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.