Transcriptional hyperactivity of human progesterone receptors is coupled to their ligand-dependent down-regulation by mitogen-activated protein kinase-dependent phosphorylation of serine 294

Breast cancers often exhibit elevated expression of tyrosine kinase growth factor receptors; these pathways influence breast cancer cell growth in par t by targeting steroid hormone receptors, including progesterone receptors (PR). To mimic activation of molecules downstream of growth factor-initiate d signaling pathways, we overexpressed mitogen-activated protein kinase (MA PK; also known as extracellular signal-regulated kinase) kinase kinase 1 (M EKK1) in T47D human breast cancer cells expressing the B isoform of PR. MEK K1 is a strong activator of p42 and p44 MAPKs. MEKK1 expression increased p rogestin-mediated transcription 8- to 10-fold above normal PR-driven transc ription levels. This was dependent on the presence of a progesterone respon se element and functional PR. PR protein levels were unchanged by MEKK1 alo ne but were extensively down-regulated by MEKK1 plus the progestin R5020. M EKK1 expression resulted in phosphorylation of PR on Ser294, a MAPK consens us site known to mediate ligand-dependent PR degradation. MEK inhibitors bl ocked phosphorylation of Ser294 and attenuated PR transcriptional hyperacti vity in response to MEKK1 plus R5020; stabilization of PR by inhibition of the 26S proteasome produced similar results. T47D cells stably expressing m utant S294A PR, in which serine 294 is replaced by alanine, fail to undergo ligand-dependent down-regulation and are resistant to MEKK1-plus-R5020-ind uced transcriptional synergy but respond to progestins alone. Similarly, c- myc protein levels are synergistically increased by epidermal growth factor and R5020 in cells expressing wild-type PR, but not S294A PR. Thus, highly stable mutant PR are functional in response to progestins but are incapabl e of cross talk with MAPK-driven pathways. These studies demonstrate a para doxical coupling between steroid receptor down-regulation and transcription al hyperactivity. They also suggest a link between phosphorylation of PR by MAPKs in response to peptide growth factor signaling and steroid hormone c ontrol of breast cancer cell growth.