Interplay between calcium, diacylglycerol, and phosphorylation in the spatial and temporal regulation of PKCalpha-GFP.
The function of protein kinase C (PKC) is closely regulated by its subcellular localization. We expressed PKCalpha fused to green fluorescent protein (PKCalpha-GFP) and examined its translocation in living and permeabilized cells of the human parotid cell line, HSY-EB. ATP induced an oscillatory translocation of PKCalpha-GFP to and from the plasma membrane that paralleled the appearance of repetitive Ca2+ spikes. Staurosporine attenuated the relocation of PKCalpha-GFP to the cytosol and caused a stepwise accumulation of PKCalpha-GFP at the plasma membrane during ATP stimulation. Diacylglycerol enhanced the amplitude and duration of the ATP-induced oscillatory translocation of PKCalpha-GFP. Ionomycin induced a transient translocation of PKCalpha-GFP to the plasma membrane despite the continuous elevation of cytosolic Ca2+. The ionomycin-induced transient translocation of PKCalpha-GFP was prolonged by staurosporine, diacylglycerol, and phorbol myristate acetate. Experiments using permeabilized cells showed that staurosporine or the elimination of ATP and Mg2+ decreases the rate of dissociation of PKCalpha-GFP from the membrane. Diacylglycerol slowed the dissociation of PKCalpha-GFP from the membrane regardless of the Ca2+ concentration. The effect of diacylglycerol was attenuated by ATP plus Mg2+ at low concentrations of Ca2+ (<500 nm) but not at high concentrations of Ca2+ (>1000 nm). These data suggest a complex interplay between Ca2+, diacylglycerol, and phosphorylation in the regulation of the membrane binding of PKCalpha.
Tanimura
Akihiko
A
Department of Dental Pharmacology, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan. tanimura@hoku-iryo-u.ac.jp
Nezu
Akihiro
A
Morita
Takao
T
Hashimoto
Noboru
N
Tojyo
Yosuke
Y
D000255:Adenosine Triphosphate / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002273:Carcinogens; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004075:Diglycerides / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012503:Saponins / Q000494:pharmacology; D019311:Staurosporine / Q000378:metabolism* / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D002273:Carcinogens; D004075:Diglycerides; D004791:Enzyme Inhibitors; D007476:Ionophores; D007527:Isoenzymes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D012503:Saponins; D049452:Green Fluorescent Proteins; D015759:Ionomycin; D000255:Adenosine Triphosphate; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D019311:Staurosporine; D008274:Magnesium; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium