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.
February 1, 2000 ?
vol. 97 ?
routinely seeded at 1 ? 106cells per dish, cultured in 10-cm
dishes, and incubated in 5% CO2 at 37°C in a humidified
environment as described (16). Stably transfected cells were also
maintained in 700 mg?ml neomycin analog G418 (Life Tech-
nologies, Gaithersburg, MD). For MAPK activation studies, 30
ng?ml epidermal growth factor (EGF), was added to cells
cultured in serum-free medium for 16–18 h before growth factor
addition. Phosphospecific and total p42?p44 MAPK antibodies,
phosphospecific p38 MAPK antibodies, and the MEK1 inhibitor
(PD98059) were purchased from New England Biolabs. The p38
MAPK inhibitors (SB202190 and SB203580) were purchased
from Upstate Biotechnology (Lake Placid, NY). Horseradish
peroxidase-conjugated secondary antibodies were obtained
from Collaborative Biomedical Products (Bedford, MA). Hem-
agglutinin (HA) epitope tag antisera were purchased from
Babco (Richmond, CA). R5020 was obtained from NEN and
used at 20 nM. Lactacystin, calpain inhibitor I (LLnL, N-acetyl-
Leu-Leu-Nle-CHO), calpain inhibitor II (ALLM, N-acetyl-Leu-
Leu-Met-CHO), and geldanamycin (GA) were purchased from
Immunoblotting. For detection of PR-A- and -B isoforms or
MAPKs in whole cells lysates, cells growing in 10-cm dishes were
washed twice in 4 ml of PBS and lysed by scraping in extraction
buffer [1% (vol?vol) Triton X-100?10 mM Tris?HCl, pH 7.4?5
mM EDTA?50 mM NaCl?50 mM NaF?20 ?g/ml Aprotinin?1
mM PMSF?2 mM Na3VO4]. Lysates were clarified by centrifu-
gation for 10 min at maximum speed in a refrigerated microfuge.
Soluble proteins in clarified lysates were quantified by the
method of Bradford (GIBCO?BRL), and equal amounts of
protein were resolved by SDS?PAGE (10% acrylamide for
MAPKs; 7.5% acrylamide for PRs) and detected by immuno-
Coimmunoprecipitation. HeLa cells were transiently transfected
with 3.0 ?g of the ?-galactosidase expression plasmid pCH110
(Amersham Pharmacia) to monitor transfection efficiency, 1.5
?g of cDNA encoding flag-tagged PR-B (17), and 2.0 ?g of
cDNA encoding HA-tagged ubiquitin (6), by using calcium
phosphate precipitation as described (19). Immunoprecipitation
of PR:flag proteins from cell lysates (1.2 mg) and immunoblot-
ting were performed as decribed by Richer et al. (17).
Results and Discussion
Ubiquitination and the 26S Proteasome.Weexaminedtheregulation
of PR protein abundance by the ubiquitin-proteasome pathway
naturally express both the A and B isoforms of PR (20), were
treated without or with the synthetic progestin R5020 to induce
ligand-dependent receptor down-regulation (Fig. 1A). Disappear-
ance of both PR isoforms was complete by 12 h of progestin
treatment. However, R5020 had no effect on PR degradation in
cells treated with lactacystin, a specific inhibitor of the 26S protea-
some (ref. 21; reviewed in ref. 22). Similar results were obtained
with progesterone (data not shown). Thus, human PRs appear to
be substrates for regulated degradation by the 26S proteasome.
Blockade of progestin-induced PR degradation by lactacystin also
occurred in T47D-YB human breast cancer cells and in HeLa:B
human cervical carcinoma cells, both of which stably express
(SV40) promoter (Fig. 1A). Because endogenous, naturally ex-
whose stable expression is driven by heterologous promoters, are
efficiently down-regulated, protein degradation rather than tran-
scriptional regulation must account for this effect. The increased
apparent molecular weight of lactacystin-stabilized, R5020-
occupied PRs, as measured by their up-shift on SDS?PAGE gels
(Fig. 1), reflects increased phosphorylation at multiple serine
PR degradation in the presence of R5020 appears to be
specifically mediated by the 26S proteasome, because ALLM,
which inhibits the activity of several degradative enzymes but not
breast cancer cells, which express the natural B and A isoforms of PRs consti-
tutively (15), and T47D-YB (16) and HeLa:B cells (17), which stably express the
recombinant B isoform of PRs, were treated without or with the progestin
R5020 (10 nM) for 12 h in the absence (DMSO solvent) or presence of lacta-
cystin [Lact. (10 ?M)], and PR protein (100 ?g of total protein per lane) was
detected by immunoblotting with PR-specific monoclonal antibodies. (B)
Inhibition of PR down-regulation by calpain inhibitor I, but not calpain
inhibitor II. T47D-YB cells were treated as in A, except with calpain inhibitor II
no effect on PR abundance. (C) PR-ubiquitin conjugates in cells transiently
overexpressing ubiquitin and PR-B. HeLa cells were transiently cotransfected
with expression vectors encoding HA-tagged ubiquitin and epitope-flag-
tagged wild-type PR-B (PR-B:flag) and treated for 4 h without (lane 2) or with
R5020 (lane 3; 10 nM) in the absence or presence of lactacystin (lane 4; 10 ?M)
or LLnL (lane 5; 25 ?M). PRs were immunoprecipitated by using anti-flag M2
affinity gel and visualized by immunoblotting with either HA- or flag-specific
antibodies. Lane 1, nonspecific antibody and similar affinity gel. High-
molecular-weight ubiquitinated forms of PR-B:flag are indicated (arrows).
PR down-regulation is mediated by the 26S proteasome. (A) T47Dco
Lange et al.
February 1, 2000 ?
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no. 3 ?
that of the 26S proteasome, had no effect on PR degradation
(Fig. 1B). In contrast, LLnL, a related compound that inhibits
several neutral cysteine proteases, including the cysteine pro-
tease activity of the 26S proteasome, effectively blocked PR
degradation (Fig. 1B).
Although ubiquitin-independent degradation of substrates by
the 26S proteasome is known (24), most proteins are targeted for
destruction through this pathway by ligation to 76-amino acid
ubiquitin molecules, assembled as polyubiquitin chains (re-
viewed in ref. 25). Polyubiquitinated proteins are often difficult
to visualize. Only ?3% of cyclin proteins exist as ubiquitin
conjugates during early mitosis; these species exhibit a 90-s
half-life (8). The proportion of PRs that exist as ubiquitin
conjugates at a given time point may be even lower, because the
receptors have a much longer half-life of several hours (4). To
demonstrate ubiquitin-conjugated PR, flag-tagged PR-B and
HA-tagged ubiquitin were transiently overexpressed in HeLa
cells by cotransfection of the respective expression vectors (17).
Cells were treated with R5020 for 4 h in the absence or presence
of 26S proteasome inhibitors, to allow accumulation of PR-
ubiquitin conjugates. flag-tagged PR-Bs were immunoprecipi-
tated with anti-flag antibodies and visualized by immunoblotting
with either HA- or flag-specific antibodies (Fig. 1C). Note that
transiently overexpressed PRs are often incompletely down-
regulated in response to R5020 and remain visible in immuno-
precipitates; a 4-h time course was used to trap PR-ubiquitin
conjugates. Additionally, PR:flag species of large molecular size
accumulated in lysates from cells treated with R5020 in the
presence of lactacystin or LLnL (Fig. 1C). These PR-B species
were also recognized by HA-specific antibodies, indicating that
they represent polyubiquitinated forms of B-receptors (Fig. 1C).
Similar, large-molecular-sized chick PR complexes were recently
identified in ubiquitin immunoprecipitates from progesterone-
treated oviducts (26). Fig. 1C shows that ubiquitin conjugates
were also present in immunoprecipitates containing unliganded
occurs in this overexpression system (see below).
MAPK and Phosphorylation. The yeast E2 ubiquitin-conjugating
enzyme Cdc34 targets phosphorylated substrates for proteolytic
degradation (reviewed in refs. 9 and 27), and phosphorylation is
a positive signal for the ubiquitination and targeting of several
D1 (30), and I?B? (31). PRs are basally phosphorylated at
multiple serine residues, and their phosphorylation is enhanced
in response to hormone binding (ref. 32; reviewed in refs. 33 and
34). Specific functions for PR phosphorylation remain unas-
signed (35). At least two phosphorylation sites common to the
A and B isoforms of PR, serine-294 and serine-345, are pre-
dominantly and latently phosphorylated after treatment of cells
with progestins (36). Both of these sites are serine–proline
consensus sites, PXX-S?T-P, for proline-directed kinases of the
MAPK superfamily (37), but whether MAPK phosphorylates
them is unknown. Furthermore, the ability of progestins, as well
as estrogens, to activate MAPK in T47D breast cancer cells has
been well-documented (38–40). We find that treatment of
serum-starved T47D-YB cells with R5020 for 5 min modestly
activates p42 and p44 MAPK, but not p38 MAPK, as measured
by the presence of phosphorylated active kinase in immunoblots
(data not shown).
Because progestins activate MAPK (39) and phosphorylation
of PRs occurs at two serine–proline MAPK consensus sites in
PR down-regulation depended on MAPK activation. R5020 was
added to T47D-YB cells pretreated with either the MEK1
inhibitor, PD (41), or a putative specific inhibitor of p38 MAPK,
SB202190 (SB190; ref. 42). The effects of these inhibitors on
ligand-dependent PR down-regulation were assayed by immu-
noblotting (Fig. 2A). PD (100 ?M) blocked PR protein loss by
?70% in T47D-YB cells treated with R5020 for 10 h, and SB190
(40 ?M) blocked down-regulation completely. To ensure the
effectiveness and specificity of each inhibitor at the concentra-
tions used, we examined p42 and p44 MAPK activity in whole-
cell lysates from EGF-treated T47D-YB cells (Fig. 2A). PD (100
?M) effectively inhibited the robust activation of p42 and p44
MAPKs by EGF at 5 min, compared with vehicle-treated
controls. Surprisingly, however, this effect was transient, and
MAPK activity was only weakly inhibited by PD in cells treated
with EGF for 15 min. In contrast, treatment of cells with EGF
of p42 and p44 MAPKs at all time points tested, indicating that
this compound is not a selective inhibitor of p38 MAPK alone.
Total MAPK levels remained constant and unchanged under
these conditions, as measured by immunoblotting (data not
shown). These results suggest that activation of either p42 and
p44 MAPKs or p38 MAPK or activation of both contribute to
ligand-dependent PR down-regulation.
To distinguish between p42 and p44 MAPKs vs. p38 MAPK,
we tested SB203580 (SB580; ref. 42), an additional inhibitor of
p38 MAPK (Fig. 2B). The activities of p42 and p44 MAPKs and
p38 MAPK were concomitantly assayed in EGF-treated cells
(A) MEK inhibitors block ligand-induced PR down-regulation. T47D-YB cells
were pretreated with vehicle (DMSO) or with PD98059 [PD (100 ?M)] or
SB202190 [SB190 (40 ?M)] for 30 min before challenge with R5020 [10 nM
(Upper) and MAPK activity (Lower) were measured by immunoblotting with
PR- or MAPK-phosphospecific antibodies, respectively. Total MAPK levels
remained unchanged (data not shown). (B) Ligand-induced PR down-
regulation requires activation of p42 and p44 MAPKs, but not p38 MAPK.
T47D-YB cells were pretreated with vehicle (DMSO), SB202190 [SB190 (40
nM (Upper)] or EGF [30 ng?ml (Lower)] for the indicated times. PR-B protein
levels (Upper) were measured with specific antibodies, and p42?p44 and p38
MAPK activities (Lower) were measured with phosphospecific antisera for
each kinase; 100 ?g of protein was loaded per lane.
Activation of MAPK triggers ligand-dependent PR down-regulation.
www.pnas.orgLange et al.
from the same experimental set. Again, SB190 blocked PR
down-regulation. This compound inhibited both EGF-
stimulated p42 and p44 MAPKs and p38 MAPK activities after
5 and 15 min of EGF treatment. In contrast, SB580 did not block
PR degradation. This compound weakly inhibited activation of
p42 and p44 MAPKs in cells treated for 5 min with EGF, but was
unable to inhibit MAPK activity in cells treated with EGF for 15
min. Under the same conditions, SB580 fully inhibited p38
MAPK activity. Thus, protection of PRs from ligand-induced
down-regulation correlated with the ability of each inhibitor to
block p42 and p44 MAPK activation. Several experimental
conditions and inhibitor concentrations led to similar conclu-
sions (data not shown). PD was a more effective inhibitor of p42
and p44 MAPKs than SB580 at 5 min, also the peak of MAPK
activation by progestins (39). Thus, early activation of p42 and
p44 MAPKs, but not p38 MAPK, may be required for progestin-
induced PR down-regulation. Because PR phosphorylation is
also a rapid event (23), neither kinase activation nor PR phos-
phorylation appears to be rate limiting for PR degradation.
Phosphorylation of Serine-294. To assess whether the requirement
for MAPK results from a direct effect on PR phosphorylation,
with alanine in both the A and B isoforms of PR. Ligand-
dependent receptor down-regulation of the mutants was then
measured in transient expression systems (Fig. 3 A and B).
Mutant A and B receptors were fully functional as measured by
transcriptional assays (data not shown; see ref. 35). HeLa cells
transiently expressing either wild-type or mutant PR-A (Fig. 3A)
or PR-B (Fig. 3B) were treated without or with R5020, and the
amount of PR protein was measured in whole-cell lysates by
immunoblotting. PR-A and PR-B, containing mutations at
serine-294 alone or at both phosphorylation sites, were com-
pletely resistant to R5020-induced down-regulation. Thus, mu-
tation of the single amino acid serine-294 to alanine was
sufficient to stabilize both PR isoforms in the presence of R5020.
Mutant PRs still underwent the characteristic ligand-induced
upward mobility shifts on SDS?PAGE gels, which was caused by
phosphorylation at multiple sites (23, 35). Clearly, phosphory-
lation at serine-294 is not required for this up-shift (ref. 36; Fig.
3). It is interesting that the basal expression levels of mutant
PR-A were consistently stabilized, even in the absence of ligand
(Fig. 3A), suggesting that PR-As undergo a low level of ligand-
independent turnover (see Figs. 5 and 6).
We noted that progestin-induced PR down-regulation is
slowed in transient expression systems (Fig. 3 A and B), probably
because excessive amounts of receptor proteins are produced.
The ubiquitin-conjugating enzymes (E2) and ubiquitin ligases
limiting in such overexpression systems (8, 9, 43). To analyze PR
down-regulation under more physiological conditions, either
S294A mutant or S344A?S345A mutant PR-B receptors were
stably introduced into PR-negative T47D-Y cells (16) and
compared with wild-type PR-B, also stably expressed in T47D-Y
cells (Fig. 3C). Cells were treated with R5020 for 2–10 h, and the
abundance of receptor protein was monitored by immunoblot-
ting. The S294A PR-B mutant was completely resistant to
progestin-induced down-regulation over the 10-h time course,
whereas both S344A?S345A and wild-type receptors were
are resistant to R5020-induced receptor down-regulation. HeLa cells were
transiently transfected with cDNA vectors (1 ?g) encoding either wild-type
PR-A or the S294A or S294?344?345A mutants of PR-A and then treated
without or with R5020 (10 nM) for 8 h. PR protein was measured in whole-cell
lysates (100 ?g) by immunoblotting. (B) Mutant S294A and S294?344?345A
PR-Bs are resistant to R5020-induced receptor down-regulation. Duplicate
cultures of HeLa cells were transfected with wild-type PR-B or each phospho-
mutant of PR-B and treated without or with R5020 for 18 h. Protein (100 ?g
per lane) was loaded, and PR levels were measured by using PR-specific
monoclonal antibodies. (C) The S294A PR-Bs stably expressed in T47D-Y cells
are resistant to ligand-induced down-regulation. PR-negative breast cell lines
stably expressing either an S344?345A (B-S345A) mutant or the S294A (B-
S294A) mutant PR-B receptors were produced by transfection of receptor
expression vectors containing the neomycin-resistance gene into T47D-Y cells
and selected for growth in G418. Neoresistant clonal cell lines were screened
for PR expression, and S344?345A or S294A PR-containing cells or wild-type
PR-B-containing T47D-YB cells (wt-PR-B) were treated without or with R5020
(10 nM) for 2–10 h; protein levels were measured with PR-specific antibodies.
150–200 ?g of protein was loaded per lane.
Mutation of serine-294 to alanine at a MAPK consensus site stabilizes
transiently cotransfected with expression vectors encoding HA-tagged-
ubiquitin and either epitope-flag-tagged wild-type PR-B [wt-PR-B:flag (lanes
1–3)] or epitope-flag-tagged S294A mutant PR-B [S294A:flag (lanes 4–6)] and
treated for 4 h without (lanes 2 and 5) or with R5020 (lanes 3 and 6). PRs were
immunoprecipitated by using an anti-flag M2 affinity gel and were visualized
by immunoblotting with either HA- or flag-specific antibodies. Lanes 1 and 3,
nonspecific antibody and similar affinity gel. High-molecular-weight ubiqui-
but not S294A mutant PR-Bs are indicated by arrows.
Mutant S294A PR-Bs fail to undergo ubiquitination. HeLa cells were
Lange et al.
February 1, 2000 ?
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no. 3 ?
largely degraded after 6–8 h (Fig. 3C). Mutant S294A PR-B
receptors were resistant to degradation for as long as 42 h of
R5020 treatment (data not shown). Other independently derived
To test whether S294A mutant PR were ubiquitinated in
response to R5020 treatment, flag-tagged wild-type or S294A
mutant PR-B and HA-tagged ubiquitin were transiently over-
expressed in HeLa cells (ref. 17; as in Fig. 1). Cells were treated
with R5020 for 4 h, and flag-tagged wild-type or mutant PR-Bs
were immunoprecipitated with anti-flag antibodies and visual-
ized by immunoblotting with either HA- or flag-specific anti-
bodies (Fig. 4). Transiently overexpressed wild-type PRs were
observed (Fig. 1 and 3). Immunoblotting with HA-specific
antibodies revealed PR:flag species of large molecular size in
lysates from cells transiently overexpressing wild-type PR-Bs,
but not S294A mutant PR-Bs, indicating that S294A mutant
PR-B fails to undergo efficient ubiquitination (Fig. 4). Immu-
noblotting with flag-specific antibodies demonstrated much
higher levels of unconjugated S294A mutant PR-B protein
compared with wild-type PR-B, reflecting the greater stability of
S294A mutant PR-B (Fig. 3). Antiflag antibodies also recog-
nized ubiquitin-conjugated wild-type PR-B species (data not
Thus, specific phosphorylation of PR on serine-294 by MAPK
appears to be required for progestin-induced PR ubiquitination
and degradation by the 26S proteasome. Ligand binding may
in response to progestin-activated MAPKs, or in response to
basally activated or preactivated MAPKs, is unknown. Previous
studies to define a function for PR phosphorylation have relied
heavily on the use of transient transfection assays (35, 44), but
the behavior of such overexpressed receptors may differ signif-
icantly from that of natural or stably expressed proteins. This
study is the first demonstration of a clear and specific role for any
phosphorylated site on human PRs, in which at least nine sites
have been documented (33, 34).
Ligand-Independent Degradation. Because expression of S294A
mutant PR-As appeared to be stabilized in a ligand-independent
manner (Fig. 3A), we determined whether PR protein degrada-
tion via the 26S proteasome can also proceed in the absence of
ligand. We took advantage of the fact that unliganded mature
PRs exist in heteromeric complexes with several heat-shock
protein (hsp) chaperones, including p23, hsp 70, and hsp 90. GA
is a benzoquinone that blocks addition of the chaperone-
assembly factor, p23, to the PR?hsp90 complex and thereby
blocks PR maturation to a ligand-binding form (45). In the
presence of GA, unliganded immature PR-Bs are degraded at an
even faster rate than that induced by ligand (Fig. 5A). However,
PRs in GA-treated cells fail to undergo the characteristic
upshifts associated with ligand-induced receptor phosphoryla-
tion, indicating that they are not similarly phosphorylated (Fig.
5A; 2–4 h). To determine whether GA-induced loss of immature
PRs is also mediated by the 26S proteasome, cells were treated
with GA in the absence or presence of lactacystin (Fig. 5B).
Lactacystin blocked GA-induced PR protein loss compared with
controls containing either GA or lactacystin alone, indicating a
role for the 26S proteasome in ligand-independent PR degra-
dation. PR levels were not significantly raised by lactacystin,
indicating minimal accumulation of newly synthesized PRs over
are targeted to the 26S proteasome in the absence of ligand and
whether phosphorylation plays a role in this process. Indeed, we
found that S294A mutant PR-Bs were also rapidly degraded in
the presence of GA (data not shown), suggesting that the
targeting mechanism for unliganded PRs differs from that of
liganded PRs. Rapid degradation of other hsp 90-chaperoned
proteins, including c-erbB2 (5), Raf-1 (46), and glucocorticoid
receptors (47), in the presence of GA also occurs by an ubiquitin-
dependent proteasomal mechanism (reviewed in ref. 48).
Summary. Substrates for the ubiquitin-proteasome pathway often
contain specific amino acid sequence motifs required for their
degradation (8). Examination of the amino acid sequences
immediately surrounding serine-294 of PRs revealed the pres-
are rapidly degraded in the absence of ligand. T47D-YB cells were treated
without [C (EtOH vehicle)] or with R5020 alone [R (10 nM)] or with the hsp
in whole-cell lysates was measured by immunoblot analysis. (B) Ligand-
independent degradation is mediated by the 26S proteasome. Duplicate
or with R5020 [R (10 nM)] alone, GA [G (10 ?g?ml)] alone, or with GA plus
lactacystin [Lt. (10 nM; 30-min pretreatment)], GA plus R5020, or lactacystin
measured by immunoblot analysis with PR-specific antisera.
ligand-independent and ligand-dependent PR down-regulation. Mature PR
complexes bind progesterone, leading to ligand-dependent phosphorylation
of serine 294 by p42?p44 MAPKs, which serves as a signal for PR degradation
by the 26S proteasome. Inhibition of p42?p44 MAPKs (PD98059 or SB190) or
the 26S proteasome (lactacystin) blocks loss of wild-type PR in the presence of
progestins. S294A-mutant PRs are resistant to ligand-dependent down-
regulation. In the presence of GA, rapid ligand-independent degradation of
www.pnas.orgLange et al.
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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