Regulation of retinoic acid receptor α by protein kinase C in B16 mouse melanoma cells

We have previously found that retinoic acid stimulates the expression of protein kinase Calpha (PKC) in B16 mouse melanoma cells. Because it has been reported that PKC can phosphorylate retinoic acid receptor (RAR) and alter its function, we determined whether changes in the level and/or activity of PKC could affect the expression or function of the RAR in B16 melanoma. Using in vivo phosphorylation and band shift techniques, we could not demonstrate that altering PKC activity and/or protein level changed the in vivo phosphorylation of RARalpha. However activation of PKC resulted in increased RARalpha protein. Increased receptor protein correlated with a phorbol dibutyrate-stimulated increase in receptor activation function-2 (AF-2)-dependent transcriptional activity. Use of enzyme inhibitors and dominant-negative PKCs indicated that enzyme activity was required for elevation in the RARalpha. The PKC-mediated increase in RARalpha was due to a 2.5-fold increase in the half-life of this protein. In contrast, the down-regulation of PKC diminished RARalpha protein half-life and markedly inhibited AF-2-dependent transcriptional activity. The down-regulation of PKC also inhibited the binding of RAR to a retinoic acid response element and the retinoic acid induction of RARbeta expression. These findings suggest that PKC can influence retinoic acid signaling by altering the stability of RAR protein without directly phosphorylating this receptor.