PHOSPHORYLATION OF 17-BETA-HYDROXYSTEROID DEHYDROGENASE IN BEWO CHORIOCARCINOMA CELLS

OBJECTIVE: The purpose of this study was to test whether 17 beta-hydro xysteroid dehydrogenase might exist in a phosphorylated form. STUDY DE SIGN: Phosphorylation of 17 beta-hydroxysteroid dehydrogenase was eval uated in BeWo choriocarcinoma cells. The phosphorylation of 17 beta-hy droxysteroid dehydrogenase expressed in Escherichia coli as a glutathi one transferase fusion protein was also studied. RESULTS: Human BeWo c horiocarcinoma cells were metabolically labeled with phosphorus 32 ort hophosphate. Immunoprecipitates were prepared with rabbit anti-17 beta -hydroxysteroid dehydrogenase antiserum from the labeled cells and sep arated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A phosphorylated protein with a molecular size of 35 kd was obtained fr om anti-17 beta-hydroxysteroid dehydrogenase immunoprecipitates, which suggested that 17 beta-hydroxysteroid dehydrogenase was phosphorylate d in BeWo cells. The predominant phosphoamino acid was phosphoserine. 17 beta-Hydroxysteroid dehydrogenase expressed in E. coli as a glutath ione transferase fusion protein was a substrate of protein kinase A in vitro. Protein kinase A phosphorylated the recombinant 17 beta-hydrox ysteroid dehydrogenase exclusively on serine. Incubation of BeWo cell lysates with bacterial alkaline phosphatase led to a decrease in the o xidative activity of 17 beta-hydroxysteroid dehydrogenase. Incubation of the alkaline phosphatase inhibitor levamisole with BeWo cell lysate s resulted in a higher estradiol-to-estrone conversion rate, compared with cell lysates without any treatment. CONCLUSION: Our data suggest that 17 beta-hydroxysteroid dehydrogenase may exist in phosphorylated forms acid that phosphorylation may regulate the activity of 17 beta-h ydroxysteroid dehydrogenase in vivo.