Abstract
We have previously found that retinoic acid stimulates the expression of protein kinase C alpha (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 RAR alpha. However activation of PKC resulted in increased RAR alpha 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 RAR alpha. The PKC-mediated increase in RAR alpha was due to a 2.5-fold increase in the half-life of this protein. In contrast, the down-regulation of PKC diminished RAR alpha 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 RAR beta expression. These findings suggest that PKC can influence retinoic acid signaling by altering the stability of RAR protein without directly phosphorylating this receptor.
Highlights
We have previously found that retinoic acid stimulates the expression of protein kinase C␣ (PKC) in B16 mouse melanoma cells
When these extracts were treated with -protein phosphatase for different periods of time, the two retinoic acid receptor (RAR)␣ bands of higher molecular mass collapsed into the lowest molecular mass band (Fig. 1) indicating that the His6-RAR␣ protein is phosphorylated in B16 cells
We have previously found that treatment of B16 mouse melanoma cells with retinoic acid induces a 6- to 8-fold increase in PKC␣ and that this increase appears to be required for growth arrest and differentiation [36]
Summary
Cell Culture—B16 mouse melanoma cells were grown in DMEM (Invitrogen, Rockville, MD) supplemented with 10% bovine calf serum (HyClone Laboratories, Logan, UT) at 37 °C, in a 5% CO2/95% air, humidified atmosphere. -Phosphatase Treatment of RAR␣-His—B16 cells were transiently transfected with the plasmid encoding the RAR␣-His protein as described above, harvested in buffer A and disrupted by sonication. Immunoprecipitation and Determination of RAR␣-His Protein Halflife—B16 cells were transfected with the RAR␣-His construct as described previously. At 44 h post-transfection, media was removed and replaced with methionine-free DMEM (Invitrogen, Rockville, MD) for 1 h This medium was replace with fresh methionine-free medium to which 100 Ci/ml [35S]methionine (PerkinElmer Life Sciences, Boston, MA) was added, and the cells were incubated for an additional 2 h. At different times of experimental treatment, medium was removed and cells were washed with PBS and harvested in 0.9 ml of radioimmune precipitation buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 1% SDS, 100 g/ml PMSF, 30 g of aprotinin, 1 mM sodium orthovanadate in PBS). All experiments described under “Results” were repeated three separate times with similar qualitative results
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