Phosphorylation of steroid hormone receptors.

0163-769X/92/1301-0105$03.00/0 Endocrine Reviews Copyright © 1992 by The Endocrine Society

Vol. 13, No. 1 Printed in U.S.A.

Phosphorylation of Steroid Hormone Receptors* EDUARDO ORTI, JACK E. BODWELL, AND ALLAN MUNCK Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire 03756

I. Introduction II. Cellular Forms, Structure and Functions of Steroid Hormone Receptors A. Cellular forms B. Structure and functions III. Indirect Evidence for Regulation of Receptor Function by Phosphorylation A. Early metabolic studies B. Effects of phosphatases and phosphatase inhibitors C. Effects of kinase activators and inhibitors D. Steroid hormone receptors as putative kinases and as substrates for phosphorylation and dephosphorylation in vitro E. Heterogeneity of receptors F. Effects of cell transformation IV. Direct Evidence for Steroid Hormone Receptor Phosphorylation in Vivo and Relation to Receptor Function A. Glucocorticoid receptors 1. Basal and ligand-dependent phosphorylation 2. Location of phosphorylated sites 3. Phosphorylation and receptor activation B. Progesterone receptors 1. Basal and ligand-dependent phosphorylation 2. Location of phosphorylated sites 3. Phosphorylation and receptor-DNA binding C. Estrogen receptors D. Androgen receptors E. Vitamin D receptors F. Thyroid and other members of the steroid hormone receptor family G. Problems associated with analysis of receptor phosphorylation V. A Cyclic Model for Glucocorticoid Receptor Phosphorylation VI. Regulation by Phosphorylation of Other Transcription Factors VII. Discussion: Significance of Steroid Hormone Receptor Phosphorylation

A. General aspects of receptor phosphorylation B. Receptor phosphorylation and transcriptional regulation C. Receptor phosphorylation and interactions with other molecules D. Looking ahead VIII. Summary

I. Introduction

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OST members of the steroid hormone receptor superfamily, which includes glucocorticoid, mineralocorticoid, progestin, estrogen, androgen, 1,25-dihydroxyvitamin D3, thyroid, and retinoic acid receptors, are ligand-dependent transcription factors. Probably all of them are phosphorylated. Several have been shown to become hyperphosphorylated on binding of their ligands, suggesting important connections between the levels of phosphorylation and their functions as transcriptional regulators. Our main goal in this review is to describe the current state of the rapidly advancing field of steroid hormone receptor phosphorylation, a subject on which general reviews have already begun to appear (1, 2). A subsidiary goal will be to summarize results emerging from closely related studies on connections between phosphorylation and functions of several other important transcription factors, and to draw on this information in considering how phosphorylation may regulate functions of the receptors. We begin with a brief outline of steroid hormone receptor structure and function. With that as a background, we proceed to a discussion of receptor phosphorylation and its possible significance for receptor function.

Address requests for reprints to: Dr. Allan Munck, Department of

II. Cellular Forms, Structure, and Functions of

Physiology, Dartmouth Medical School, Hanover, New Hampshire 03756 *Preparation of this review and the work of the authors cited herein was supported by Research Grant DK-03535 from the National Institutes of Health, and by the Norris Cotton Cancer Center Core Grant CA23106. E.O. was supported by fellowships from the Consejo Nacional de Investigaciones Cientificas y Tecnicas de la Republica Argentina and from the U.S. Public Health (1FO5 TW03923-01).

Steroid Hormone Receptors Except for recent work, most of the information in this section comes from the following reviews, which should be consulted for more detail and references to primary sources (3-13). 105

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A. Cellular forms

Unliganded receptors for most of the steroid hormones appear to be present in cells as parts of large oligomers (~ 300K mol wt, 7-10S sedimentation constant) that can be isolated in cytosols from cells disrupted in hypotonic media. The oligomers are formed by noncovalent association of a monomeric or dimeric receptor protein with a dimer of the 90K heat shock protein (Hsp90)\ Hsp70 and a 55-60K protein that appears to be a heat shock protein (Hsp56) (14) have also been identified in many of these oligomers. Whereas thyroid hormone receptors are tightly bound in the cell nucleus in the absence or presence of hormone, the cellular location of unliganded receptors for the steroid hormones has been controversial. There is now fair agreement, however, that except for glucocorticoid and mineralocorticoid receptors, which in most cells appear to be predominantly cytoplasmic, they reside mainly within the nuclear envelope, from which they escape when cells are disrupted by conventional methods. Whether cellular localization is an intrinsic property of each type of receptor in normal cells, or is contingent on underlying variables such as relative rates of transport in and out of the nucleus (8), is unclear. For example, in cells in which glucocorticoid receptors are overexpressed, the unliganded receptors are predominantly nuclear (15, 16). Hormone binding to unliganded receptors initially results in formation of nonactivated hormone-receptor complexes. Under physiological conditions these complexes immediately start to be transformed to activated complexes, most of which rapidly become bound to the nuclei. Activated complexes can be characterized in vitro by their ability to bind to DNA and other polyanionic substances. They appear to consist of only the hormonebinding proteins (~ 50-100K, 4-5S), although they have been found in association with Hsp70. Activation (or transformation, as this process is also called) involves conformational changes accompanied by dissociation of the oligomeric complex and exposure of the DNA binding site on the receptor. Most evidence indicates that activation is a thermodynamically irreversible process. Reconstitution of receptors with Hsp90 and Hsp70 has so far been achieved only in rabbit reticulocyte lysates and appears to require ATP (17,18). Most activated nuclear-bound hormone-receptor complexes can be solubilized with high salt concentrations (0.3-0.4 M NaCl), but a small fraction generally remains unextractable. Salt-unextractable receptors are not covalently bound to nuclear structures since they can be extracted with sodium dodecyl sulfate (SDS), and exhibit no major change in molecular weight as determined by nonreducing SDS-poly aery lamide gel electrophoresis (PAGE). The majority of the nuclear-bound complexes

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appear to be bound nonspecifically, and may be in rapid equilibrium with cytosolic activated complexes. In hormone-treated cells under steady state conditions there are generally measurable amounts of all forms of hormone-receptor complexes (cytosolic nonactivated, cytosolic activated, nuclear-bound extractable, and unextractable) in relative amounts that vary with receptor, ligand, and cell type. Complexes that directly stimulate transcription of particular genes constitute a small subpopulation of nuclearbound receptors that become tightly and specifically bound to hormone response elements (HREs), which are enhancer-like nucleotide sequences associated with those genes. Negative transcriptional control also occurs through association with specific nucleotide sequences, which are related to HREs in ways that have not been clearly defined.

B. Structure and functions Members of the steroid hormone receptor superfamily share a common domain structure, illustrated schematically in the upper part of Fig. 1. There are three principal domains. At the C-terminal end is the hormone-binding domain, which comprises 220-250 amino acids that contain two regions with substantial sequence homology among receptors for different hormones. Next, separated from the hormone-binding domain by a short so-called hinge region, is the DNA-binding domain, comprising 65-70 amino acids that display high sequence homology among receptors. The N-terminal domain, sometimes known as the immunogenic domain, is the least conserved. It varies in length from more than 600 amino acids for the mineralocorticoid receptor to around 25 for the vitamin D receptor. N-terminal 20-600 aa

DNA binding 65-70 aa

Hormone binding 220-250 aa

Transactivation Hsp90 binding

—

Nuclear localization

Dimerization ^-^——— FIG. 1. Schematic linear representation of a receptor of the steroid hormone receptor superfamily. The domain structure shown at the top is common to all these receptors, but as indicated, the N-terminal domain for different receptors varies in size from about 20 to 600 amino acids. The shaded area designates the DNA-binding domain, next to which is the short hinge region followed by the hormone-binding domain. Activities schematically assigned in the lower part of the figure to regions of the receptor apply most closely to glucocorticoid receptors, but comparable regions, often in similar locations, have been identified with other receptors.


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STEROID HORMONE RECEPTOR PHOSPHORYLATION

The domains are modular in that they can retain their functions when moved to other proteins. Modular structures are a feature of many transcription factors (19). For example, if the DNA-binding domain of the estrogen receptor is replaced by that of the glucocorticoid receptor, the resulting chimeric receptor responds to estrogens but regulates genes with glucocorticoid response elements (GREs). In the absence of hormone the steroid-binding domains can be regarded as repressors of receptor function, since if they are deleted, the truncated receptors become constitutive gene activators. When attached to unrelated transcription factors, steroid-binding domains can make such factors hormone-dependent. Some of the sites in the hormone-binding domain with which steroids make contact have been identified by affinity labeling with bound steroids. With the glucocorticoid receptor, for example, it has been shown that dexamethasone mesylate becomes attached covalently to a single site, cysteine-656 in the rat receptor and the homologous cysteine-644 in the mouse receptor. Triamcinolone acetonide can be photoaffinity linked to both methionine-622 and cysteine-754 in the rat receptor, suggesting that the binding site is composed of regions that are distant in the linear sequence but come together by protein folding. For the estrogen receptor, cysteine530 has been localized in the hormone binding site. The DNA-binding domain bears similarities to certain other DNA-binding proteins. It contains two zinc finger structures, in each of which four cysteines can coordinate one zinc atom. Several steroid hormone receptors have been shown to bind to HREs, which are roughly palindromic in structure with a dyad axis of symmetry, as dimers. Some are also dimerized in solution. One HRE serves a family group of receptors for positive transcriptional control. For example, glucocorticoid, mineralocorticoid, progesterone, and androgen receptors all bind with high affinity to the same HRE. Three-dimensional models (reviewed in Ref. 20) for glucocorticoid and estrogen receptor dimer-HRE complexes based on nuclear magnetic resonance-derived structures of the DNA-binding domains in solution, and direct X-ray crystallographic analysis of complexes between the glucocorticoid receptor DNA-binding domain and GRE-containing DNA (21), have established a distinctive structure for these complexes that agrees well with biochemical observations. Much evidence also indicates that receptor-HRE transcription complexes can involve other transcription factors that may bind directly to the DNA, the receptor, or both (5, 12, 22-28). In fact, a distinction has been made between 'simple HREs' to which receptors can bind and enhance transcription by themselves, and 'composite HREs' at which receptors require other factors for hormonal regulation (28). Rapid advances in this area can be expected as hormone effects are reproduced in

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vitro. So far, hormone-dependent activation of gene transcription in vitro has been reported with progesterone receptors (29) and with an estrogen-responsive system (30). Receptor-dependent activation has been reported with glucocorticoid receptors (31, 32). Within the general domain structure of the receptors there are several regions that are associated with particular activities. They vary among receptors for different hormones and appear to display some species specificity. Locations of some of these are indicated roughly in the lower part of Fig. 1 for glucocorticoid receptors, which are among the most intensively studied in this respect. Acidic 'transactivation' or 'enhancement' regions are found within the N-terminal domain and between the DNA- and steroid-binding domains. They are necessary for full transcriptional enhancer activity of the receptor and can increase activity of other transcriptional activators when coupled to their DNA-binding regions (3335). A region within the steroid-binding domain has been implicated as the binding site for Hsp90 (36, 37). Other regions are important for nuclear localization and for receptor dimerization. III. Indirect Evidence for Regulation of Receptor Function by Phosphorylation A. Early metabolic studies The observation that the hormone binding capacity of glucocorticoid receptors in rat thymus cells falls and rises with cellular ATP levels (38-40), even in the absence of protein synthesis (41), led to the hypothesis that the functions of receptors in normal cells are regulated by a hormone- and ATP-dependent phosphorylation-dephosphorylation cycle, which in cells lacking ATP causes the receptors to become dephosphorylated and lose the ability to bind hormone (42). Somewhat similar results and conclusions were derived from observations with glucocorticoid receptors in mouse fibroblasts (L cells) (43), and energy dependence was found with these receptors in chick embryo retina (44). Suggestive evidence for a phosphorylation cycle that also involved binding to RNA was obtained with androgen receptors (45, 46). A nonhormone-binding form of the glucocorticoid receptor, termed the 'null' receptor, has since been identified by Western blotting in ATP-depleted cells (47, 48). It will be discussed in Section V along with a model of a phosphorylation cycle. B. Effects of phosphatases and phosphatase inhibitors Evidence for receptor phosphorylation subsequently came from experiments showing that treatment of L cell cytosols with alkaline phosphatase inactivated the hormone binding capacity of the glucocorticoid receptors


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(49). Similar results were obtained with rat liver cytosols (50). Binding activity of thyroid hormone receptors in rat liver nuclear extracts also was reduced by alkaline phosphatase (51). Acid phosphatase inhibitors such as sodium fluoride and orthovanadate improved recovery of dihydrotestosterone binding activity from fractions of rat ventral prostate (52). Alkaline phosphatase treatment of cytosols containing glucocorticoid-receptor complexes was found by several groups (53-55) to activate some of the complexes, supporting the proposal that activation is associated with dephosphorylation of the receptor (53). Results of experiments to be described in Section IV.A.3, however, do not favor this idea. Okadaic acid (56), a potent inhibitor of serine/threonine phosphatase 1 (PPl) and 2A (PP2A), has recently become available. It can activate transcription in monkey kidney CV-1 cells of a transfected reporter gene containing progesterone response elements (PREs). Activation of transcription did not require progesterone but did require progesterone receptors (57). Okadaic acid similarly activates glucocorticoid receptor-dependent transcription of a GRE-containing reporter gene, but overrides transcriptional repression induced through a negative GRE (58). In a rat fibroblast cell line, okadaic acid leads to inefficient nuclear retention and redistribution to the cytoplasm of glucocorticoid-receptor complexes, which are then unable to recycle to the nucleus. These and related observations suggest that inhibition of phosphatase activity during nuclear export of the complexes is responsible for blocking recycling of the receptors (58). C. Effects of kinase activators and inhibitors

Rapid changes in the estradiol-binding capacity of estrogen receptors in cytosols from endometrial cells or endometrial cancer cells were observed after addition of cyclic nucleotides (59,60). Binding activity was increased by cGMP and decreased by cAMP. The effects were dependent on the presence of ATP, divalent cations, and particulate fractions, suggesting that they were mediated either by allosteric interactions of these compounds with the receptors or by phosphorylations involving cyclic nucleotide-dependent kinases (60). Other studies on interconversion of estrogen receptor forms are described in Section III.D. Similar results suggested a role for phosphorylation of glucocorticoid receptors from rat placenta. Cytosolic glucocorticoid binding was decreased by cAMP, binding and endogenous cAMP concentrations were inversely correlated in early and term placentas, and epinephrine treatment of tissue slices, which stimulated cAMP production, also inhibited glucocorticoid binding (61). In lymphoma

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cell lines, cAMP was found to promote receptor function and increase the levels of steroid binding (62). Here, in addition to directly modulating the function of the receptor protein, the cyclic nucleotides seem to regulate receptor gene expression (63). Transcription of a transfected PRE-containing reporter gene was activated by the cAMP analog, 8-BrcAMP, to a level comparable to that achieved with progesterone. Activation required progesterone receptors. Activation by progesterone was inhibited by the protein kinase inhibitor PKI, a synthetic peptide that is a potent inhibitor of c AMP-dependent protein kinase (protein kinase A) (57). These results, together with the okadaic acid results described in the previous section, suggested that transcriptional activity of the progesterone receptor is regulated by kinases and phosphatases, being increased by phosphorylation and decreased by dephosphorylation, and that progesterone binding converts the receptor from a poor to a good substrate for kinases (57). Activators and inhibitors of protein kinase C have been reported to affect functions of steroid receptors. With glucocorticoids, phorbol esters enhanced hormone induction of hepatic enzymes (64), and inhibitors reduced this induction as well as nuclear translocation of the receptors (65). Protein kinase C activators, however, inhibited glucocorticoid-dependent transcription in NIH-3T3 cells (66). With estrogens, activators reduced hormone-binding capacity in breast cancer cells (67) and inhibited hormone-mediated transcription in A431 cells (68). With T3, H-8 and other kinase inhibitors blocked accumulation of a group of lipogenic enzymes and their mRNAs in chick embryo hepatocytes (69). Whether mechanisms underlying these results involve indirect effects of protein kinase C or changes in receptor phosphorylation is not known. With WEHI-7 cells we have found no inhibition of basal or hormone-induced glucocorticoid receptor phosphorylation by the protein kinase C inhibitor staurosporine (our unpublished results). D. Steroid hormone receptors as putative kinases and as substrates for phosphorylation and dephosphorylation in vitro With the availability of purified preparations of steroid hormone receptors, phosphorylation reactions can be analyzed in vitro. Receptors have proved to be good substrates for various kinases, and in some cases kinase activity has been proposed for the receptor itself. Although these studies provide useful information on properties of the receptors and on mechanisms that may operate in. vivo, their physiological meaning will remain in doubt until they can be correlated with observations in intact cells. Glucocorticoid receptors have been reported by several


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groups to copurify with protein kinase activity. Affinitypurified receptors could be phosphorylated by the addition of 32P-labeled ATP and magnesium (70). Receptors purified by a different procedure did not themselves become phosphorylated, but did cause hormone-dependent phosphorylation of other proteins such as histones and myosin light chain kinase in the presence of calcium or magnesium (71). Purified glucocorticoid receptors have been claimed to have intrinsic kinase activity, undergoing calcium and hormone-dependent autophosphorylation at threonine residues (72). Other evidence, however, suggests that steroid receptors are not protein kinases. First, the amino acid sequences of steroid receptors have revealed no kinase related structures. Glucocorticoid receptor preparations have been found to contain kinase activity when purified with an antiserum against the receptor, but not when purified with a monoclonal antibody (73). The kinase activity could be recovered by adding preimmune serum to the monoclonal antibody during immunopurification, suggesting that the kinase was nonspecifically adsorbed by the antiserum (73). In another study a contaminant of a glucocorticoid receptor preparation was reported to be photoaffinity-labeled with 8-azido-ATP, and to phosphorylate various protein substrates (74). A magnesiumdependent serine/threonine kinase, which phosphorylated glucocorticoid receptors and other substrates, copurified with receptor preparations but could be separated by DEAE-Sepharose chromatography (75). The progesterone receptor purified from chick oviducts was also initially believed to include protein kinase activity (76), but subsequently the same investigators found that this activity was due to a distinct enzyme that copurified with the receptor (77, 78). Similarly, the estrogen receptor from human breast cancer cells was initially reported to be associated with kinase activities (79, 80), but later this activity was found in a distinct copurified component (81). Progesterone and glucocorticoid receptors can be phosphorylated in vitro by several kinases. Protein kinase A phosphorylates purified A and B forms of the chick oviduct progesterone receptor (82) at a serine different from those phosphorylated in vivo (83). It phosphorylates nonactivated 8S progesterone receptors at serine residues (84, 85). As shown in our laboratory (unpublished results), protein kinase A also phosphorylates the glucocorticoid receptor in vitro. The oviduct progesterone receptor was found to be a good substrate for phosphorylation with purified epidermal growth factor receptor (86). This phosphorylation was exclusively at tyrosine residues, located in two major tryptic phosphopeptides. Similarly, insulin receptor tyrosine kinase phosphorylated the progesterone receptor (87). A proline-directed serine/threonine kinase phosphorylated in vitro a cloned

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fragment of the glucocorticoid receptor containing part of the N-terminal domain and the DNA-binding domain (88). Whether the sites phosphorylated this way correspond to those phosphorylated in vivo at proline-directed kinase consensus sequences (Section IV.A.2) has not been determined. Cell-free phosphorylation of uterine estrogen receptors has been found to occur at tyrosines (reviewed in Ref. 1). A nuclear phosphatase from calf uterus inactivated hormone-binding, 32P-labeling of phosphotyrosines, and antiphosphotyrosine antibody binding of the receptor (89-94). The inactive form of the receptor was transformed to an estradiol-binding form by phosphorylation with a cytosolic calcium-calmodulin-dependent tyrosine kinase (92, 95-100). The kinase could be separated from the receptor protein itself, and was activated by complexes of the receptor with estrogens but not with antiestrogens (100-102). Tyrosine phosphorylation of human receptors synthesized in vitro by purified endogenous calf uterus kinase promoted hormone binding without change in affinity. Studies with truncated mutant receptors indicated that the phosphotyrosine was located within or near the steroid-binding domain (101, 102). Interconversion of estrogen receptors has also been examined in chick oviduct cytosol, where the receptor exists in three forms: two of them bind estradiol with different affinities, and the third does not bind hormone (103, 104). Conversion of the nonbinding to the low affinity form in vitro requires magnesium and the yphosphoryl moiety of ATP (103, 104). This conversion involves a 40-kilodalton cytosolic activation factor (105, 106) that apparently is associated with both kinase and phosphatase activity (107). Although the immunopurified nonbinding receptor could be labeled on serine residues by incubation with [32P]ATP, conversion to the hormone-binding form by the partially purified activation factor was accompanied by a reduction in phosphorylation of the receptor (105,107). Androgen receptors have been phosphorylated in vitro. Receptors purified from rat prostate could be phosphorylated by a cAMP-independent nuclear kinase, but not by a cAMP-dependent kinase or by other nuclear and cytosolic kinases including casein kinase. In addition, no autophosphorylation of the receptor was detected (108). The vitamin D receptor is phosphorylated in vitro by casein kinase II, apparently in the same region indicated by mutational analysis to be phosphorylated in vivo (109). Thyroid hormone receptors are phosphorylated in vitro by protein kinase A and casein kinase II at sites that also appear to correspond to those phosphorylated in vivo (110, 111). E. Heterogeneity of receptors

Posttranslational modifications, including phosphorylation, of proteins can be detected by resolving isoforms


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of different molecular weight and/or charge. For steroid receptors a common finding is that the apparent molecular weight obtained by SDS-PAGE is higher than that deduced from the amino acid sequence, a discrepancy that could be due to posttranslational modifications such as phosphorylation, glycosylation, and acylation. Proteolytic cleavage or association with other cellular components can also result in receptor heterogeneity. It is thus important to keep in mind whether isoforms are analyzed under denaturing and reducing conditions, which determine only variations in the covalent structure, or under nondenaturing conditions in which the behavior of a complex depends mostly on subunit composition. The best characterized example of modifications that produce alterations in apparent molecular weight is given by the progesterone receptor. The photoaffinity-labeled receptor gave a doublet on SDS-PAGE (112). After hormone binding, activation, and association with nuclear structures, the higher molecular weight form increased in amount and the lower form gradually disappeared. The possibility that these isoforms were produced by proteolysis was ruled out by analysis of receptor stability after extraction and by inclusion of protease inhibitors (113, 114). The increase in apparent molecular weight was thought to involve phosphorylation and was shown to be accompanied by increased 32P labeling. Similar changes were observed with the vitamin D receptor (115, 116). A doublet was also found in immunoblots of the glucocorticoid receptor after SDS-PAGE (117), but no hormone-dependent changes were reported. Microheterogeneity of human glucocorticoid receptors has been detected by two-dimensional gel electrophoresis [isoelectric focusing and SDS-PAGE (118)]. About five discrete forms were found with isoelectric points ranging from 6.5 to 7.5, suggesting the presence of posttranslational modifications. These results were supported by analysis of the receptors using nonequilibrium pH gradient electrophoresis in the first dimension (119). With this system no covalent charge modification was found after receptor activation, but only the more basic of two major receptor isoforms bound to DNA-cellulose (120). Analysis of tryptic fragments suggested that the modifications that create charge heterogeneity are present in the approximately 27-kilodalton carboxy-terminal peptide (meroreceptor) encompassing the steroid-binding domain (121). By high resolution ion exchange chromatography of glucocorticoid receptors, at least five peaks could be resolved within the low salt peak (usually ascribed to activated receptors) obtained by DEAE-Sepharose chromatography (122). Treatment of the receptors with heat or alkaline phosphatase promoted increased DNA-cellulose binding and a change in the elution profile toward more basic forms (123). Microheterogeneity was also

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determined by isoelectric focusing under nondenaturing conditions, and a shift toward more basic forms was observed after activation (124). In these cases, both posttranslational modifications and interaction with other proteins might account for the heterogeneity and the changes after activation. We have found that glucocorticoid receptor isoforms, labeled in intact cells with 32P; and separated by SDSPAGE (125) or by pH-gradient electrophoresis, show step wise progression in 32P content (unpublished results). This observation provides direct evidence that differences in phosphorylation underlie receptor heterogeneity. Isoforms of estrogen receptor have been detected by SDS-PAGE and by chromatographic methods. Receptors from mouse uteri displayed a doublet by SDS-PAGE, with the higher molecular weight form being induced by estrogen agonists, suggesting hormone-dependent processing of the receptor protein (126). By ion exchange chromatography, isoelectric focusing, and HPLC, polymorphism was observed in rat and human receptors, with different patterns during development (reviewed in Ref. 127). These different forms of the receptor were proposed to result from association with other macromolecules or from varying levels of phosphorylation. F. Effects of cell transformation Oncogenic transformation can modify responses of cells to steroid hormones. Introduction of the v-mos oncogene into NIH-3T3 mouse fibroblasts repressed glucocorticoid-induced transcription of transfected genes. Repression was mediated by the oncogene product. That product is a cytoplasmic serine/threonine kinase, raising the possibility that transformation may have affected the phosphorylation state of the glucocorticoid receptor (128). In a rat cell line that is temperature sensitive for expression of the p85gagmos oncoprotein, glucocorticoid induction of metallothionein-1 messenger RNA in the transformed cells (cells in which p85gagmos was expressed) was transient compared to induction in the untransformed cells (129). This 'desensitization' of transformed cells to continued stimulation by glucocorticoids was associated with inefficient nuclear retention of the glucocorticoid receptors. Desensitized receptors accumulated in the cytoplasm and could not be reutilized and recycled to the nucleus. It was suggested that these were normal intermediate receptor forms that were blocked in their progression through a receptor cycle, possibly as a result of altered phosphorylation (130). In both untransformed and transformed cells, the antagonist RU486 efficiently translocated glucocorticoid receptors to the nucleus, but the receptors remained nuclear even when


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RU486 was removed. They returned to the cytoplasm only after treatment with a glucocorticoid agonist, which appeared necessary for proper processing and recycling of the receptors (131).

IV. Direct Evidence for Steroid Hormone Receptor Phosphorylation in Vivo and Relation to Receptor Functions A. Glucocorticoid receptors 1. Basal and ligand-dependent phosphorylation. The first evidence for glucocorticoid receptor phosphorylation came from experiments showing that the receptor protein could be labeled in vivo with 32P;. Housley and Pratt (132) affinity-purified receptors from mouse L fibroblasts and identified two receptor-associated phosphoproteins. Receptors also appeared to be phosphorylated in intact rats, since 32P comigrated with the 3H-labeled receptor ligand after injection of 32 P ; and purification of the receptors (133, 134). After Hsp90 was identified as an intrinsic non-hormone-binding component of the receptor complex, both the approximately 100-kilodalton steroid-binding protein and the Hsp90 with which it was complexed were shown to be phosphorylated (135-137). It is likely that in some of the earlier studies Hsp90 comigrated with the receptor. The stoichiometry of receptor phosphorylation has been determined by comparing the incorporation of 32P (after long-term steady state labeling) with the amount of covalent bound [3H]dexamethasone 21-mesylate. The specific activity of the incorporated phosphate was obtained by measuring the specific activity of the 7-phosphate in ATP. An average number of 2.6 phosphates per ligand binding site was obtained (138), indicating that there are two to three fully phosphorylated sites or a larger number of partly phosphorylated sites. Although an early report indicated that receptor phosphorylation was unchanged after addition of hormone to L cells (139), subsequent experiments demonstrated that the receptor phosphate content increased after addition of glucocorticoids to WEHI-7 thymoma cells (140), NIH 3T3 fibroblasts (141, 142), and FTO 2B hepatoma cells (143). This so-called hyperphosphorylation was induced by glucocorticoid agonists, but not by the antagonist RU486 (140, 141), suggesting a functional role for phosphorylation. The magnitude of the increase in phosphate ranged from about 70% (140) to 3- to 4-fold (141, 142), and was mostly due to phosphorylation of the N-terminal domain (143). Analysis of nuclear receptors after hormone treatment indicated that whereas the salt-extractable fraction was phosphorylated to the same extent as the cytosolic receptors (138-140), the small fraction of receptors bound

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to the nuclear matrix, which is resistant to extraction with salt and nucleases, had only about 60% of the phosphate of the cytosolic receptors (140). From these results it appears that hyperphosphorylated receptors become partly dephosphorylated in the unextractable nuclear form. It cannot be excluded, however, that the unextractable fraction consists of receptors that have failed to become hyperphosphorylated. 2. Location of phosphorylated sites. Phosphoamino acid analysis revealed that both the steroid-binding protein and Hsp90 from a variety of cells are phosphorylated on serine residues (132, 144-146). Results of Hoeck and Groner (143) with rat hepatoma cells and our own observations with mouse thymoma cells and Chinese hamster ovary (CHO) cells with overexpressed receptors (147) indicate that minor phosphorylation also occurs on threonine. Small amounts of phosphotyrosine have been reported to be present in the receptor from human breast epithelial cells (148). It has also been claimed that the receptor can be immunoprecipitated with antiphosphotyrosine antibodies (94). By limited proteolysis of the receptor, phosphorylated sites have been mapped to the functional domains. Most appeared to be in the N-terminal domain (143,145,146). A minor fraction of the 32P label was associated with chymotryptic fragments containing the DNA and steroid-binding domains. This label was localized to the steroid-binding domain in receptors from WEHI-7 cells (146), and to the DNA-binding domain (isolated as a ~ 16-kDa tryptic fragment with the BuGR antiglucocorticoid receptor antibodies) in receptors from L fibroblasts (145) and hepatoma cells (143). In a separate study, the radioactivity associated with the DNA-binding fragment was ascribed to an impurity of similar molecular mass (149). Recently we have found that a highly purified tryptic fragment containing the steroid-binding domain is not phosphorylated (147), indicating that the earlier result from our laboratory on labeling of this domain (146) may have been due to a contaminant. By phosphopeptide mapping and sequencing of tryptic peptides from 32P-labeled receptor, we have identified seven phosphoryated sites in the mouse glucocorticoid receptor (147). These sites appear to be nearly identical, with respect both to location and relative phosphorylation levels, in CHO cells in which the receptor has been overexpressed (150) and in WEHI-7 mouse thymoma cells where the endogenous receptor is normally expressed (147). As shown in Fig. 2, all of these sites are in the N-terminal domain, and all are on serines except for the one at threonine 159. Serines 122, 150, 212, 220, and 234, and the sequences surrounding them, are conserved in the homologous regions of the rat and human receptor, but threonine 159 and serine 315 have no homologs


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400

200

600 HORMONE

DNA 106

p

122

0-J

pp

PP

150 159

p

320

212 220

234

315

1

1

I

1

0

50

100

150

1

200

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Fraction Number

FlG. 2. Relationship of phosphorylated sites in the glucocorticoid receptor to the tryptic phosphopeptide map. At the top is a diagram of the mouse glucocorticoid receptor, with numbered residues. The shaded region, which is expanded below, contains all seven phosphorylated sites that have been identified so far. Positions of individual phosphopeptides are shown by the solid black bars. The letters S, T, and P designate serine, threonine, and phosphate, respectively. At the bottom of the figure is an HPLC phosphopeptide map of phosphorylated receptors from hormone-treated WCL2 cells [CHO cells in which the glucocorticoid receptor is overexpressed (150)], with peaks and corresponding phosphopeptides indicated by arrows. [Adapted with permission from Ref. 147.]

in the human receptor. All but serine 315 are within transactivation domains identified in human or rat receptors (34, 35). Serines 212, 220, and 234 are in a highly acidic region that in the mouse receptor reduces nonspecific DNA binding and is required for full transcriptional activity (33). These seven sites account for about 80% of the receptor-associated 32 P. Therefore most of the major sites have been identified, but other sites may still be uncovered (147). All the phosphorylated sites except serines 150 and 315 are in known kinase consensus sequences (147). Serine 122 is in a consensus sequence for casein kinase II (XXX-Ser-XXX-XXX-Glu) (151, 152). Serines 212, 220, and 234 and threonine 159 are in consensus sequences (XXX-Ser/Thr-Pro-XXX) for proline directed

protein kinase or PDPK (p34cdc2-p58cyclinA) (153, 154), sequences that have been found to occur with high frequency in gene-regulatory proteins (155). Serines 212 and 220 also fit the very similar sequence for the p34cdc2p62cyciinB k i n a s e ( 152> ^Q)} which is important in regulation of the cell cycle (157). The predominance of PDPK sites is also a feature of the phosphorylated sites in the progesterone receptor, which will be described below. 3. Phosphorylation and receptor activation. As noted in Section III.B, the effects of phosphatases in cell-free systems led to the proposal that activation of glucocorticoid receptors involves dephosphorylation (53-55, 158, 159). This idea has been tested by directly comparing the phosphate contents of nonactivated and activated hor-


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mone-receptor complexes labeled in intact cells, normalizing 32P-incorporation with incorporation of [35S]methionine incorporated in the receptor protein (138). No decrease in phosphorylation was seen after activation either in intact cells or cell-free systems (138, 139). Dephosphorylation in vitro of the receptor has not been found to affect the ability to bind to DNA (145). Furthermore, analysis of the Hsp90 component of the nonactivated receptor indicated that this protein has the same amount of phosphate whether it is free in the cells or complexed with the receptor, and that no change in phosphorylation occurred during its dissociation upon activation in a cell-free system (125). Our kinetic studies (Section V) show that hyperphosphorylation probably does not begin until after activation. It appears, therefore, that during activation of the receptor there is no change in its level of phosphorylation. B. Progesterone receptors 1. Basal and ligand-dependent phosphorylation. The nonactivated receptor from chick oviduct tissue minces incubated with 32Pi has been resolved by ion exchange chromatography into two components, one form consisting of the approximately 80-kDa receptor A and Hsp90, the other form consisting of the approximately 110-kDa receptor B (which includes the sequence of receptor A (3)) and Hsp90 (160, 161). Phosphoamino acid analysis of the receptor from chick oviduct and T47D human breast cancer cells has revealed only phosphoserine (160, 162, 163). Addition of progestins to intact cells generates hyperphosphorylated forms of A and B receptors with retarded mobility on SDS-PAGE (114, 162-167). Both cytosolic and nuclear-bound receptors are hyperphosphorylated. In one study, which has not been confirmed, it was found that the nuclear-bound receptors are not phosphorylated (168). Hormone-induced hyperphosphorylation is evident after 5 min, and therefore is an early event in receptor action (114). The antiprogestin and antiglucocorticoid RU486 promotes receptor activation, nuclear binding, and hyperphosphorylation, but does not induce agonist-dependent processing and down-regulation of the receptor protein (163). These results indicate that phosphorylation by itself does not cause receptor processing. Increased phosphorylation and number of steroid-binding sites of progesterone receptors was observed after treatment of human breast cancer cells with a protein kinase C activator (169). Phosphorylation of the rabbit progesterone receptor has been studied in COS-7 cells transfected with wild type and mutant receptors. The wild type, as well as mutants lacking DNA-binding activity, exhibited hormone-dependent hyperphosphorylation, showing that

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hyperphosphorylation did not depend on binding to DNA. It did, however, depend on nuclear localization, as demonstrated with mutants lacking nuclear localization signals. A constitutively active mutant without the steroid binding domain showed only a low level of phosphorylation, whereas inactive complexes of wild type receptor with the antagonist RU486 became hyperphosphorylated. From both these results, it was concluded that hyperphosphorylation was not required for biological activity. Hyperphosphorylation was also shown to be unnecessary for receptor down-regulation (170). 2. Location of phosphorylated sites. Two-dimensional gel analysis of tryptic phosphopeptides from affinity-purified chick oviduct receptors after in vivo 32P-labeling yielded five identically migrating major phosphopeptides common to the A and B forms and one unique to the B form (171). Reverse phase HPLC of phosphopeptides from immunopurified chick oviduct (167) and human (172) A and B receptors yielded about six phosphopeptides. By analysis of cyanogen bromide cleavage fragments, all phosphorylated sites were mapped to the Nterminal half of the chick oviduct receptor (167) and within the N terminus and 28 residues of the DNAbinding domain in the human receptor (172). Whereas with the chick oviduct receptor hormone treatment caused a general increase in phosphorylation of all phosphopeptides (167), with the human receptor several different phosphopeptides appeared (172). In rabbit receptors transfected into COS-7 cells, phosphorylated sites were shown both by chemical cleavage and analysis of mutants to be confined to the N-terminal domain, with or without hormone treatment (170). By phosphopeptide mapping and sequencing, three hormonally regulated phosphoserines have been identified in both A and B forms of the chick oviduct receptor after short-term 32P labeling (83). Two of these, at positions 211 and 260, are in the N-terminal domain. The third, at position 530, is in the hinge region between the DNA-binding and steroid-binding domains, a region known to contain one of two transactivation domains in the receptor. The first two of these sites are about 20% phosphorylated under basal conditions, a level that is increased 1.5- to 2-fold by progesterone treatment. The site in the hinge region shows marked hormone dependence, increasing from below 2% phosphorylation in the basal state to more than 33% after hormone treatment. All three sites are in consensus sequences for PDPK. Such a consensus sequence is also found in the hinge region of many other members of the steroid receptor family (83). 3. Phosphorylation and receptor-DNA binding. An effect of hormone-induced hyperphosphorylation on in vitro binding of the receptor to DNA has been proposed (173).


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Progesterone receptors hyperphosphorylated in vivo by progesterone treatment of chick oviduct slices have greater affinity for calf thymus DNA, as determined by analysis of elution from DNA-cellulose, than receptors bound to the hormone in vitro under conditions in which their phosphorylation is not increased (173). By analysis of nitrocellulose filter binding and DNase I footprinting, no differences in the interaction of the rabbit uterine progesterone receptor with a PRE of the uteroglobin gene were found for receptors that had been activated (and presumably hyperphosphorylated) by hormone treatment in vivo, or by in vitro treatments (174). C. Estrogen receptors Studies on phosphorylation of estrogen receptors are in a somewhat unsettled state, with the question of the nature of the predominant phosphoamino acid present in urgent need of resolution. A striking series of observations by Auricchio and colleagues have indicated that calf, rat, and human receptors are phosphorylated on tyrosine residues (reviewed in Refs. 1 and 94). Experiments by this group with cell-free systems, which were described in Section III.D, suggested that a nuclear phosphatase inactivates the steroid-binding capacity of estrogen receptors, and that a cytosolic kinase restores this activity by phosphorylating tyrosines. Phosphorylation in vivo by incubation of rat uteri with 32P; yielded receptors labeled exclusively on phosphotyrosine (93). Furthermore, calf uterine receptors reacted with antiphosphotyrosine antibodies, a reaction that was abolished by treatment with nuclear phosphatase (93). Contrasting sharply with these observations, it has been found that mouse uterine receptors labeled in vivo are phosphorylated only on serines (175). In addition, according to a preliminary report phosphorylation of estrogen receptors in calf uterine cells and in the MCF7 human breast cancer cell line also occurs exclusively on serines (176). Phosphorylation of estrogen receptors in a Leydig cell line has been reported (177), but phosphoamino acids were not analyzed. Estrogen treatment of the mouse uterus causes hyperphosphorylation of nuclear receptors, giving rise to a receptor form that migrates more slowly on SDS-PAGE than the cytosolic form (175). With MCF-7 cells, however, estrogen treatment apparently gives nuclear receptors that are dephosphorylated (176). D. Androgen receptors Androgen receptors in intact human lymph node carcinoma of the prostate (LNCaP) cells and in transiently transfected COS-7 cells can be labeled with 32P (178181). In LNCaP cells, phosphorylation occurred in either the presence or absence of androgen (178). In COS-7

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cells the receptor is apparently less phosphorylated in the presence of hormone (179). Preliminary reports indicate that truncated receptors encompassing either the steroid- and DNA-binding domains (179) or the N-terminal domain (182), become phosphorylated. Results with various point mutations and deletions suggest phosphorylated sites in the hormone binding domain and within the hinge region (183). In particular, mutation of serine 735 to alanine in the hormone binding domain eliminated high affinity androgen binding and diminished phosphorylation. Phosphoamino acids were not directly analyzed in these studies. Nuclear, but not cytosolic activated androgen receptors from rat prostate were reported to react with a polyclonal antiphosphotyrosine antibody, raising the possibility that these receptors or some associated protein contain phosphotyrosines (52, 184). E. Vitamin D receptors The effects of vitamin D are mediated by the receptor that binds the active metabolite 1,25-dihydroxyvitamin D3. Hormone-dependent hyperphosphorylation was observed in mouse fibroblasts, where the receptor protein had reduced mobility on SDS-PAGE gels after addition of 1,25-dihydroxyvitamin D3, and the retarded form of the receptor was the only one labeled with 32P; (115). Similarly, hormone-dependent hyperphosphorylation was seen with embryonic chick duodenal organ culture (116). In this system the two receptor forms resolved by SDS-PAGE had increased 32P-label after exposure to 1,25-dihydroxyvitamin D3. Basal phosphorylation in the absence of added hormone was also observed. Hyperphosphorylation was detected as early as 15 min after addition of hormone, preceding the effects on calcium metabolism. From the phosphorylation in presence of hormone of transfected constructs containing the human receptor and various deletion mutants, it has been inferred that the major site of phosphorylation lies within a 37-residue sequence in the hormone binding domain (109). F. Thyroid and other members of the steroid hormone receptor family The thyroid hormone receptor is encoded by the protooncogene c-erbA. By peptide mapping and mutational analysis of the receptor labeled in intact transformed erythroblasts, two major potential phosphorylated sites have been identified (110, 111). Phosphorylation occurred on serine residues and was stimulated by activators of protein kinases A and C. One of the sites, which has a consensus sequence for c AMP-dependent protein kinase phosphorylation, is in the N-terminal region of the receptor near the DNA-binding domain. This site is


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phosphorylated in vitro by purified protein kinase A and is also phosphorylated in the protein encoded by the oncogene v-erbA, which lacks hormone-binding capacity. Phosphorylation may be required for full transforming activity of the v-erb protein. The other potential site is at serine 12. Serine 12 lies in a consensus sequence for casein kinase II, which phosphorylates the protein in vitro at that site. Hormone dependence of phosphorylation was not analyzed. Mineralocorticoid receptors overexpressed in Sf9 insect cells are reported to be phosphorylated. Phosphoaminoacids and hormone dependence were not studied (185). The NGFI-B protein is a short-lived phosphorylated member of the steroid receptor family. It is rapidly induced by nerve growth factor (NGF) and other stimuli and may function as a nuclear messenger (186). In rat PC12 pheochromocytoma cells, NGF stimulates hyperphosphorylation of NGFI-B. As determined by cell fractionation, NGFI-B is associated with the nuclear fraction in the absence of NGF. Treatment with NGF causes partial redistribution to the cytosol. The hyperphosphorylated form predominates in the cytosol (186). G. Problems associated with analysis of receptor phosphorylation

The survey in the previous sections on receptor phosphorylation discloses numerous minor and some major apparent inconsistencies among results. Here we briefly mention a number of technical problems that can give rise to such discrepancies, possibly including some of those above. A question often not addressed is whether labeling in. vivo with 32P has reached a steady state, an essential requirement for quantitative comparison of phosphorylation levels at different sites. For comparison of receptors from cells exposed to different treatments, an accurate measure of protein is also necessary. Results obtained with short term 32P labeling can be useful, but give prominence to sites with rapidly turning over phosphates. They may also reflect transient changes in specific activities of cellular ATP, a problem that is compounded in cells treated with hormones or other agents that affect ATP metabolism. It is important to minimize hormonal activity derived from serum or phenol red in incubation media, since such activity can be expected to alter receptor phosphorylation. Because concentrations of receptors in cells are very low, even when receptors are overexpressed, massive amounts of 32P and cells must be used to obtain sufficient labeled receptor for analysis. The ubiquity in cells of other phosphoproteins that become labeled places exceptional demands on the purity of receptor preparations.

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Beyond gross impurities, contaminants can be entrained if the antireceptor antibodies used for purification crossreact with other proteins. Receptor-associated proteins are another source of contamination. Antiphosphotyrosine antibodies may recognize a receptor-associated protein rather than the receptor itself. During purification, endogenous phosphatase activity must be rigorously controlled to avoid losing labeled phosphates from receptors. Endogenous kinases may be copurified as contaminants with receptors and phosphorylate them in in vitro phosphorylating systems. Contaminating kinases may also be present in antibody preparations. When receptors are phosphorylated in vitro with added kinases or with endogenous kinases in broken cell preparations, the sites that are phosphorylated may be different from those phosphorylated in vivo. Treatment of cells with activators or inhibitors of protein kinases and phosphatases may cause changes in phosphorylation of receptors in ways unrelated to normal receptor function. Failure of a mutant to become phosphorylated is an indication that the normal receptor is phosphorylated in the mutated region but can also be due to disruption of phosphorylation at a distant site. Partial digests of receptors can be useful for assigning major phosphorylated sites to regions of the receptor, but the results may be highly dependent on subtle variations in procedure. Obtaining a reproducible limit digest for phosphopeptide mapping is difficult and can be complicated by the presence of protected cleavage sites in the receptor. Extreme care is required in the handling of a digest to avoid large losses and to obtain uniform recovery of peptides. A high resolution system is necessary to separate cleanly the numerous peptides obtained, especially if the phosphopeptides are to be sequenced. Finally, the sensitivity of assays used for measuring 32 P can affect conclusions on whether a receptor, a peptide, or an amino acid is phosphorylated. A long autoradiographic exposure, for example, may reveal radioactivity where a shorter exposure leaves no trace. V. A Cyclic Model for Glucocorticoid Receptor Phosphorylation Early metabolic studies led to the hypothesis that glucocorticoid receptors (and possibly other steroid hormone receptors) traverse an irreversible hormone- and ATP-dependent cycle involving phosphorylation and dephosphorylation (Section III.A). Although conclusive evidence for this hypothesis is still lacking, it has been supported by numerous subsequent observations. Thus, receptors are indeed phosphorylated {Section IV), their levels of phosphorylation in cells are in many cases rapidly increased after hormone binding and may be decreased in certain nuclear-bound forms (Section IV),


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and their biological activities appear to be regulated by both kinases and phosphatases (Section HI). Support for an energy-dependent cycle also comes from other sources. The apparent thermodynamic irreversibility of the activation step (Section II.A), coupled with evidence that glucocorticoid receptors are recycled and reutilized (42, 130, 131, 159, 187, 188), implies that a continuous supply of energy is required to maintain levels of active receptors in cells. An irreversible activation step, moreover, is a key element in a simple cyclic mathematical model that reproduces accurately the complex kinetics of glucocorticoid-induced receptor transformations in thymus cells (188). A prediction of the hypothesis that has been partly borne out is that in ATP-depleted cells the receptor accumulates in a non-hormone-binding form, which in normal cells is present as a short-lived intermediate product of nuclear receptors that is regenerated to hormone-binding form via an ATP-dependent reaction (42). Such a non-hormone-binding form has been identified in ATP-depleted WEHI-7 cells, and accounts quantitatively for the disappearance of hormone-binding receptors (47, 48, 189). We have termed it the 'null' receptor. It is bound to the nuclei, is formed in the presence or absence of hormone, is unextractable even with high salt concentrations, and appears to be partly dephosphorylated. These characteristics are shared by the salt-unextractable nuclear receptors that constitute about 5% of the receptors in normal hormone-treated WEHI-7 cells (140, 189). Whether unextractable receptors in normal and ATP-depleted cells are similar in other respects remains to be determined. The null receptor can be restored rapidly to hormone-binding form by raising cellular ATP levels. No protein synthesis is required. Loss of hormone binding capacity may be due to dephosphorylation, but could also be accounted for by the apparent requirement for ATP to restore the receptor to hormone binding state by reconstitution with Hsp90 (17, 18). It is known that after activated glucocorticoid-receptor complexes dissociate, the resulting receptor cannot bind hormone again until reassociated with Hsp90 (190). Formation of null receptors even in the absence of hormone has suggested to us that receptors continuously traverse an ATP-dependent cycle between soluble and nuclear-bound forms, whether or not hormone is present (48,189). Our current model of the hormone-dependent glucocorticoid receptor phosphorylation cycle is shown in Fig. 3. It embodies many of these observations along with some new experimental results and is an updated version of a previous model (189). Initially the hormone, H, binds reversibly to an unliganded receptor R (shown at lower left) to form the nonactivated complex HR, which becomes activated to HR' with dissociation of Hsp90 and

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other components of the nonactivated oligomer. HR' rapidly binds to the nucleus and establishes a quasiequilibrium with its nuclear-bound form HR'n, the saltextractable nuclear complex (Section II.A). Hormoneinduced hyperphosphorylation appears to begin at this stage. As noted in Section IV.A.3, no change in receptor phosphorylation accompanies activation. In recent kinetic experiments we have found that after addition of hormone to cells, activated receptors (both HR' and HR'n) are formed without being hyperphosphorylated. They become hyperphosphorylated shortly after being formed, and well before hyperphosphorylation can be detected in the nonactivated receptors HR (our unpublished results). The phosphorylation step indicating incorporation of P; into the receptor is shown between HR' and HR'n, to indicate uncertainty about which of these two activated forms is substrate for hyperphosphorylation. Continuing around the outer cycle, HR'n is assumed to be the precursor of the salt-unextractable complex HR"n. For simplicity, the nuclear forms of the receptor are shown in Fig. 3 as monomers, although it is known that receptors bind to GREs as dimers. On dissociation of H, HR"n gives the unextractable receptor R"n. To account for the relatively dephosphorylated state of unextractable receptors, dephosphorylation is shown as occurring at one or more of these steps (140). R"n is the hypothetical counterpart in normal cells of the null receptor formed in ATP-depleted cells. By analogy with the null receptor, we assume that R"n can be reconstituted to the soluble unliganded receptor, R, through ATP-dependent reactions involving reassociation with Hsp90 and other subunits as well as rephosphorylation. These reactions, which complete the cycle, may resemble those undergone by newly synthesized receptors. Two difficulties, both based on kinetic observations, have led us to conclude that the phosphorylation cycle described so far, in which all hormone-receptor complexes entering the cycle exit through HR"n and R"n, is incomplete (189). One difficulty arises from the cyclic mathematical model mentioned above. An important assumption in that model, which agrees well with experimental data, is that hormone dissociates from all hormone-receptor complexes at a rate equal to that for HR, after which the receptors are recycled to R (188). If all complexes are funneled through HR"n, which comprises only about 5% of total receptors, then HR"n would have to dissociate much faster. The second difficulty is that whereas hormone-induced hyperphosphorylation occurs rapidly, with a half-time of about 15 min, dephosphorylation after a cold chase with or without hormone has a half-time of more than 60 min (our unpublished results). Thus it appears that only a fraction of hyperphosphory-


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STEROID HORMONE RECEPTOR PHOSPHORYLATION OOOOC

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FIG. 3. Model of glucocorticoid receptor phosphorylation-dephosphorylation cycle. R, Unliganded receptor; H, hormone; HR, cytosolic nonactivated hormone-receptor complex; HR', cytosolic activated hormone-receptor complex; HR'n, salt-extractable nuclear-bound activated hormone-receptor complex; HR"n, unextractable nuclear-bound activated hormone-receptor complex; R"n, unextractable nuclear-bound unliganded receptor; Pi, inorganic phosphate; Hsp90, 90 kDa heat shock protein. Other receptor-associated heat shock proteins (Hsp70, Hsp56) are presumed to accompany Hsp90. Question marks indicate possible requirements for ATP and other ingredients to reconstitute the unliganded receptor R. See text for explanation.

lated receptors undergoes dephosphorylation before recycling. Both these difficulties are overcome by assuming that, as in the mathematical model, HR' and HR'n dissociate at rates similar to HR and are then immediately recycled. Some, of course, will be converted to HR"n before dissociating. Most hyperphosphorylated receptors would therefore be recycled without traversing the unextractable fraction and undergoing partial dephosphorylation. This alternative is shown in Fig. 3 by the vertical path leading directly from HR' and HR'n to R. ATP would again be required for reassociation with Hsp90 and other subunits to restore the receptor to hormone binding form. Supporting the idea that most hyperphosphorylated receptor complexes recycle without dephosphorylation, we find that after exposure of cells for some time to subsaturating concentrations of hormone, even the unliganded receptor R becomes hyperphosphorylated (our unpublished results). This form of R, which in the model is within the cycle, is presumably converted by dephosphorylation back to the original R at lower left in Fig. 3. The model is consistent with all our results, but involves some unverified assumptions. We have no experimental evidence that HR'n is the precursor for HR"n. This relationship, however, is in accord with the general view that among nuclear-bound hormone-receptor complexes only a few reach specific, high affinity sites on the genome (HREs) where they exert biological activity. The rest, which are considered to be nonspecifically bound to chromatin, can be thought of as 'searching' for those sites (191); when the hormone dissociates they are recycled. Our main reason for assuming that HR"n and R"n are recycled is that they resemble null receptors, which can certainly be recycled. However, the small number of receptors in this fraction would probably have undetect-

HR'n

R"n

able influence on the measurements of overall receptor kinetics and turnover rates which underlie the conclusion that most receptors are reutilized, leaving open the possibility that they are not recycled but degraded. Finally, although the cycle implies that HR"n and R"n are bound in some functional relation to hormonally regulated genes, we do not know where in the nucleus the unextractable receptors are located. The same reservation applies to null receptors. In fact, we cannot exclude the possibility that these receptors are trapped in nuclear pores, transport through which requires ATP (192). Though the cyclic model is designed mainly to account for our own data with glucocorticoid receptors in lymphoid cells, it is broadly consistent with results from other laboratories. In particular, it bears close relationship to observations (Section III) that have been made with glucocorticoid receptor recycling in other systems (58, 130,131,193), with androgen receptors (45, 46) and with estrogen receptors (1, 107).

VI. Regulation by Phosphorylation of Other Transcription Factors It is becoming evident that phosphorylation may modulate the activity not only of steroid hormone receptors but also of many other transcriptional regulators, in systems including mammalian cells, yeast, and bacteria. The effects that have been observed with these other factors and the mechanisms that have been suggested, may well furnish examples for understanding how phosphorylation can modulate activities of steroid receptors. Phosphorylation alters functions of the factor that mediates cAMP induction of eukaryotic genes such as the somatostatin gene. This cAMP response element binding protein (CREB), as it is called, is phosphorylated


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in vivo. Its increased phosphorylation by protein kinase A in response to cAMP seems a requirement for transcriptional induction (194) and affects multiple functions. Protein kinase A phosphorylates a site which, possibly through allosteric modifications, confers transactivating functions to a separate domain of the protein (195). In addition, CREB has regions with consensus sequences for protein kinase C and casein kinase II (196). In an in vitro system, induction of transcription has been shown to be mediated by dimerization of the protein. This dimerization is stimulated by protein kinase C but not by protein kinase A, which stimulates transcription without affecting dimer formation (197). Three sets of phosphoproteins were found to bind to the cAMP response element. Their DNA-binding activity was increased by phosphorylation by protein kinase A, which for one of these proteins also regulated its site-specificity (198). CREB also mediates gene induction by calcium in response to membrane depolarization. It is a substrate for depolarization-activated Ca2+-calmodulin-dependent protein kinases (CaM kinases) I and II. Mutation to alanine of the serine that is the major site of phosphorylation after depolarization decreases transcriptional activity in response to membrane depolarization or cAMP by about 80%. CaM kinases and protein kinase A apparently phosphorylate the same serine in vitro (199). Other mammalian transcription factors that so far have been found to be regulated by phosphorylation include serum response factor (SRF), myc intron factor (MIF), Octl, Oct2, IKB (an inhibitor of the /cB-binding nuclear factor NF-KB), cJun, cFos, IP-1 (an inhibitor of API), and Pit-1. SRF binds to the serum response element which mediates serum and growth factor regulation of cFos gene transcription. It is phosphorylated on serine residues in vivo. Dephosphorylation abolishes its DNAbinding activity in vitro (200). MIF binds to a regulatory region in intron I of the c-myc gene, and its DNA-binding ability is abolished by phosphatase treatment (201). Octl, a member of the POU homeodomain family, appears to be required for transcription of several genes including those for histone H2b. Analysis of Octl through the cell cycle has shown that it undergoes a complex temporal program of phosphorylation and dephosphorylation, suggesting the involvement of multiple kinases and phosphatases in its regulation. Its activity is apparently decreased at mitosis by hyperphosphorylation, which inhibits its binding to DNA from the promoter region of the H2b gene, and increased on entry into Gl by removal of most phosphates (202). Oct2 is a POU homeodomain protein that shows a specific phosphorylation pattern (determined by upshifts in 32P-labeled bands in SDS-PAGE gels) that correlates with enhanced transcriptional activation (203). In contrast to

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SRF and MIF, where phosphorylation directly affects DNA-binding, for Oct2 it is proposed that the effects of phosphorylation are due to creation of an acidic region, or to an allosteric change resulting in a functional activation region (203). NF-/cB, which regulates the immunoglobulin K light chain gene and participates in the induction of interleukin 2 and its receptor, is found as a cytosolic inactive complex bound to an inhibitor protein, LcB. Phosphorylation in vitro inactivates IKB, which dissociates from the complex. The active NF-KB can then migrate to the nucleus and bind to the KB site (204, 205). The phosphoproteins cJun and cFos are components of the transcription complex API. They form stable heterodimers, and cJun (but not cFos) forms homodimers. These dimers mediate effects on transcription of many genes by protein kinase C through a phorbol response element to which they bind. cJun and cFos have been found to be associated with glucocorticoid receptors in activating and repressing expression of certain genes (22-25, 28). Activation of protein kinase C rapidly dephosphorylates cJun at sites adjacent to its DNA-binding domain and increases its DNA binding activity, changes that may contribute to API-mediated gene activation. Mutations of cJun that block phosphorylation increase its transactivating activity. Whether dephosphorylation increases DNA binding by increasing dimer formation or by directly affecting the DNA-binding domain is not known (206). Ha-Ras increases cJun-mediated transactivation by increasing the transactivating potency of the N-terminal activation domain; at the same time it stimulates hyperphosphorylation of that domain (207), apparently via mitogen-activated protein-serine kinases pp54 and pp42/44 (208). Thus, full activity of cJun may require hyperphosphorylation at some sites and dephosphorylation at others. With cFos, mutation of serines to alanines near its Cterminus decreases phosphorylation at the C-terminus and abolishes the ability to repress transcription of the c-fos promoter. The mutant, however, fully retains the ability to activate genes containing a phorbol response element. A mutant in which the serines were replaced by aspartic and glutamic acid did not lose repressor activity, suggesting that the function of the phosphorylations was to provide negative charges at the C-terminus (209). IP-1 is an inhibitor of API activity that is found in both nucleus and cytoplasm. It associates with cFos/ cJun dimers and specifically blocks DNA binding of API and cFos/cJun dimers. It is inactivated by phosphorylation and reactivated by dephosphorylation (210). IP-1 resembles IKB in that it represses a transcriptional activator and is inactivated by phosphorylation. Pit-1 is a POU homeodomain pituitary-specific transcription factor that has been implicated in activation of


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GH and PRL genes by phorbol esters, cAMP, and epidermal growth factor. In response to phorbol esters and cAMP, its phosphorylation increases at several sites, which are also phosphorylated in vitro by protein kinase C and A. Two of these sites, which are adjacent to each other, interact so that mutation of threonine to alanine at one site (threonine 220) blocks phosphorylation at the other. Phosphorylation modifies the conformation it assumes when bound to its DNA recognition elements, decreasing affinity for certain GH and PRL sites, increasing affinity for others, and increasing dimerization on the GH element GH-2 and the PRL 3D site. How phosphorylation affects binding depends on the DNA sequences next to the core Pit-1 binding motif. These responses appear to depend on a single residue (threonine 220), which is located in the POU homeodomain within a sequence conserved throughout the POU-domain family (211). Transcriptional inductions that follow viral infections are also subject to regulation by phosphorylation. For adenovirus these inductions are mediated by the El A protein. ElA-mediated activation of transcription factors IIIC, E2F, and E4F appears to involve phosphorylation of the factors (212-214). The hyperphosphorylated proteins acquire increased transcriptional activity, probably through enhanced DNA-binding activity. Spl is a mammalian transcription factor that activates transcription by interacting with GC boxes (specific GCrich DNA elements) that are present in a variety of cellular and viral promoters, including those of SV40 (Simian virus 40) early genes. SV40 infection induces hyperphosphorylation of Spl. With an in vitro transcription system it was found that for phosphorylation to occur, Spl must be bound to DNA that contains GC boxes. The kinase, a DNA-binding protein, is a dsDNAdependent protein kinase. Phosphorylation does not affect DNA binding of Spl. Nuclear extracts contained not only this kinase activity, but also activity that quantitatively dephosphorylates Spl, suggesting that in vivo Spl oscillates between phosphorylated DNA-bound forms and dephosphorylated free forms (215). In yeast, high temperature increases the activity and phosphorylation (as determined by the phosphatase-sensitive upshift in SDS-PAGE) of the heat shock factor responsible for the induction of heat shock genes. Phosphorylation does not seem to affect DNA-binding activity of the factor, but may create an acidic region required for interaction with other components of the transcriptional machinery (216). Similarly GAL4, which mediates galactose induction of the galactose/melibiose regulon, shows increased activity and phosphorylation upon galactose treatment, changes that are reversed after glucose-induced repression (217). Yeast ADRl protein, an activator of alcohol dehydrog-

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enase II gene expression, is inactivated by phosphorylation. It is phosphorylated in vitro by protein kinase A, and mutations that enhance its activity decrease its phosphorylation. Activation of protein kinase A in vivo inhibits transcription of the alcohol dehydrogenase II gene, suggesting that phosphorylation of ADRl mediates the physiological repression by glucose (218). Transcription in bacteria also appears to be regulated by phosphorylation and dephosphorylation mechanisms that turn the activity of regulatory proteins on or off in response to external signals (219-221). Regulatory effects of phosphorylation on transcription are not limited to transcription factors. They directly influence RNA polymerase. RNA polymerase II (RNAP II) is present in mammalian cells in two forms, IIA and IIO. They differ in the extent of phosphorylation of the C-terminal domain of their largest subunit, which consists of 52 tandem repeats of a serine- and threoninerich heptapeptide sequence. In IIA this domain is unphosphorylated, in IIO it is heavily phosphorylated. RNAP IIO is responsible for elongation, and RNAP IIA may be involved in initiation. Phosphorylation of IIA has been shown to occur after interaction with the promoter to form a preinitiation complex. The kinase that converts RNAP IIA to IIO is an integral component of the promoter-associated complex, from which it has been isolated (222-224). Its relation to p34cdc2 kinase, which can phosphorylate the heptapeptide in vitro (225), is uncertain (223).

VII. Discussion: Significance of Steroid Hormone Receptor Phosphorylation A. General aspects of receptor phosphorylation

From the results we have reviewed on steroid receptor phosphorylation in vivo, some general features begin to emerge. The receptors are basally phosphorylated in unliganded form. They become hyperphosphorylated after association with hormone. Of the phosphorylated sites directly identified so far on the chicken progesterone and mouse glucocorticoid receptors, most lie in the N-terminal domain. One site, on the progesterone receptor, is in the hinge region, and shows striking hormone dependence in short-term labeling experiments. There are reports of phosphorylation of the DNA-binding domain of the glucocorticoid receptor. Indirect evidence indicates that the vitamin D receptor, and possibly the androgen receptor, have major phosphorylated sites in the hormone-binding domain, and that the thyroid hormone receptor is phosphorylated in the N-terminal domain. Cell and species specificity of receptor phosphorylation have not yet been explored in depth. However, the observation that mouse glucocorticoid receptors, whether normally expressed in WEHI-7 mouse thymoma


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cells or overexpressed in CHO cells, yield nearly identical phosphopeptide maps (147) suggests that phosphorylation exhibits little cell specificity. Almost all reported analyses of phosphoamino acids, as well as information from direct identification of phosphorylated sites, point to serine as the main phosphorylated amino acid in receptors, with minor amounts of threonine. The outstanding exception to this generalization is the estrogen receptor. As discussed in Section IV. C, there is controversy as to whether it is phosphorylated predominantly on serines or tyrosines. Phosphorylation on tyrosines would appear to place this receptor in a class by itself in relation to other steroid hormone receptors and transcription factors. Most of the sites that have been identified are only partly phosphorylated. Each of the three serines on the progesterone receptor is phosphorylated on less than half the receptor molecules, even after hormone treatment. The same can be said for almost all the sites on the glucocorticoid receptor. From these considerations, which are in accord with findings on receptor microheterogeneity, it follows that within a cell or group of cells there are several subpopulations of receptors, each with different numbers or patterns of phosphorylated amino acids and possibly with different biological specificities. Therefore, a population of receptors has the potential for simultaneously exerting transcriptional control through several regulatory sites with different specific requirements. Preliminary information on the kinds of kinases that may phosphorylate receptors comes from the phosphorylated sites directly identified so far. Most of the sites on progesterone and glucocorticoid receptors are in consensus sequences for the predominantly cytosolic PDPK, which is composed of p34cdc2 associated with p58cyclinA. Such a kinase has been shown to phosphorylate in vitro a fragment of the glucocorticoid receptor that includes the N-terminal domain (88). The glucocorticoid receptor also has two sites in consensus sequences for the closely related M-phase specific p34cdc2-p62cyclinB kinase. Consensus sequences for these two types of kinases are very similar, and occur with high frequency in transcription factors and other gene regulatory proteins (155). For example, two such sequences lie within each multiply repeated heptapeptide sequence that becomes heavily phosphorylated in RNA polymerase II (Section VI). The central role of the p34cdc2p62cyclinB kinase in regulation of the cell cycle suggests a possible connection to cell cycle-dependence such as has been observed with glucocorticoid receptors (226). In addition to these phosphorylated sites, the glucocorticoid receptor has one casein kinase II site and two sites that correspond to no recognized consensus sequences. Future studies may therefore uncover new kinases. An intriguing question is whether

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phosphorylation or dephosphorylation of receptors occurs within transcription complexes. Some transcription factors are phosphorylated in such complexes by DNAdependent kinases (Section VI). Regarding the kinds of phosphatases that might be involved, little is known for either receptors or other transcription factors. Few results on receptor hyperphosphorylation have been obtained with hormone antagonists. Studies with the glucocorticoid and progesterone antagonist RU486 provide contrasting results (Sections IV.A 1 and IV.B.I). When bound to glucocorticoid receptors in WEHI-7 thymoma and NIH-3T3 fibroblasts, RU486, which in some cells is ineffective in causing glucocorticoid receptor activation and nuclear translocation (227), fails to induce hyperphosphorylation (Section IV.A.l). When bound to progesterone receptors, however, it promotes activation, nuclear binding, and hyperphosphorylation (Section IV.B.I). These results, like those on the kinetics of hyperphosphorylation in relation to activation of glucocorticoid receptors (Section V), as well as observations with mutant progesterone receptors (Section IV.B.I), indicate that activation and perhaps nuclear localization are prerequisites for hyperphosphorylation. A liganddependent conformational change of the receptor protein may also be necessary. Whether receptor translocation or other mechanisms are required to bring the receptor into contact with the kinases, and whether specific kinases must be activated, is not known. Many in vivo and in vitro results have suggested that receptor phosphorylation is necessary for hormone binding. Such regulation would seem most likely for receptors that are phosphorylated in the hormone binding domain, as appears to be the case with the vitamin D and possibly the androgen receptor. With glucocorticoid receptors no phosphorylated site has been identified in the hormone binding domain. Changes that have been observed in hormone binding could be secondary to other perturbations of the receptor. Null receptors (generated by ATP depletion of cells) and phosphatase-treated receptors appear to be partly dephosphorylated and lack hormone binding capacity. Despite this association of loss of binding with dephosphorylation, in both cases loss of binding could be due primarily to dissociation of Hsp90, since glucocorticoid receptors cannot bind hormone unless they are associated with Hsp90 (190). Phosphatase treatment can activate these receptors (Section IV.A.3), and would therefore be expected to cause dissociation of Hsp90. As discussed in Section V, loss of binding in ATP-depleted cells may be accounted for by inability to reconstitute the receptor with Hsp90. How significant a role receptor phosphorylation cycles will be found to play remains to be determined. Since the time phosphorylation-dephosphorylation cycles were first postulated from results of metabolic experiments,


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much evidence has accumulated in their support, and they have been invoked to account for numerous other observations. From the discussion in Section VI, the question of the role of phosphorylation cycles can be seen to apply equally well to transcription factors. Several transcription factors appear to be regulated by kinases and phosphatases, cycling through states of hyperand hypophosphorylation. Nevertheless, for those factors as for receptors, the evidence is still inconclusive. However, if receptors and other transcription factors that become hyperphosphorylated are eventually reutilized, then cyclic phosphorylation and dephosphorylation becomes almost an obligatory requirement. B. Receptor phosphorylation and transcriptional regulation Definitive evidence that phosphorylation regulates transcriptional activity of steroid hormone receptors is still lacking, leaving open the possibility that phosphorylation at some sites is neutral, in the sense of having no influence on receptor function. The temporal association of hormone-induced hyperphosphorylation with DNA binding and induction of transcription, however, is highly suggestive. Furthermore, the studies on phosphorylation of other transcription factors reviewed in Section VI offer ample precedent for such regulation. A general lesson from those studies is that phosphorylation may exert either positive or negative control: activity appears to be enhanced in some cases by kinases through hyperphosphorylation, in other cases by phosphatases through dephosphorylation, and yet in others by both. Several comparisons between transcription factors and receptors have already been made. Some further parallels and differences will now be noted. CREB and numerous other transcription factors become hyperphosphorylated in conjunction with stimulation to transcriptional activity, as do receptors. Phosphorylation by protein kinase A is thought to confer transactivating functions to a domain in CREB separate from the site of phosphorylation; in progesterone and glucocorticoid receptors the phosphorylated sites lie in or close to regions with transactivating function. CREB, like the receptors, appears to stimulate transcription as a dimer. Its dimerization can be induced by phosphorylation, something that has not yet been tested with receptors. IKB and IP-1 bind to and inhibit the activities of their respective factors, NF-KB and API, much as Hsp90 binds to receptors and inhibits their transcriptional activity; but whereas LcB and IP-1 are released from the factors by phosphorylation, Hsp90 is released by binding of hormone to the receptor, and as far as is known, no

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change in its phosphorylation state is required for dissociation (125). Phosphorylation increases binding to DNA of several transcription factors. Evidence of such an increase has been reported for nonspecific binding of chick oviduct progesterone receptors to DNA. No effect of phosphorylation, however, was observed on specific binding of rabbit uterine receptors to PREs (Section IV.B.3). Dephosphorylation of some transcription factors increases their binding to DNA. With glucocorticoid receptors, partial dephosphorylation did not affect nonspecific binding to DNA (145). However, phosphorylation of glucocorticoid receptors occurs in an acidic region which, by mutational analysis, has been implicated in reduction of nonspecific binding to DNA, with an increase in transcriptional activity (33). Unextractable nuclear glucocorticoid receptors are relatively dephosphorylated. They are extremely tightly bound, but it has not been shown that they are bound to DNA or that their tight association with nuclei is due to dephosphorylation. C. Receptor phosphorylation and interactions with other molecules

As they fulfill their cellular functions, steroid hormone receptors become associated transiently with numerous other molecules in specific and nonspecific interactions that might be influenced by basal and hormone-induced phosphorylation. Before activation, receptors bind specifically with one or more heat shock proteins and with the hormone. After activation they interact nonspecifically with chromatin or DNA, and in the transcription complex they become bound specifically to HREs and to other transcription factors. In addition, they bind to each other to form dimers. Along the way they come in contact with kinases and probably phosphatases. Undoubtedly they participate in many other interactions that have not yet been defined, such as, perhaps, with nuclear pore complexes (228), where phosphorylation is known to play a role (229). Multisite phosphorylation (230), which appears to be the rule for steroid hormone receptors both in their basal state and after hormone-induced hyperphosphorylation, can be expected to influence these interactions in several ways. In general terms, the influence of hyperphosphorylation may be to either enhance or diminish certain interactions, which would then be respectively diminished or enhanced by dephosphorylation. Random phosphorylation can increase the negative charge and acidity of a region on a protein, thereby modifying nonspecific interactions with other proteins or DNA. Negatively charged transactivation regions are common in eukaryotic transcription factors; in some cases transactivating activity increases progressively as



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negative charge is increased by point mutations (231, 232). A candidate for such control would be the transactivating region in the N-terminal domain of glucocorticoid receptors, which contains most of the phosphorylated sites on the receptor. It carries a net negative charge even when unphosphorylated. As already mentioned, that region has been proposed to increase transcriptional activity by reducing nonspecific binding of the receptor to DNA. By decreasing such nonspecific interactions in dose-dependent fashion, hormone-induced hyperphosphorylation could shift the distribution of receptors toward phosphorylation states with higher transcriptional activity. The level of random phosphorylation could also determine the conformational state of a protein, regulating specific interactions such as dimer formation, and binding to other transcription factors and to DNA. Specific interactions would be more directly controlled through phosphorylation of particular amino acids in the vicinity of a binding site, acting as on-off switches. A phosphorylation site exhibiting high hormone dependence, such as that in the hinge region of the progesterone receptor, could have such a function. However, even phosphorylated sites with only moderate hormone dependence could participate in specific interactions as on-off switches through a subpopulation of receptors possessing a particular arrangement of sites and biological activity, as suggested earlier. The potential for fine-tuning of transcriptional activity through precise changes in phosphorylation is well illustrated by the Pit-1 transcription factor, in which phosphorylation of a single threonine increases its binding to some targets and decreases them to others (211). Pit-1 also illustrates the phenomenon of hierarchal phosphorylation (230), in which phosphorylation of one site influences phosphorylation at another. Such interactions between sites might take place in glucocorticoid receptors, which have a high concentration of sites in a limited region. D. Looking ahead

The study of steroid hormone receptor phosphorylation has expanded rapidly in recent years, as has that of phosphorylation of other transcription factors. With receptors, the focus initially was on their biochemistry and cellular transformations, but it has now swung toward regulation of transcriptional activity. With other transcription factors the focus all along has been squarely on transcription. The two areas have so much in common that it seems likely they will eventually merge. Advances in both will depend on the accomplishment of several more-or-less well defined steps: 1) Identifying the sites that are phosphorylated under basal and stimulated conditions; 2) Associating the state of phosphorylation of

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the sites individually or collectively with interactions and reactions involved in regulation of particular genes; 3) Identifying and purifying the kinases and phosphatases that regulate the state of each functional phosphorylation site, and correlating their properties in vitro with those in vivo; 4) Defining how the kinases and phosphatases are regulated in vivo by hormones and other agents, and how particular sites on a receptor or transcription factor become exposed to those enzymes. These steps differ greatly in relative difficulty. Some can be accomplished expeditiously; others will require years of work. The molecular and cell biological tools required to undertake them are at hand, however, so one can be optimistic about a successful outcome. VIII. Summary Many observations with intact cells as well as cell-free systems suggest that receptors of the steroid hormone superfamily, along with other transcription factors, are regulated by phosphorylation. All receptors that have been analyzed carefully so far have turned out to be phosphoproteins. They are basally phosphorylated in the absence of ligand, and in many cases become hyperphosphorylated in the presence of hormone or other agonists, and sometimes of antagonists. Several studies indicate that hyperphosphorylation of receptors follows activation, and may require nuclear binding of the receptor. Serines are the predominant phosphorylated residues detected in receptors, with minor amounts of threonine. Tyrosine phosphorylation of the estrogen receptor is a subject of controversy. With various receptors, evidence has been found for phosphorylation in vivo of the N-terminal, hormonebinding, and DNA-binding domains, as well as of the hinge region. All but one of the phosphorylated sites identified in progesterone and glucocorticoid receptors by phosphopeptide mapping and sequencing are in the N-terminal domain; one is in the hinge region. Even after hormone treatment most of those sites are only partly phosphorylated, which means that several subpopulations of receptors, characterized by different states of phosphorylation and potentially different biological activities, must coexist. The majority of identified phosphorylated sites lie in consensus sequences for the PDPK. Many parallels can be discerned between phosphorylation of receptors and of other transcription factors. For example, several transcription factors become hyperphosphorylated on stimulation, and much indirect evidence points to regulation of both receptors and transcription factors by kinases and phosphatases, with cycling between different phosphorylated states. Functions of receptors that are regulated by phosphorylation are


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only beginning to be investigated. With transcription factors a substantial body of information is already available, and functions that appear to be thus regulated include dimerization, interactions with other proteins, binding to DNA, nuclear-cytoplasmic localization, and transcriptional activity. These and other functions may be found to be regulated by phosphorylation of receptors.

Acknowledgment We are grateful to Dr. Reed Detar for computer-assisted graphics design.

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Steroid hormone receptors and in vitro transcription.

Steroid hormone receptors in human adipose tissues.

Steroid hormone receptors and the differentiation of myeloid leukemic cells.

Steroid hormone receptors in the regulation of differentiation. A review.

Antitumor activity of naltrexone and correlation with steroid hormone receptors.

Covalent modification of proteins by ligands of steroid hormone receptors.

Immunohistochemical analysis of steroid hormone receptors in hidradenitis suppurativa.

Demonstration of steroid specific hormone receptors by chromatography.

[Thermolabile characteristics of steroid hormone receptors in breast cancer].

Role of microsomal receptors in steroid hormone action.

Steroid hormone receptors in pelvic muscles and ligaments in women.

pS2 protein and steroid hormone receptors in invasive breast carcinomas.

Steroid hormone receptors as prognostic markers in breast cancer.

Identification of a conserved region required for hormone dependent transcriptional activation by steroid hormone receptors.

Steroid receptors.

Characteristics of steroid hormone receptors in cultured MC3T3-E1 osteoblastic cells and effect of steroid hormones on cell proliferation.

Effects of hormone and cellular modulators of protein phosphorylation on transcriptional activity, DNA binding, and phosphorylation of human progesterone receptors.

Inhibitory role of sex steroid in the regulation of ovarian follicle-stimulating hormone receptors during pregnancy.

Identification of receptors for retinoids as members of the steroid and thyroid hormone receptor family.

Therapy for cancer of the breast. Current status of steroid hormone receptors.

Thyroid - hormone effects on steroid - hormone metabolism.

Expression of steroid hormone receptors in the genital structures of a true hermaphrodite pug dog.

Steroid hormone receptors in the thymus: a site of immunomodulatory action of melatonin.

Characteristics and biological role of steroid hormone receptors in neuroepithelial tumors.