Identification of Phosphorylated Sites in the Mouse Glucocorticoid Receptor*

phosphorylated in the become hyperphosphor-ylated in the presence of but not As a preliminary step to elucidating the functional signif-icance of receptor phosphorylation, we have identified seven phosphorylated sites the phosphopeptides

Identification of Phosphorylated Sites in the Mouse Glucocorticoid Receptor* (Received for publication, October 15, 1990) Jack E. Bodwell Glucocorticoid receptors in vivo are phosphorylated in the absence of hormone and become hyperphosphorylated in the presence of glucocorticoid agonist but not antagonists (Orti, E., Mendel, D. B., Smith, L. I., and Munck, A. (1989) J. Biol. Chem. 264,9728-9731). As a preliminary step to elucidating the functional significance of receptor phosphorylation, we have identified seven phosphorylated sites on the mouse receptor. Tryptic phosphopeptides from S2P-labeled receptors were purified from glucocorticoid-treated mouse thymoma cells (WEHI-7) and from stably transfected Chinese hamster ovary cells (WCL2) that express large numbers of mouse receptors. Phosphopeptide maps of receptors from these two cell types were almost indistinguishable. Solid phase sequencing revealed phosphorylation at serines 122, 150, 212, 220, 234, and 315 and threonine 159. Serines 122, 150, 212, 220, and 234 and the sequences surrounding them are conserved in the homologous regions of the rat and human receptors, but threonine 159 and serine 315 have no homologues in the human receptor. The seven phosphorylated sites are in the amino-terminal domain of the receptor. All but serine 315 are within transactivation domains identified in the human and/or rat receptors. Serines 212, 220, and 234 are in a highly acidic region that in the mouse receptor is necessary for full transcription initiation activity and reduces nonspecific DNA binding. Serines 212, 220, and 234 and threonine 159 are in consensus sequences for proline-directed kinase and/or ~34"~"' kinase. Serine 122 is in a consensus sequence for casein kinase I1 whereas serines 150 and 315 do not appear to be in any known kinase consensus sequence. The location of many of these sites suggests a role of phosphorylation in transactivation.
Many glucocorticoid target cells respond to changes in hormone levels by altering the expression of specific genes. Transduction between hormone levels and gene expression is mediated by the GR,' a -100-kDa steroid-binding protein *This research was supported by National Institutes of Health Research Grant DK 03535 and by Norris Cotton Cancer Center Core Grant CA 23108. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ To whom correspondence should be sent.
Mutagenesis studies have determined a number of functions for different portions of the GR (for a general review see Carson-Jurica et al., 1990). DNA binding has been localized to a central cysteine-rich area. This area contains two zinc fingers that are thought to be necessary for binding to DNA. The carboxyl-terminal portion contains the hormone binding domain, nuclear localization signals, and a transactivation region. Transactivation regions have also been identified in the amino-terminal domain of the human (Hollenberg and Evans, 1988), rat (Godowski et al., 1988), and mouse (Danielsen et al., 1987) receptors.
Glucocorticoid and other steroid receptors are known to be phosphorylated (Housley and Pratt, 1983;Carson-Jurica et al., 1990). The unliganded receptor from WEHI-7 mouse thymoma cells contains 2-3 mol of phosphate/mol of protein . The addition of glucocorticoid agonists, but not of the antagonist RU 486, increases phosphorylation by 50-70% (Orti et al., 1989a). These changes imply a role for phosphorylation/dephosphorylation in the function of the GR. As a step toward understanding how phosphorylation may affect function of the GR we have determined the location of seven in vivo phosphorylated sites on the mouse GR.

EXPERIMENTAL PROCEDURES
Materials-Sources for the materials used in this study have been listed previously Smith et al., 1988;Smith et al., 1989;Orti et al., 1989a). Methylated trypsin was obtained from Promega Biotec, Madison, WI. The WCL2 cell line was a generous gift from Dr. Margaret A. Hirst (Hirst et al., 1990).
Whole Cell Labeling and Preparation of Cytosol-This procedure has been described in detail ( Orti et al., 1989a). WEHI-7 cells were collected by centrifugation and incubated twice with phosphate-free Dulbecco's modified Eagle's medium containing 10% dialyzed fetal bovine serum (100 m1/2 X lo6 cells, 30 min, 37 "C). Cells were distributed to 162-cm2 flasks (Costar, Cambridge, MA) in 200 ml of medium at a concentration of 1.3 X lo6 cells/ml. [32P]Orthophosphoric acid was added to four flasks (6.25 mCi/flask), after which the cells were incubated for approximately 14 h at 37 "C. In order to obtain enough counts, a second set of four flasks was prepared similarly the following day, and the two preparations were combined after the immunopurification step. Triamcinolone acetonide (10 mM stock in ethanol) was added to the cultures at a final concentration of 2.5 X M for the last 30 min of the incubation with 32P. Cells were collected by centrifugation (2.5 min at 700 X g), washed with 10 ml of cold phosphate-free KRBGH, and the cytosol prepared by the freeze/thaw procedure (4 volumes of FT buffer/volume of packed cells) described previously (Orti et al., 198913).
WCLZ cells (80-100% confluent) in 162-cm2 flasks were incubated twice with phosphate-free Dulbecco's modified Eagle's medium containing 2% dialyzed fetal bovine serum (75 ml/flask, 30 min, 37 "C). Phosphate-free Dulbecco's modified Eagle's medium (50 ml) containing 10% dialyzed fetal bovine serum was added to each flask. ["PI Orthophosphoric acid was added to four flasks (6.25 mCi/flask) and incubated for approximately 4 h. There was no difference in the shape of the phosphopeptide tryptic map if incubations went for 14 h as was done with the WEHI-7 cells. Triamcinolone acetonide (2 mM stock in ethanol) was added to a final concentration of 2.5 X M to the cultures for the last 30-60 min of the incubation. Cells were washed once with 15 ml of HBS buffer (-23 "C) after which they were treated with 2 ml of 0.1% trypsin/flask (-23 "C). After 5 min the cells were dislodged by directing the flow of 10 ml of medium from a pipette toward the cells on the bottom of the flask. The medium was withdrawn and expelled repeatedly until the cells were thoroughly resuspended. Cells were centrifuged as above, washed once with ice-cold medium and once with cold phosphate-free KRBGH. The cytosol was prepared as described for the WEHI-7 cells except that FTT buffer was used for the freeze/thaw cell lysis step.
In order to increase the total amount of receptor in the preparation, unlabeled cells were processed in parallel. Typically, 120 ml of packed cells was used for the WEHI-7 and 2-10 ml of packed cells with WCLP cells. Labeled and unlabeled preparations were pooled just prior to the SDS-polyacrylamide gel electrophoresis step.
Immunopurification of Receptor-Dissociation of the 90-kDa heat shock protein subunits from the GR was accomplished as described previously (Orti et al., 1989a) by making the cytosol 1.6% in SDS, placing in boiling water for 5 min, and adding a 5-fold excess of Triton X-100 to scavenge the SDS. The cytosol was cooled on ice for 15 min. All subsequent steps were performed at 3 "C. FIGR antibody was added to a 5-fold molar excess over the estimated amount of receptor (number of cells X the number of receptors/cell) in the Preparation. The FIGR antibody is a monoclonal antibody that reacts against both rat and mouse GR and behaves exactly as the BUGRZ antibody (Gametchu and Harrison, 1984) used previously? FIGR and BUGR2 antibodies appear to have similar epitopes as both antibodies will inhibit the binding by the other, and both recognize the same 16-kDa tryptic fragment of the GR. After incubating from 4 to 14 h all of the 32P-labeled cytosol was applied to a Sepharose 4B column (0.7cm inner diameter X 0.7-cm bed height). Carrier cytosol was applied to a larger (1.7 X 0.7-cm) column. After the cytosol had drained to the top of the bed, the column was washed with 1 column volume of FT buffer. "P-Labeled cytosol that passed through the column was collected in 15-ml centrifuge tubes. Carrier cytosol was collected in 50-ml tubes. Protein A-Sepharose was added to a 5-fold excess of binding capacity over the amount of FIGR antibody, and the tube was twirled slowly for 4-14 h. Protein A-Sepharose was collected by centrifugation (3 min at 200 X g), supernatant was removed, and the J. E. Bodwell protein A-Sepharose was resuspended in 40 volumes of FTN buffer. After collection by centrifugation the protein A-Sepharose was washed 6 more times in FTN buffer and transferred to a syringe (sized for a bed height of 1-2 cm) with a 70-pm polypropylene disc fitted to the bottom. The column was washed with 25 column volumes of FTNT and 10 column volumes of FT buffer. Gentle air pressure was applied to the top of the column to remove excess liquid and then the protein A-Sepharose was expelled into 0.7 volume of TSTG buffer and the mixture placed in boiling water for 2 min. After cooling to room temperature the liquid was separated from the protein A-Sepharose by centrifuging through a column with a polypropylene disc fitted to the bottom (5 min at 200 X g). Subsequent steps were done at room temperature. Iodoacetamide (1 M stock) was added to a final concentration of 100 mM, and the receptor preparation was left at room temperature for 45 min before loading onto SDS-polyacrylamide gel electrophoresis.
SDS-Polyacrylamide Gel Electrophoresis and Transblotting of Receptor-The alkylated receptor was loaded onto a vertical slab gel of 7% acrylamide and run for 14-16 h at 8 mA (Smith et al., 1989). The gel was incubated twice for 10 min in fresh electrode buffer without SDS and transblotted in the same buffer to a 15 X 15-cm PVDF membrane (Immobilon, Millipore, Milford, MA) for 10-12 h at 100 mA (Trans-blot cell, Bio-Rad). The cassette holding the blot was allowed to drain for 5 min, and then the gel was removed from the membrane. Where protein has been transferred, the membrane has a matt or dull appearance that allows the position of the band to be marked (the bands are only visible for less than a min). The receptor band was excised from the membrane, cut into slices with an area of 2-20 mm', and extracted from the membrane by incubating with 300-900 pl of TST buffer for 4-16 h (Szewczyk and Summers, 1988). The extracted receptor was removed from the slices and the slices washed once in 200-300 pl of TST. Only a single band in the area of the GR was observed either following Coomassie Brilliant Blue staining of the PVDF membrane or after analysis of SDSpolyacrylamide gel electrophoresis gel slices of GR purified from cells labeled with [35S]methionine (see Fig. 3 in Orti et al., 1989a).
Trypsin Digestion-A 30O-pl sample of the combined extract and wash was placed in a 1.5-ml centrifuge tube with 4 volumes of acetone at -80 "C. After 45 min at -20 "C the tube was centrifuged for 10 min at 12,000 X g (-10 "C) and the supernatant decanted. This procedure was repeated until all of the receptor bad been precipitated. The pellet was washed once with -80 "C acetone, the supernatant decanted, and the pellet air dried until the odor of acetone could not be detected. The pellet was dissolved in 50 MI of UAB buffer (Stone et al., 1990) by warming to 37 "C, vortexing, and sonicating for one to three 10-s intervals. After adding 15 MI of 1 M ammonium bicarbonate and 135 pl of water, the tube was vortexed, and methylated trypsin was added at a ratio of 120 (w/w, trypsin:receptor). The trypsin, dissolved in 50 mM acetic acid, was at a concentration such that 5-12 p1 was added to the receptor preparation. Digestion was at 37 "C for 20-24 h. The WCL2 preparation used for the results in Fig.  1 showed the presence of peptide 45, a sign of incomplete hydrolysis (see "Results"). This was probably caused by failure to dissolve the GR from the acetone pellet thoroughly. The sonication step was omitted for this preparation, and the GR that dissolved slowly during the digest incubation may not have been hydrolyzed completely. Current preparations are always sonicated and show no signs of peptide 45. Trifluoroacetic acid (2 p l ) was added to the digest, the sample vortexed, centrifuged (2 min at 12,000 X g), and injected into the HPLC.
Protein Determination-To determine the amount of receptor extracted from the Immobilon membrane, a small sample (approximately 10 mm2) of the receptor band was extracted with PST buffer (PST buffer has no amines to react with fluorescamine). The amount of receptor in the extract was determined with fluorescamine (Bodwell and Meyer, 1981) using bovine serum albumin as the standard. The radioactivity in the samples extracted with PST and TST buffers was determined by Cerenkov counting. The ratio of protein to Cerenkov counts for the PST samples was then used to determine the amount of receptor protein extracted from the membrane with the TST buffer.
Purification of Phosphopeptides-HPLC analysis was performed on a Waters 840 HPLC work station equipped with two model 510 pumps, a U6K injector, and a Beckman model 165 multiwavelength detector. Samples were chromato raphed on a Vydac C4 column (2.1 X 250 mm, 5-pm beads with 300 x pores) using 0.06% trifluoroacetic acid (solvent A) as the ion pairing agent and 75% acetylnitrile in 0.056% trifluoroacetic acid (solvent B) as the organic modifier (Stone et al., 1990). The flow rate was 0.15 ml/min, and the peptides were FIG. 1. Comparison of tryptic phosphopeptide maps from WEHI-7 and WCL2 cells. Purified GR (10 pg from WEHI-7 and 11 pg from WCL2 cells) labeled with [32P]orthophosphoric acid was digested with trypsin and subjected to reverse phase chromatography. Phosphopeptides from WEHI-7 (3,000 cpm, lower graph) and WCL2 (115,000 cpm, upper graph) cells were injected into the HPLC. After Cerenkov counting, fractions from each phosphopeptide were pooled and sequenced as described under "Experimental Procedures." The number near the apex of each peak identifies each phosphopeptide.  Fig. 1 were subjected to solid phase sequencing as described under "Experimental Procedures." Initial yields were 1-5 pmol except for phosphopeptide 37 from WEHI-7 cells (all other phosphopeptides were from WCL2 cells) which was less than 1 pmol. The amount of radioactivity applied to the sequenator was 330, 2,780, 1,320, 548, 1,030, 1,078, and 725 cpm for phosphopeptides 37 (WEHI-7), 37 (WCLP), 8, 28, 36, 29 (obtained from a different preparation than that those in Fig. l), and 45, respectively.
eluted with a linear gradient from 0 to 48% solvent B at a rate of increase of 0.25% solvent B/min. Fractions were collected every 0.7 min, and radioactivity was determined by Cerenkov counting. Radioactivity and absorbance at 210 nm in the fractions were compared so that the 32P-containing peaks could be pooled to maximize purity. The purified "'P-labeled phosphopeptides were dried in a Speed-Vac (Savant, Farmingdale, NY) to 50% of the original volume, lyophilized, and subjected to solid phase sequencing.
Solid Phase Sequencing of Tryptic Phosphopeptides-Each lyophilized phosphopeptide was dissolved in 20 p1 of 50% acehnitrile containing 0.1% trifluoroacetic acid by heating the sample tube at 55 "C for 5 min and then sonicating for 30 s. The peptide solution was applied to a disc of arylamine PVDF membrane (Sequelon-AA, MilliGen/Biosearch, Burlington, MA) which was placed on a piece of plastic film resting on a 55 "C heat block. In most experiments, more than 90% of the peptide was transferred to the membrane disc as   Fig. 1). The results are from five experiments done subsequent to those described in Fig. 1. The amount of peptide 16 in these experiments was much less than in Fig.  1, and peptide 45 was absent in all five of the experiments.
Average for five experiments with WCLZ cells (see "Experimental Procedures"). Values were calculated from the percent of total radioactivity in the individual peptides after assuming that Total phosphates, 3.4.
determined by analysis of Cerenkov counts remaining in the tube. Once the solution had evaporated (about 5 min), the plastic film and disc were removed from the heat block and allowed to cool to room temperature. The membrane was wetted subsequently with 5 p1 of 0.1 M MES, pH 5.0, containing 15% aqueous acetonitrile and 10 mg/ml 1 -(3dimethylaminopropyl) -3 -ethylcarbodiimide hydrochloride (Coull et al., 1991). After 20 min at room temperature, the disc was placed in the reaction chamber of a MilliGen/Biosearch 6600 covalent protein sequenator. A portion of the phenylthiohydantoin derivative generated during each 34-min Edman cycle was injected automatically into an on-line HPLC system. The remainder was diverted to a fraction collector so that radioactivity could be measured. The proportion of sample used for radioactivity and phenylthiohydantoin derivative analysis depended on the amount of radioactivity in the phosphopeptide. Fifty percent of the sample was collected to determine radioactivity if the phosphopeptide had less than 500 cpm whereas 20% was collected for phosphopeptides with greater than 500 cpm. In some cases, after the peptide had been identified the entire phenylthiohydantoin derivative sample was used to measure radioactivity. Radioactivity was determined by liquid scintillation after adding 5 ml of scintillation fluid (Biofluor, Du Pont-New England Nuclear) and counting for 5 min. Initial sequence yields for the various phosphopeptides was determined by integration of phenylthiohydantoin derivative peak areas observed in the HPLC profiles. The values were between 1 and 5 pmol for the WCLZ phosphopeptides and less than 1 pmol for peptide 37 from WEHI-7 cells.

RESULTS
We have used two different cell lines to determine the location of phosphates on the mouse receptor. Our basic procedures and initial results were developed with WEHI-7 cells, with which all of our previous data on phosphorylation were obtained. With these cells, which have about 30,000 receptors/cell, massive preparations of labeled cells are required for sequencing work. WCL2 cells are Chinese hamster ovary cells that have been transfected stably with the mouse GR gene (Hirst et al., 1990). They express about lo6 receptors/ cell. When we established that WCL2 cells gave the same phosphopeptide map as WEHI-7 cells (Fig. I), the same location for a phosphoserine (Fig. 2, A and B ) , and exhibit similar hormone dependence and kinetics of phosphorylation: we used them for most of the sequencing work. Our strategy has been to identify first the phosphorylated sites in hormone-treated cells, leaving for later the question of which of these are influenced by hormone treatment. Fig. 1 (lower graph) shows the elution of WEHI-7 phosphopeptides from reverse phase HPLC. Each peak and corresponding phosphopeptide preparation is named by the approximate percentage of buffer B at the apex of the peak after allowing for a 12-min gradient delay time (the time it takes for changes in the gradient to appear at the detector). The general location in the mouse GR of the phosphopeptides and phosphorylated sites we have identified is shown in Fig. 3. Peptide 37, the major phosphopeptide from the WEHI-7 cells, was collected and sequenced. Results are shown in Fig.  2 A . The sample contained 330 cpm, and less than 1 pmol of GR was sequenced. Despite these constraints 5 of the first 9 residues were identified, enough to identify the phosphopeptide unambiguously as a tryptic peptide starting from serine 224 in the mouse GR (Danielsen et al., 1986). The radioactivity eluted from the sequenator with a peak at cycle 11, which corresponds to serine 234. Peptide 37 probably extends past lysine 254 to lysine 264 since lysines 254 and 262 are followed by proline, which is known to make cleavage sites almost completely resistant to trypsin (Wilkinson, 1986). There are no phosphorylation sites in this additional sequence since we have determined by phosphoamino acid analysis that peptide 37 contains only phosphoserine (data not shown). Thus the major phosphorylated site on the WEHI-7 GR is serine 234.
Peptide 37 is also the major phosphopeptide for the WCLB cells (Fig. 1, upper graph). In fact, the tryptic map for WCL2 cells is almost indistinguishable from that for WEHI-7 cells. Peptide 45, present only in the WCLB cells, is an incomplete digestion product that has not appeared in more recent preparations (see below and "Experimental Procedures"). Peptide 37 from the WCLB cells was the same tryptic peptide, with a phosphorylated serine 234, as that from WEHI-7 cells (Fig.  2B).
Peptides 8, 28, 29, 36 and 45 from WCL2 cells were then isolated and sequenced. The partial sequence of peptide 8 (Fig. 2C) identified it as the tryptic peptide from serine 150 to lysine 163. It has two phosphorylated sites, radioactivity appearing at serine 150 (cycle 1) and threonine 159 (cycle 10). The sequence for peptide 28 (Fig. 2 0 ) matched the tryptic fragment from leucine 305 to lysine 323. Radioactivity eluted at cycle 11, which corresponds to serine 315.
There is a clear shoulder on the peak for pept.ide 28 from the WCL2 preparation shown in Fig. 1. In more recent preparations this shoulder has been resolved to give two distinct peaks, peptides 28 and 29, identical to the two peaks seen in the WEHI-7 preparation (Fig. 1). The partial sequence of peptide 29 from a different WCL2 preparation (chromatograph not shown) matched the tryptic peptide from valine 106 to arginine 129 (Fig. 2F). Radioactivity eluted at cycle 17, identifying serine 122 as the phosphorylated site.
Peptides 34 (data not shown), 35 (data not shown), 36, and 45 all had partial sequences corresponding to the tryptic peptide starting with leucine 192 (Fig. 2, E and G), and all had radioactivity that eluted at serine 212. A second site, common to peptides 35,36, and 45, was identified from peptide 45, which showed radioactivity not only at serine 212 but also at serine 220 (Fig. 2G), 5 residues past the end of the tryptic peptide from leucine 192 to lysine 215. It is clear that in this peptide the bond at lysine 215 was not cleaved. Serine 220 is in the next tryptic peptide from glutamic acid 216 to arginine 223.
The phosphorylated sites at serines 212 and 220 probably account for all of the radioactivity in peptides 34, 35, 36, and 45, but because of incomplete hydrolysis the two sites appear in four peptides. These peptides may have originated as follows. Peptide 34 is the tryptic peptide from leucine 192 to lysine 215 which contains the phosphorylated serine 212. Although barely detectable in the preparation that was sequenced ( Fig. 1, upper graph), this peptide is more abundant in recent preparations (see Table  I). Peptides 35 and 36 encompass peptide 34 and the next tryptic peptide from glutamic acid 216 to arginine 223. Both have the same primary sequence but differ in the distribution of phosphate between serines 212 and 220. The failure of trypsin to cleave at lysine 215 is probably a result of glutamic acid 216. Adjacent negative charges are known to make cleavage sites very resistant to hydrolysis by trypsin (Wilkinson, 1986). An experiment that used a much higher than usual ratio (1:4) of trypsin to receptor demonstrated that peptides 35 and 36 contain two phosphorylation sites. The larger amount of trypsin produced a substantial decrease in radioactivity in the peak containing peptides 35 and 36, an increase in radioactivity in peptide 34, and a new phosphopeptide, peptide 9, that was not sequenced.
Peptide 45 elutes at a position in the HPLC which is much more hydrophobic than would be expected if it contained only the two tryptic peptides encompassing serines 212 and 220. This preparation was hydrolyzed incompletely (see "Experimental Procedures"), and the bonds at serines 215 and 223 were not cleaved, with the result that peptide 45 contains both peptides 35/36 and 37.
Most of the phosphopeptides were sequenced a t least twice and some three times. In all cases the same sequences were identified, and the radioactivity eluted at the same position. Peptide 45 was sequenced only once, but the results clearly show phosphorylation a t serines 212 and 220.
Peptide 16 has virtually disappeared from recent preparations that are hydrolyzed more completely whereas peptide 5 has increased. Peptide 5, however, is not homogeneous. Rechromatography on a C18 reverse phase column separated peptide 5 into at least two peaks. We have not had enough material to sequence either of these two peaks nor peptide 23. Peptide 38 has the same initial sequence as peptide 37 with a phosphorylation site at serine 234 (data not shown), but the exact cleavage product is unknown. Table I shows the relative abundance of each of the phosphopeptides recovered from the HPLC, with averages and ranges from five experiments. The distribution of phosphate among the sites is reflected in the relative amounts of radioactivity in the individual phosphopeptides. If recoveries of the individual phosphopeptides are similar, the seven phosphorylated sites account for greater than 80% of the phosphate in the receptor.
Serine 234 (peptide 37) is the most heavily phosphorylated site. If it is assumed to be fully phosphorylated and thus to account for 1 mol of phosphate/mol of receptor, then the sum of all the identified phosphopeptides account for 3.4 mol. This rough estimate is reasonably consistent with the range of 4-5 that we determined previously by a different method (Orti et al., 1989a). DISCUSSION We have identified seven phosphorylated sites on the mouse G R serines 122, 150, 212, 220, 234, and 315 and threonine 159. Although most of the sites were determined from WCL2 cells, the almost identical patterns of phosphorylation found with the mouse receptor, whether in WEHI-7 mouse thymoma cells or overexpressed in Chinese hamster ovary (WCL2) cells, suggest strongly that phosphorylation is not cell type specific.
Although solid phase sequencing of picomol levels of 32Plabeled phosphopeptides has been reported previously (Wettenhall et al., 1991;Aebersold et al., 1989), this is the first study to use covalent attachment to an arylamine PVDF membrane in sequencing these levels of phosphopeptides. We were able to locate phosphoserine and phosphothreonine residues using 1-5 (pmol of peptide that contained only a few hundred cpm of radioactivity. These results could be obtained with small quantities of radioactive material because of the efficient way the solid phase sequenator handles the ["PI inorganic phosphate that is released during the Edman degradation. With a gas phase sequenator the phosphate from the amino acid normally remains in the reaction chamber because it is largely insoluble in the nonpolar solvents that are used to extract the amino acid derivatives (Soderling and Walsh, 1982). Covalent attachment of the peptide to arylamine PVDF membranes allows the use of liquid trifluoroacetic acid to remove the phosphate from the reaction vessel efficiently for subsequent analysis (Coull et al., 1991).
In the mouse GR, phosphoserine accounts for approximately 90% of the total phosphate recovered from the HPLC. Originally we reported only phosphoserine, but our recent experiments' using longer exposure times for autoradiograms reveal small amounts of phosphothreonine. Likewise, Dalman et al. (1988) found only phosphoserine in the rat GR, but a more recent study (Hoeck and Groner, 1990) shows a small amount of phosphothreonine. One group (Rao and Fox, 1987) has reported 11% phosphothreonine in the human receptor. However, this phosphothreonine must be a t a different site from that equivalent to threonine 159 in the mouse since the amino acid at the homologous position in the human GR is alanine (see below).
Location of the seven phosphorylated sites in the aminoterminal domain between residues 122 and 315 is consistent with previous reports (Dalman et aL., 1988;Smith et al., 1989;Hoeck and Groner, 1990) that most of the phosphates are located in that domain. Originally, we detected a phosphorylation site in the carboxyl-terminal domain (Smith et al., 1989), but recent experiments2 using a more rigorous purification procedure with two different monoclonal antibodies have failed to confirm this site.
Proteolytic cleavage studies (Dalman et al., 1988) of the GR in mouse L cells showed that the 15-kDa tryptic fragment containing the DNA binding domain was phosphorylated. We did not identify any phosphorylated sites in that portion of the GR, but such a site could be contained in one of the phosphopeptides that we were not able to sequence.
Transactivation domains have been identified within the amino-terminal domain of the human, rat, and mouse receptors. In the human receptor (Hollenberg and Evans, 1988) a transactivation domain designated as tau 1 is contained in amino acid residues 77-262, homologous to residues 86-270 in the mouse receptor. All the phosphorylated sites except serine 315 are in this region. The rat transactivation domain designated enh2 (Godowski et al., 1988) lies between amino acids 237 and 318, corresponds to mouse region 225-306 that includes the major phosphorylated site, serine 234. In the mouse receptor (Danielsen et al., 1987) a highly acidic region between 196 and 293 has been shown to be necessary for maximum initiation activity and also to decrease nonspecific binding to DNA. This region contains three of the phosphorylated sites, serines 212, 220, and 234, increasing its acidity even more. The location of these phosphorylated sites in the transactivation regions suggests a role for phosphorylation in modulating these functions.
The sequences around the mouse GR phosphorylation sites at serines 122, 150,212,220, and 234 are conserved in the rat (homologous serines at 134,161,224,232, and 246) and human (homologous serines a t 113, 141, 203, 211, and 226) GR. The site for serine 150 is also conserved in the human mineralocorticoid receptor (corresponding to serine 214). The phosphorylation site at threonine 159 is conserved in the rat but not in the human receptor, in which the threonine is replaced by an alanine. Likewise, the homologue to serine 315 is replaced by a proline in the human GR. It therefore seems unlikely that serine 315 and threonine 159 are important for modulating function.
All the phosphorylated sites except serines 150 and 315 are in consensus sequences of known kinases. Serine 122 is the only one of four potential casein kinase II sites (XXX-Ser-XXX-XXX-Glu) (Kemp and Pearson, 1990) that is phosphorylated. There are 11 potential phosphorylation sites that fit the consensus sequence (XXX-Ser/Thr-Pro-XXX) of the proline-directed kinase (Vulliet et al., 1989), and four of them are phosphorylated (serines 212, 220, and 234 and threonine 159). Serines 212 and 220 also fit the consensus sequence for the P3FdC2 kinase (Ser/Thr-Pro-XXX-Arg/Lys) (Moreno and Nurse;1990); which is important in regulation of the cell cycle (Nurse, 1990).
Recently three phosphorylated sites have been identified in the progesterone receptor from chick oviduct (Denner et al., 1990). There is no extensive homology between the sites on the GR and the progesterone receptor. One of the progesterone receptor sites is located in the hinge region between the DNA and hormone binding domains. However, all three sites on the progesterone receptor have the same motif of a serine followed by proline seen in three of the seven sites found in the mouse GR (serines 212, 220, and 234).
We are currently determining which phosphorylated sites of the GR are influenced by hormone and how phosphorylation a t those sites affects GR activity.