Differential localization of protein kinase C delta by phorbol esters and related compounds using a fusion protein with green fluorescent protein.
Enzyme localization often plays a controlling role in determining its activity and specificity. Protein kinase C (PKC) has long been known to translocate in response to physiological stimuli as well as to exogenous ligands such as the phorbol esters. We report here that different phorbol derivatives and related ligands, selected for differences in chemical structure and profile of biological activity, induce distinct patterns of redistribution of PKC delta. Localization of a PKC delta-green fluorescent protein (GFP) fusion construct was monitored in living Chinese hamster ovary cells as a function of ligand, concentration, and time using confocal laser scanning microscopy. delta-PKC-GFP was expressed predominantly in the cytoplasm, with some in the nucleus and perinuclear region. Phorbol 12-myristate 13-acetate (PMA) induced plasma membrane translocation followed by slower nuclear membrane translocation. As the concentration of PMA increased, the proportion of nuclear to plasma membrane localization increased markedly. In contrast to PMA, bryostatin 1, a unique activator of PKC that induces a subset of PMA-mediated responses while antagonizing others, at all doses induced almost exclusively nuclear membrane translocation. Like PMA, the complete tumor promoter 12-deoxyphorbol 13-tetradecanoate induced plasma membrane and slower nuclear membrane translocation, whereas the inhibitor of tumor promotion 12-deoxyphorbol 13-phenylacetate, which differs only in its side chain, induced a distinctive distribution of PKC delta-GFP. Finally, the novel constrained diacylglycerol derivative B8-DL-B8 induced a slow Golgi localization. We speculate that differential control of PKC delta localization may provide an interesting strategy for producing ligands with differential biological consequences.
Wang
Q J
QJ
Molecular Mechanisms of Tumor Promotion Section, Laboratory of Cellular Carcinogenesis and Tumor Promotion, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Bhattacharyya
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D
Garfield
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Nacro
K
K
Marquez
V E
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Blumberg
P M
PM
D000818:Animals; D054713:Bryostatins; D016466:CHO Cells; D006224:Cricetinae; D004075:Diglycerides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D049452:Green Fluorescent Proteins; D007527:Isoenzymes / Q000032:analysis*; D007783:Lactones / Q000494:pharmacology; D008164:Luminescent Proteins / Q000032:analysis*; D018942:Macrolides; D011493:Protein Kinase C / Q000032:analysis*; D011993:Recombinant Fusion Proteins / Q000032:analysis; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection
D054713:Bryostatins; D004075:Diglycerides; D007527:Isoenzymes; D007783:Lactones; D008164:Luminescent Proteins; D018942:Macrolides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C046785:bryostatin 1; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate