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The effect of retinoic acid on cyclic-AMP-binding proteins in mouse melanoma cells
Abstract:
We have previously reported [(1980) J. Biol. Chem. 255, 5999-6002] that retinoic acid inhibited growth and increased cyclic-AMP-dependent protein kinase activity in mouse melanoma cells. A variant melanoma line having depressed levels of cyclic-AMP-dependent protein kinase was not growth-inhibited by retinoic acid. In this report we describe the effect of retinoic acid on cyclic AMP binding proteins in B16 mouse melanoma cells. Using the technique of photoaffinity labeling, we found three major proteins of Mr 49 000, 52 000, and 55 000 which were specifically labeled with 8-N3-[32P]AMP in both control and treated cells. Based upon their molecular weight, relative affinity for 8-N3-[32P]AMP and comigration with standards, we have designated the 49 000-Mr and 55 000-Mr species as RI and RII respectively. The position of the intermediate band (Mr 52 000) was not affected by pre-incubation with ATP or alkaline phosphatase, and two-dimensional gel analysis indicated that it had the same pI as RI. Retinoic acid increased the 8-N3-[32P]AMP labeling of RI within 24 h, reaching a maximal six fold increase by 48 h. These increases were limited to the 40 000 X g supernatant fraction and occurred prior to any growth inhibition. By using increasing concentrations of 8-N3-cAMP we were able to construct a saturation curve for RI binding. Calculation of apparent Kd values from these curves showed nearly identical affinities for RI binding of 8-N3-cAMP from control and retinoic-acid-treated cells. Therefore we conclude that retinoic acid is increasing the amount of RI rather than altering its properties. Corroboration of these results was obtained by DEAE-cellulose chromatography. Peak I (corresponding to type I protein kinase) from retinoid-treated cells was increased about six fold in binding activity.
Insights
Retinoic acid increases the amount of cyclic AMP binding protein RI in mouse melanoma cells, leading to growth inhibition. This effect occurs before observable changes in cell growth.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Previous studies indicated retinoic acid inhibits melanoma cell growth and increases cyclic-AMP-dependent protein kinase activity.
- A variant melanoma line with low cyclic-AMP-dependent protein kinase levels was resistant to retinoic acid's growth inhibition.
Purpose of the Study:
- To investigate the effect of retinoic acid on cyclic AMP binding proteins in B16 mouse melanoma cells.
- To determine if retinoic acid alters the properties or amount of cyclic AMP binding proteins.
Main Methods:
- Photoaffinity labeling using 8-N3-[32P]cAMP to identify and quantify cyclic AMP binding proteins.
- Two-dimensional gel electrophoresis and DEAE-cellulose chromatography for protein characterization and analysis.
- Saturation curve analysis to determine binding affinities (Kd values) of cyclic AMP binding proteins.
Main Results:
- Three major cyclic AMP binding proteins (Mr 49,000, 52,000, and 55,000) were identified, designated RI and RII based on molecular weight and affinity.
- Retinoic acid treatment significantly increased the labeling of RI (regulatory subunit type I) by approximately six-fold within 48 hours.
- The affinity of RI for cyclic AMP remained unchanged, indicating an increase in the total amount of RI protein.
Conclusions:
- Retinoic acid increases the quantity of cyclic AMP binding protein RI in mouse melanoma cells.
- The observed increase in RI precedes growth inhibition, suggesting a role in retinoic acid-mediated cellular responses.
- The mechanism involves an upregulation of RI protein synthesis or stability rather than modification of its binding properties.