Ligand-dependent down-regulation that leads to rapid and extensive loss of protein is characteristic of several nuclear steroid receptors, including human progesterone receptors (PRs). In breast cancer cells, >95% of PRs are degraded 6 h after the start of progestin treatment. The mechanism for down-regulation is unknown. We examined the role of PR phosphorylation by mitogenactivated protein kinases (MAPKs) in this process. Lactacystin and calpain inhibitor I, specific inhibitors of the 26S proteasome, blocked progestin-induced down-regulation, and ubiquitinated conjugates of PR accumulated in cells. Ligand-dependent PR degradation was also blocked by specific inhibition of p42 and p44 MAPKs. To define the targets of phosphorylation by this kinase, two serine͞proline MAPK consensus sites on PR were mutated. We demonstrate that mutation of PR serine-294 to alanine (S294A) specifically and completely prevents ligand-dependent receptor down-regulation. We also find that rapid, ligand-independent degradation of immature PR intermediates occurs by a proteasome-mediated pathway. These results demonstrate that PR destruction, by either of two alternate routes, is mediated by the 26S proteasome. Specifically, down-regulation of mature PRs occurs by a mechanism in which ligand binding activates PR phosphorylation by MAPKs at a unique serine residue, which then targets the receptors for degradation.

Breast cancers often exhibit elevated expression of tyrosine kinase growth factor receptors; these pathways influence breast cancer cell growth in part by targeting steroid hormone receptors, including progesterone receptors (PR). To mimic activation of molecules downstream of growth factor-initiated signaling pathways, we overexpressed mitogen-activated protein kinase (MAPK; also known as extracellular signal-regulated kinase) kinase kinase 1 (MEKK1) in T47D human breast cancer cells expressing the B isoform of PR. MEKK1 is a strong activator of p42 and p44 MAPKs. MEKK1 expression increased progestin-mediated transcription 8-to 10-fold above normal PR-driven transcription levels. This was dependent on the presence of a progesterone response element and functional PR. PR protein levels were unchanged by MEKK1 alone but were extensively down-regulated by MEKK1 plus the progestin R5020. MEKK1 expression resulted in phosphorylation of PR on Ser294, a MAPK consensus site known to mediate ligand-dependent PR degradation. MEK inhibitors blocked phosphorylation of Ser294 and attenuated PR transcriptional hyperactivity in response to MEKK1 plus R5020; stabilization of PR by inhibition of the 26S proteasome produced similar results. T47D cells stably expressing mutant S294A PR, in which serine 294 is replaced by alanine, fail to undergo ligand-dependent down-regulation and are resistant to MEKK1-plus-R5020-induced 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 incapable of cross talk with MAPK-driven pathways. These studies demonstrate a paradoxical coupling between steroid receptor down-regulation and transcriptional hyperactivity. They also suggest a link between phosphorylation of PR by MAPKs in response to peptide growth factor signaling and steroid hormone control of breast cancer cell growth.Many solid tumors, including breast cancers, exhibit elevated mitogen-activated protein kinase (MAPK) expression and/or activities (13, 40), presumably due to increased expression of growth factor receptors that couple to MAPK activation. Overexpression of type I tyrosine kinase growth factor receptors in the epidermal growth factor (EGF) receptor/cErbB family is believed to contribute to proliferative signaling in breast cancer and to be indicative of a poor prognosis. Analogous to other members of the steroid receptor superfamily, human estrogen receptors (ER) and progesterone receptors (PR) are highly phosphorylated and therefore sensitive to growth factor-initiated signaling pathways. Indeed, the same phosphorylation sites on ER and/or PR can be regulated in response to steroid hormone or growth factor treatment of cells (reviewed in references 18 and 49). Although the role of direct phosphorylation of steroid hormone receptors and the exact kinase-signaling pathways involved remain large...

During late stages of breast cancer progression, tumors frequently acquire steroid hormone resistance with concurrent amplification of growth factor receptors; this alteration predicts a poor prognosis. We show here that following treatment with the progestin, R5020, breast cancer cells undergo a "biochemical shift" in the regulation of epidermal growth factor (EGF)-stimulated signaling pathways: R5020 potentiates the effects of EGF by up-regulating EGFR, c-ErbB2 and c-ErbB3 receptors, and by enhancing EGF-stimulated tyrosine phosphorylation of signaling molecules known to associate with activated type I receptors. Independently of EGF, R5020 increases Stat5 protein levels, association of Stat5 with phosphotyrosine-containing proteins, and tyrosine phosphorylation of JAK2 and Shc. Furthermore, progestins "prime" breast cancer cells for growth signals by potentiating EGF-stimulated p42/p44 mitogenactivated protein kinase (MAPK), p38 MAP kinase, and JNK activities. Although the levels of cyclin D1, cyclin E, and p21 WAF1 , are up-regulated by R5020 alone, they are synergistically up-regulated by EGF in the presence of R5020. Up-regulation of cell cycle proteins by EGF is blocked by inhibition of p42/p44 MAPK only in the presence of R5020, supporting a shift in the regulation of these cell cycle mediators from MAPK-independent to MAPK-dependent pathways. In summary, progesterone selectively increases the sensitivity of key kinase cascades to growth factors, thereby priming cells for stimulation by latent growth signals. These data support a model in which breast cancer cell growth switches from steroid hormone to growth factor dependence.Estradiol and progesterone are involved in breast cancer development, but at the time of diagnosis only one-third of tumors are steroid hormone-dependent. As they progress, these tumors often acquire steroid hormone resistance, yet retain nuclear steroid receptors. Indeed, nuclear receptor loss or mutation accounts for only 10 -20% of steroid hormone-resistant tumors (1). Thus, it has been postulated that in the majority of resistant tumors, control over growth is assumed by locally acting autocrine or paracrine peptide growth factors. As a result, invasive cancers with the worst prognosis are those that are growth factor receptor positive and steroid hormone resistant (2). Growth factors regulate cell growth by activating intracellular signal transduction pathways after binding to high affinity tyrosine kinase receptors on the cell surface. Steroid hormone receptors are ligand-activated nuclear transcription factors. Herein, we sought to understand how these two separate pathways converge to regulate breast cancer cell growth.The mechanisms by which growth factors stimulate cell proliferation are complex. Growth factors induce receptor dimerization and activation of intrinsic tyrosine kinase activity. Autophosphorylation of tyrosine residues located in the cytoplasmic domain of the receptors stimulates binding of specific regulatory proteins via their SH2 1 and SH3 domains, which ...