Phosphorylation of 17 beta-hydroxysteroid dehydrogenase in BeWo choriocarcinoma cells.
OBJECTIVE
The purpose of this study was to test whether 17 beta-hydroxysteroid dehydrogenase might exist in a phosphorylated form.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Barbieri
R L
RL
Department of Obstetrics, Gynecology, and Reproductive Biology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115.
Gao
X
X
Frost
R A
RA
D015067:17-Hydroxysteroid Dehydrogenases / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D002822:Choriocarcinoma / Q000201:enzymology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005260:Female; D005982:Glutathione Transferase / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011247:Pregnancy; D011993:Recombinant Fusion Proteins; D014407:Tumor Cells, Cultured; D014594:Uterine Neoplasms / Q000201:enzymology*
D011993:Recombinant Fusion Proteins; D015067:17-Hydroxysteroid Dehydrogenases; D005982:Glutathione Transferase; D017868:Cyclic AMP-Dependent Protein Kinases