Effects of Okadaic Acid, a Protein Phosphatase Inhibitor, on Glucocorticoid Receptor-Mediated Enhancement

Jeremy

P. Somers*

and

Donald

B. DeFrancot

tory proteinsthat has the capacity to both enhanceand repress transcription (1, 2). The enhancementof promoter activity by GR requires a sequence-specificinteraction of the receptor with a linked glucocorticoid response element (GRE) (3-5). Various mechanisms account for transcriptionalrepressionmediatedby the GR, includingdirect interactions of the receptor with a negative GRE (6) competition between GR and other frans-acting factors for overlapping binding sites (7) and interaction of the receptor with c-fos and c-jun proteins (8-l 1). Although the precise mechanism through which GRs influencethe activity of RNA polymerase-11promoters remains obscure, various approaches(12-14) implicatea role for GR in facilitating the recruitment of transcriptionfactors into active transcriptionalcomplexes.Given the multitudeof promoters that acquire hormonal responsivenesswhen linked to GREs (15) it seemslikely that the activity of a number of different transcription factors could be regulated by the GR, and that various physiologicalfactors could influencethe efficacy of these interactions. GRs are phosphoproteins whose phosphorylation state is quantitatively altered by hormone treatment (16, 17). The mouse GR is predominantlyphosphorylated on five serines and one threonine, all clustered within a 193-aminoacid NH,-terminalsegment of the receptor (i.e. from amino acids 122-315) (18). The kinasesthat phosphorylatethese sitesin viva have not been identified as yet. Recent evidence suggeststhat protein phosphatasetype 2A (PP-2A) and/or type 1 (PP-1) may act on a specific subsetof phosphorylation sites in the rat GR (19). Although phosphorylationhas been implicated in the regulation of GR nucleocytoplasmicshuttling (19), the influenceof phosphorylation on other GR functions has not beenestablished.Phosphorylation has been shown to both positively and negatively influence the DNA-binding activity of other transcriptional regulatory proteins (20-22) and to be requiredfor efficient transactivation (23). Various approacheshave been usedto probefor the functional significance of protein phosphorylation. Through the use of agents that activate or inhibit specific protein kinases and phosphatases,the relative

The effects of okadaic acid (OA), a protein phosphatase inhibitor, on transcriptional enhancement activity of rat glucocorticoid receptor (GR) were examined in transiently transfected cells. In the absence of hormone, GRs expressed in CV-1 and COS-1 fibroblasts were capable of enhancing transcription from cotransfected chloramphenicol acetyltransferase reporter plasmids in response to OA treatment. Synergistic enhancement resulted from combined hormone and OA treatment. The effects of OA on GR-mediated enhancement required the presence of linked glucocorticoid response elements and were observed with reporter plasmids that contained different promoters and glucocorticoid response elements. Since OA did not affect nuclear translocation of the receptor, enhancement mediated by unliganded GR was most likely accounted for by the accumulation of some unliganded GRs within nuclei of transfected CV-1 and COS-1 cells. Deletion of individual GR transactivation domains and point mutations within DNA- and hormone-binding domains severely reduced the response of receptors to OA, although some mutant receptors retained the capacity to elicit a synergistic response when exposed to OA and hormone. The effects of OA on transcriptional enhancement did not appear to correlate with major changes in GR phosphorylation, as visualized by two-dimensional tryptic mapping of in viva 32Plabeled GRs. Thus, phosphorylation of various components of the GR signal transduction pathway, and not necessarily the receptor itself, may influence its transcriptional enhancement activity. (Molecular Endocrinology 6: 26-34, 1992)

INTRODUCTION

The glucocorticoid receptor (GR) is a member of a superfamily of ligand-activated transcriptional regulaOSSS-6609/92/0026-0034$03.00/O M&cular Endocrinology Copyright 0 1992 by The Endocrine

Society

26

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Department of Biological Sciences University of Pittsburgh Pittsburgh, Pennsylvania 15260

Effects

27

of OA on GR

Y

DeX

OA

Dex+OA

TG%l~C~t

Fig. 1. OA Uncovers Transcriptional Enhancement Activity of Unliganded GR and Potentiates Enhancement Mediated by Hormone-Activated Receptor CV-1 cells were cotransfected with a GR expression plasmid (VARO) (47) and a CAT reporter plasmid (GMCO) (44) in which CAT expression is driven by the linked MMTV LTR. Transfected cell cultures were either untreated or treated with 1 O-’ M dexamethasone (Dex), OA, or Dex plus OA. Average CAT activity (n = 4) expressed relative to that in untreated controls (-), was determined using a TLC assay (45).

cv-I

cos- 1

RESULTS OA Uncovers Transcriptional Enhancement Activity of Unliganded GR and Potentiates Enhancement Mediated by Hormone-Activated Receptor

To assay for the effects of OA on GR-mediatedtranscriptional enhancement, we transiently transfected CV-1 monkey kidney fibroblasts with a GR expression plasmid(VARO) and a chloramphenicolacetyltransferase (CAT) reporter plasmid(GMCO), whose expression is driven by the GRE and promoter containedwithin the mouse mammary tumor virus long terminal repeat (MMTV LTR). To insure that transfected GR was not activated by endogenous glucocorticoids present in serum used for cell growth, transfected cells were grown for at least 24 h in charcoal-stripped serum before harvesting(seeMaterials and Methods). As can be seenin Fig. 1, dexamethasonetreatment led to over a lOO-fold induction of CAT activity in extracts from cells cotransfected with GMCO and VARO. Interestingly, treatment of cotransfected cells with 100 nM OA alone also led to induction of CAT activity that was approximately 10% as effective as dexamethasone(Figs. 1 and 2). A dose-responseanalysis revealed that 100 nM OA was the most effective concentrationfor induction of GMCO promoter activity that produced minimal cytotoxic effects (data not shown). Thus, all of our subsequentstudies used this dose of OA. Induction of GMCO promoter activity by OA was completelydependenton the presenceof GR, sinceGMCO promoter activity was not affected by OA treatment in the absenceof cotransfected GR (data not

lxx

CA

Dex * CA

-

cex

OA

Dex + OA

Fig. 2. OA Affects GR-Mediated Enhancement in CV-1 and COS-1 Cells CV-1 and COS-1 cells were cotransfected with a GR expression plasmid (VARO) (47) and a CAT reporter plasmid (GMCO) (44) in which CAT expression is driven by the linked MMTV LTR. Transfected cell cultures were either untreated or treated with 10e7 M dexamethasone (Dex), OA, or Dex plus OA. The autoradiograph of a typical thin layer chromatogram shows the position of [14C]chloramphenicol substrate (lower spots) and ‘%-acetylated chloramphenicol product (upper spots) derived from the CAT assay.

shown). In addition to uncovering the transcriptional enhancementactivity of unligandedreceptors, OA also affected enhancementmediatedby hormone-activated GR. As shown in Figs. 1 and 2, combineddexamethasone and OA treatment exerted a synergisticeffect on GMCO promoter activity. Analogouseffects of OA on transcriptional enhancement mediated by unliganded and hormone-activatedGR were observedintransiently transfected COS-1 cells (Fig. 2). The uncovering of GR transcriptionalenhancement activity by OA alonewas unexpected, sincein previous

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extent of phosphorylation at specific amino acid residues within the target proteins may be altered. Cyclic AMP appears to influence GR activity via effects on receptor synthesis (24) although changes in GR phosphorylation that might result from such treatments have not been analyzed. Likewise, although CAMP and inhibition of protein phosphatase types 1 and 2A by okadaic acid (OA) lead to hormone-indepedent transactivation by progesterone receptor in transfected cells (25) the effects of these agents on receptor phosphorylation have not been determined. We have sought to investigate whether PP-1 and PP2A influence the transcriptional enhancement activity of rat GR in transiently transfected cells through the use of OA, a potent intracellular inhibitor of PP-1 and PP2A (26). The effects of OA on GR enhancement were observed in both the presence and absence of hormone. OA uncovers the transcriptional enhancement activity of unliganded GRs and potentiates enhancement mediated by ligand-activated receptors. The results of GR phosphorylation in viva suggest that phosphorylation of various components of the GR signal transduction machinery, and not necessarily the receptor itself, may influence the transcriptional enhancement activity of the receptor.

Vol6 No. 1

MOL ENDO. 1992 28

OA Activates

GREs Fused to Various

Promoters

The results presented above demonstrate GR-mediatedactivation of the MMTV GRE by OA in transiently transfected monkey kidney fibroblasts. The GMCO reporter plasmidused in these experiments contains the MMTV LTR in which the promoter and GRE driving CAT expressionare linked in their native configuration. To assess whether the effects of OA are GRE or promoterspecific,other CAT reporter plasmidscontaining linked GREs were used in cotransfectionsinto CV1 cells with the GR expression plasmidVARO. GBCO (27) containsa 46-basepair (bp) synthetic MMTV GRE clonedup-streamof the rabbit @-globinpromoter, while TAT-3 (12) contains three copies of a 26-bp GRE derived from the rat tyrosine aminotransferase(TAT) gene fused up-stream of a Drosophila alcohol dehydrogenase(Adh) promoter (12). As shown in Fig. 4, both GREs were functional in transiently transfected CV-1 cells,as dexamethasonetreatment led to 60- and g-fold inductions of promoter activity from TAT3 and GBCO DNA, respectively. Treatment of VARO-trans-

CV-I

6rn2 kndogenous

GR’sl

ttranslected

cosGR’SI

ttranslected

I GR’s)

f oex

Fig. 3. Subcellular Distribution of Endogenous GR in 6m2 Rat Fibroblasts and Transfected GR in CV-1 and COS-1 Monkey Kidney Fibroblasts Indirect immunofluorescence analysis. The 6m2 cells and CV-1 and CO.51 cells transiently transfected with a GR expression plasmid (VARO) (47) were either untreated or treated for 1 h with lo-’ M dexamethasone (Dex). Cells were fixed and processed for anti-GR immunofluorescence, as described previously (46).

”

Del

Promoter:

Drosophila

OA

DsrcOA

Del

Adh I

Rabbit

OA

Dcr+OA

B&bin

Fig. 4. OA Activates GREs Fused to Various Promoters CV-1 cells were cotransfected with a GR expression plasmid (VARO) (47) and CAT reporter plasmids that contain either a TAT GRE sequence fused to the Drosophila Adh promoter (12) or a synthetic GRE derived from the MMTV LTR fused to the rabbit @-globin promoter (27). Transfected cell cultures were either untreated or treated with 1O-’ M dexamethasone (Dex), OA, or Dex plus OA. Average CAT activity (n = 4) expressed relative to that in untreated controls (-), was determined using a TLC assay (45). The 2-fold induction of ,3globin promoter activity by OA was judged to be statistically significant (0.01 -z P < 0.02) by a two-tailed Student’s t test.

fected CV-1 cells with OA alone led to induction of TAT3 promoter activity that was approximately 10% effective as dexamethasone alone, and a significant (0.01 < P < 0.02) 2-fold induction of GBCO promoter activity (Fig. 4). Induction of promoter activity from TAT3 and GBCO by OA was completelydependenton the presence of cotransfected GR (data not shown). Likewise, in the absence of linked GRE sequences, neither Adh nor P-globin promoters exhibited any significant responseto OA, even in the presenceof cotransfected GR (data not shown). For both transfected GBCOand TAT3 DNAs, combineddexamethasoneand OA treatment led to synergistic induction of promoter activity (Fig. 4). This synergismwas particularly striking with GBCO, in which OA and dexamethasonetreatment led to a 170-fold induction of promoter activity, which represented a nearly 20-fold increase over that observed with dexamethasonealone(Fig. 4). In summary, the effects of OA on both hormone-dependentand independenttranscriptional enhancementmediated by the GR can be exerted on at least two different GREs and three different promoters. Deletion of Any GR Transactivation Domain Severely Reduces the Effects of OA on Transcriptional Enhancement

The GR containsat leastthree transactivationdomains, one each in the NH2-and Cog-terminalregions(28) and one interdigitated within the centrally located DNAbinding domain (29). It is conceivable that individual transactivation domainsalone or specific combinations thereof could mediate the effects of OA on transcriptional enhancement.We, therefore, cotransfectedthree

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studiesOA did not enhancetranscription from GMCO DNA stably integratedinto the temperature-sensitivevmos-transformed6m2 rat fibroblast cell line (19). Indirect immunofluorescenceanalysisrevealed that differences in the effects of OA on enhancementactivity of transfected vs. endogenousGRs were most likely due to differences in the subcellularlocalization of GR. In nonhormone-treated6m2 cells, receptors were exclusively cytoplasmic, while a considerableproportion of GR was detected in nuclei of VARO-transfected COS1 and CV-1 cells grown under identical hormone-free conditions(Fig. 3). OA treatment exerted no effect on the subcellularlocalizationof GR in either 6m2cells(19) or transiently transfected CV-1 and COS-1 cells (data not shown).Thus, the presenceof nuclearGRs appears to be a prerequisite for the effects of OA on GRmediatedenhancementin the absenceof hormone.

29

Effects of OA on GR

DFJX CR Mutant:

OA

Dex+OA

VA407C

OA 1

VAN556

OA GR Mutant:

VAX556

Fig. 5. Deletion of Any GR Transactivation Domain Severely Reduces the Effects of OA on Transcriptional Enhancement CV-1 cells were cotransfected with the GMCO CAT reporter plasmid (44) and GR expression plasmids VA407C and VAN556 (a) and VAX556 (b), which possess deletions of various segments of the receptor (30). Transfected cell cultures were either untreated or treated with 1 O-’ M dexamethasone (Dex), OA, or Dex plus OA. Average CAT activity (n = 4), expressed relative to that in untreated controls (-), was determined using a TLC assay (45). Apparent OA effects on enhancement activity of VA407C (‘) and VAN556 (*‘) were judged not to be statistically significant (P > 0.05) by a twotailed Student’s t test.

unique interactions with the transcriptional machinery that could differ in its sensitivity to OA from components that interact with intact GR. Alternatively, NHP- and Con-terminal deletions may disrupt some aspect of GR structure that is required for the effects of OA to be exerted. Distinguishing between these two possibilities and others will require detailed knowledge of the targets of OA action that participate in GR-mediated transcriptional enhancement.

Single Point Mutations in the DNA- and HormoneBinding Domains Severely Impair the Effects of OA on GR-Mediated Enhancement The GR deletion mutants tested above possess at least one intact transactivation domain and are competent to bind DNA. To examine whether other GR functional

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different GR deletion mutants along with the GMCO reporter plasmid to assess the effects of OA on distinct GR transactivation domains. In the GR NHP-terminal deletion mutant VA407C, 406 NHn-terminal amino acids of the receptor along with transactivation domain enh2 are deleted, while in mutant VAN556, amino acids from 557 to the Cop-terminus of the receptor are missing (30). VAN556, therefore, no longer contains the hormone-dependent transactivation domain and, as a result of the deletion of the hormone-binding domain, is a constitutive activator of transcription (30). Finally, VAX556 possess GR amino acids from 407-556 and is missing both NH2- and COP-terminal transactivation domains (30). VAX556 is also a constitutive activator, albeit a weak one, since it contains only the enhl transactivation domain located within the DNA-binding domain (30). Expression of all mutants tested was similar, as judged by Western blot analysis, and was unaffected by OA treatment (data not shown). As shown in Fig. 5A, VA407C effectively enhanced GMCO promoter activity over lOO-fold in response to dexamethasone, while VAN556 was a potent constitutive activator of GMCO (>l OO-fold increase over basal activity of VA407C). We have no explanation for the relatively high dexamethasone-dependent enhancement activity of VA407C in our transfections compared to previous results (30). Although dexamethasone induction of GMCO transcription by both VARO and VA407C was reproducibly high, the absolute induction levels varied between experiments. It is, therefore, difficult to make meaningful comparisons between the high levels of induction observed with different transfected GRs. The amounts of VARO and VA407C protein expressed in transfected cells appeared similar, as judged by Western blot analysis (data not shown), suggesting that selective overexpression of VA407C does not contribute to its relatively high enhancement activity. Nonetheless, in contrast to OA induction of GMCO promoter activity by intact GR (Figs. 1 and 2) no significant induction was observed in response to OA alone for GR mutants VA407C and VAN556 (Fig. 5A). The synergistic response to combined dexamethasone and OA treatment was weak with VA407C and amounted to less than a 2-fold increase over the response to dexamethasone alone. The reduction in the OA responsiveness of GR mutants was not due to alterations in GR nuclear translocation, as a considerable proportion of VA407C and VAN556 mutant GRs were localized within nuclei in the absence of hormone and presence of OA (data not shown). Although these results suggest that significant responses to OA require three intact GR transactivation domains, deletion mutant VAX556, which contains only the enhl transactivation domain, exhibited a significant 9.5-fold induction of GMCO promoter activity in response to OA treatment (Fig. 58). It is possible that the effective response of VAX556 to OA is related to the fact that VAX556 is a crippled constitutive activator (activity reduced 1 O-fold relative to constitutive activator VAN556). However, we cannot exclude the possibility that transcriptional enhancement by VAX556 involves

MOL 30

ENDO.

Vol6

1992

10 x .Z .: 4 2 u 0 .B + rz

8 6.1

6 4 2

-

GR Mutant:

Dcr

VARO

OA

CSXIY

Dcx+OA

Dcx

I

VARO

0.4

Der+OA

L584S

Fig. 6. Single Point Mutations in the DNA- and HormoneBinding Domains Severely Impair the Effects of OA on GRMediated Enhancement CV-1 cells were cotransfected with the GMCO CAT reporter plasmid (44) and GR expression plasmids VARO C5OOY (29) and VARO L584S, which possess single point mutations within the DNA- and hormone-binding domains of the receptor, respectively. Transfected cell cultures were either untreated or treated with lo-’ M dexamethasone (Dex), OA, or Dex plus OA. Average CAT activity (n = 4) expressed relative to that in untreated controls (-), was determined using a TLC assay (45). Dex (‘) and OA (‘) did not significantly affect (P > 0.05) the enhancement activity of VARO L584.S. as judged by a two-tailed Student’s t test.

point mutations within the DNA- and hormone-binding domains render the GR unresponsive to OA treatment alone and reduce, but do not completely abolish, the ability of hormone-activated receptor to synergistically enhance transcription when exposed to OA. The Phosphorylation State of Transfected Altered by OA Treatment

GR Is not

In various cell types, OA enhances specific protein phosphorylation events through inhibition of PP-1 and PP9A (31). GRs are phosphorylated at multiple sites (18) some of which have been shown to be substrates for PP-1 and PP9A in vitro (19). Alterations in GR phosphorylation in vivo upon OA treatment provide a plausible explanation for the observed effects of OA on the transcriptional modulatory activity of the receptor. The fact that some of the phosphorylation sites in the mouse GR map within an NHn-terminal segment of the receptor that includes the transcription activation domain enh2 (18) lends support to the notion that phosphorylation of GR may modulate the activity of this domain. Therefore, we have analyzed the effects of OA on phosphorylation of transfected rat GR using a twodimensional tryptic mapping procedure (19). Gel- and immunopurified 32P-labeled GRs were isolated from VARO transfected COS-1 cells that were either untreated or treated for 2 h with lo-’ M OA, dexamethasone, and OA plus dexamethasone. The 12 32P-labeled GR phosphopeptides that were resolved by the two-dimensional mapping procedure (19) are shown in the autoradiographs in Fig. 7. As can be seen from comparing the autoradiographs in Fig. 7, a and c, the extent of 32P phosphorylation within at least one peptide was dramatically increased by dexamethasone treatment (designated by arrowhead in Fig. 7~). In contrast, GR phosphorylation in transfected COS-1 cells does not appear to be significantly altered by OA treatment. The tryptic maps of [32P]GR phosphopeptides isolated from untreated (Fig. 7a) and OA-treated cells (Fig. 7b) are virtually indistinguishable. Therefore, the effects of OA on GR-mediated enhancement in the absence of hormone agonist do not appear to correlate with any major qualitative or quantitative changes in GR phosphorylation that can be detected by our two-dimensional mapping procedure. Likewise, since the tryptic maps of [32P]GR isolated from dexamethasone-treated and OA- plus dexamethasone-treated cells appear to be indistinguishable (Fig. 7, c and d), OA-mediated synergism of hormone-dependent transcriptional enhancement does not appear to correlate with detectable alterations in GR phosphorylation.

DISCUSSION It has become increasingly apparent over the past several years that the activity of RNA polymerase-II promoters is sensitive to protein phosphorylation

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domains play a role in the effects of OA on transactivation, we transfected GR mutants that possessed intact transactivation domains, but had point mutations in the DNA- and hormone-binding domains. In the GR mutant VARO CZOOY, a tyrosine has been substituted for the cysteine at amino acid position 500 in the second zinc finger of the receptor (29). As a result of this mutation, transactivation in yeast and DNA binding in vitro (29) are severely reduced. In the VARO L584S mutant, a leucine at amino acid position 584 in the hormone-binding domain has been changed to a serine. Hormone-binding activity in vitro and in vivo and transactivation activity in mammalian cells are severely compromised by this mutation (Garabedian, M., and K. Yamamoto, unpublished observations). Western blot analysis confirmed that expression in transfected cells of these two mutant GRs was identical to that of intact GR (data not shown). As shown in Fig. 6, GMCO promoter activity in transfected CV-1 cells was not significantly induced by dexamethasone with mutant VARO L584S. The GR mutant VARO C5OOY was capable of weakly enhancing GMCO promoter activity in response to dexamethasone treatment (Fig. 6). Although both VARO L584S and VARO C5OOY were unresponsive to OA alone, they could still act synergistically to induce GMCO promoter activity upon combined OA and dexamethasone treatment (Fig. 6). It is noteworthy that hormone and OA uncover enhancement activity of the hormone-binding mutant VARO L584S, since neither agent alone generated detectable enhancement activity. In summary, single

No. 1

Effects

of OA on GR

events. The effects of phosphorylation are exerted on various steps in the transcription process, as both DNAbinding (20-22) and transactivation (23) activities of particular transcription factors have been shown to be influenced by phosphorylation. In this report we have shown that alterations in protein phosphorylation that result from OA inhibition of two major protein phosphatases, PP-1 and PP9A, affect the transcriptional enhancement activity of the GR. However, the effects of OA on GR-mediated enhancement do not appear to correlate with major qualitative or quantitative changes in GR phosphorylation. We cannot exclude the possibility that OA affects the phosphorylation of a minor subpopulation of receptors that are participating in transcriptional enhancement. Such minor changes in GR phosphorylation would not be detected by our analysis. Nonetheless, our results suggest that phosphorylation of other components of the transcriptional machinery may be important in modulating the contribution of the receptor to transcriptional control. Since unliganded nuclear GRs in OA-treated transfected cells are capable of enhancing transcription in the absence of detectable alterations in receptor phosphorylation, hormone-induced phosphorylation of GR (16, 17) appears to be dispensable for GR-mediated enhancement. Furthermore, the fact that hormone-induced phosphorylation (DeFranco, D., unpublished), but not transcriptional enhancement, was observed

with the hormone-binding mutant VARO L584S suggests that hormone-induced phosphorylation is not sufficient on its own to cause receptor-dependent transcriptional enhancement. Transcriptional enhancement by unliganded GR that is uncovered by OA is relatively inefficient compared to that mediated by hormoneactivated GR, leaving open the possibility that phosphorylation of the receptor could influence the absolute efficiency of GR-mediated enhancement or be important under some physiological conditions. Analysis of mutant GRs with substitutions at phosphorylated amino acid residues (18) should definitively establish the role of phosphorylation in transcriptional enhancement and reveal whether the effects of OA on GR-mediated enhancement are exerted via subtle changes in receptor phosphorylation. If hormone-induced phosphorylation of GR contributes to transcriptional enhancement, synergistic enhancement that results from hormone and OA treatment might reflect the combined contributions of hormone-induced phosphorylation of the receptor and distinct OA-induced phosphorylation events. Synergism in this case would be distinguished from enhancement mediated by OA alone by its sensitivity to GR phosphorylation. This hypothesis is directly testable, because it predicts that GR mutants that lack specific phosphorylation sites would be capable of enhancing transcription in response to OA, but unable to exert a synergistic effect in response to OA and hormone. Regardless of the precise mechanism involved, synergism imparted by OA and hormone can be particularly striking, as observed with a GRE linked to the 8-globin promoter for wild-type GR and with the GR point mutant VARO L584S, which was an effective inducer of GMCO transcription only in the presence of both hormone and OA. What is the nature of the components of the transcriptional machinery that interact with GR whose phosphorylation state may be regulated by PP-1 and PP9A and, therefore, potentially altered by OA? GR-mediated enhancement in response to OA alone requires the presence of a linked GRE and can be imparted upon a number of different promoters, including those contained within the MMTV LTR and the rabbit @globin and DrosophilaAdh genes. Since these promoters contain binding sites for different transcription factors (e.g. NF-1, Sp-1 , and OTF-l), OA could influence the phosphorylation state of either the various transcription factors themselves or coactivators that may mediate interactions with the GR (32, 33). Both Sp-I and NF-1 are phosphorylated in response to specific DNA binding (34) but it has not been established whether their phosphorylation is altered in OA-treated cells. Alternatively, the phosphorylation state of some component(s) of the basic RNA polymerase-II transcription machinery may be influenced by OA. The fact that a minimal Adh promoter, which contains only 33 bp of 5’-flanking sequences and linked GREs, responds to GRmediated activation by OA implies that one target of OA action could be some component of the basal RNA

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Fig. 7. The Phosphorylation State of Transfected GR Is not Altered by OA Treatment 3ZP-Labeled GR was isolated from CO.51 cells transfected with the GR expression plasmrd VARO (47) and subjected to two-dimensional tryptic mapping on thin layer cellulose plates. Autoradiographs are shown of separated GR phosphopeptides from either untreated cells (a) or cells treated for 2 h with lo-’ M OA (b), dexamethasone (Dex) (c), or Dex plus OA (d). The arrowhead (c) points to a major Dexinduced phosphopeptide.

31

MOL

ENDO.

1992

Vol6

No. 1

32

MATERIALS Cell Culture

AND METHODS and Transfection

CV-1 and COS-1 monkey kidney fibroblasts were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum. The 6m2 cells, a temperature-sensitive vmos-transformed normal rat kidney (NRK) fibroblast cell line (43) were grown in McCoy’s 5a medium supplemented with 15% fetal bovine serum. Transfections were performed essentially as described previously, using the calcium phosphate

precipitation method (8). Typically, 2.5 x lo5 cells/60 mM plate were cotransfected with 3 rg each of reporter and expression plasmids. Transfected DNA was removed from cells after an overnight incubation, and cells were incubated for an additional 8 h before hormone or drug treatments. All medium that was used for cell growth after the removal of DNA contained serum that had been stripped of endogenous steroids using dextrancoated charcoal. Typically, dexamethasone and/or OA were then added to attain a final concentration of lo-’ M, and transfected cells cultures were incubated for an additional 16 h. CAT Assays Cell extracts were prepared and assayed for CAT activity, as previously described (44). Relative CAT activity, normalized to total protein in cell extracts, was quantified by determining the percent conversion of [“‘CJchloramphenicol to acetylated [‘“Cl chloramphenicol, which were separated by TLC (45). A promoterless CAT expression vector (44) was used in all transfections to provide a measure of background CAT activity. Each experiment was performed at least four times, and average values are presented. Where indicated, the significance of the data was assessed by determination of P values using a Student’s two-tailed t test. This analysis was necessary, since in some cases, minor inductions of CAT activity were judged to be statistically significant. “Phosphate Labeling Peptide Mapping

and Two-Dimensional

Tryptic

Methods for the in viva labeling of GR with [32P]orthophosphate and mapping of =P-labeled tryptic phosphopeptides have been described in detail previously (19). Brieflv, lo6 transfected COS-1 cells were incubated for 2-h with 3 mCi 10.5 mCi/ml) f3’Plorthoohosohate (carrier free: 7000 CilmM: New England Nuclear, Boston, MA) in phosphate-free D&e& co’s Modified Eagle’s Medium without serum. Dexamethasone and OA were added where indicated, and the 32P labeling continued for an additional 2 h. 32P-Labeled GR was immunoprecipitated from whole cell lysates, as described previously (19) and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-Tosyl-L-phenylalanine chloromethyl ketone-treated trypsin (20 pg) was added to excised gel slices containing 32P-labeled GR, and resultant peptides were separated by electrophoresis in the first dimension and chromatography in the second dimension on thin layer cellulose plates (19). Dried plates were autoradiographed. Indirect

lmmunofluorescence

Indirect immunofluorescence staining of transfected cell cultures was performed as described previously (46). For these experiments, 2.5 pg GR expression plasmid were transfected onto 1 O5 cells on 35-mm plates. After incubations with transfected DNAs, hormone, and/or OA, cells were fixed and processed for immunofluorescence staining. CAT Reporter

Plasmids

The following CAT reporter plasmids were used: 1) GMCO (44) contains the MMTV LTR linked to the CAT gene; 2) GBCO (27) contains a 48-bp synthetic MMTV GRE linked up-stream of the rabbit @-globin promoter at position -125 relative to the start site of transcription; and 3) TAT3 (12) contains three copies of a 26-bp synthetic GRE sequence derived from the rat TAT gene linked up-stream (at position -33) of a Drosophila Adh minimal promoter sequence. GBCO and TAT3 were kindly provided by Keith Yamamoto and co-workers (Department of Biochemistry, University of California, San Francisco).

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polymerase-II transcription machinery that interacts either directly or indirectly with GR, or RNA polymeraseII itself. The largesubunit of RNA polymerase-IIis phosphorylated predominantlywithin a domainof a heptad amino acid repeats in its Con-terminalregion (35) which in yeast, plays an important role in the responseof the polymeraseto various transcriptionalactivator proteins (36, 37). If transactivation by the GR protein involves interactionswith the Con-terminalrepeats of RNA polymerase-II, alterations in phosphorylation within this domain,perhapsbrought about by OA, could influence the efficacy of GR interactions. The ability of a PP9A homologin S. cerevisiae to suppressa HIS4 transcriptionaldefect providessupportfor the notion that protein phosphatasesinhibited by OA (i.e. PP-1 and PP9A) could regulate RNA polymerase-IIphosphorylationand activity (38). The uncoveringof GR-mediatedenhancementby OA in the absence of hormone suggeststhat unliganded receptors possesssomeintrinsic,albeit weak, capacity for activating transcription. Furthermore, the absolute requirementfor the presenceof a linked GRE for the effects of OA necessitatesthe occupancy of target GREsby unligandedreceptor in vivo. These unliganded receptors may be poised on the GRE and competent to bring about changesin transcriptionalactivity from a linked promoter.We hypothesizethat in additionto the binding of hormoneligand to the receptor, alterations in protein phosphorylation that may not include the receptor itself could trigger the subsequentbiochemical events that lead to the effects of GR on RNA polymerass-11 activity. Although the resultsof independentanalyses of chromatin structure suggest that unliganded GRs do not bind to GREs in vivo (39, 40) resultsfrom transfectionexperimentsare consistentwith the notion that unligandedsteroid receptors could bind to the appropriatehormone-responsiveelementin vivo (41). It would be of interest to determinewhether activation of GREs by unligandedGRs in OA-treated cells is correlated with analogousalterations in chromatin structure that accompany hormone-inducedtranscriptional activation (39, 42). The putative role of OA-inhibited phosphatases PP-1 and PPQA in hormonal regulation of transcription may be relevant to many unanswered questionsconcerningthe interaction of GR with chromatin and the RNA polymerase-IItranscription machinery.

Effects

of OA on GR

GR Expression

33

Plasmids 9.

10.

11.

12.

13. We would like to thank Drs. Keith Yamamoto, Michael Garabedian, and Mark Diamond for the kind gifts of CAT reporter and GR expression plasmids. Drs. Michael Garabedian, Didi Robins, and Todd Evans are thanked for their critical reading of the manuscript.

Received August 16, 1991. Revision received October 7, 1991. Accepted October 15, 1991. Address requests for reprints to: Donald B. DeFranco, Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260. This work was supported in part by grants from the NIH (CA-43037) and the NSF (DMB-8902897). Current address: Division of Biology and Medicine, Section of Biochemistry, Brown University, Providence, Rhode Island 02912. T Recipient of a Research Career Development Award from the NIH. l

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