hormone-dependent phosphorylation of the glucocorticoid

THE JOURNAL OF BIOLOGICAL. CHEMISTRY Vol. 265, No. 10, Issue of April 5, pp. 5403-5408,199O (0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Prmted in U.S. A.

Hormone-dependent Phosphorylation of the Glucocorticoid Receptor Occurs Mainly in the Amino-terminal Transactivation Domain*

(Received for publication, November 6, 1989)

Wolfgang HoeckS and Bernd Groner From the Friedrich-Miescher Institute, P. 0. Box 2543, CH-4002 Basel, Switzerland

Phosphorylation of glucocorticoid receptors is increased by hormone binding and has been implicated in transcriptional regulation. We performed a phosphoamino acid analysis and identified the phosphorylated regions of the glucocorticoid receptor with respect to its functional domains before and after hormone activation. Receptor was isolated by immunoprecipitation from [32P]orthophosphate-labeled FTO 2B rat hepatoma cells grown in the absence or presence of glucocorticoids. The receptor contained mainly phosphoserine, with little phosphothreonine and no phosphotyrosine. Partial proteolysis of receptor from hormone-treated or control cells revealed a similar phosphopeptide pattern. Chemical cleavage with hydroxylamine and cyanogen bromide or digestion with trypsin and chymotrypsin localized the majority of receptor phosphorylation sites to a transactivation domain amino-terminal of the DNA-binding domain. Phos- phorylation of this region, termed rl/enh%, was increased 2-3-fold by hormone treatment. The DNA- binding domain itself is weakly phosphorylated; no phosphorylation was found in the hormone-binding domain. Phosphorylated regions were also identified in receptor deletion mutants stably transfected into CV- 1 monkey kidney cells. Hormone-independent phosphorylation was observed with a strong constitutively active mutant lacking the hormone-binding domain. No phosphorylation was detected in a mutant lacking the amino-terminal region, which showed only weak, hormone-dependent activity. These results sup- port the idea that phosphorylation is important for the strength of the glucocorticoid receptor as a transcriptional regulator.

The action of glucocorticoid hormones is mediated by sol- uble receptor proteins. After binding the hormone they become activated and assume the role of transcription factors. The glucocorticoid receptor (CR)’ is a member of the steroid/ thyroid hormone receptor superfamily, which share structural and functional features (1, 2). It contains a central DNA- binding domain that interacts with DNA sequences in the

* 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.

$ Supported by funds of the Ludwig Institute for Cancer Research, Zurich, Switzerland. To whom correspondence should be addressed.

1 The trivial names and abbreviations used are: GR, glucocorticoid receptor; dexamethasone, 9~-fluoro-16Lu-methyl-ll~,l701,21-trihy- droxvpregn-1,4-diene-3,20-dione; RU486, 17&hydroxy-11&(4-di- meth;laminophenyl)-l7a-(l-propynyl)-oestra~4,9-~ien-3-one; TLCK: N”-p-tosvl-L-lvsine chloromethvl ketone: TPCK, tosylphen- ylalanyl chioromkthyl”ketone; CAT, chioramphenicol acetyltiansfer- ase; PVDF, polyvinylidene difluoride.

promoter of hormone-regulated genes, a carboxyl-terminal hormone-binding region and an amino-terminal domain that is believed to modulate the activity of the receptor in the transcription process.

Covalent modification of proteins by phosphorylation is a central mechanism in hormonal regulation of gene expression (3). Experiments in bacteria (4), yeast (5-7) and mammalian cells (8) have demonstrated that activation as well as inacti- vation of transcription factors can be mediated by phosphorylation. For example, the GAL4 protein in yeast regulates expression of the galactose/melibiose regulon and is activated by sugar-induced phosphorylation (7). The transcription factor ADRl, controlling the alcohol dehydrogenase II gene, is inactivated by CAMP-dependent phosphorylation (6). Several steroid receptors, including the GR, have been demonstrated to be phosphoproteins (9), but evidence for functional changes resulting from modification is, however, still limited. Mendel et al. (10) and Migliaccio et al. (11) suggested that phosphorylation might be important for hormone-binding, but DNA- binding activity of mouse GR appears to be independent of phosphorylation (12). Recently, we (13) and others (14) demonstrated a hormone-dependent increase in GR phosphorylation which can be prevented by addition of the glucocorticoid antagonist RU486. Since RU486 inhibits transactivation of hormone-responsive genes as well as increased GR phosphorylation, it has been suggested that phosphorylation might play a role in regulating the transcriptional activity of the GR. Hormone-dependent phosphorylation has also been found in progesterone receptors (15, 16). Progesterone receptor phosphorylation in the absence and presence of progestins seems to occur on the same peptides, which are localized NH2- terminal to the DNA-binding domain (17). The location of phosphoamino acids in tryptic and chymotryptic fragments of the mouse GR has been investigated by Dalman and co- workers (18). They demonstrated that hormone-free receptors were phosphorylated in the DNA-binding domain, but not in the hormone-binding domain. The amino-terminal region could not be analyzed due to its rapid digestion by proteases. It was speculated that this region contains the majority of receptor phosphoamino acids.

We used proteases and chemical cleavage reagents on wild- type rat GR and GR deletion mutants transfected into CV-1 cells to locate the phosphorylation sites both in hormone-free and hormone-activated GR. GR phosphorylation could be demonstrated mainly in a potent transactivating domain, termed ~1 (19) or enh2 (20). Phosphorylation of this region is strongly increased by hormone treatment. Furthermore, phosphorylation of the DNA-binding domain as well as the absence of phosphorylation within the hormone-binding domain strongly suggest that this modification plays a role in the ability of the GR to work as a transcription factor.

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5404 Hormone-dependent GR Phosphorylation

EXPERIMENTAL PROCEDURES

(‘(‘II Cu/lurc and ‘I’ran,Jectrons-FTO 2B rat hepatoma cells and (‘V-1 monkey kldneb epithelial cells were maintained in Dulbecco’s modlfled Eagle’s medium supplemented with 10% fetal calf’ serum. Tran\fectiom- Mere performed accordmg to the modlf’ied CaPO,-DNA copreclpitation method of Chen and Okayama (21). Stable transf’ec- tanth were ohtalned 1, cotransfectmg 400 ng of pSV2neo (22) either Irith 10 pg of pSTC .&556 (2X), codmy f’or a GR lacking the hormone- bmdm:: domam. or with 10 pg of pSTC 407-795. coding fbr an amino- terminal deletion mutant. SelectIon for Gal&resistant colonies was done m Dulbecco’i modified Eagle’s medium containing 10% fetal calf serum and 1.6 mg/ml G418. Single colonies were isolated and further propagated. Transient transfectlons were done with 10 pg of pMMTV-LTR tkC.4T (24). The precipitate was removed after 16 h t, washmg the cells twice with Dulbecco’s modified Eagle’s medium. The cells were suhsequentl\ cultivated for 48 h with or wlthout 0.1 PM dexamethasone.

I’iP/Orthool~osohnt~ LabPline-Cells were ureincubated f’or 1 h in phosphate-free medium containing 1’~ dlalyzeh fetal calf serum. Then fresh phosphate-free medium including between 100 and 4000 pCi/ ml [ ‘~I’lorthophosphate iamersham Corp.) was added, and the cells i\ere further Incubated for 4 h.

Antr-.:‘(cccoturt~co/d Receptor Arztlbodle,j-Three antibodies were used in these studies. clP1 IS a polyclonal antiserum raised against a bacterially expressed GR fragment containing the DNA- and hor- mane-bmding domalns of’ the rat GR (13). One major epitope was localized to ammo acids 510-560.’ IgG 39/Y is a mouse monoclonal antihod\ described hv Westphal ct al. (X). Its epitope lies within amino &ids 120-160.of the ;at GR. HCGIi is a mouse monoclonal antibody raised aaainst uartiallv uurif’ied GR from rat liver (26). The epltope‘has been localized to amyno acids 405-423 (27).

fc o-dlmcvwonal I-‘hoaphoom~no Acid Anal\,szs-Phosphorylated GR was isolated by lmmunoprecipltatlon with oP1, electrophoresed on 8’~ polyacrylamlde gels, and blotted onto PVDF membranes (Mlllipore Corp.). Acid hydrolysis was performed on the membrane according to Kamps and Sef’ton (28). Phosphoammo acids were analyzed by two-dimensional electrophoresis at pH 1.9 and pH 3.5 on cellulose thin-layer plates (28).

I’hphopeptlde Mapptng-One-dimensional analysis of phospho- peptldes was done accordmg to the method of’ Cleveland et al. (29). Briefly. phosphor>lated GR was isolated from FTO 2B cells (0.5 mCi/ ml) b> immunoprecipltatlon and electrophoresed on 8% polyacrylamide gels. The GR band was cut out and o\erlayed with chymotrypsin or V8 protease m the slot of a 12.55 polyacrylamide gel. Phos- phopeptides were separated on the gel and visualized by autoradiog- raph?

?‘np.srn and Ch~motr.vpph~n I)gestro,z-Cell extracts were prepared by lysls in radioimmune precipitation huf’fer (13). Cleared extracts were treated with either 2-5 &ml trypsin (Boehringer Corp.) or 2 &ml chqmotr-ypsin for 30 min on ice. 0.1 mM TLCK or 0.2 mM TPCK were used to stop the action of trypsin or chymotrypsin, respectively.

Immunopreclpltatrons and Immunoblottrng-Immunoprecipita- tlons were done bv mcubatmg untreated or protease-treated cell extracts with the indicated antibodies for 1 h on ice. Protein A bacteria (Pansorbin. Behring Diagnostics, Somerville, NJ) were added f’or a further 30 mm on ice. Pelleted proteins were washed up to four times \\ith radiolmmune precipitation buffer, eluted by boiling m sodium dodeql sulfate sample buffer and analyzed on 12.5% polyacrylamlde gels. For immunoblottmg. proteins were separated on pol\acrvlamide gels and electroblotted onto PVDF membranes. Mem- branes were incubated \vith either (#I and ““I-protein A (Amersham Coru.) or with I&-19/9 or HI:CK and ““I-sheeo anti-mouse IaG F(A’B’)?-fragment (Amersham Corp.),

H~drouxlamlnc and Cl\anogen Rromlde Cleacag(J-For hydroxylamine cleavage ive employed the method of Saris et al. (30). Briefly. phosphorylated GR \vas isolated b> immunopreclpltatlon using oP1 antlrerum and electrophoresed on srr pol)acrylamlde gels. Gel pieces containing the GR were treated with hydroxylamme for :s h at 45 “C. After desslcatlon In a Speed Vat, GR fragments were analyzed on a l’L.nS polvacr\lamlde gel. Cvanogen bromide cleavage was done with GR blotted onto nitrocellulose. The membrane piece containing the GR was incubated for 90 mm m .iO mg/ml qanogen bromide in 70% formic acid at room temperature. The mtrocellulose was then thor- oughlv washed with water. and the supernatants were ecaporated m

’ LV. Hoeck. unpublished data

a Speed Vat. Phosphopeptides were analyzed ah described above. Ashays for ~hloramphemcol Acc,tylrrcln.sf(,rcl\l, (CA’I’)-CAT assays

were performed as described hy Gorman et al. (31). Protein concentration was determined with the Bio-Rad protein assay. 2.5 pg of protein from each sample were analyzed for CAT actlvlty.

RESULTS

Hormone-dependent Increase in Glucocorticoid Receptor Phosphorylation Occurs Mainly on Serine Residues-The extent of phosphorylation and the phosphoamino acids in glu- cocbrticoid receptors from FTO 2B cells grown in the absence or presence of dexamethasone were investigated. Dexameth- asone caused a 2-3-fold increase in receptor phosphorylation within 1 h, as reported earlier (13) (Fig. 1A). In the absence of hormone, the GR was phosphorylated mainly on serine residues, with some phosphothreonine (Fig. 1B). We did not detect phosphotyrosine even on long exposures. Dexametha- sane treatment caused an increase in both phosphoserine and phosphothreonine, but again no phosphotyrosine was observed.

GR Phosphopeptide Mapping-We employed the method of Cleveland et al. (29) to investigate the phosphopeptide pattern of the GR in the absence or presence of hormone after partial digestion with chymotrypsin or V8 protease (Fig. 2). Chymo- trypsin digestion of hormone-free GR generated three phosphopeptides of approximately 55, 40, and 26 kDa. A IO-fold increase in protease concentration resulted in only two phosphopeptides of 26 and 24 kDa. Hormone treatment led to an increase in signal intensity, but no new phosphopeptides were visible. Using V8 protease three peptides of 23, 21, and 16 kDa were detected. Dexamethasone treatment again led to an increase in phosphorylation, but no change in the pattern of labeled peptides. Thus, no unique phosphopeptides characteristic of hormone-induced phosphorylation were detected.

Localization of GR Phosphorylation Sites before and after Hormone Treatment Using the Proteases Chymotrypsin and Trypsin-Digestion of mouse and rat GR with chymotrypsin and trypsin yields defined fragments of the GR (18, 32). The

A NRS a2 I

wnl* - 94 kDa

Dex - - +

B 2nd b

1%

- Dex + Dex - FIG. 1. Phosphorylation of glucocorticoid receptors in the

absence or presence of hormone. A, hormone-dependent increase in GR phosphorylation. GR was isolated by immunoprecipitation from “‘P-labeled FTO 2B cells grown for 1 h in the absence or presence of 0.1 pM dexamethasone. <,[‘I, polyclonal GR antiserum; NKS, normal rabbit serum; arrow, rat GR. H, Phosphoamino acid analysis. The 94.kDa protein shown in A was subjected to hydrolysis in 6 N HCI f’or 4 h. Phosphoamino acids were separated and visualized by autoradiography. S, phosphoserine; 7’, phosphothreonine; Y, phosphotyrosine.


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Hormone-dependent GR Phosphorylation 5405

chymotrypsin V8 protease

500 ng 5000 ng 2000 ng

1 . 94K’ e fi 94K* b b 94K. ’ ’ 94K’ t 9

55K*

40K.

26K’ SK. 24K’

2 23K: ! (

?&* l -

Dex - + - + - + - +

FIG. 2. Cleveland mapping of glucocorticoid receptors in the absence or presence of dexamethasone. “‘P-Labeled receptor was subjected to partial proteolysis with chymotrypsin or V8 prot.ease arcording to Cleveland et al. (29) (see “Experimental Procedures”). Phosphopeptides were separated on 12.5% polyacrylamide gels and visualized by autoradiography.

DNA- and hormone-binding domains are present in a 42.kDa chymotryptic fragment as well as in a 44-kDa tryptic fragment. The tryptic fragment can be further digested to a 15- kDa peptide containing the DNA-binding domain and a 27/ 30.kDa peptide containing the hormone-binding region. The amino-terminal half of the GR is digested to small peptides, which cannot be analyzed on polyacrylamide gels. We used this information to investigate the phosphorylation state of these fragments before and after hormone activation of the GR. Extracts from ‘“P-labeled FTO 2B cells were treated with proteases. GR fragments were immunoprecipitated with either the cvP1 antiserum, which recognizes the hormone- binding domain, or the BUGR antiserum, which binds to a short peptide amino-terminal of the DNA-binding domain (see “Experimental Procedures”). The peptides were trans- ferred onto PVDF membranes and visualized by autoradiography (Fig. 3, left panels) and by immunoblot analysis using rvP1 or BUGR antiserum (Fig. 3, right panels), allowing us to determine the phosphorylation state as well as the presence of GR fragments in the same experiment. Immunoblots were four times less exposed than the membranes for phosphate analysis.

Fig. 3A demonstrates the phosphorylation state of intact GR in the absence or presence of hormone and confirms the resu1t.s shown in Fig. 1A. Chymotrypsin treatment generated a 42-kDa fragment, which showed a faint phosphorylation signal at the expected position (asterisk) in the 3rd lane of Fig. 3B. This result indicates that at least one phosphorylation site is contained within this GR fragment. Comparison of the signals obtained with intact GR and the 42-kDa fragment indicates that the majority of phosphorylation must occur in the amino-terminal half of the protein. The immunoblot in Fig. 3B confirms the presence of the 42-kDa GR fragment.

Trypsin digestion generated three receptor fragments of 44, 27130, and 15 kDa, depending on the amount of trypsin used (Fig. 3, C and D). The aP1 antiserum immunoprecipitated the 44- and 27/30-kDa fragments (Fig. 3C). We observed a faint phosphorylation signal for the 44-kDa fragment, but none for the 27/30-kDa fragment. Analysis of the 15-kDa fragment in Fig. 30, however, using the BUGR antibody indicated t.he presence of a phosphorylation site within the 15-kDa DNA-binding domain. Due to lower background signals in the 15-kDa region, it was possible to expose this gel longer than t,he one in Fig. 3C.

In summary, we observe phosphorylation of the DNA- binding domain retained in the 42-kDa chymotryptic and in the 44- and 15.kDa tryptic GR fragments. The hormone- binding domain does not seem to be phosphorylated. Experi-

no protease chymotrypsin (2000 ng)

32P, Ippt. lmmunoblot 32P, ippt. immunoblot (&x - - + - - + --+ -_*

MODULATOR DNA HORMONE

i--II ~- 94 kDa

C

trypsin (2000 ng)

32P, ippt. immunoblot I-Jex --• --+

.44kDa * 27kDa

B

DNA HORMONE

- 42 kDa -

D trypsln (5000 ng)

32 P. IPPt lmmunoblot

D

m O-.ldkDa I \/ I \/

NRS aP1 NRS aP1

- . I 11 I \/

NRS BUGR NRS BUGR

DNA HORMONE

fzm - TI ~ 44 kDa -

- 15 kDa - -27130 I

kDe-

1

FE. 3. Proteolytic digestion of phosphorylated CR with chymotrypsin or trypsin. A, no protease treatment. GR was Im- munoprecipitated from ‘“P-labeled cells (0.5 mCi/ml), stimulated for 1 h with or without 0.1 PM dexamethasone, and analyzed on a 12.5% polyacrylamide gel. Proteins were blotted onto PVDF membrane and visualized by autoradiography (left panel). The right panel shows the corresponding immunoblot. NRS, normal rabbit serum; <uPI, GR- specific antiserum; ippt., immunoprecipitate. At the bottom a schematic picture of wild-type rat GR is shown. H, chymotrypsin treatment. A lysate of cells grown in “P-containing medium with or without hormone was digested with 2 &ml chymotrypsin. The reaction was stopped by adding 0.2 mM TPCK. Fragments of the GR were immunoprecipitated with ~~6’1 and analyzed on a 12.55; polyacrylamide gel. The left panel shows the autoradiogram of the phosphorylated fragments, the right panel the corresponding immunoblot. A schematic picture of the 42.kDa chymotryptic GR fragment is shown at the bottom. C, trypsin treatment. Digestion and immunoprecipitation was done as in H using 2 &ml trypsin instead of chymotrypsin. 0.2 mM TLCK was used to stop the action of trypsin. A schematic picture of tryptic GR fragments is shown at the bottom D, trypsin treatment. Digestion was done as in C using 5 &ml trypsin, but immunoprecipitation and immunoblots were performed with RUGR antiserum. The 15kDa tryptic GR fragment is shown.

ments with the isolated 27/30-kDa GR fragment gave no indication that this domain is phosphorylated (data not shown).

Analysis of Phosphorylation of the Amino-terminal Half of the GR Using the Chemical Cleavage Agents Hydroxylamine and Cyanogen Bromide-To examine phosphorylation of the amino terminus we used chemical cleavage reagents, which generate fragments of the GR containing this region (Fig. 4). Hydroxylamine cleaves proteins between asparagine and gly- tine residues (30). Cleavage of rat GR at amino acids 200, 411, and 785 generates three fragments which can be separated on polyacrylamide gels. The largest fragment of 374 amino acids contains the DNA- and hormone-binding domains and is recognized by tvPI antiserum. The fragment from amino acid 1 to 200 is detected by the monoclonal


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Hydroxylamine Cyanogen bromide

Mr 116 84 - _ .- Y’, Mr 1;: = 58 - 58 -

X 4*- ;, - aP1 X 16” *d IgG 49/9 16” ;,“I H#& -/gG49/9 36 . . _.. - -

26 - 26 -

Dex - + Dex - +

Hydroxylamine 200 411 IgG 49/9 1 n ( aP1

785

I pL--r-pt___. -’

Cyanogen bromide 119 335

FIG. 1. Chemical cleavage of phosphorylated GR. a, hydroxylamine cleavage. GR wan immunoprecipitated from “ZP-labeled cell extracts (2 mCi/ml) usin g (\I’1 and separated on an 85 polyacrylamide gel. The GR-containing band was cut out and subjected to hydrosylamine cleavage according to Saris P[ al. (:30). Cleavage prod- ucts were separated on a 1’2.5’; polyacrylamide gel and autoradiographed. Arrorc~s indicate the position of the bands reacting with anti-GR antibodies in the immunoblot (not shown). R, cyanogen bromide cleavage. GR was isolated as in A but blotted onto nitrocellulose. The GR-containing piece of nitrocellulose was incubated with cyanogen bromide for 90 min at room temperature. GR peptides were resolved on a l’Z.~5~; polvacrvlamide gel and autoradiographed. The bottom shows a schemaiic picture of the chemical cleavage sites in the GR and indicates the reactivitv of the fragments with receptor antibodies. Hatchc,d has, DNA-bind& domain; shnded boa, hormone- binding domain.

antibody IgG 49/9 (see “Experimental Procedures”). GR was isolated from ‘“P-labeled cells grown in the absence or presence of dexamethasone and subjected to hydroxylamine cleav- a.Pe. In a parallel sample we cleaved nonlabeled GR under the same conditions; this material was used for immunoblot analysis (data not shown). A phosphopeptide with an appar- ent molecular mass of approximately 38 kDa was recognized by antibody ZgG 49/9 (Fig. 4A). The molecular mass of this peptide is bigger than the sequence predicts (approximately 23 kDa); we assume that the high number of acidic residues in this region causes abnormal migration on the gel. Dexa- methasone treatment led to a significant increase in phosphorylation of this peptide. Antibody CUPI recognized a peptide in the immunoblot which was not visible as a phosphorylated band and migrated just above the phosphorylated 46- kDa band, which is probably an intermediate cleavage prod- uct. The remaining phosphorylated band at about 90 kDa represents noncleaved GR. Hydroxylamine cleavage of the GR generates a second peptide (amino acids 200-411) of similar molecular weight as the one recognized by antibody ZgG 49/9. No antibody directed against this peptide is available, so we were unable to determine its exact position on t.he gel. It might comigrate with the ZgG 49/9-recognized peptide; therefore, we cannot clearly say which one of these peptides is phosphorylated.

Cyanogen bromide cleaves proteins at methionine residues and has several recognition sites in the GR. Only one fragment, spanning amino acids 119-335, can be detected on polyacrylamide gels. Inactive or hormone-activated GR was subjected to cyanogen bromide treatment. Fig. 4B shows a phosphorylated fragment of about 40 kDa, whose phosphorylation is increased 2-3-fold by hormone treatment. This fragment is recognized by monoclonal antibody ZgG 49/9 and comprises a transactivation domain of the GR, termed rl/

enh2. It also shows a retarded migration on the gel, like the phosphorylated hydroxylamine fragment. The signal intensity of these peptides is comparable. Equal amounts of ‘“P-labeled GR were used in both experiments, leading to the conclusion that the majority of phosphorylation sites are located between amino acids 119 and 335.

Stable Expression of GR Deletion Mutants in CV-1 Cells and Analysis of Their Phosphorylation State-Protease digestion and chemical cleavage studies on phosphorylated GR indicated that most of the phosphate residues are located in its amino-terminal domain. To investigate the specificity of this phosphorylation pattern, we studied transfected receptor genes in a heterologous system and correlated phosphorylation with transcriptional enhancement by the GR.

CV-1 monkey kidney cells lack functional receptors and are widely used to analyze delet.ions in the GR molecule (19, 20). They were stably transfected with rat GR, lacking either the hormone-binding domain or the amino-terminal 407 amino acids. Transient transfection analysis has shown that deletion of the hormone-binding domain creates a constitutively active receptor, whereas the amino-terminal deletion influences the magnitude of transactivation by the GR mutant (19, 20). Colonies of G418-resistant cells were isolated and screened for expression of GR mutants. The immunoblot in Fig. 5A demonstrates expression of the COOH-terminal deletion mutant (*60 kDa) in clone CVC7 and expression of the NH,- terminally truncated mutant (*42 kDa) in clone CVN3. A protein of about 90 kDa is recognized by the tvP1 antiserum but not by the two monoclonal antisera BUGR and IgG 4919 (data not shown). It represents the endogenous GR (see

()ex + *

NRS - aP1 - NRS - aP1 -

CVC7 aa 3 556 CVN3 aa 407 - 795

C chymotrypsin 1 2 1 2

.i b .55K .40K .26K 7 b :;:I:

500 “cl 6000 ng

FIG. 5. Stable expression of GR deletion mutants in CV-1 cells. A, immunoblot. GR was immunoprecipitated from nontransfected CV-1 cells, CVC’i, and CVNB clones, either with normal rabbit serum (NRS) or GIi-specific antiserum ((~1’1). Proteins were separated on a 12.5’:; polyacrylamide gel and blotted onto I’VDF membrane and reacted with cvl-‘l and ““l-protein A, followed by autoradiography. H, phosphate labeling. Cells were grown in [.“P]orthophosphate-containing medium (0.1 mCi/ml) and lysed in radioimmune precipitation buffer. GR mutants were immunoprecipitated with normal rabbit serum or ~1’1 and analyzed on a 12.;i!i polyacrylamide gel. C’V-I, nontransfected control cells (‘V(‘7, CO-kDa mutant; (‘VN3, 4%kDa mutant. (‘, phosphopeptide mapping. Intact GR or GR mutants were isolated from ‘“I’-labeled cells and subjected to partial proteolysis accordin, CT to Cleveland c’t al. (29). Lanrts I, CVC7; [anes 2, FTO 23.


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Hormone-dependent GR Phosphorylation 5407

% acelylaled ChloramphenlCOl 100

enh2 and 72, characteristic of transactivation domains (34), whereas enhl is largely basic. We found that Tl/enh2 contains the majority of phosphorylation sites. The extent of phosphorylation of this region is increased after hormone treatment. The DNA-binding domain/enhl probably contains one

sphorylation site, whereas the hormone-binding region is phosphorylated. These observations extend previous re- s obtained by Dalman et al. (18), who analyzed GR phos- rylation only in the absence of hormone. They demon-

ated one nhosnhorvlation site within the DNA-binding

reporter RSV-CAT

cell clone CV-1 MMTV-CAT MMTV-CAT MMTV-CAT

cv-1 CVN3 cvc7

m - Dex f Dex

FIG. 6. Activity of GR mutants on a transiently transfected MMTV-LTR tkCAT reporter construct. The MMTV-LTR ktCAT reporter plasmid was transiently transfected into wild-type CV-1 cells (CV-I ) or into clones CVN3 and CVC7. Cells were cultured for 48 h in the absence or presence of 0.1 pM dexamethasone. RSV- CAT was transfected into wild-type CV-1 cells which were grown in the absence of hormone. 25 rg of protein were used to measure CAT activities. MMTV-CAT = MMTV-LTR tkCAT.

“Discussion”). Fig. 5B shows immunoprecipitated proteins from ““P-labeled cells. Only the BO-kDa mutant is phosphorylated, which is independent of hormone treatment. Phos- phorylation of the 42-kDa mutant was not observed. Cleve- land analysis performed on 32P-labeled 60-kDa mutant expressed in CV-1 cells and wild-type rat GR from FTO 2B cells shows a conserved phospeptide pattern (Fig. 5C). These results demonstrate that GR can be phosphorylated on distinct residues in a heterologous system. Phosphorylation of the 60- kDa but not of the 42-kDa mutant confirn-s the protease digestion and chemical cleavage data obtained with wild-type rat GR. We would like to mention that we were unable to obtain stable transfectants expressing intact receptors.

domain of the GR-and iound that the hormone-binding region is not phosphorylated. Estimates for the number of phosphate residues in the GR range between 4 and 5 in the absence of glucocorticoids (18). This means that 3 to 4 phosphate residues are located in the rl/enh2 domain. At this point we cannot distinguish between two possibilities: 1) hormone treatment might result in the phosphorylation of serine/ threonine residues not phosphorylated in the absence of hormone or 2) hormone treatment might result in the phosphorylation of a larger fraction of GR molecules. A similar picture became evident from studies with the progesterone receptor. Progesterone receptor phosphorylation occurs both in the absence and presence of progestins excl.usively in its amino-terminal domain (17). So far no function for this domain has been identified, but it exhibits a high degree of acidity, like Tl/enh2 of the GR, and is therefore reminiscent of a transactivation domain.

Functional Analysis of GR Mutants in CV-1 Cells-Phos- phorylation of a transactivating domain of the GR might modulate its transcriptional efficiency. We analyzed the ability of both receptor mutants to activate transcription of a transiently transfected MMTV-LTR-tkCAT reporter gene (Fig. 6). CAT assays performed on extracts from untrans- fected CV-1 cells showed only basal transcription from the tk promoter and no effect of hormone treatment. CVN3 cells exhibit a 3-fold increase in MMTV-CAT transcription after hormone treatment. CVC7, however, showed a strong, hormone-independent activation of the MMTV-CAT reporter gene. RSV-CAT was included in this assay to allow comparison of the activity of GR mutants with a relatively strong viral promoter. At this point we cannot distinguish whether deletion of receptor amino acids or whether lack of phosphorylation or both are responsible for the differences in the activity of GR mutants.

DISCUSSION

We demonstrate in this paper that GR phosphorylation occurs both in the absence and presence of hormone mainly in transactivating regions of the GR and could possibly be involved in transcriptional regulation. Glucocorticoid receptors contain at least three regions, which are able to independently function as “transcriptional enhancement domains.” rl/enh2 has been localized to about 200 amino acids in the amino-terminal half of the GR (19, 20, 33), enhl co- localizes with the DNA-binding domain (20), and 72 has been mapped to 30 amino acids in the hormone-binding domain (19). A large number of acidic residues is contained in Al/

Phosphorylation of a number of enzymes, e.g. phosphoryl- ase kinase, which is active in glycogen utilization in muscle cells, has been demonstrated to effectively alter their activity (35). Functional relevance of this covalent modification for transcription factors has been demonstrated only in a few examples. Heat-shock transcription factor (HSTF), for in- stance, is important for expression of heat-shock genes in yeast and occurs in several phosphorylated forms (5). In vitro studies demonstrated that heat-induced phosphorylation of heat-shock transcription factor does not alter its affinity for DNA nor its dimerization capability. It has been speculated that phosphorylation might modulate the overall acidity of its transactivation domain, thereby allowing a rapid and con- tinuous regulation of the activity of heat-shock transcription factor according to the physiological needs of the cell. Studies on the mammalian transcription factor CAMP response element binding protein (CREB) indicate that dimerization and transcriptional activity of CAMP element binding protein in in vitro assays is regulated by phosphorylation (8, 36). Con- ceptually, phosphorylation could influence each one of the known functions of the GR, i.e. it could alter the hormone- or DNA-binding ability or the transactivation function. The available evidence suggests that phosphorylation does not influence the ability of the GR to bind to DNA or to dimerize. Experiments with mouse GR have shown that GR binds to DNA independently of its phosphorylation state (12). This is corroborated by experiments with Escherichia coli-expressed CR fragments, which bind to DNA and also show the ability to form dimers (37,38). Circumstantial evidence suggests that GR phosphorylation might regulate its potency as a transac- tivator. Freedman and Yamamoto (39) have been able to establish a GR-dependent in uitro transcription system based on Drosophila nuclear extracts and E. coli-expressed GR fragments. They demonstrated a 3-fold activation of transcription using a rat GR fragment, which contains only the DNA- binding domain/enhl. A further 2-fold enhancement could be observed by using a protein consisting of the Tl/enhB-“en- hancer” domain connected to the DNA-binding domain. Analysis of the activity of similar constructs transfected into


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CV-1 cells, however, showed an at least ZO-fold increase in the ability to activate transcription (19, 20). This difference in the in vitro and in vivo activity of the GR domains could possibly be ascribed to regulation by phosphorylation. Mu- tants containing the Tl/enh2 domain, but lacking the hormone-binding domain, work almost as efficiently as wild-type receptors, except that they act hormone-independently (19, 20). This is also demonstrated with GR mutants used in this report. The phosphorylated mutant “3-556” is able to maxi- mally activate transcription from the MMTV LTR promoter. The mutant lacking the Tl/enh2 domain is only weakly active, despite the presence of a hormone-inducible transactivation function (19, 20). Phosphorylation might play a role in modulating protein-protein interactions, which are believed to happen through the amino-terminal domain of the GR.

It has been suggested that tyrosine phosphorylation is necessary for full hormone binding activity of the estrogen receptor (11). Mendel et al. (10) demonstrated that ATP- depleted cells contain glucocorticoid receptors (“null receptor”) in the nucleus, which are seemingly nonphosphorylated and unable to bind hormone. These results led to the sugges- tion that phosphorylation is important for hormone binding of steroid receptors. We only find phosphoserine and -threonine, but no tyrosine phosphorylation both in the absence or presence of hormone. Furthermore, no phosphorylation of the hormone-binding domain of the GR could be observed (Ref. 18 and this paper). These results would argue against an involvement of GR phosphorylation in hormone binding and define an important difference between the GR and the estrogen receptor.

CV-1 monkey kidney cells do not show a functional response to glucocorticoids. Therefore it has been assumed that they lack endogenous GR. Using the aP1 polyclonal antiserum, which is directed against a well conserved region of the GR, we detected a protein of 90 kDa, which can be proteolytically digested into fragments characteristic of glucocorticoid receptors (data not shown). This protein also shows hormone-dependent phosphorylation. Thus, endogenous GR is present in CV-1 cells but might be defective in one of the essential GR functions (nuclear translocation, DNA binding, transactivation). Comparison of the phosphopeptide pattern of the transfected phosphorylated mutant and the wild-type rat GR indicates that GR phosphorylation occurs on distinct residues. If phosphorylation is important for the full activity of the GR, it will become interesting to analyze phosphorylation sites in the GR expressed in yeast, where it is able to work as a “bona fide” transcription factor (40).

The results presented substantiate the hypothesis that phosphorylation is involved in modulating the strength of the transactivating activity of the GR. A further evaluation of the role of phosphorylation in GR regulation will be based on the identification of phosphorylated amino acids by phosphopeptide sequencing and mutation analysis within the context of GR expression constructs.

Acknowledgments-We are grateful for the help of Dr. Sandro Rusconi (Ziirich, Switzerland), who provided the GR expression plas- mids and CV-1 cells. FTO 2B cells and pMMTV-LTR tkCAT were kindly provided by Dr. G. Schiitz (Heidelberg, Federal Republic of Germany). We would like to thank Drs. R. Ball, N. Hynes, K. Ballmer, and L. Ballou (Friedrich-Miescher Institute, Basel, Switzerland) for critical reading of the manuscript and for their technical advice.

REFERENCES 1. Beato, M. (1989) Cell 56,335-344 2. Evans, R. M. (1988) Science 240,889-895 3. Cohen; P. (1988) Proc. R. Sot. Lo&. B. 234, 115-144 4. Amster-Choder, 0.. Homman, F.. and Wright, A. (1989) Cell 58.

847-855 -

5. Sorger, P. K., and Pelham, H. R. B. (1988) Cell 54,855-864 6. Cherry, J. R., Johnson, T. R., Dollard, C., Shuster, J. R., and

Denis, C. L. (1989) Cell 56,409-419 7. Mvlin. L. M.. Bhat. J. P.. and Honner. J. E. (1989) Genes & Deu.

3, 1157-11’65 ’ ’ -- ’ ’ 8. Yamamoto. K. K.. Gonzales. G. A.. Biees. W. H.. III. and Mont-

miny, M.‘R. (1988) Nature 334,‘494-468 ’ 9. Auricchio, F. (1989) J. Steroid Biochem. 32, 613-622

10. Mendel, D. B., Bodwell, J. E., and Munck, A. (1986) Nature 324, 478-480

11. Migliaccio, A., DiDomenico, M., Green, S., deFalco, A., Kajtaniak, E. L., Blasi, F., Chambon, P., and Auricchio, F. (1989) Mol. Endocrinol. 3, 1061-1069

12. Tienrungroj, W., Sanchez, E. R., Housley, P. R., Harrison, R. W., and Pratt, W. B. (1987) J. Biol. Chem. 262, 17342-17349

13. Hoeck, W., Rusconi, S., and Groner, B. (1989) J. Biol. Chem. 264,14396-14402

14. Orti, E., Mendel, D. B., Smith, L. I., and Munck, A. (1989) J. Biol. Chem. 264,9728-9731

15. Logeat, F., Le Cunff, M., Pamphile, R., and Milgrom, E. (1985) Biochem. Biophys. Res. Commun. 131,421-427

16. Sheridan, P. L., Krett, N. L., Gordon, J. A., and Horwitz, K. B. (1988) Mol. Endocrinol. 2,1329-1342

17. Sullivan, W. P., Madden, B. J., McCormick, D. J., and Toft, D. 0. (1988) J. Biol. Chem. 263, 14717-14723

18. Dalman, F. C., Sanchez, E. R., Lin, A. L., Perini, F., and Pratt, W. (1988) J. Biol. Chem. 263, 12259-12267

19. Hollenbere. S. M.. and Evans. R. M. (1988) Cell 55.899-906 20. Godowski:P., Pi&d, D., ad Yamamoto,‘K. R. (i988) Science

241,812-816 21. Chen, C., and Okayama, H. (1987) Mol. Cell. Biol. 7,2745-2752 22. Southern, P. J., and Berg, P. (1982) J. Mol. Appl. Genet. 1,327-

341 23. Severne. Y.. Wieland. S.. Schaffner. W.. and Rusconi. S. (1988)

EMBb J.‘8,2503-i508 24. Miksicek, R., Heber, A., Schmid, W., Danesch, U., Posseckert,

G., Beato, M., and Schlitz, G. (1986) Cell 46,283-290 25. Westphal, H. M., Moldenhauer, G., and Beato, M. (1982) EMBO

J. 1, 1467-1471 26. Gametchu, B., and Harrison, R. W. (1984) Endocrinology 114,

274-279 27. Rusconi, S., and Yamamoto, K. R. (1987) EMBO J. 6,1309-1315 28. Kamps, M. P., and Sefton, B. M. (1989) Anal. Biochem. 176,22-

27 29. Cleveland, D. W., Fischer, S. G., Kirschner, M. W., and Laemmli,

U. K. (1977) J. Biol. Chem. 252, 1102-1106 30. Saris, C., van Eenbergen, J., Jenks, B., and Bloemers, H. (1983)

Anal. Biochem. 132, 54-67 31. Gorman, C. M., Moffat, L. F., and Howard, B. H. (1982) Mol.

Cell. Biol. 2. 1044-1051 32. Carlstedt-Duke, J., Stromstedt, P.-E., Wrange, O., Bergmann, T.,

Gustafsson, J.-A., and Jornvall, H. (1987) Proc. Natl. Acad. Sci. U. S. A. a&4437-4440

33. Danielsen, M., Northrop, J. P., Jonklaas, J., and Ringold, G. M. (1987) Mol. Endocrinol. 1. 816-822 ~~~~, ~~ ~

34. Mitchell, P. J., and Tjian, i. (1989) Science 245, 371-378 35. Snranc. S. R.. Acharva. K. R.. Goldsmith. E. J.. Stuart. D. I..

‘Va&, K., ‘FletterGk; R. J.,‘Madsen, N: B., and John&on, L. N. (1988) Nature 336, 215-221

36. Gonzales, G. A., Yamamoto, K. K., Fischer, W. H., Karr, D., Menzel, P., Biggs, W., III, Vale, W. W., and Montminy, M. R. (1989) Nature 337, 749-752

37. Freedman, L. P., Luisi, B. F., Korszum, R. Z., Basavappa, R., Sigler, P. B., and Yamamoto, K. R. (1988) Nature 334, 543- 546

38. Tsai, S. Y., Carlstedt-Duke, J., Weigel, N. L., Dahlman, K., Gustafsson, J.-A., Tsai, M.-J., and O’Malley, B. W. (1988) Cell 55,361-369

39. Freedman, L., Yoshinaga, S., Vanderbilt, J., and Yamamoto, K. R. (1989) Science 246, 298-301

40. Schena, M., and Yamamoto, K. R. (1988) Science 241, 965-967


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W Hoeck and B Gronermainly in the amino-terminal transactivation domain.

Hormone-dependent phosphorylation of the glucocorticoid receptor occurs

1990, 265:5403-5408.J. Biol. Chem.

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