IN-VIVO AND IN-VITRO PHOSPHORYLATION OF THE HUMAN ESTROGEN-RECEPTOR

We report here that the human estrogen receptor (hER) overexpressed in Sf9 insect cells is phosphorylated similarly to hER from the human MC F-7 mammary carcinoma cell line. The recombinant and native hER labele d to steady-state with [P-32]phosphate were purified to homogeneity us ing specific DNA-affinity chromatography followed by SDS-gel electroph oresis. Resolution of the hER tryptic digests by reverse phase-high pe rformance liquid chromatography revealed that five [P-32]phosphopeptid es from the hER expressed in the Sf9 cells had retention times identic al to five of the seven [P-32]phosphopeptides from the hER in MCF-7 ce lls. Uniquely, a dephosphorylation of a single P-32-labeled peptide oc curred in response to estradiol treatment of MCF-7 cells. In vitro pro tein kinase assays with the purified recombinant hER revealed that the DNA-dependent protein kinase (DNA-PK) phosphorylated the receptor and induced a decrease in the receptor's mobility as demonstrated by SDS- gel electrophoresis. In contrast, protein kinases A and C did not phos phorylate the purified recombinant hER. These results suggest that in the process of becoming transcriptionally active the estrogen receptor undergoes a dephosphorylation after estrogen-binding and subsequent p hosphorylations, in part by the DNA-PK.