Phosphorylation of the 27-kDa gap junction protein by protein kinase C in vitro and in rat hepatocytes.
We previously demonstrated that the 27-kDa major component protein in rat liver gap junctions was phosphorylated by protein kinase C in vitro (Takeda, A. et al. (1987) FEBS Lett. 210, 169-172). In this study, we examined this further and examined the phosphorylation of the 27-kDa gap junction protein in rat hepatocytes by metabolically labeling cells with [32P]orthophosphate and using a monoclonal antibody to immunoprecipitate the protein. The in vitro phosphorylation was inhibited by monoclonal antibodies recognizing the carboxyl- (C-)terminal domain of the 27-kDa protein. Protease digestion analysis revealed that phosphorylation occurred at the C-terminal domain. In rat hepatocytes, the phorbol esters, 12-O-tetradecanoylphorbol-13-acetate and phorbol-12,13-dibutyrate, stimulated the 27-kDa protein phosphorylation, whereas 4 alpha-phorbol-12,13-didecanoate did not. 1-Oleoyl-2-acetyl-sn-glycerol also stimulated the 27-kDa protein phosphorylation. In addition, norepinephrine stimulated the phosphorylation and pretreatment of hepatocytes with staurosporine, a potent inhibitor of protein kinase C, inhibited this stimulatory effect of norepinephrine. Both in vitro and in hepatocytes, analysis of chemical cleavage of the 27-kDa phosphoprotein revealed that phosphorylation occurred mainly at a 10-kDa fragment which the antibodies recognized. These results indicate that protein kinase C phosphorylates the 27-kDa gap junction protein, not only in vitro but also in hepatocytes, at the C-terminal domain of the protein.
Takeda
A
A
Department of Laboratory Medicine, Ehime University School of Medicine.
Saheki
S
S
Shimazu
T
T
Takeuchi
N
N
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D017630:Connexins; D004075:Diglycerides / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism*; D009638:Norepinephrine / Q000494:pharmacology; D010703:Phorbol Esters / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D013997:Time Factors
D000596:Amino Acids; D017630:Connexins; D004075:Diglycerides; D008565:Membrane Proteins; D010703:Phorbol Esters; D011493:Protein Kinase C; D009638:Norepinephrine