Retinoic acid specifically increases nuclear PKCα and stimulates AP-1 transcriptional activity in B16 mouse melanoma cells

Abstract

B16 melanoma cells differentiate upon treatment with retinoic acid (RA). This differentiation process is accompanied by an increase of protein kinase Ca (PKCα) mRNA and protein levels. Overexpression of PKCα 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 PKCα, whether the RA-induced increase in PKCα 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 PKCα protein levels in all subcellular compartments examined, but it also induced a selective enrichment in nuclear-associated PKCα levels. Treating cells with an active phorbol ester induced translocation of PKCα to membrane fractions, but had no effect on nuclear PKCα 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 PKCα 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 PKCα, 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 PKCα 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. © 1995 Academic Press, Inc.