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The effect of retinoic acid on protein phosphorylation in mouse melanoma cells
Abstract:
Vitamin A inhibits growth and increases the activity of cAMP-dependent protein kinase in B16 mouse melanoma cells. In this report we show that retinoic acid (RA) treatment of intact cells alters their subsequent in vitro protein phosphorylation, but we could not demonstrate any changes in in vivo protein phosphorylation. A 48-h treatment with RA results in a concentration-dependent decrease of protein phosphorylation of a 95K molecular weight (MW) protein in both supernatant and particulate fractions. The phosphorylation of this protein does not appear to be regulated by cAMP. Proteins at 92K and 82K MW in the supernatant fraction are increased in phosphorylation. The former (but not the latter) is regulated by cAMP. In the particulate fraction a variety of proteins 12K-68K MW are increased in phosphorylation, as the cells are treated with increasing amounts of RA. The phosphorylation of most of these proteins is regulated by cAMP. Another inhibitor of B16 cell growth, melanocyte-stimulating hormone (MSH) also alters protein phosphorylation. At short incubation periods (1 h), this hormone stimulates phosphorylation of a number of proteins (17-40K MW), while in longer incubation periods (48 h) phosphorylation is inhibited. All of these phosphorylations appear to be regulated by cAMP. We attempted to repeat these observations using intact-cell phosphorylation with 32PO4. In two experiments we saw small changes in the phosphorylation of proteins. In most experiments, however, we could find no change in the phosphoproteins. Further experiments have led us to question the in vivo phosphorylation, since treatment of the cells with MSH, cholera toxin, or db-cAMP also did not affect intact-cell protein phosphorylation. We have previously documented that under these latter conditions cAMP levels are greatly elevated and cAMP-dependent protein kinase is activated. The in vitro phosphorylation results suggests that in RA-treated cells, kinase activities and/or protein substrate levels are changing. However, the physiological significance of the particular MW phosphoproteins changes we have described must await resolution of the in vivo phosphorylation data.
Insights
Retinoic acid (RA) alters protein phosphorylation in B16 melanoma cells in vitro, affecting specific proteins. However, in vivo phosphorylation changes were not consistently observed, questioning the physiological significance of these in vitro findings.
Area of Science:
- Biochemistry
- Cell Biology
- Cancer Research
Background:
- Vitamin A derivatives, like retinoic acid (RA), are known to inhibit B16 mouse melanoma cell growth.
- Cyclic adenosine monophosphate (cAMP)-dependent protein kinase activity is increased by Vitamin A in these cells.
Purpose of the Study:
- To investigate the effects of retinoic acid (RA) on protein phosphorylation in B16 mouse melanoma cells.
- To determine if observed in vitro phosphorylation changes correlate with in vivo phosphorylation events.
Main Methods:
- Treatment of intact B16 mouse melanoma cells with varying concentrations of retinoic acid (RA).
- Analysis of protein phosphorylation in both supernatant and particulate fractions using in vitro assays.
- Attempted replication of phosphorylation changes using intact-cell phosphorylation with 32PO4.
Main Results:
- RA treatment decreased phosphorylation of a 95K MW protein and increased phosphorylation of 92K and 82K MW proteins in vitro.
- In vitro phosphorylation of several other proteins (12K-68K MW) in the particulate fraction increased with RA.
- In vivo phosphorylation studies showed minimal or no consistent changes in response to RA or other stimuli.
Conclusions:
- Retinoic acid (RA) treatment leads to alterations in protein kinase activities and/or substrate levels in B16 melanoma cells, as evidenced by in vitro phosphorylation changes.
- The physiological relevance of the observed in vitro protein phosphorylation changes remains uncertain due to the lack of consistent in vivo evidence.