Growth-dependent subcellular redistribution of protein kinase C in cultured porcine aortic endothelial cells.
We have previously observed major differences in the phosphorylation of membrane proteins in sparse, proliferating versus confluent, quiescent pig aortic endothelial cells (EC) (Kazlauskas and DiCorleto, 1987). In the present study we examined whether EC growth state can influence the activity of a specific phosphorylating enzyme, protein kinase C (PKC) in cytosolic and membrane fractions of pig aortic EC. Levels of PKC were measured using two methods: 1) Ca2+ and phospholipid-dependent phosphorylation of exogenous histones using gamma-labeled [32P]ATP, and 2) [3H]phorbol-12,13-dibutyrate (PDBu) binding activity. The total amount of PKC activity in the quiescent versus proliferating cells was similar but the percentage of PKC activity in the membrane fraction correlated with the proliferative index of the cells: confluent, quiescent cultures exhibited a majority of PKC activity in the cytosolic fraction (67%), whereas sparse, proliferating cultures contained principally membrane-bound PKC (70%). We also examined the role of PKC in the mitogenic response of pig aortic EC to fetal calf serum. Following serum stimulation of sparse, serum-deprived pig aortic EC, PKC activity redistributed from the cytosolic to the membrane fraction in a rapid process that correlated with subsequent DNA synthesis. A potent activator of PKC, 12-O-tetradecanoylphorbol-13-acetate (TPA), induced a minimal mitogenic response in pig aortic EC when added alone but acted synergistically with low concentrations of fetal calf serum to greatly stimulate DNA synthesis. Furthermore, pig aortic EC treated with TPA for 24 h to down-regulate PKC exhibited only 25% of the serum-stimulated mitogenic activity of control cultures. These results suggest a role for PKC activation and translocation in the proliferation of pig aortic EC.
Uratsuji
Y
Y
Department of Brain and Vascular Research, Cleveland Clinic Research Institute, Ohio 44195.
DiCorleto
P E
PE
D000818:Animals; D002455:Cell Division; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D004789:Enzyme Activation; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D011493:Protein Kinase C / Q000032:analysis*; D013347:Subcellular Fractions / Q000201:enzymology*; D013552:Swine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate