Retinoic acid specifically increases nuclear PKC alpha and stimulates AP-1 transcriptional activity in B16 mouse melanoma cells.
B16 melanoma cells differentiate upon treatment with retinoic acid (RA). This differentiation process is accompanied by an increase of protein kinase C alpha (PKC alpha) mRNA and protein levels. Overexpression of PKC alpha in these cells results in a more differentiated phenotype, suggesting the importance of this protein in the control of differentiation by RA. The purpose of the study reported here was to determine the subcellular distribution of the RA-induced PKC alpha, whether the RA-induced increase in PKC alpha protein levels was accompanied by an increase in in situ enzyme activity, and whether RA altered AP-1 transcriptional activity. We found that RA treatment increased PKC alpha protein levels in all subcellular compartments examined, but it also induced a selective enrichment in nuclear-associated PKC alpha levels. Treating cells with an active phorbol ester induced translocation of PKC alpha to membrane fractions, but had no effect on nuclear PKC alpha levels. RA also increased PKC enzymatic activity in intact cells as determined by phosphorylation of the PKC-specific endogenous substrate MARCKS. However, while RA induced a five- to eightfold increase in total cellular PKC alpha protein levels, it only increased MARCKS phosphorylation by twofold. In light of the increase in in situ PKC enzyme activity and the enrichment of nuclear PKC alpha, we determined whether AP-1 activity might be increased in RA-treated cells. Use of luciferase reporter gene constructs with or without AP-1 elements transfected into B16 cells indicated that RA induced a four- to fivefold increase in AP-1 transcriptional activity. These results suggest a hypothesis whereby RA-induced nuclear PKC alpha might lead to increased AP-1 activity and show that RA-induced growth inhibition and differentiation are not always accompanied by an inhibition of AP-1 activity as has been proposed by other investigators.
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology; D002467:Cell Nucleus / Q000201:enzymology; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008545:Melanoma / Q000201:enzymology* / Q000378:metabolism; D008565:Membrane Proteins; D051379:Mice; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010766:Phosphorylation; D011493:Protein Kinase C / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism; D051571:Protein Kinase C-alpha; D011506:Proteins; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D014212:Tretinoin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; C000619711:Marcks protein, mouse; D008565:Membrane Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D018808:Transcription Factor AP-1; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D014212:Tretinoin; D008156:Luciferases; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D013755:Tetradecanoylphorbol Acetate