecanoylphorbol-13-acetate (TPA), but it recovers spon taneously a few hours later and becomes refractory to TPA (Yada et al. , J. Membr. Biol. 88, 217-232 (1985)). We now report that gap junction s between C9 cells contain at least two junctional proteins, connexin2 6 (Cx26) and connexin43 (Cx43), and that the TPA-induced changes in IG JC correlate temporally to changes in the state of phosphorylation of Cx43. The latter changes were prevented by inhibition of protein kinas e C. Phosphoamino acid analysis and two-dimensional tryptic peptide ma ps of P-32-labeled Cx43 showed that during the TPA-induced phosphoryla tion at least two of the phosphorylated forms of Cx43 were differentia lly phosphorylated in seryl residues as compared to control. TPA induc ed a drastic reduction in junctional conductance as well as a redistri bution of unitary gap junction channel event sizes seen in control cel ls. These changes were associated with retrieval of Cxs from the plasm a membrane. Reappearance of gap junctions formed by Cx43 but not by Cx 26 accounted for the spontaneous recovery in IGJC. It is proposed that gap junctions between C9 cells contain two types of channels each for med by Cx43 or Cx26 and that they are differentially affected during t he action of TPA.