The effect of retinoic acid on cyclic‐AMP‐binding proteins in mouse melanoma cells

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 M r 49000, 52000, and 55000 which were specifically labeled with 8‐N 3 ‐ 32 P] AMP in both control and treated cells. Based upon their molecular weight, relative affinity for 8‐N 3 ‐[ 32 P]AMP and comigration with standards, we have designated the 49000 ‐ M r and 55000‐ M r species as R I and R II respectively. The position of the intermediate band ( M r 52000) was not affected by pre‐incubation with ATP or alkaline phosphatase, and two‐dimensional gel analysis indicated that it had the same pI as R I . Retinoic acid increased the 8‐N 3 ‐[ 32 P]AMP labeling of R I within 24 h, reaching a maximal six fold increase by 48 h. These increases were limited to the 40000 × g supernatant fraction and occurred prior to any growth inhibition. By using increasing concentrations of 8‐N 3 ‐cAMP we were able to construct a saturation curve for R I binding. Calculation of apparent K d values from these curves showed nearly identical affinities for R I binding of 8‐N 3 ‐cAMP from control and retinoic‐acid‐treated cells. Therefore we conclude that retinoic acid is increasing the amount of R I 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.