Phosphorylation of 17 beta-hydroxysteroid dehydrogenase in BeWo choriocarcinoma cells.

Abstract

The purpose of this study was to test whether 17 beta-hydroxysteroid dehydrogenase might exist in a phosphorylated form. Phosphorylation of 17 beta-hydroxysteroid dehydrogenase was evaluated in BeWo choriocarcinoma cells. The phosphorylation of 17 beta-hydroxysteroid dehydrogenase expressed in Escherichia coli as a glutathione transferase fusion protein was also studied. Human BeWo choriocarcinoma cells were metabolically labeled with phosphorus 32 orthophosphate. Immunoprecipitates were prepared with rabbit anti-17 beta-hydroxysteroid dehydrogenase antiserum from the labeled cells and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A phosphorylated protein with a molecular size of 35 kd was obtained from anti-17 beta-hydroxysteroid dehydrogenase immunoprecipitates, which suggested that 17 beta-hydroxysteroid dehydrogenase was phosphorylated in BeWo cells. The predominant phosphoamino acid was phosphoserine. 17 beta-Hydroxysteroid dehydrogenase expressed in E. coli as a glutathione transferase fusion protein was a substrate of protein kinase A in vitro. Protein kinase A phosphorylated the recombinant 17 beta-hydroxysteroid dehydrogenase exclusively on serine. Incubation of BeWo cell lysates with bacterial alkaline phosphatase led to a decrease in the oxidative activity of 17 beta-hydroxysteroid dehydrogenase. Incubation of the alkaline phosphatase inhibitor levamisole with BeWo cell lysates resulted in a higher estradiol-to-estrone conversion rate, compared with cell lysates without any treatment. Our data suggest that 17 beta-hydroxysteroid dehydrogenase may exist in phosphorylated forms and that phosphorylation may regulate the activity of 17 beta-hydroxysteroid dehydrogenase in vivo.