Department of Physiology Dartmouth Medical School Lebanon, New Hampshire 03756-0001
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ABSTRACT |
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INTRODUCTION |
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To date, reported effects of steroid hormone receptor phosphorylation on activity are subtle and varied, serving modulatory rather than obligatory functions. For example, phosphorylation of serine 118 in the human estrogen receptor (9) modulates enhancement of transcriptional activation by growth factors (10); mutation of this serine to alanine decreases estrogen-induced transactivation by 15 to 75%, depending on cell type and reporter gene (11, 12). In the chicken progesterone receptor, mutation of serine 530 to alanine reduces transcriptional activity by 7085% at low, but not at high, hormone concentrations (13). Mutation of serine 211 to alanine reduces transcriptional activity by 25 to 80%, again depending on cell type and reporter gene (14). Comparable effects are seen with vitamin D (15) receptors. Mouse GRs mutated at single and multiple phosphorylated residues have been reported to give hormone-induced activity almost indistinguishable from that of wild type GRs (WTGRs) when transiently transfected into COS cells with a reporter gene under a mouse mammary tumor virus promoter (16). However, when in similar experiments a minimal promoter with simple glucocorticoid response elements is used, most GRs with one or more phosphorylated sites mutated to alanine are 5075% less active than WTGRs (J. A. Cidlowski, personal communication). These and related GR phosphorylation mutants behave like WTGRs with respect to subcellular localization (17) and superactivation of transcription by cAMP (18) but are much less sensitive to hormone-induced down-regulation (19).
A largely unexplored role for GR phosphorylation is regulation of hormone-induced GR activity through the cell cycle. Synchronized cells with endogenous WTGRs generally are sensitive to glucocorticoids in late G1 and S, but resistant in G2, M, and early G1. Effects measured include induction of alkaline phosphatase activity (20) and epidermal growth factor receptors (21) in HeLa cells, of tyrosine aminotransferase in hepatoma cells (22), and of the metallothionein-I gene in L cell fibroblasts (23). This cell cycle dependence has some specificity for glucocorticoid activity because with cells arrested in G2, transcription of the endogenous metallothionein-I gene is resistant to glucocorticoid induction but remains sensitive to induction by heavy metals (23). In GrH2 cells glucocorticoid resistance in G2 is selective, affecting transactivation from a simple glucocorticoid response element but not repression from a composite glucocorticoid response element (24).
Much evidence links glucocorticoid activity through the cell cycle to GR phosphorylation. Cidlowski and colleagues found that GRs in HeLa cells are more negatively charged in G2 and early G1 than in late G1 and S (21, 25) and proposed that increased GR phosphorylation or glycosylation causes glucocorticoid resistance in G2 (26). Hsu et al. (23) detected alterations in two-dimensional phosphopeptide maps of GRs from L cells arrested in G2, and advanced a similar proposal. Most phosphorylated residues in mouse GRs lie in consensus sequences for cell cycle-associated kinases (2, 27). Furthermore, basal GR phosphorylation is 3-fold higher in G2/M than in S (reflecting a 3-fold higher frequency of phosphorylation of the same set of sites, since no new phosphorylated sites appear in G2/M) (27). Most significantly, hormone-induced hyperphosphorylation nearly doubles GR phosphorylation in S (a glucocorticoid-sensitive phase) but is absent in G2/M (glucocorticoid-resistant phases) (27).
This parallel between cell cycle dependence of hormone-induced GR hyperphosphorylation and of glucocorticoid activity prompted us to determine why GR hyperphosphorylation can occur in S but is blocked in G2/M (27). We have tested the following three hypotheses: hormone-induced hyperphosphorylation is controlled 1) by overall negative charge from basal phosphorylation, being permitted by the relatively low charge in S and blocked by the high charge in G2/M; 2) by the presence in S and absence in G2/M of required kinases; 3) by the presence in S and absence in G2/M of phosphorylatable sites, which are unfilled in S but filled by basal phosphorylation in G2/M. Our results strongly favor the first hypothesis and are inconsistent with the other two.
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RESULTS AND DISCUSSION |
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Cells stably transfected with the mutant GRs were synchronized in S and G2/M (27). Hormone-induced hyperphosphorylation within each phase was measured by comparing 32P/35S ratios of GRs, purified by immunoprecipitation and SDS-PAGE, from cells that had been incubated with [35S]methionine and [32P]orthophosphoric acid, and during the last hour of labeling were treated with 500 nM triamcinolone acetonide (TA) or were untreated (controls). Incorporation of 35S served to normalize 32P incorporation, with and without TA treatment, to the same amount of GR protein (3, 7, 27).
Figure 1 shows that A7-GRs, in contrast to WTGRs, become
hyperphosphorylated in both S and G2/M. This is the outcome predicted
by the first hypothesis, because with A7-GRs the negative charge in the
N-terminal domain is limited throughout the cell cycle by an almost
complete lack of phosphorylatable sites and therefore cannot be raised
through basal phosphorylation to levels postulated to block
hyperphosphorylation of WTGRs in G2/M.
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The conclusions drawn from the results in Fig. 1 assume that no major
new sites, i.e. sites that are not present in WTGRs, are
phosphorylated in the A7GRs. The HPLC phosphopeptide maps in Fig. 2
validate this assumption by establishing that no
significant phosphorylated sites other than those present in WTGRs
appear in A7GRs, either before or after hormone treatment. The
top map, of WTGRs from unsynchronized cells, is included for
comparison. It was obtained by our current procedures (8, 28), which
differ slightly from those used originally (2). Locations are given of
previously identified phosphopeptides (2) and of a peak we have
recently identified tentatively with a tryptic peptide containing
serine 412.
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Figure 3 shows that with E7GRs no hyperphosphorylation
occurs in either S or G2/M. This, again, is the outcome predicted by
the first hypothesis, since throughout the cell cycle the glutamate
residues in the E7GR contribute a high, fixed negative charge in the
N-terminal domain, which cannot be lowered to a level comparable to
that postulated to permit hyperphosphorylation of wild type GRs in S
phase. These results also provide evidence against the second
hypothesis, because it fails to predict absence of hyperphosphorylation
in S.
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Second, the HPLC phosphopeptide map of WTGRs from cells synchronized in
G2/M (bottom map in Fig. 2) is practically indistinguishable
from the maps of WTGRs from unsynchronized cells (top map in
Fig. 2
) and of WTGRs from cells synchronized in S (27). It gives no
indication that in G2/M, all sites on the WTGRs are fully
phosphorylated. If they were fully phosphorylated, they would be
equally labeled after reaching steady state with 32P. Rates
of GR phosphorylation and dephosphorylation (3, 7, 8, 28) indicate that
the 4-h incubations with 32P used here will label GRs to
7080% of steady state, sufficient to give HPLC-labeling patterns
closely approximating those at steady state. The sum of 32P
in peaks for individual sites from HPLC maps of unsynchronized WTGRs
obtained after 14 h incubation with 32P (2), as well
as from the top HPLC map in Fig. 2
, show, for example, that
the ratio of steady state labeling of serine 234 to serine 315 is about
4. The same ratio is found from HPLC maps of WTGRs from cells
synchronized in G2/M (bottom map of Fig. 2
) and synchronized
in S (27), despite the fact that basal GR phosphorylation is about 3
times higher in G2/M than in S (27). If all sites in WTGRs from G2/M
were filled, the ratio would be about 1. Even if all contributions to
each site were not included in the sums, substantial differences
between the ratios for WTGRs in S and G2/M would still be expected.
Thus, absence of hormone-induced hyperphosphorylation of WTGRs in G2/M
cannot be explained by all sites being fully phosphorylated at basal
levels.
We are left, then, with the conclusion that hormone-induced
hyperphosphorylation of GRs is modulated by overall negative charge in
the N-terminal domain. If that conclusion is valid it follows that
qualitatively, at least, the charge carrier is not critical, since
negative charges carried in E7GRs by glutamates (Fig. 3) can block
hyperphosphorylation as effectively as those in WTGRs carried by
phosphates (27). Quantitatively there may be differences, however: E7GR
molecules are negatively charged at all seven mutated sites, whereas
WTGR molecules on average are phosphorylated, and therefore negatively
charged, at only a fraction of those sites. The region from about
residues 195 to 260, which includes the most heavily phosphorylated
sites in WTGRs (2), is strongly acidic even without phosphorylation
(29), suggesting that phosphorylation modulates negative charge above
and below a threshold.
Whether particular phosphorylated sites or combinations of sites are more effective than others in blocking hyperphosphorylation is not known. The fact that the same set of sites on WTGRs is phosphorylated throughout the cell cycle and after hormone treatment, despite large changes in overall phosphorylation levels (8, 27), and that the sites are neither cell type-specific (2, 7) nor, apparently, species-specific (30), is consistent with a concerted role of the phosphorylated sites to modulate overall negative charge in the N-terminal domain. How, in turn, the high negative charge in G2/M might block hormone-induced hyperphosphorylation is an open question. Possible mechanisms include the alternatives that the negative charge hinders hormone-induced activation of GRs (which, as mentioned earlier, appears to be required for hyperphosphorylation), or causes activated GRs to be sequestered beyond reach of kinases, or interferes with proper contact between the N-terminal domain of the GR and the active sites of kinases.
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MATERIALS AND METHODS |
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Cells and Cell Culture
Cells with A7GRs and E7GRs were grown at 37 C with 5%
CO2 in DMEM (4.5 g glucose/liter) containing 10%
charcoal-stripped calf serum supplemented with 39.5 mg proline/liter
and 100 nM methotrexate. WCL2 cells were grown in the same
medium but with 3 µM methotrexate.
Charcoal-Stripped Serum
A suspension of dextran-coated charcoal was prepared as
described previously (31), except that 10-fold higher concentrations of
charcoal and dextran were used, and PBS was substituted for
MgCl2. The pellet obtained by centrifuging 25 ml of the
suspension at 10,000 x g for 10 min was resuspended
with 250 ml of iron-supplemented calf serum and incubated for 30 min at
2325 C. After centrifugation at 10,000 x g for 10
min, the serum was decanted from the charcoal, then treated once more
with dextran-coated charcoal before sterile filtering through a
0.2-µm filter.
Mutation of GRs
Mouse GR phosphorylation mutants with phosphorylated sites
changed to either alanine or glutamate were produced by the Kunkel
method (32, 33) as described previously (28). Two mutant GRs, A7GR and
E7GR, were generated for this study. In the A7GR, serines 150, 212,
220, 234, 315, 412, and threonine 159 were changed to alanine. Serine
122 was not mutated: it remained as a reporter of phosphorylation. In
the E7GR the same sites were changed to glutamate, again leaving serine
122 intact.
Generation of Stably Transfected Cell Lines
The general procedures have been described by Hirst et
al. (34). Briefly, mutant GR cDNA and a selectable dhfr cDNA
(pSV2dhfr) were cotransfected into DG44 cells [a Chinese hamster ovary
cell line missing both alleles of the dhfr gene (35)] by calcium
phosphate precipitation. After 48 h the cells were exposed to
different concentrations of methotrexate starting from 100
nM. Only cells that express dhfr survive in the presence of
methotrexate. Clones expressing relatively high numbers of GRs were
selected by [3H]TA binding assay (27). Those used for the
present studies had approximately 600,000 A7GRs and about 800,000 E7GRs
per cell. SDS-PAGE and Western blot analyses showed that the mutant GRs
had the same molecular mass (100 kDa) as WTGRs and that no other
receptor forms were present. Binding affinities of the mutant GRs for
TA were indistinguishable from that of wild type GRs.
Cell Synchronization and Metabolic Labeling
Procedures used were those described for synchronization of WCL2
cells (27). Briefly, cells with mutant GRs were incubated under serum
deprivation conditions (0.1% calf serum) for 48 h at 37 C, which
partially synchronized them in G0/G1. They were
then synchronized in late G1/early S phase by incubation in 1
mM hydroxyurea for 24 h. To lower phosphate and
methionine levels for labeling, during the last hour they were
incubated in phosphate-free, low methionine (5.4 mg/liter) medium with
1 mM hydroxyurea. Hydroxyurea was washed out (time =
0), and the cells were divided equally among four flasks (4 x
106 cells per flask), to each of which was added
[35S]methionine in phosphate-free, low methionine
labeling medium. Culture in this medium (as compared with medium with
normal methionine and phosphate) did not affect cell progression
through the cell cycle.
[32P]orthophosphoric acid was added to one pair of flasks at 0.5 h for S phase labeling and to the other pair at 6.5 h for G2/M labeling. TA (500 nM) was added to one of each pair of flasks 3 h later. After 1 h the cells were harvested and the cell pellets frozen with liquid nitrogen in FTT buffer (freeze-thaw buffer + 0.2% Triton X-100).
For cell cycle analysis, in parallel with the cells used for labeling, some cells were cultured in labeling medium without radioactivity and incubated for various times, after which they were harvested and fixed in ethanol. To measure relative DNA content the cells were treated with RNase A, stained with propidium iodide (36), and analyzed by flow cytometry (FACScan, Becton Dickinson, Franklin Lakes, NJ). The synchronization procedure yielded approximately 85% of cells in S phase from 0 to 5 h after release from hydroxyurea, and 85% in G2/M from 8 to 12 h after release.
Purification and HPLC Phosphopeptide Mapping of GRs
As described for WTGRs (2, 28), A7GRs and E7GRs were extracted
and purified by immunopurification and SDS-PAGE. To determine the
magnitude of hormone-induced hyperphosphorylation, the total
32P incorporation into GRs was normalized to total
35S incorporation, the latter providing a relative measure
of GR protein (3). The purified A7GRs and E7GRs were digested with
trypsin, and the resulting phosphopeptides were analyzed by HPLC as
detailed previously (2, 28).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This research was supported by Research Grants DK-03535, DK-47329, and DK-45337 from the NIH and by the Norris Cotton Cancer Center Core Grant CA-23108. J.-M. H. was supported by a predoctoral fellowship from the Norris Cotton Cancer Center.
1 Abbreviations and trivial names used: A7GR,
mutant GR with all but one phosphorylated site mutated to alanine;
E7GR, mutant GR with all but one phosphorylated site mutated to
glutamate; hsp90, approximately 90-kDa heat shock protein; G1 phase,
gap 1 phase of cell cycle; S phase, DNA synthesis phase of cell cycle;
G2, gap 2 phase of cell cycle; M, mitosis phase of cell cycle; G2/M,
gap 2 and mitosis phases of cell cycle; RU486,
17ß-hydroxy-11ß,4-dimethylaminophenyl-17-propynyl
estra-4,9-diene-3-one; TA, tri-amcinolone acetonide,
9
-fluoro-11ß,16
,17
,21-tetrahydroxypregna-1,4-diene-3,20-dione-16,17-acetonide;
dhfr, di-hydrofolate reductase.
Received for publication October 29, 1996. Revision received December 2, 1996. Accepted for publication December 5, 1996.
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REFERENCES |
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