Hormone-induced progesterone receptor phosphorylation consists of sequential DNA-independent and DNA-dependent stages: analysis with zinc finger mutants and the progesterone antagonist ZK98299.

摘要

Human progesterone receptors (hPRs) are phosphorylated at multiple serine residues, first in a basal step and then in a hormone-induced step. To determine whether hormone-induced phosphorylation precedes or follows the interaction of hPRs with DNA two strategies were used. (i) DNA binding was prevented or altered with site-specific mutants of the A form of hPR; (ii) DNA binding of wild-type hPR forms A and B was prevented with the progesterone antagonist ZK98299. Two hPRA mutants were constructed: DBDCys, which lacks a critical cysteine residue in the first zinc finger, and DBDsp, which is mutated at three discriminatory amino acids to change its DNA binding specificity from a progesterone response element to an estrogen response element. Receptors were transiently expressed in PR-negative cells and were intranuclear. DBDCys did not bind DNA in vitro and DBDsp bound only the estrogen response element. Transiently expressed hPRA and DBDsp showed the upward shift in electrophoretic mobility characteristic of hormone-induced phosphorylation; it was absent with DBDCys. Hormone-induced [32P] orthophosphate incorporation into transiently expressed DBDCys was reduced 60% compared to hPRA and DBDsp but was not eliminated. ZK98299 binds hPRs but prevents their interaction with DNA. Compared to R5020, the antagonist reduced phosphorylation of hPRB and hPRA in T47D breast cancer cells by 60% and totally prevented the mobility shift. We conclude that the hormone-induced phosphorylation of hPR includes DNA-independent and DNA-dependent stages and that only DNA-dependent sites contribute to the mobility shift.