HORMONE-INDUCED PROGESTERONE-RECEPTOR PHOSPHORYLATION CONSISTS OF SEQUENTIAL DNA-INDEPENDENT AND DNA-DEPENDENT STAGES - ANALYSIS WITH ZINC FINGER MUTANTS AND THE PROGESTERONE ANTAGONIST-ZK98299

Human progesterone receptors (hPRs) are phosphorylated at multiple ser ine residues, first in a basal step and then in a hormone-induced step . To determine whether hormone-induced phosphorylation precedes or fol lows the interaction of hPRs with DNA two strategies were used. (i) DN A 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 prev ented with the progesterone antagonist ZK98299. Two hPR(A) mutants wer e constructed: DBD(Cys), which lacks a critical cysteine residue in th e first zinc finger, and DBD(sp), which is mutated at three discrimina tory amino acids to change its DNA binding specificity from a progeste rone response element to an estrogen response element. Receptors were transiently expressed in PR-negative cells and were intranuclear. DBD( Cys) did not bind DNA in vitro and DBD(sp) bound only the estrogen res ponse element. Transiently expressed hPR(A) and DBD(sp) showed the upw ard shift in electrophoretic mobility characteristic of hormone-induce d phosphorylation; it was absent with DBD(Cys). Hormone-induced [P-32] orthophosphate incorporation into transiently expressed DBD(Cys) was r educed 60% compared to hPR(A) and DBD(sp) but was not eliminated. ZK98 299 binds hPRs but prevents their interaction with DNA. Compared to R5 020, the antagonist reduced phosphorylation of hPR(B) and hPR(A) in T4 7D 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.