Inhibition of the specific DNA binding activity of the dioxin receptor by phosphatase treatment.

The dioxin receptor stimulates transcription of the cytochrome P-450IA1 gene in response to dioxin. Exposure of the intracellular dioxin receptor to dioxin leads to a rapid conversion of the receptor from a latent form to a DNA binding species which specifically recognizes dioxin-responsive positive control elements in vitro. In this report, we show that treatment of in vivo or in vitro ligand-activated receptor with potato acid phosphatase significantly reduced or abolished its specific DNA binding activity. This effect was inhibited in the presence of sodium phosphate. In control experiments, the ligand-activated glucocorticoid receptor was not inactivated by phosphatase treatment. Moreover, phosphatase treatment did not induce any detectable degradation of covalently labeled dioxin receptor, arguing against protease contamination as a cause for receptor inactivation. Finally, phosphatase-inactivated dioxin receptor exhibited bona fide levels of ligand binding activity. Taken together, these data suggest that phosphorylation may regulate the DNA binding activity of the ligand-occupied dioxin receptor.

It is possible to reconstitute ligand-dependent activation of the dioxin receptor to a DNA binding form i n vitro (Fujisawa-Sehara et al., 1988;Nemoto et al., 1990;Cuthill et al., 1991). Under these condkions, the XRE binding activity of the dioxin receptor is regulated by ligand in a manner that directly reflects the affinity of the ligand for the receptor (Cuthill et al., 1991). We have recently shown that conversion of the latent form of dioxin receptor to an XRE binding species can be achieved by release from an inhibitor protein, possibly the 90-kDa heat shock protein, hsp90 (Wilhelmsson et al., 1990). To further explore the mechanism of regulation of dioxin receptor activity, we have investigated the effect of phosphatase treatment on ligand and DNA binding activities of the receptor. We show that a dephosphorylated form of dioxin receptor exhibited control levels of ligand binding activity. However, the XRE binding activity could be significantly reduced or abrogated by phosphatase treatment of either crude i n uiuo or i n vitro ligand-activated forms of receptor.
Ligand cross-linking experiments demonstrated that the phosphatase-treated, non-DNA binding receptor form was a physically intact 95-kDa protein. Thus, we propose that specific DNA binding activity of the dioxin receptor may require receptor phosphorylation.

EXPERIMENTAL PROCEDURES
Cells and Preparation of Cellular Extracts-Wild-type Hepa lclc7 hepatoma cells (Hankinson, 1979), obtained from Dr. Oliver Hankinson (UCLA), were used throughout. Cells were grown and treated with 1 nM dioxin for 1 h as described (Cuthill and Poellinger, 1988;Wilhelmsson et al., 1990). Nuclear extracts from untreated or dioxintreated cells were prepared as described by Dignam et al. (1983) with minor modifications (Wilhelmsson et al., 1990). Cytosolic extracts from untreated cells were prepared as described previously (Wilhelmsson et al., 1990).
Phosphatase Treatment of Nonactivated and Ligand-activated Dioxin Receptor-The cytosolic form of dioxin receptor in Hepa lclc7 cells was activated to a DNA binding form by ligand treatment in vitro (Nemoto et al., 1990). I n viuo-activated dioxin receptor was recovered in a nuclear extract from Hepa lclc7 cells treated with dioxin as described above. Enzymatic dephosphorylation of dioxin receptor was performed by incubation of the receptor with the indicated concentrations of potato acid phosphatase (Boehringer Mannheim) a t 25 "C for 20 min in 20 mM Tris-HC1, pH 7.2, 1 mM EDTA 10% (w/v) glycerol, and 2 mM 2-hydroxyethylmercaptan.
D N A Binding Assay-The specific DNA binding activity of the dioxin receptor was monitored by a gel mobility shift assay employing as specific probe a "'P-labeled, double-stranded oligonucleotide XRE (5"AATTCCAGGC TCTTCTCACG CAACTCCGGG GC-3') spanning the dioxin-responsive XREl element (Fujisawa-Sehara et al., 1987) from positions -1,026 to -999 relative to the transcription start site of the rat cytochrome P-450IA1 gene. The DNA binding reactions were performed at 30 "C for 30 min, and bound and free DNA was electrophoretically separated under conditions described previously (Hapgood et al., 1989;Nemoto et al., 1990). A similar gel Dioxin Receptor Activation mohility shift assay was used to characterize the DNA binding activity of purified glucocorticoid receptor. In these experiments, a radiolaheled, double-st randed oligonucleot ide ( 5 ' -A G C T T C T A G A -GGATCTGTAC AG(;A'I'(;'l''ITT A(;A'I'CGAATT-3') spanning the palindromic (;HE ofthe rat tyrosine aminotransferase gene (.Jantzen r t 01.. 1987) was used as specific prohe. In indicated DNA hinding competition experiments.a syntheticoctamerelement from the HCL1 immunoglohulin promoter (Poellinger ct nl.. 1989) was used as an unrelated sequence motif.
Lignnd Binding I.:.rprrimrnts-The cytosolic dioxin receptor was labeled hy inruhation of crude cytosol for 3 h at 25 "C with 10 nM [ 'Hldioxin (Chemsyn, Lenexa, KS) in the ahsence or presence of a 200-fold molar excess of the unlaheled high affinity receptor ligand 2,:1,~,8-tetrachlorodit)enzofuran. A hydroxylapatite adsorption assay (Poellinger c f nl., 1985) was used to quantitate the levels of proteinhound [ 'Hldioxin. Specific binding activity was determined by subtracting the binding in the presence of an excess of the unlabeled ligand (nonspecific binding) from the levels of totally bound.
I'urification nnd Immunohlot Annlvsis of Glucocorticoid Rrccptor-Glucocorticoid receptor was purified from rat liver (Wrange 41 01.. 1986) and treated with potato acid phosphatase as descrihed ahove for the dioxin receptor. Following electrophoretic separation on SDSpolyacrylamide gels, proteins were electrophoretically transferred to nitrocellulose filters. Immunoromplexes were identified t)v incuhation with anti-glncocorticoid receptor monoclonal antibodies recognizing epitopes in either the amino terminus (Okret rt al.. 1984;Rusconi and Yamamoto, 1987) or in the central DNA binding domain' of the receptor. Immunocomplexes were visualized by incuhation with horseradish peroxidase-conjrlgated anti-mouse immunoglohulins. Snfrty I'rrcnntions-In cell culture and biochemical experiments involving the use of dioxin, special handling procedures were employed (Wilhelmsson rt nl.. 1990, and references therein). Contaminated materials were disposed of hy high temperature incineration.

RESULTS
Inactivation of the Dioxin Receptor by Phosphatase Treatment-The dioxin receptor was activated to a nuclear, DNA binding form by treatment of Hepa lclc7 cells with dioxin (Fujisawa-Sehara Pt al., 1988;Hapgood et al., 1989). The ability of the receptor to bind to DNA was monitored by a gel mobility shift assay using as probe a synthetic XRE sequence. This XRE probe matches the XREl sequence of the rat cytochrome P-450IA1 gene (Fujisawa-Sehara et al., 1987). Fig.  1A shows the dioxin-induced, receptor-dependent XRE binding activity (lane I, receptor-XRE complex indicated by an arrow) in nuclear extract from treated cells. Binding could be abolished by the addition of excess unlabeled probe to t,he binding reaction, but not by addition of an excess of an unrelated sequence motif (compare lanes 1-3). We next added potato acid phosphatase to the extract containing in uiuo activated receptor to remove 0-linked phosphates. This treatment resulted in a concentration-dependent decrease in formation of the specific recept,or-XRE complex ( Similar results were obtained when we examined the effect of phosphatase treatment on the XRE binding activit.y of the cytosolic, in vitro-activat,ed dioxin receptor. In vitro activation of the receptor was achieved by incubation of crude Hepa lclc7 cytosol with 10 nM dioxin for 3 h a t 25 "C prior to assembly of the DNA binding reaction (Nemoto et al., 1990;Cuthill et al., 1991). We confirmed that the in uitro-induced XRE binding activity was sequence-specific by competition with unlabeled oligonucleotides. In the presence of an excess of the unlabeled XRE sequence, dioxin-induced XRE complex ' G. G. F. Mason and .I. Rergman, manuscript in preparation.  ' r 2 ) 0 C. XRE hinding activities were monitored by gel mohility shift formation was abrogated, whereas no effect on XRE binding was observed in the presence of an excess of an unrelated sequence motif (Fig. 2.4, compare lanm I-.?). The relative mobility of the XRE-specific complex generated bv in vitro-activated receptor (Fig. 2 A , lane 1 ) is identical with that generated by the in uiuo-activated receptor (Wilhelmsson et al., 1990). Moreover, the DNA binding properties of the in vitro ligand-activated receptor form are indistinguishable from those of the in uiuo-activated nuclear receptor (Cuthill et al., 1991), arguing that all the components required for receptor-XRE interaction are present in the cytosolic extract. Phosphatase treatment nearly abolished the XRE binding activity of the in vitro-activated receptor. This effect was, however, inhibited by addition of 10 mM phosphate (Fig. 2R,  compare lanes 1-3).
These results could be explained if our conditions for dephosphorylation of the receptor simply resulted in accidental degradation of the receptor protein. T o address this issue, the dioxin receptor was covalently labeled with [ l"I]dioxin prior t o incubation with phosphatase. The ['"'I]dioxin-labeled receptor was treated with or without phosphatase and analyzed in parallel for ligand binding and DNA binding activities by nondenaturing gel electrophoresis. Although the phosphatase-treated, ["'I]dioxin-labeIed receptor did not generate a complex with the .'?P-labeled XRE probe (Fig. 3A, lane 2 ) , the high specific activity of the iodinated ligand permitted us t o visualize the ligand-receptor complex by autoradiography and to qualitatively compare the relative mohilities of untreated and phosphatase-treated [""Ildioxin-receptor complexes, respectively, following nondenaturing gel electrophoresis in the ahsence of the radiolabeled XRE prohe. Under these conditions, a significant amount of the [""!]dioxinreceptor complex appeared to be aggregated and was recovered in the application slots of the gel (Fig. 3A, lanes 3 and 4 ) . However, the untreated and phosphatase-treated ["r'I]dioxinreceptor complexes which penetrated the gel exhibited iden- tical relative mohilit,ies and very similar levels of ligand binding activity (compare lanes . ? and 4 ) . Importantly, these ligand-receptor complexes were detected in the very same region of the gel as the "'P-laheled XRE complex generated by the phosphatase-untreated ["'Ildioxin-receptor complex (compare lanes 1,3, and 4 ) .
We next examined the M , of untreated and phosphatasetreated forms of [".'I]dioxin-laheled receptor bv SDS-polyacrylamide gel electrophoresis. Although the XRE hinding activity of the treated form of receptor was virtuallv undetectable (Fig. 3A, lane Z ) , both the untreated and treated forms migrated as 95-kDa proteins on SDS-polyacrylamide gels (Fig. 3B). Moreover, no change in apparent amounts of the [""I]dioxin-receptor complex was detected hv this assay after phosphatase treatment. Thus, these experiments strongly argue against simple protein degradation (possihlv due to contaminating proteases) as the cause of phosphataseinduced loss in XRE hinding activity of the receptor.
A number of DNA binding transcription factors appear to be differentially modified hy proteases, kinases, or phosphatases in a DNA-hound state as opposed to an unbound configuration (Prywes et al., 1988; Schreiher at a/., 1988; . Jackson et al., 1990). In the case of the ligand-activateddioxin receptor, preformation of the receptor-XRE complex prior to addition of phosphatase did not significantly protect the receptor from enzyme-dependent inactivation (data not shown). Thus, the configuration of either unbound or DNA-hound receptors appears to permit access of the enzvme to kev phosphorylated residues.
The Specific DNA Binding Activity of tha Glucocorticoid Receptor Is Not Altered h-v Phosphatasa Traatrnant-In control experiments, we examined the effect of phosphatase treatment on the DNA hinding activity of the glucocorticoid receptor which represents a protot-ypical ligand-activated intracellular receptor (reviewed in Gustafsson P t n/.. Evans, 1988;Reato, 1989). Importantly, the glucocorticoid receptor has been shown to he phosphorylated in a hormonedependent manner a t amino acids within its DNA binding domain (Hoeck and Groner, 1990). Moreover, the rat. glucocorticoid receptor is very sensitive to proteolysis, generating several distinct protease-induced degradation products (Carlstedt-Duke et al., 1987 and references therein), and thus serves as a control of the possible level of protease contamination in the phosphatase preparation used.
In agreement with the results obtained with affinity-laheled dioxin receptor (Fig. 3 R ) , treatment of purified, ligand-activated glucocorticoid receptor with phosphatase resulted in no change in the apparent M , (94 kDa) or amount of the receptor, as assessed by immunohlot analysis of untreated and treated receptor forms using monoclonal antibodies directed against either the amino terminus (Fig. 4 A ) or the centrally located DNA binding domain (data not shown) of the receptor. Furthermore, the specific DNA binding activitv of the glucocorticoid receptor toward a synthetic GRE probe was not altered by phosphatase treatment (Fig. 4R). Thus, although the glucocorticoid and dioxin receptors both represent ligand-activated DNA binding factors, the effect of phosphatase appears to he specific for the dioxin receptor.
Dephosphorylated Dioxin Racaptor Bxhihits Diffarmtial Ligand and XRB Binding Actiuitim-To address the mechanism underlying the phosphatase-induced reduction in XRE hinding activity of the dioxin receptor, we treated the ligandunoccupied form of cLytosolic receptor with phosphatase. This form or receptor shows very low levels of specific XRE hintling activity in gel mobility shift experiments (Nemoto at al., 1990;  latent form of receptor was treated without or with phosphatase prior to exposure to dioxin. In the absence of phosphatase treatment, the receptor was efficiently in vitro-activated to a DNA binding form (Fig. 5R, compare lanes 2 and 3 ) after interaction with ligand ( Fig. !X, columns 1 and 2 ) . However, the XRE binding activity of the phosphatase-treated form of receptor was not induced upon addition of ligand ( Fig. N 3 ,  compare lanes 4 and 5 ) , although this form of receptor showed hona fide levels of specific dioxin hinding activity (Fig. 5C, compare columns 1-4).

DISCUSSION
Regulation of the DNA Binding Activity of the Dioxin Receptor-Both XRE binding activity and nuclear localization of the dioxin receptor are modulated by ligand (Denison et al., 1988;Fujisawa-Sehara et al., 1988;Hapgood et al., 1989). In this report, we show that the XRE binding activity of both cytosolic and nuclear ligand-occupied dioxin receptor is significantly reduced or abolished by treatment with potato acid phosphatase. Inactivation of the receptor was inhibited in the presence of an excess of phosphate. Moreover, phosphatase treatment of the cytosolic, latent form of dioxin receptor selectively inhihited ligand-induced conversion to an XRE binding species hut did not alter its ligand binding activity. In agreement with these results, the ligand binding activity of the dioxin receptor has been reported to he insensitive to treatment with alkaline phosphatase (Denison et al., 1989). Finally, no proteolysis of either dioxin receptor or purified glucocorticoid receptor was detected upon incubation with phosphatase, suggesting that the ohserved effects are caused by receptor dephosphorylat,ion and not protein degradation due to contaminating proteases. Taken together, these results imply that one or more 0-linked phosphorylations may he necessary for generating the dioxin receptor species with high affinity for the XRE target sequence. In this context, it is int.eresting to note that a conserved pattern of charge heterogeneity has recently been ohserved for hoth the mouse and rat dioxin receptors (Perdew and Hollenhack, 1990). Several steroid hormone receptors, including the glucocorticoid receptor, have been demonstrated to he phosphorylated a t multiple sites (reviewed in Auricchio, 1989: Denner ef al.. 1990). In the case of the glucocorticoid and estrogen receptors, it has been suggested that phosphorylation may he important for hormone binding activity (Mendel et al.. 1986;Auricchio et al., 1987;Migliaccio et al., 1989). Moreover, phosphorylation of the amino-terminal trans-activating domain of the glucocorticoid receptor is strongly increased by hormone treatment (Hoeck and (ironer, 1990), indicating that this modification may he important for the function of the receptor as a transcriptional regulator. In contrast, the nonspecific DNA binding activity of the glucocorticoid receptor has been reported to he independent of phosphorylation (Tienrungroj ~t al., 1987), in agreement with our observation that binding of the receptor to a GRE sequence was not altered by phosphatase treat,ment. Thus, phosphorylation appears to affect different functional properties of ligand-activated intracellular receptor proteins.
Phosphorylation of Transcription Factors-Phosphorylation has been implicated in transcriptional activation by a number of transcription factors. The effect of protein phosphorylation on functional properties of these factors appears to fall into two classes. For instance, protein phosphorylation which leads to increased DNA binding activity has been shown for the serum response factor (Prywes et al., 1988;Manak et al., 1990), and the adenovirus-induced E4F (Raychaudhuri et al., 1989) and E2F factors . On the other hand, phosphorylation of the heat shock (Sorger and Pelham, 1988), CREB (Yamamoto et al., 1988), GAL4 (Mylin et al., 1990), and Oct-2 (Tanaka and Herr, 1990) transcription factors correlates with transcriptional activation, rather than DNA binding activity. In summary, phosphorylation appears to be an important mechanism of regulation of eukaryotic transcription factors. Our present data raise the possibility that the dioxin receptor is also regulated by this mechanism. Thus, it will be interesting to establish which sites on the receptor are modified and to see which protein kinases are involved. Finally, the question whether receptor phosphorylation itself is regulated by dioxin must be addressed.