INTRODUCTION

In contrast to peptide hormones whose signals are transduced into the nucleus through membrane receptor-activated signal transduction pathways, steroid hormones act through intracellular receptors which themselves are ligand-regulated transcription factors (1-7). The lipophilic hormones diffuse through the cell membrane, bind the receptors inside the cell, and transform them into active transcription factors. In this way, the signal carried by steroids is directly transduced into long term changes in gene expression without an absolute requirement for a phosphorylation cascade to mediate the effect.

Studies in recent years have provided increasing evidence that there is cross-talk between the signaling processes induced by growth factors and steroids, and phosphorylation plays an important role in these processes. First, most, if not all, steroid receptors are phosphoproteins (8-14). Second, functional analyses of phosphorylation site mutants have demonstrated that phosphorylation regulates the transcriptional activity of many steroid receptors (14-19). Third, modulators of various signal transduction pathways have been shown to regulate the ligand-stimulated transcriptional activity of the receptors (20-29). Most importantly, many steroid receptors have been shown to be activated in the absence of their cognate ligands by modulation of protein kinase or phosphatase activity (22, 24, 25, 30-35). Ligand-independent activation of steroid receptors may have important physiological and clinical implications for the study and treatment of the tumors of hormone-responsive organs. Very little is known about the molecular mechanism of ligand-independent activation for any of the receptors. Since steroid receptors are phosphoproteins, it is possible that alteration of receptor phosphorylation in response to different treatments mediates the ligand-independent activation.

Chicken progesterone receptor (cPR)¹ is one of the steroid receptors that has been shown to be activated in a ligand-independent manner by modulators of kinases and phosphatases such as 8-bromoadenosine 3:5-cyclic monophosphate (8-bromo-cAMP), okadaic acid, vanadate, growth factors (e.g. EGF), and neurotransmitters (e.g. dopamine) (25, 30, 32). The phosphorylation sites in cPR have been identified (8, 36), making it possible to answer the question of whether the phosphorylation of the receptor is involved in or mediates the ligand-independent activation of the receptor.

cPR is expressed as two forms, cPR_B and cPR_A, which lack the amino-terminal 128 amino acids of cPR_B. Like the other members of the steroid/thyroid superfamily, cPR is composed of separable domains such as domains for DNA binding, hormone binding, and transcriptional activation (37-39). Different from the DNA binding and hormone binding domains which are not further separable, two separable transcriptional activation domains have been identified in chicken progesterone receptor (37, 40). The activation function 2 (AF-2, previously named transactivation function 2 or TAF-2), within the hormone binding domain is known to be regulated by hormone. In contrast to AF-2, the activation function 1 (AF-1, previously named transcriptional activation function 1 or TAF-1) is located in the amino-terminal region (37, 40). The fact that AF-1 is located in the sequence outside the hormone binding domain raises the possibility that the activity of AF-1 might be regulated by means such as phosphorylation rather than by ligand binding. Consistent with this idea, three out of the four phosphorylation sites identified in cPR are located in the A/B region (8, 36).

Based on the primary sequence of cPR_B, the four identified phosphorylation sites are Ser²¹¹, Ser²⁶⁰, Ser³⁶⁷, and Ser⁵³⁰ (8, 36). All four sites are in Ser-Pro motifs (8, 36), and they account for all the Ser-Pro motifs in the receptor. The same phosphorylation pattern was detected in both cPR_A and cPR_B and is conserved between the endogenous receptor isolated from chicken oviduct and the recombinant receptor expressed in and purified from yeast (41). Among the four sites, Ser²¹¹ and Ser²⁶⁰ are basally phosphorylated, but their phosphorylation is enhanced in response to hormone stimulation; Ser³⁶⁷ and Ser⁵³⁰ are phosphorylated primarily in response to progesterone treatment (8, 36).

To examine whether any of the known phosphorylation sites in cPR is involved in or mediates the ligand-independent activation of the receptor, the four phosphorylation sites were mutated either individually or simultaneously to nonphosphorylatable alanines, and the responses of these mutant receptors to 8-bromo-cAMP and a dopamine agonist were assayed and compared with that of the wild type receptor. Our data show that receptor phosphorylation is not absolutely required for ligand-independent activation, raising the possibility that the ligand-independent activation of cPR might be mediated through changes in the phosphorylation of receptor-associated proteins such as coactivators and chaperone proteins that are known to play important roles in receptor function.

EXPERIMENTAL PROCEDURES

All cell culture reagents were purchased from Life Technologies, Inc. [³H]Chloramphenicol and carrier-free [³²P]H₃PO₄ were purchased from DuPont NEN. N-Butyryl-coenzyme A and Protein-A Sepharose were purchased from Pharmacia Biotech Inc. The T7-Gen in vitro mutagenesis kit and Sequenase version 2.0 sequencing kit were purchased from United States Biochemical Corp. The oligonucleotides used in the mutagenesis and sequencing were synthesized by GenoSys (The Woodlands, TX). Triethylamine, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, 8-bromo-cAMP, Triton X-100, 8-methoxypsoralen, sequencing grade trifluoroacetic acid, poly-L-lysine and 2,6,10,14-tetra-methylpentadecane were purchased from Sigma. Xylene was purchased from Fisher. Tosylphenylalanyl chloromethyl ketone-treated trypsin was obtained from Worthington. Phenylisothiocyanate and HPLC reagents were obtained from J. T. Baker Inc. The D1 dopamine agonist, SKF82958 (6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide) was obtained from Research Biochemicals Inc. (Natick, MA). Monoclonal antibody to the chicken progesterone receptor (PR22) was kindly provided by Dr. David Toft. All other chemicals are of reagent grade.

Single site mutations were generated using a conventional, non-polymerase chain reaction mutagenesis strategy as described previously (15). The mutant in which all four phosphorylation sites are changed to alanine was generated by exchanging the restriction fragments between the single site mutants. All mutations were confirmed by direct sequencing using a Sequenase version 2.0 sequencing kit. The subcloning of the mutant and the wild type cPR_A into the expression vector, p91023B, has been fully described (15, 38). GRE₂e1bCAT (provided by Dr. John Cidlowski) is a simple promoter-based reporter composed of two progesterone/glucocorticoid response elements (GREs), the TATA box from the adenovirus e1b gene, and the cDNA sequence for chloramphenicol acetyltransferase (CAT) (42).

cPR_A was expressed in cells using a nonrecombinant adenoviral-mediated DNA transfer technique (31, 43) with a few modifications. Replication-deficient adenovirus, dl312, was grown in human 293 embryonic kidney cells. The virus was released from the cells by three successive freeze/thaws of the cell pellet in phosphate-buffered saline. Following centrifugation at 2,500 rpm in a clinical centrifuge, the resulting supernatant was layered on top of a cesium chloride step gradient (1.5, 1.35, and 1.25 g/ml CsCl). Following centrifugation of the CsCl gradient at 150,000 × g for 1 h at 10 °C, the adenovirus band (between the 1.25 and 1.35 g/ml layers) was removed and brought to a final concentration of 1.35 g/ml CsCl. The adenovirus was centrifuged again at 150,000 × g for 5 h at 10 °C, and the adenovirus band was collected and dialyzed against HEPES-buffered saline (150 mM NaCl, 20 mM HEPES, pH 7.3) for 16 h at 4 °C with 50,000 molecular weight cutoff dialysis tubing. The dialysate was exposed to short wavelength UV light (254 nm) for 3 min. 8-Methoxypsoralen was added to the supernatant at a final concentration of 0.33 mg/ml, and the sample was exposed to long wavelength UV light (366 nm) for 20 min. The preceding treatments inactivated the viral genome without significant loss of infectivity. These treatments were a modification from a previously published method designed to inactivate adenovirus particles (44, 45). 8-Methoxypsoralen was removed from the virus preparation by G25 gel filtration chromatography. Adenovirus (1.4 × 10¹¹ particles) was mixed with 160 µl of 10 mg/ml poly-L-lysine and 6.5 µl of 80 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in a sterile polystyrene tube in a final volume of 4 ml of HEPES-buffered saline and incubated for 4 h on ice with mixing every 30 min. The coupled virus was brought to a final concentration of 1.35 g/ml CsCl. The adenovirus was centrifuged at 150,000 × g for 5 h at 10 °C, and the adenovirus band was collected and dialyzed against HEPES-buffered saline for 16 h at 4 °C. Viral DNA was quantitated by measuring the optical density at 260 nm. Briefly, 25 µl of purified virus was mixed with 465 µl of phosphate-buffered saline and 10 µl of 5% SDS. The mixture was vortexed for 2 min and then centrifuged for 2 min at room temperature in a microcentrifuge. The optical density of the resulting supernatant was measured at 260 nm (1 optical density unit equals approximately 1 × 10¹² virus particles/ml).

Cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with fetal bovine serum (FBS) and antibiotics (penicillin and streptomycin). Twenty-four hours before transfection, cells were plated in 6-well plates at a density of 1 × 10⁵ cells per well in the same medium. Before transfection, cells were rinsed with Hank's balanced salt solution, and 2 ml of DMEM was added to each well. DNA and polylysine-coupled viruses were incubated for 30 min at room temperature, and additional polylysine (polylysine/DNA ratio is 1:1.3) was added. The DNA-virus-polylysine complex was incubated at room temperature for another 30 min. During the incubations, the DNA-virus complex was kept in the dark. After the incubations, the complex was added dropwise to the cells. Cell were incubated with DNA-virus complex for 2 h. Then 2 ml of DMEM supplemented with 10% stripped FBS was added to each well. On the next day, progesterone and compounds such as 8-bromo-cAMP at the indicated concentrations were added to the cells, and the cells were incubated for an additional 24 h before being harvested and assayed for CAT activity.

For CAT assays, cells were harvested by scraping and subsequently lysed by three cycles of freeze-thawing. The protein concentrations were determined using the Bio-Rad protein assay reagent according to the manufacturer's protocols. Equal amounts of protein (usually 5-10 µg) were then used for a liquid CAT assay that has been described (46) and used in our previous studies (15, 16, 32). The samples were heated at 60 °C for 8 min before the addition of the substrates, and the reactions were terminated and extracted before the substrates become a limiting factor to ensure the accurate determination of the CAT activity. Duplicate samples were assayed for each data point.

CV-1 monkey kidney cells were plated on 150-mm dishes (2 × 10⁶ cells/dish) and cultured at 37 °C with 5% CO₂ in DMEM with 10% FBS that had been stripped of steroid hormones by treatment with dextran-coated charcoal. After 24 h in culture, cells were subjected to adenovirus infection (virus to cell ratio, 750:1) with 0.2 µg of cPR_A expression vector/plate and cultured for an additional 24 h. To remove endogenous phosphate pools from the cells, the culture medium was removed, and phosphate-free DMEM was added to the cells for 1 h at 37 °C. Subsequently, this medium was removed, and cells were cultured in phosphate-free DMEM with 1% dialyzed, stripped FBS. 6 mCi of [³²P]H₃PO₄ (2 mCi/ml) was added to each plate of CV-1 cells, and cells were cultured for 1 h at 37 °C. Treatment was as follows. For 10-h treatments, either 8-bromo-cAMP (final concentration 2 mM) or progesterone (final concentration 10⁸ M) was added to cell cultures 1 h after [³²P]H₃PO₄ addition, and cells were cultured for 10 h prior to harvest. For 30-min treatments, 8-bromo-cAMP (final concentration 2 mM) was added to cell cultures 10.5 h after [³²P]H₃PO₄ addition, and cells were cultured for 30 min prior to harvest.

cPR protein was purified and subjected to trypsin digestion and HPLC analysis as described previously (8) with the following modifications. Briefly, cells were scraped from the plates in phosphate-buffered saline and centrifuged at 500 × g for 10 min at 4 °C. Cell pellets were resuspended in homogenization buffer (50 mM potassium phosphate, pH 7.4, 10 mM sodium molybdate, 50 mM sodium fluoride, 2 mM EDTA, 2 mM EGTA, 0.4 M sodium chloride, 5 mM -monothioglycerol) (8) with 1% Triton X-100 and vortexed for 30 s. Following centrifugation at 100,000 × g for 30 min at 4 °C, 3 volumes of homogenization buffer without Triton X-100 was added to the supernatant to reduce the detergent concentration. The receptor was then purified by passing the entire supernatant over a 1-ml PR22-Protein-A-Sepharose immunoaffinity column as described previously (8). Purified receptor was electrophoresed on a 6.5% SDS-PAGE gel, and the wet gel was exposed to X-AR film (Eastman Kodak) for 2-4 h at 4 °C. The phosphorylated receptor band was cut out of the gel, and the gel slice was digested with trypsin to prepare the tryptic cPR_A peptides (8). HPLC analysis of tryptic peptides was performed as described previously (8). Briefly, phosphopeptides were loaded onto a C-18 reverse phase HPLC column in HPLC-grade H₂0 containing 0.1% trifluoroacetic acid. Phosphopeptides were separated by elution from the C-18 column using a 0-45% gradient of acetonitrile containing 0.1% trifluoroacetic acid over a period of 90 min.

Purified cPR_A was analyzed by SDS-PAGE and transferred to a nitrocellulose membrane. The receptor was detected using the monoclonal antibody, PR22, followed by chemiluminescent detection using enhanced chemiluminescence reagent (Amersham Corp.) as described (47).

RESULTS

To determine the molecular mechanism of the ligand-independent activation of cPR by 8-bromo-cAMP, we individually mutated each of the four known phosphorylation sites to alanine, and the transcriptional activity of each of the four single-site cPR_A mutants was analyzed and compared with the wild type receptor after treatment with either 2 mM 8-bromo-cAMP or 10 nM progesterone. As shown in Fig. 1, the transcriptional activity of the Ala²¹¹ and Ala²⁶⁰ mutants in response to either progesterone or 8-bromo-cAMP is significantly lower than that of the wild type. As reported previously (15), the activity of Ala⁵³⁰ is not statistically different from that of the wild type at this hormone concentration. The mutation of Ser³⁶⁷ to alanine appears not to affect the transcriptional activity of the receptor. From Fig. 1, it is obvious that the transcriptional activity of Ala²¹¹ and Ala²⁶⁰ in response to 8-bromo-cAMP is affected by the mutations to a magnitude similar to the activity in response to progesterone, suggesting that the receptor phosphorylation at these two sites can regulate the ligand-independent activation. The fact that all four mutants can still be activated by 8-bromo-cAMP suggests that phosphorylation at any of the individual sites is not absolutely required for the ligand-independent activation. However, this experiment did not rule out the possibility that phosphorylation of cPR at the remaining sites compensates for the loss at one position in mediating the receptor activation.

To investigate the possibility that phosphorylations at the four known sites compensate for one another during the ligand-independent activation of the receptor, all four sites were mutated simultaneously to alanines, and the activity of this quadruple mutant (AAAA) was compared with that of the wild type after stimulation with either 8-bromo-cAMP or 10 nM progesterone. As shown in Fig. 2, the transcriptional activity of the AAAA mutant in response to progesterone as well as to 8-bromo-cAMP is lower than that of the wild type. However, it is obvious that the AAAA mutant can still be activated by 8-bromo-cAMP in the absence of hormone. Together with the data shown in Fig. 1, this experiment suggests that phosphorylation of the four sites modulates but is not absolutely required for the ligand-independent activation of cPR by 8-bromo-cAMP.

Dopamine is another compound that induces ligand-independent activation of cPR_A (25). Previous studies have also indicated that the activation by dopamine is mediated through the D1 subtype of dopamine receptor (25). To determine whether phosphorylation at any of the four known sites is required for the ligand-independent activation of cPR_A by dopamine, cPR mutants were analyzed for their ability to be activated by SKF82958, an agonist for the D1 type of dopamine receptor. As shown in Fig. 3, the change in the transcriptional activity of the mutant receptors in response to stimulation with SKF82958 is very similar to that in response to progesterone and 8-bromo-cAMP. However, all mutants including the AAAA mutant can still be activated by SKF82958, suggesting that none of the phosphorylation sites is required for the ligand-independent activation of cPR_A by SKF82958.

The identification of the four phosphorylation sites was achieved by [³²P]H₃PO₄ labeling and purification of cPR protein from tissue minces of chicken oviducts. It is conceivable that the receptor expressed in cultured cells could be phosphorylated differently from the endogenous receptor. Theoretically, it is also possible that mutation of the known phosphorylation sites could cause the phosphorylation of the receptor at alternate sites that might counteract the mutation of the known sites and mediate the ligand-independent activation of the mutant receptor. To address these questions, phosphopeptide mapping of the wild type or the AAAA mutant receptors expressed in CV-1 cells was performed. As shown in Fig. 4, panel A, three major phosphorylation peaks were detected in the wild type receptor in response to progesterone treatment after HPLC analysis using a C-18 reverse phase column. These are indistinguishable from the phosphorylation pattern of the endogenous cPR isolated from chicken oviducts (8, 36). From our previous analysis (8, 36), it is known that the first peak corresponds to the phosphorylation at Ser²⁶⁰; the peak in the middle contains both Ser²¹¹ and Ser³⁶⁷ sites, and the last peak is Ser⁵³⁰. In contrast to the wild type receptor, no significant phosphorylation was detected in the AAAA mutant in either the presence (Fig. 4, panel B) or the absence (Fig. 4, panel C) of 10 nM progesterone. The multiple peaks of wild type cPR_A that eluted at 50-60 min after loading the sample (Fig. 4, panel A) are likely due to incomplete digestion because they are not present in the AAAA mutant. Similar heterogeneity around the 260 peak has been detected in phosphopeptide mapping of the recombinant cPR_A expressed in yeast (41). These mapping analyses suggest that no additional phosphorylation occurs in cPR_A expressed in cultured cells, and the mutation of the four known sites to alanine did not cause alternate phosphorylation.

It is possible that ligand-independent activation of steroid receptors could result in early, transient alterations in phosphorylation or prolonged alterations in phosphorylation of cPR_A. To assess these possibilities, cPR_A expressed in CV-1 cells was labeled in vivo with [³²P]H₃PO₄ followed by both a short treatment period (30 min) and a long treatment period (10 h) with 8-bromo-cAMP. When phosphorylation of cPR_A following different treatments was assessed in the same experiment, it was clear that only treatment with progesterone resulted in a significant enhancement of the receptor phosphorylation (Fig. 5, panel A; compare lane 4 with lanes 1-3). The increase in the amount of receptor protein detected following steroid treatment (Fig. 5, panel B, lane 4) is not significant enough to account for the large increase in hormone-dependent phosphorylation. The two time points with 8-bromo-cAMP as well as no treatment showed a roughly equivalent degree of phosphorylation when normalized for protein level. To ensure that treatment with 8-bromo-cAMP resulted in ligand-independent activation of cPR_A, CV-1 cells were assayed for activation of the GRE₂e1bCAT reporter (using normal conditions for infection of cells) at the same time that the labeling experiment was performed. Although there is negligible receptor activation as measured by CAT activity following 30 min of treatment with 8-bromo-cAMP (Fig. 6, lane 2), receptor activation following 10 h of drug treatment was comparable with receptor activation following 10 h of steroid treatment (Fig. 6, compare lane 3 and lane 5).

Although treatment with 8-bromo-cAMP did not enhance the overall level of cPR_A phosphorylation, it was possible that site-specific phosphorylation in the receptor was altered by treatment with 8-bromo-cAMP. Following in vivo labeling of cPR_A in CV-1 cells with [³²P]H₃PO₄ and treatment of cells with 10⁸ M progesterone, separation of cPR_A tryptic peptides on C-18 reverse phase HPLC columns revealed four major ³²P-labeled peaks (Fig. 7, panel A). The pattern for three of the peaks was consistent with previous studies that identified Ser²⁶⁰ in the first peak, Ser²¹¹ and Ser³⁶⁷ in the second peak, and Ser⁵³⁰ in the third peak (as labeled in Fig. 7, panel A). The fourth broad peak on the HPLC profile (between the Ser²⁶⁰ peak and the Ser²¹¹/Ser³⁶⁷ peak) was not identified and could represent incomplete tryptic digestion of peptides in the other peaks. As expected, in the absence of hormone, only the Ser²⁶⁰ and the Ser²¹¹/Ser³⁶⁷ peaks were present on the HPLC profile (Fig. 7, panel B). Further analysis of the Ser²¹¹/Ser³⁶⁷ peak by alkaline peptide gel electrophoresis revealed that only the peptide for Ser²¹¹ was present in this peak (data not shown). Following treatment with 2 mM 8-bromo-cAMP, two phosphopeptide peaks were detected (Fig. 7, panel C) that are identical to the peaks for Ser²⁶⁰ and Ser²¹¹/Ser³⁶⁷ detected under conditions of no treatment (compare Fig. 7, panels B and C). Analysis of the Ser²¹¹/Ser³⁶⁷ peak by alkaline peptide gel electrophoresis showed that the Ser²¹¹ peptide instead of the Ser³⁶⁷ peptide was present in the peak (data not shown). Because the preceding experiments necessitated the use of so many cells, it was not feasible to do the different treatments and subsequent phosphopeptide analysis concurrently. Hence the magnitude of the peaks shown in different panels of Fig. 7 can only be compared qualitatively.

To ensure that ligand-independent activation of cPR_A occurred in this experiment, CV-1 cells were assayed for activation of the GRE₂e1bCAT reporter following treatment with 2 mM 8-bromo-cAMP concurrently with the [³²P]H₃PO₄ labeling experiment. As shown in Fig. 8, treatment with 8-bromo-cAMP resulted in significant activation of cPR_A with CAT activity approximately 40% of that following progesterone treatment.

Taken together, these results show that ligand-independent activation of cPR_A induced by treatment of CV-1 cells with 8-bromo-cAMP does not alter the overall level or sites of receptor phosphorylation relative to conditions of no treatment.

DISCUSSION

Based on our previous studies, phosphorylation of cPR in response to progesterone stimulation can be separated into two classes, the hormone-dependent phosphorylation at Ser⁵³⁰ and Ser³⁶⁷ which occurs only in the presence of hormone and the phosphorylation at Ser²¹¹ and Ser²⁶⁰ which occurs before hormone treatment and whose level is enhanced by progesterone treatment (8, 36). In the present study, our phosphopeptide mapping analyses showed that cPR_A is only phosphorylated at Ser²¹¹ and Ser²⁶⁰ during the ligand-independent activation by 8-Br-cAMP. The differential phosphorylation of cPR_A in hormone-dependent and ligand-independent activation suggests that the two activation processes use distinct mechanisms.

Our previous functional analyses have suggested that Ser⁵³⁰ phosphorylation in cPR enhances the response of the receptor to low levels of progesterone (15), whereas Ser²¹¹ phosphorylation is required for the receptor to achieve its full transcriptional potential at all hormone concentrations (16). In the present study, we show that the mutation of Ser²⁶⁰ to alanine also decreased the transcriptional activity of the receptor to a similar degree as the mutation of Ser²¹¹. Mutation of Ser³⁶⁷ to alanine did not affect the transcriptional activity of the receptor significantly in the experiments shown in Figs. 1 and 3. In some experiments, the activity of Ala³⁶⁷ is significantly higher than that of the wild type (data not shown), indicating that phosphorylation at Ser³⁶⁷ might down-regulate the activity of the receptor. The transcriptional activity of cPR_A in response to 8-bromo-cAMP or dopamine agonist is not affected by mutation of either Ser⁵³⁰ or Ser³⁶⁷ to alanine. This is consistent with our phosphopeptide mapping results which show that these two sites are not phosphorylated following ligand-independent activation by 8-Br-cAMP. The activity of the AAAA mutant in response to either progesterone or 8-Br-cAMP is not decreased to the degree of some of the single site mutations. Whether this is due to the positive effect of mutation of Ser³⁶⁷ to alanine remains to be determined.

The transcriptional activity of the receptor in response to 8-Br-cAMP is reduced by mutation of either Ser²¹¹ or Ser²⁶⁰ to alanine to a similar degree as that in the response to progesterone. This suggests that receptor phosphorylation at either Ser²¹¹ or Ser²⁶⁰ regulates certain aspects of the activation process common to both ligand-dependent and ligand-independent pathways. This is also consistent with the observation that Ser²¹¹ and Ser²⁶⁰ are the two sites phosphorylated in receptors activated by either 8-bromo-cAMP or progesterone. The localization of Ser²¹¹ and Ser²⁶⁰ to the A/B region suggests that they both might be required for activation function 1 to achieve its maximal transcriptional potential.

Regarding the molecular mechanism of the ligand-independent activation of cPR, our data showed that mutation of either one or all of the phosphorylation sites to alanine did not abolish the ligand-independent activation by 8-bromo-cAMP. Mutation of all four sites did not result in alternate phosphorylation of cPR_A. In addition, in concurrent experiments in which ligand-independent activation of the receptor is observed, 8-bromo-cAMP treatment failed to either increase the overall phosphorylation or cause alternate phosphorylation of the receptor. Based on these results, we conclude that phosphorylation of cPR is not absolutely required for ligand-independent activation of the receptor by 8-bromo-cAMP.

Dopamine receptor activation is known to activate multiple signal transduction pathways including the protein kinase A and the protein kinase C pathways (48-51). Thus, it is possible that ligand-independent activation of cPR in response to dopamine agonists is mediated through the same molecular mechanism that 8-bromo-cAMP uses to induce receptor activation. This is consistent with our finding that receptor phosphorylation is not absolutely required for ligand-independent activation by an agonist for the D1 type of membrane dopamine receptor.

Our conclusion that the ligand-independent activation of cPR in response to 8-bromo-cAMP or dopamine is not mediated by receptor phosphorylation appears contrary to a report which showed that the ligand-independent activation of human estrogen receptor in response to EGF is mediated through receptor phosphorylation at Ser¹¹⁸ (52). However, another group has shown that phosphorylation at the same site by mitogen-activated protein kinase in response to EGF enhanced the estrogen-stimulated activity of the receptor but did not cause the activation of the receptor in the absence of estrogen (53). It remains to be determined whether human estrogen receptor and cPR represent two types of receptors that are ligand-independently activated through different mechanisms. An alternative explanation is that 8-bromo-cAMP and EGF are using different mechanisms to induce ligand-independent activation of steroid receptors, with EGF using direct receptor phosphorylation as part of its mechanism to induce receptor activation while 8-bromo-cAMP does not.

Because receptor phosphorylation is not essential for the activation of cPR by 8-bromo-cAMP, it is possible that phosphorylation of receptor-associated proteins may mediate the ligand-independent activation. In this regard, the phosphorylation of two classes of proteins that interact with steroid receptors is worth further investigation. The first class of proteins is heat shock proteins. Many of the heat shock proteins are known to be phosphoproteins. Studies have shown that the homologous deletion of a heat shock protein, dnaJ, from yeast resulted in the activation of both estrogen and glucocorticoid receptors in the absence of their corresponding ligands (54). It must be pointed out that the mechanism of steroid receptor activation in yeast might be different from the mechanism in higher organisms. For example, the glucocorticoid receptor has not been shown to be ligand-independently activated in mammalian cells, and the activation of human androgen receptor by androgen in yeast was shown to be down-regulated by deletion of the same dnaJ protein (55). However, it is possible that the signal pathway activated by 8-bromo-cAMP in mammalian cells results in the phosphorylation of certain heat shock proteins. This phosphorylation could cause an alteration in either the expression level of the heat shock proteins or the affinity of the heat shock proteins toward steroid receptors, leading to receptor activation. Interestingly, the phosphorylation of a heat shock protein, GroEL, in bacteria has been shown to regulate its interaction with substrates (56).

In addition to heat shock proteins, other proteins that are known to interact with and to be involved in the activation process of steroid receptors are transcriptional coactivators such as TAF_II30 (57), transcriptional intermediary factor-1 (58), CREB binding protein (59), and the recently identified steroid receptor coactivator including SRC-1 (60) and p140/p160 (61-63). So far, very little is known about the phosphorylation of these cofactors. It will be interesting to determine whether alterations in phosphorylation of coactivators is induced by 8-bromo-cAMP treatment and whether these alterations may contribute to ligand-independent activation of steroid receptors.