RETINOIC ACID SPECIFICALLY INCREASES NUCLEAR PKC-ALPHA AND STIMULATESAP-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 prote in 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 differen tiation by Rk 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-I trans criptional activity, We found that RA treatment increased PKC alpha pr otein levels in all subcellular compartments examined, but it also ind uced a selective enrichment in nuclear-associated PKC alpha levels, Tr eating cells with an active phorbol ester induced translocation of PKC alpha to membrane fractions, but had no effect on nuclear PKC alpha l evels, RA also increased PKC enzymatic activity in intact cells as det ermined by phosphorylation of the PKC-specific endogenous substrate MA RCKS, However, while RA induced a five- to eightfold increase in total cellular PKC alpha protein levels, it only increased MARCKS phosphory lation by twofold. In light of the increase in in situ PMC enzyme acti vity 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 int o B16 cells indicated that RA induced a four- to fivefold increase in AP-1 transcriptional activity, These results suggest a hypothesis wher eby RA-induced nuclear PKC alpha might lead to increased AP-1 activity and show that RA-induced growth inhibition and differentiation are no t always accompanied by an inhibition of AP-1 activity as has been pro posed by other investigators. (C) 1995 Academic Press, Inc.