Phosphorylation of human progesterone receptors at
serine-294 by mitogen-activated protein kinase
signals their degradation by the 26S proteasome
Carol A. Lange*, Tianjie Shen, and Kathryn B. Horwitz†
†Department of Medicine, The Molecular Biology Program, and The Colorado Cancer Center, University of Colorado Health Sciences Center,
Denver, CO 80262
Edited by Ronald M. Evans, The Salk Institute for Biological Studies, San Diego, CA, and approved November 30, 1999 (received for review
September 1, 1999)
Ligand-dependent down-regulation that leads to rapid and exten-
sive 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 un-
known. We examined the role of PR phosphorylation by mitogen-
activated 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 deg-
radation was also blocked by specific inhibition of p42 and p44
MAPKs. To define the targets of phosphorylation by this kinase,
two serine?proline 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 protea-
some-mediated pathway. These results demonstrate that PR de-
struction, 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.
gesterone receptors (PRs) are key markers of steroid hormone
dependence and indicators of disease prognosis in breast cancer;
their loss signals development of an aggressive tumor phenotype
associated with acquisition of enhanced sensitivity to growth
factors (1, 2). Among the factors that regulate PR levels are their
ligands. Within 6 to 8 h after occupancy by progestins, the
receptors are extensively down-regulated, by mechanisms that
Regulation of PR expression by ligands occurs at both the
protein and mRNA levels. At the mRNA level, the effects of
human breast cancer cells (3, 4). PR mRNA levels decrease
gradually between 4 and 20 h after progestin treatment and then
return to pretreatment levels 24 to 48 h later. However, the
relationship between PR mRNA fluctuation and levels of the
two PR protein isoforms is unknown, because of the number and
heterogeneity of PR transcripts, which can encode either one or
both receptor isoforms (3). In addition to these fluctuations in
PR mRNA levels, PR protein levels are also extensively and
rapidly down-regulated in response to ligand binding. Endoge-
nous PRs, labeled by biosynthetic incorporation of2H,13C, and
cells, compared with 6 h in progestin-treated cells (4). The rate
and extent of PR protein decrease reflects the time course of
receptor occupancy by ligand and the fractional saturation of
receptors (4). Antiprogestins also induce PR down-regulation
but with much slower kinetics than agonists, suggesting a rela-
dvanced-stage breast cancers often lack steroid hormone
receptors and?or are resistant to endocrine therapies. Pro-
tionship between transcriptional activity of PR induced by ligand
and receptor down-regulation (C.A.L. and K.B.H., unpublished
may expend the energy to clear activated receptors is to atten-
uate their own transcriptional responses. Alternatively, nuclear
receptor turnover may provide a mechanism to ‘‘reset’’ the
transcriptional apparatus after each stimulus, so that previously
modified receptors can be replaced with newly synthesized, fully
functional molecules. Thus, at steady state in tissues in which
PRs are constantly exposed to changing levels of physiological
progesterone, receptor down-regulation may allow for continual
reactivation of transcription at PR-regulated genes.
To define the mechanisms for ligand-dependent PR down-
regulation, we studied the role of phosphorylation and receptor
degradation by the 26S proteasome. The timed destruction of
regulatory proteins by the ubiquitin-proteasome pathway is
emerging as an important mechanism for the tight control of
diverse cellular processes, including signal transduction from cell
surface receptors (5), gene transcription (6), angiogenesis (7),
and cell cycle progression (ref. 8; reviewed in refs. 9 and 10).
Aberrations in this pathway or its protein substrates are impli-
cated in several disease states ranging from Alzheimer’s disease
(11) to cancer (reviewed in ref. 12), and inhibitors of ubiquiti-
nation are candidates for cancer clinical trials (13, 14).
We now demonstrate that PRs are targeted for down-
regulation by phosphorylation. A highly specific inhibitor of the
26S proteasome blocks ligand-dependent PR protein loss. The
same result is produced by inhibition of p42 and p44 MAPKs, as
well as by mutation of a single MAPK consensus phosphoryla-
tion site on PR. These data demonstrate that liganded PRs are
substrates for MAPK-induced phosphorylation, which targets
the receptors for degradation by the 26S proteasome.
Materials and Methods
Cell Lines and Reagents. Estrogen-resistant T47Dco breast cancer
cells, their monoclonal PR-negative T47D-Y derivatives, and
T47D-YB cells, which are T47D-Y cells engineered to stably
express the PR-B isoform, were previously described (15, 16).
Construction of HeLa:B cells—HeLa cervical carcinoma cells
stably expressing PR-B—was described recently (17). Cells were
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: PR, progesterone receptor; MAPK, mitogen-activated protein kinase; EGF,
epidermal growth factor; HA, hemagglutinin; LLnL, N-acetyl-Leu-Leu-Nle-CHO; ALLM,
N-acetyl-Leu-Leu-Met-CHO; GA, geldanamycin.
*Present address: University of Minnesota Cancer Center and Department of Medicine,
University of Minnesota, Minneapolis, MN 55455.
†To whom reprint requests should be addressed. E-mail: kate.horwitz@UCHSC.edu.
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
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vol. 97 ?
ence of such a motif; a 9-amino acid consensus ‘‘destruction box’’
originally described in cyclin molecules and required for their
destruction by the ubiquitin-proteasome pathway (8, 49). It is
interesting that, in PRs, this sequence is nested within the
serine-294 MAPK consensus site. Another consensus destruc-
tion box motif is located further downstream at amino acid
positions 399–407 of human PRs, encompassing basal phosphor-
ylation site serine-400, which is an in vitro target of cyclin-
dependent kinase 2 (Cdk2) (50). In addition to other basal
phosphorylation sites in human PR, this site also undergoes a
modest increase in phosphorylation in response to ligand (36).
Although the functional significance of these two sequences is
unknown, their presence at different positions in the PR N
terminus may explain why destruction of immature receptor
intermediates and ligand-induced phosphorylation of mature
PRs, followed by their down-regulation, ultimately proceed via
targeting to a common degradation pathway. Phosphorylation of
PRs at serine-294 by MAPK may expose one destruction box
motif(s) for targeted destruction by the 26S proteasome. Phos-
phorylation of the second site may lead to degradation of
immature or unliganded receptors. Indeed, structural analysis of
the human PR-A amino terminus indicates that both phosphor-
ylation sites are exposed on the surface of the protein molecule
(D. L. Bain and K.B.H., unpublished results). Similarly, phos-
phorylation may also target mouse glucocorticoid receptors for
degradation (51). Simultaneous mutation of a minimum of three
phosphorylation sites resulted in more stable glucocorticoid
receptors in the presence of ligand; the kinases involved have not
been defined. Estrogen receptors recently have been shown to be
degraded by the ubiquitin-proteasome pathway in vitro (44), but
the role of phosphorylation, if any, is unknown.
In summary (Fig. 6), these results demonstrate that rapid,
mediated by the 26S proteasome. Additionally, we show that
ligand-dependent down-regulation of mature PRs occurs by a
mechanism involving phosphorylation of PRs by p42?p44
MAPKs at serine-294, thus targeting PRs for ubiquitination and
destruction by the 26S proteasome. This is a demonstration of a
specific effect of the MAPK pathway on a hormone-dependent
nuclear receptor phosphorylation site and a definition of the
function of this phosphorylation. Cross-talk between MAPK-
and PR-signaling pathways suggests mechanisms by which ste-
roid hormone resistance and acquisition of growth factor re-
sponsiveness are integrated in advanced breast cancer (17, 18).
Center, Seattle, WA, for the gift of the HA-tagged ubiquitin construct;
Glenn S. Takimoto, University of Colorado Health Sciences Center, for
the gift of the S344?345A PR-A mutant receptor construct; and Jennifer
K. Richer, University of Colorado Health Sciences Center, for the gift
of the HeLa:B cells stably expressing epitope flag-tagged human PR-B
by The National Institutes of Health Grants DK53825 (C.A.L.) and
DK48238 and CA26869 (K.B.H.).
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