Phosphorylation of steroid receptors

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J. steroid Biochem. Vol. 32, No. 4, pp. 613-622, 1989 Printed in Great Britain. All rights reserved

Copyright 0 1989Pergamon Press plc

GENERAL REVIEW

PHOSPHORYLATION

OF STEROID

RECEPTORS

FERDINANDDAURICCHIO II Cattedra di Patologia Generale, I Facolta’ di Medicina e Chirurgia Universita’ di Napoli, Via S. Andrea delle Dame, 2, 80138 Napoli, Italy (Receiued

INTRODUCI-ION

Several years passed between the pioneer report demonstrating the existence of glucocorticoid receptor and correlation between intracellular ATP levels and hormone binding to the receptor [l] and findings that steroid receptors are phosphorylated in whole cells or tissues or in animals (see Table 1). This long interval was due to the fact that demonstration of phosphorylation of steroid receptors required very extensive purification of these proteins which are present in target tissues in low amounts and are closely associated with other proteins, some of which are also phosphorylated. Whereas there is general agreement that steroid receptors are phosphoproteins, the function of receptor phosphorylation is still under investigation. In theory this modification might regulate different functions of the same receptor like hormone binding, interaction with other proteins and interaction with specific DNA sequences. Phosphorylation could also modulate different functions in different receptors, or not yet known receptor function(s). An aspect which is also disputed is whether steroid receptors, in analogy with peptide growth factor receptors like EGF [2], insulin [3], PDGF [4, 51 and CSF receptors [6] have intrinsic kinase activity. I will briefly review the recent work on steroid receptor phosphorylation and its related aspects. It will be evident from this analysis that there is a strong tendency to attribute to receptor phosphorylation a role in steroid action, although rarely is this tendency rewarded by conclusive experimental data. The perspectives in receptor phosphorylation research offered by new and powerful techniques like those offered by the more recent achievements of molecular endocrinology, will be discussed. PHOSPHORYLATION OF STEROID RECEPTORS IN INTACT CELLS

Phosphorylation of progesterone, glucocorticoid, estradiol and vitamin D,receptors has been observed PR, progesterone receptor; E,R, oestradiol receptor; GR, glucocorticoid receptor.

Abbreoiutions:

11 h/y

1988)

in intact cells or tissues labelled with [‘*P]orthophosphate under a variety of conditions. Glucocorticoid receptor

Phosphorylation of GR has been initially studied in mouse fibroblasts [7] and in the liver of adrenalectomized rats [8,9]. In these reports a phosphorylated 90-92 kDa protein associated with receptor ligand activity was observed. In addition, a minor 100 kDa phosphoprotein capable of binding the receptor ligand [3H]dexametasone mesylate [lo] was detected in fibroblasts. In all these reports molybdate-stabilized GR form has been utilized. Subsequent studies have shown that this form of receptor is associated with a nonhormone binding 90 kDa heat shock protein [ll]. This protein, which was also associated to molybdatestabilized androgen, progesterone and estradiol receptors [12,12a], was phosphorylated in both mouse fibroblasts [I 31 and thymoma cells [ 141.Therefore a portion of the [32P]phosphorylated 90-92 kDa protein(s) observed in these initial reports and identified with the GR, could in fact belong to the non hormone binding 90 kDa protein copurifying with the molybdate-stabilized receptor. Phosphorylation of GR purified from mouse fibroblasts [13] and thymoma cells [14] was subsequently demonstrated by improvement of separation of GR from the 90 kDa non hormone binding protein by antireceptor antibodies [13, 141 and selected electrophoresis conditions [14]. In fact GR migration on SDS-PAGE is dependent on conditions such as temperature [ 141. The phosphorylated receptor was found to be a 100 kDa protein. The 90-92 kDa hormone binding protein previously observed [7-91 might have been a proteolytic product of the 100 kDa GR[lI]. The initial phosphoaminoacid analysis of the GR showed phosphoserine [7]. Although the hormone binding protein and the non hormone binding 90 kDa protein were not separated in this study on fibroblasts, since only one phosphoaminoacid was found, it is likely that GR was phosphorylated on serine. The same finding was obtained using 613

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FERDINANWAURICCHIO

untransformed GR from a pituitary tumor cell line [14a]. Recently it has been observed that the GR (97 kDa) was phosphorylated in human breast epithelial cells [ 151. P-aminoacid analysis of this receptor showed phosphoserine (89%) and phosphotyrosine (11%). Unfortunately the total number of phosphates incorporated by the GR was not assayed to control if the amount of phosphotyrosine was significant. Progesterone receptor

Phosphorylation studies of PR must also take into consideration the association of the molybdate stabilized receptor with the non-hormone binding 90 kDa phosphoprotein [ 17,181. Phosphorylation of the two forms of chicken PR, the 75 kDa A and 110 kDa B forms, was demonstrated by incubation of chicken oviduct minces with [32P]orthophosphate and purification of PRs in the presence of molybdate from cytosol with a procedure which removed most of the 90 kDa non-hormone binding protein [17]. In a previous report from the same group phosphorylation of the 110 kDa receptor was observed. Paminoacid analysis of this form of the PR showed exclusively serine [18]. Additional recent reports on in vivo phosphorylation of PR and GR will be reviewed in the section, “Functional phosphorylation of steroid receptors”, because receptor phosphorylation was studied in those reports in relation with the process of “transformation” of receptors. Estradiol receptor

Phosphorylation of proteins on tyrosine is a very rare event involved in cell multiplication induced by growth factors [2-6] and retroviral transformation [19]. Phosphorylation of E,R has been observed in uteri of intact adult rats incubated in the presence of [32P]orthophosphate [20]. E,R was purified from cytosol using different affinity chromatographies: dietylstilbestrol-Sepharose, heparin-Sepharose and immunoaffinity chromatography, the latter utilizing monoclonal antibody against uterus E,R [21]. Phosphoaminoacid analysis of E2R showed exclusively radioactive phosphotyrosine [20]. This was the first demonstration of phosphorylation of a steroid receptor on tyrosine in intact cells. This finding was corroborated by the previous observation that in a cell-free system calf uterus E2R is phosphorylated on tyrosine by an endogenous kinase which confers hormone binding to E2R [22]. Observation of steroid receptor phosphorylation on tyrosine has been extended to GR of human breast cancer epithelial cells [15]. Orthovanadate, a phosphotyrosine-phosphatase inhibitor [23,24], has been used during the metabolic [32P]phosphorylation of these two receptors to protect receptor-tyrosine phosphorylation [15,20]. In addition in the case of ErR phosphorylation, a short incubation in the presence of [32P]orthophosphate was used. Since P-serine is

known to turn over relatively slowly [25], relatively more [32P]tyrosine would be expected to be found at shorter labeling times. Also E2R was eluted from the immunoaffinity column at alkaline pH that might have destroyed small amounts of phosphoaminoacids different from phosphotyrosine. Therefore it is possible that other phosphoaminoacids are present in the estradiol receptor which might be detected under different experimental conditions [20]. Most of proteins phosphorylated on tyrosine are also phosphorylated on different aminoacids. Phosphorylation of E,R has been observed also in a cell line derived from an estrogen-responsive mouse Leydig cell tumor [26]. No phosphoaminoacid analysis was made. Interaction of calf uterus E,R with antiphosphotyrosine antibody confirmed that phosphorylation of this receptor on tyrosine is a natural event [16,20]. Vitamin 0, receptor

The 1,25-hydroxyvitamin D, receptor which belongs to the class of steroid receptors [27] appeared to be phosphorylated in mouse fibroblasts. This phosphorylation was dependent on the presence of vitamin D3 in the medium [28]. Table 1 summarizes most of the findings on steroid receptors phosphorylation in whole cells or tissues or animals. ARE STEROID RECEPTORS KINASES?

The answer to this proposed question should take into adequate consideration the fact that proteinkinases are largely present in tissues and steroid receptors have a strong tendency towards aggregation with other proteins. Glucocorticoid receptors

Kinase activity intrinsic to rat liver GR has been reported by different groups [29-321. This receptor was purified either in the absence [29] or in the presence of molybdate and transformed before being assayed [30-321. In these 4 reports GR purified by similar procedures appeared to be associated with kinase activities showing different properties like stimulation by different cations and different ability to phosphorylate the receptor itself. This fact suggests that these GR preparations have been purified in association with different kinases and favors the possibility that the kinase activities observed were not intrinsic to the GR. This interpretation is strongly supported by three recent reports which, using different purification procedures of the GR, showed that in mouse fibroblasts as well as in rat liver GR has no intrinsic kinase activity [33-351. Progesterone receptor

The possibility that PR has intrinsic kinase activity has been investigated in several studies and found to

615

General Review Table 1. Steroid receptor phosphorylation

Receptor

in whole cells, tissues and animals

system

Stimulation by the cognate hormone

Chicken oviduct mince Chicken oviduct mince Chicken oviduct mince Rabbit uterine slices Chicken oviduct cells Human breast cancer cells Human breast cancer cells

Present Prcscnt Absent present Present

Mouse fibroblasts Mouse fibroblasts Mouse fibroblasts Mouse fibroblasts Rat liver (in viva) Rat liver (in uiuo) Mouse thymoma cells Mouse pituitary cells Mouse thymoma cells

Absent -

Propaemne

Gl-rticoid

-

P-aminoacids

References

P-serine P-mine

P-serine -

t;; 1874

1:;; t:6:

[ii

P-serine

Human breast epithelial cells

Rat uterus Leydig cell tumor cells

-

P-tyrosine -

t::;

Mouse fibroblasts

present

-

[3gl

De&radio1

-

‘“rpB1”’ [91 [I41

Absent -

P-swine

-

P-mine

[W

[791

and P-tyrosine

[131

Vitamin D3

be unlikely. No autophosphorylation of the chicken oviduct PR was observed in the 1st report of this series [36]. Subsequently it was observed that a purified preparation of the B form of the chicken oviduct PR phosphorylated the receptor itself as well as histones [37]. It was suggested that the receptor is a kinase. However when purification of PR was slightly modified by the same group, a partial separation of PR from the kinase was observed showing that the PR and kinase are different entities [38]. Chicken oviduct cytosol, molybdate-stabilized PR has been extensively purified. The final preparations contained the A and B form of the PR in addition to 90 kDA non hormone binding component [39]. Again no intrinsic kinase activity of the receptors was detected. Androgen receptor

Androgen receptor has been purified from the prostate of castrated rats using testosteroneSepharose and DNA-Sepharose chromatographies. No autophosphorylation of this receptor was observed [40]. Estradiol receptor

Estradiol receptor purified from calf uterus by heparin-Sepharose and estradiol agarose [41] does not show autophoshorylation activity [42]. In addition incubation with radiolabeled ATP analogue, [“C]fluorosulfonyl benzoyl adenosine, of the purified E,R followed by fluorography of the SDS-PAGE did not show any binding of the analogue to the ErR (Migliaccio A. and Auricchio F., unpublished data). Therefore there is no evidence that uterus E,R is a kinase [42-43]. Immunoprecipitation of the MCF-7

cell E,R with monoclonal antibody against the receptor bound to a polystyrene matrix led to immunoprecipitation of a kinase and suggested that E,R is a kinase [44]. However subsequent study from the same group has disproved this possibility [45]. In conclusion, with the exception of some reports on GR, in conflict with other reports on the same receptor, steroid receptors have not been found to have intrinsic kinase activity. The frequent association of steroid receptors with kinases, in particular with those stimulated by the cognate hormone, might have a biological role, as proposed in the case of E,R [43]. FUNCTIONAL PHOSPHORYLATION STEROID RECEPTORS

OF

Phosphorylation is by far the most studied modification regulating the activity of enzymic as well as non enxymic proteins. The problem of the role of steroid receptor phosphorylation was posed even before these proteins were found to be phosphorylated. The most studied functions in relation to phosphorylation have been hormone binding and receptor transformation. I will separately review indirect and direct evidence on the role of steroid receptor phosphorylation on these two functions. Hormone Binding Inhibition of energy production

The use of inhibitors of energy production has given useful information on the possible relation between phosphorylation and hormone binding to the receptor. The initial experiments were performed

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FERDINANWAURICCHIO

in intact thymus cells [l]. In the absence of glucose and/or oxygen the intracellular ATP levels of these cells was lowered. Specific [‘Hlcortisol binding also decreased. When the normal intracellular ATP level was restored, the hormone binding to the receptor was also recovered. Consequently Munck et al. [46] proposed that in cells deprived of ATP the receptor is present in a form that cannot bind hormone, the “null receptor” form. In intact thymoma cells in which ATP levels are lowered, null GR was detectable by immunoblot in nuclei [47]. Inhibitors of aerobic energy production also reversibly affected the glucocorticoid receptor binding of mouse fibroblasts so that it was proposed that the binding of the GR was inactivated by these inhibitors and reactivated or recycled by an energy requiring process to a form which binds hormone [48]. Respiratory poisons inactivated hormone binding of androgen receptor in rat minced prostrate. Removal of them was followed by a rapid partial recovery of hormone binding sites [49]. These and other similar reports showed that hormone binding to the receptor requires cell energy. When metabolic inhibitors were removed from the medium, the ability to bind hormones was restored and protein synthesis inhibitors did not affect this return [48,50, 511 suggesting that a protein modification was responsible for the observed effect. Exogenous enzymes

A different approach to investigate the role of receptor phosphorylation consists in studying the effects of phosphatases or kinases on the steroid receptor functions. Crude or purified endogenous preparations or purified exogenous enzymes have been incubated with steroid receptor preparations of different purities. Bovine alkaline intestinal phosphatase inactivated binding of crude hormone-free GR from mouse fibroblasts and rat liver cytosol whereas it did not significantly affect the 17/3-estradiol receptor from rat uterus cytosol[52]. A possible explanation was that phosphorylation of the GR regulates hormone binding. Also low affinity binding sites (& * 25 nM) of chicken oviduct PR were inactivated by bovine alkaline intestinal phosphatase [53]. Since inhibitors of phosphatase reduced this inactivation it was proposed that dephosphorylation of PR was responsible for the low affinity binding sites inactivation. Endogenous enzymes: indirect evidences on the role of receptor phosphorylation

Use of endogenous enzymes in the study of the role of receptor phosphorylation should be greatly preferred to that of exogenous enzymes because endogenous enzymes are responsible for natural phosphorylations. The effect of endogenous enzymes on steroid receptors has been reported. The particulate fraction sedimenting between 27,000 and 100,000 g prepared from

mouse fibroblasts, rat thymocytes and rat liver contained an activity inactivating the steroid binding of hormone-free GR. It was inhibited by phosphatase inhibitors like glucose-l-phosphate, molybdate and fluoride [54]. Furthermore, GR in cytosol of mouse fibroblasts is rapidly inactivated at 25°C. This inactivation, which was markedly slowed by glucosel-phosphate and fluoride, was partially reversed by ATP and MgCI,. These results were interpreted as evidence that the GR can acquire the hormone binding activity through a phosphorylation process [55]. In thymocyte cytosol maximal reactivation of GR was observed when in addition to ATP, dithiothreitol, molybdate and a heat-stable factor were also present [56]. In a different report from the same group it was proposed that receptor phosphorylation ensures that SH groups required for hormone binding are not oxidized [57]. A similar proposal has been made for the rat liver nuclear T, receptors [57a]. Alkaline phosphatase has an inhibitory effect on T, binding which is thiol dependent. There is a series of studies from different groups showing that endogenous enzymes modify hormone binding to E,R apparently through a phosphorylation/dephosphorylation process. Phosphorylation confers hormone binding and dephosphorylation abolishes this binding. We observed that estradiol treatment of mice initially induced the so called E,R “nuclear translocation” in uterus; thereafter a decrease of hormone binding receptor was observed together with the appearance in cytosol of a corresponding amount of dephosphorylated, non hormone binding receptor [58]. The appearance of the dephosphorylated E,R could not be attributed to newly synthesized receptor since dephosphorylated receptor was found in the absence of protein synthesis. The inactive, dephosphorylated receptor found in cytosol was reactivated by incubation with a crude preparation of the uterus kinase [58] that phosphorylates E,R on tyrosine [22]. It was proposed that in vivo E,R in complex with the hormone after high affinity interaction with chromatin is inactivated by the nuclear phosphatase that subsequent studies have shown to be able to remove phosphate from E,R-phosphotyrosine [20,59]. It was also hypothesized that in cytoplasm the E2R kinase might recycle the receptor inactivated in the nucleus [for a review on the steroid receptor cycling, see Ref. 601. Progesterone treatment of hamsters rapidly reduced the uterus nuclear hormone-occupied estrogen receptor (611. Inactivation of the receptor in nuclear extract was increased after in vivo progesterone treatment and was inhibited by low concentration of phosphatase inhibitors like molybdate and vanadate. It was proposed that progesterone induces a nuclear phosphatase that dephosphorylates and inactivates the estrogen receptor (621. Addition of molybdate to human endometrial adenocarcinoma cells HEC-1 incubated in the pres-

General Review ence of estradiol increased E,R binding activity [63]. Addition to homogenate of those cells or normal endometrium of molybdate, ATP, GTP or cGMP in the presence of estradiol also increased the binding whereas CAMP had the opposite effect. Subsequent study on HEC-1 cells showed that in cytosol incubated with hormone, addition of cGMP increased the binding sites only if the cytosol was not ATP depleted, suggesting that the positive effect of cGMP and the negative of CAMP were mediated by kinases and involved phosphorylation [64]. Chicken oviduct estrogen receptor(s) has two affinity binding sites, one, Xwith Kd 0.1 nM, the other Y with Kd 1 nM. The Y-receptor exists in a nonestradiol binding form, produced in uiuo shortly after withdrawal from estrogen and in vitro during dialysis. The non hormone binding form could be converted into the binding form in the presence of estradiol and MgZf by in vitro treatment with ATP and ADP [65]. These findings suggested to the authors that phosphorylation of either the receptor or some other cytosol factors could be involved in this conversion [66]. Endogenous enzymes: direct evidences on the role of receptor phosphorylation

In all the above mentioned studies there is no direct evidence that phosphorylation of a steroid receptor is involved in the change of hormone binding to the receptor. The role of phosphorylation on hormone binding to the uterus E,R has been investigated using cell-free systems. This has been possible because of the identification and characterization of two uterus enzymes also present in the mammary gland, a nuclear phosphatase [67] and a cytosol kinase [68] that reversibly modify the hormone binding of crude or purified E,R [22,42,43,58,69]. The phosphatase, which has several interesting properties including the ability to discrimate between receptor complexed with hormone and receptor complexed with non-steroid antiestrogens [70], inactivates the hormone binding of E,R. It is a receptor-phosphotyrosine phosphatase. In fact ErR which has been [32P]phos-phorylated on tyrosine by the uterus kinase (see below) lost a significant portion of 32P by incubation with this phosphatase [59]. Parallel loss of hormone binding and interaction with antiphosphotyrosine antibody of E,R after incubation of E,R with the phosphatase [16,20] showed that the phosphatase dephosphorylated E,R naturally phosphorylated on tyrosine (and inactivated the hormone binding). Incubation of native E,R-inactivated and dephosphorylated by the uterus phosphatase with the partially purified calf uterus kinase and [Y-~~P]ATP restored the receptor hormone binding and phosphorylated the estradiol receptor [42]. This phosphorylation occurred on tyrosine [22]. Requirement of phosphorylation on tyrosine for hormone binding to E2R has been subsequently confirmed under different

617

conditions [43], as well as with antiphosphotyrosine antibody [16,20]. The receptor tyrosine phosphorylation reaction appears from in oitro experiments to be regulated in a complex way. It was stimulated by physiological concentration of Ca2+ in complex with calmodulin [22], the intracellular receptor of Ca2+. Parallel stimulation of hormone binding activation was detected. Phosphorylation on tyrosine and hormone binding activation of E,R by this kinase was also stimulated by hormone in complex with E,R whereas it was inhibited by the occupancy of E,R by the non-steroidal antiestrogen, tamoxifen [43]. Estradiol stimulation was due to increased affinity of the kinase for its substrate [43]. Uterus estradiol receptor and uterus tyrosine kinase are two different entities: ammonium sulphate precipitation partially separated E,R from kinase [68]; E,R bound to heparin-Sepharosc whereas the kinase did not [68]; the purified kinase did not interact with monoclonal antibody against calf uterus receptor; E,R did not bind ATP analogue whereas the kinase did; the purified kinase did not bind hormone [43]; no phosphorylation of purified E,R was observed in the absence of the kinase [42]. Nevertheless phosphorylated, hormone-binding receptor and the kinase are functionally associated as shown by the stimulation of the kinase by hormone occupancy of the receptor. Therefore we are in the presence of a system which reminds us of that described for the peptide growth factors; estradiol binding to the receptor stimulates a protein-tyrosine kinase to phosphorylate substrates like the dephosphorylated, non hormone-binding form of the receptor [43] and other proteins (manuscript in preparation). The mechanism by which the estradiolreceptor complex activates the kinase must be still clarified. The following observations, regarding whether phosphorylation on tyrosine regulates the binding of only a portion or all the mammalian E,R, can be made. In all the published reports the inactivation of E2R by the phosphatase ranged from 20 to 60% depending on the animal species, on the age of the animals (uteri from young calves, rich in E,R are a good source of the enzyme), on how long tissues have been frozen (uteri loose enzymic activity after a week whereas they still retain the hormone binding of E2R). However a complete inactivation of E,R has never been observed in oitro although this fact might be due to the production of an inhibitor [71]. A second finding of interest is that the in vitro synthesized E,R binds hormone with the same affinity of the uterus receptor but at very low efficiency ,(1-4% of the expected maximum binding) [72]. This fact suggested the possibility, now being investigated, that most of the binding efficiency is conferred to the receptor by the tyrosine phosphorylation of the newly synthesized receptor. In conclusion, present findings suggest that in optimal conditions most of the E,R

requires phosphotyrosine to bind hormone. It is not proved that this is true for all the EIR. Regulation of hormone binding by receptor phosphorylation in T47 human breast cancer cells is suggested by stimulation of phospho~lation of a protein which could be the PR and increase of steroid binding as a consequence of cell exposure to phorbol-12-myristate-13-acetate [72]. Unfortunately the identification of the phosphorylated protein with the PR is not sufficiently proved. Receptor Transformation The role of phosphorylation-dephosphorylation of steroid receptors in the process by which they acquire the property to tightly bind to DNA or chromatin, has been investigated by several groups. We will call this process transfo~ation since the term activation employed by different groups instead of transformation has been used in the present article for the ability of the receptor to bind hormone. Exogenous enzymes The effect of incubation of calf intestinal alkaline phosphata~, phosphatase inhibitors and phosphorylated compounds including ATP with rat liver and kidney cytosol on the transformation of the GR has been studied [73]. Transformation was quantified by interaction with DNA-cellulose and visualized by DEAE-Sephadex resolution of non-transformed and transfo~ed receptor. Taken together the results were interpreted as an indication that a dephosphorylation process involving either the receptor or receptorrelated molecules transforms the OR receptor. Incubation of crude GR from a mouse pituitary tumor cell line with calf intestinal alkaline phosphatase also increased the rate of receptor transformation measured by a change in sedimentation rate of the GR from 9 to 5 S [74]. It was suggested that dephosphorylation of some cytosolic component is involved in the destabilization of receptor subunit interactions resulting in glucocorticoid receptor transfo~ation. Endogenous enzymes In contrast with the hypothesis suggested by the experiments in the previous paragraph that dephosphorylation transforms GR, is the ~ssibility that phospho~lation is responsible for GR transformation. This is suggested by the finding that incubation of crude rat liver glucocorticoid receptor with ATP stimulated the binding of the steroid-receptor complex to nuclei, ATP-Sepharose, phosphocellulose and DNA-cellulose [75,76]. ATP also induced transformation of the rat thrice GR [76a]. In~bation with ATP, hypothetically causing phospho~lation, transformed the purified chicken oviduct progesterone receptor from the 8 S into a 4 S form [39]. There are detailed reviews on these and other similar reports using similar approaches [77,78].

Direct studies on the role of receptor phosphorylation Two papers have been recently published on the role of phosphorylation of GR on receptor transformation [79,80]. In both reports a negative conclusion was reached. In the 1st report the receptor which has been found to contain 2-3 phosphates was transformed by incubating mouse thymoma cells with triamcinolone at 37°C. No quantitative change of the GR phosphorylation upon transformation was detected [79f. In the 2nd report fibroblast GR was transform by heating cytosol as well as by ho~one treatment of intact cells. No quantitative change in the extent of receptor phosphorylation induced by transformation was observed [80]. The fibroblast GR has been recently found to contain 4 phosphoserines, 1 of which lies in a tryptic DNA-binding fragment, No phosphate was detected in a tryptic fragment containing most of the hormone binding domain [sOa]. In contrast to GR, phosphorylation of PR has been found to be stimulated in progesterone treated mammalian tissues although apparently there is no causal connection between phosphorylation and transfo~ation. PR phosphorylation was studied in rabbit uterus slices incubated with ‘*P at 37°C in the absence and in presence of the progestin R5020[81]. In the absence of hormone, cytosol receptor was phosphorylated. After hormone administration, stimulation of phospho~lation of cytosol as well as nuclear receptor was observed. Hormonal stimulation of phosphorylation of the receptor caused a slight decrease of electrophoretic mobility of the receptor, a phenomenon observed for several proteins due to conformational change induced by phosphorylation and preserved in SDS 182,831. However no effect of this ho~one-indu~d receptor phospho~lation on the interaction of rabbit progesterone receptor with specific DNA sequences in the uteroglobulin gene have been detected [84]. Study of interaction of phosphorylated receptor with specific DNA sequences should be extended to other steroid receptors to further investigate whether receptor phospho~lation plays a role in gene activation. Phosphorylation of the human progesterone receptor has been studied in T 47 human breast cancer cells, which are progesterone receptor rich [85,86]. When cells were grown in the absence of hormone, phospho~lation of the cytosol B form of the receptor was observed [85]. After 4 h of treatment with the progestin analogue R5020 a decrease of electrophoretic mobility of the B form suggested a second round of PR phosphorylation in the nucleus when the receptor is already transformed. In a different report 1 h progesterone t~atment of cells also stimula~ PR phospho~lation [86]. Progesterone did not stimulate the PR phosphorylation in chicken oviduct cultures [87]. In contrast progesterone treatment caused a very rapid increase of phosphorylation of A and B froms of PR in cytosol of avian tissue minces [87a]

General Review

suggesting that receptor phosphorylation is a very early event during progesterone action. In oivo experiments showed that progesterone treatment of chicks caused a conversion of the A form of the receptor into a nuclear form with a slower electrophoretic mobility [87a]. Progesterone treatment of chicken oviduct minces increased phosphorylation of PR at existing rather than new sites. Phosphoaminoacids are localized to a specific region that does not include either the DNA or steroid binding domains [87b]. A recent report on E,R showed that estradiol and [ring-‘Hltamoxifen aziridine treatment induced a molecular heterogeneity of the receptor causing the appearance of a EIR form with a decreased electrophoretic mobility [88]. It is possible that this appearance reflects phosphorylation of the E,R. It should be noted that in some of the cases cited as showing stimulation of [32P]phosphorylation of receptors there may have been only a change in labeling without change in number of phosphates of receptors. PERSPECI’WES IN THE STUDY OF PHOSPHORYLATION OF THE STEROID RECEPTORS

In the near future we will know much more about the function(s) of steroid receptor phosphorylation. There is a general trend towards the acquisition by many laboratories of powerful, and in some cases simple techniques useful for the study of receptor phosphorylation. Affinity chromatographies, monoclonal and polyclonal antibodies against receptors, identification and purification of endogenous kinases and phosphatases acting on steroid receptors will certainly play an important, positive role. The use of antiphosphotyrosine antibody seems to be promising for these purposes. In fact antiphosphotyrosine antibody provides an alternative, very simple method of identifying events related to phosphorylation on tyrosine of proteins. This technique was first described by Ross et al., who produced antibody to phosphotyrosine by immunization of rabbits with aminobenzyl phosphonate, a close phosphotyrosine analogue coupled with diazotization to keyhole limpet hemocyanin (KLH) [89]. Antiphosphotyrosine antibodies covalently coupled with Sepharose have been used to isolate proteins from cells transformed by retrovirus and stimulated by EGF [90-931. Antiphosphotyrosine antibody has been also employed for the intracellular localization of proteins phosphorylated on tyrosine [94] and to study phosphorylation on tyrosine of insulin receptor [95]. As mentioned in the course of this article we have recently utilized the antiphosphotyrosine antibody coupled to Sepharose to study the phosphorylation events involving steroid receptors [16,20]. The hormone binding calf uterus receptor interacts with high affinity with these antibodies confirming that this form of receptor is phosphorylated on tyrosine. In addition the aliquot of the receptor whose hormone

619

binding has been lost by incubation with the uterus nuclear phosphatase also loses the ability to interact with these antibodies. The parallel lack of interaction with antiphosphotyrosine antibody and loss of hormone binding suggests that naturally occurring receptor phosphorylation of E,R on tyrosine is required to bind hormone. Also partially purified rat liver GR interacts with high affinity with anti-phosphotyrosine antibody [16]. This interaction followed by elution by phenylphosphate of GR from the antibody was used in the purification of small amounts of GR and suggested that rat liver GR could be phosphorylated on tyrosine [ 161. The most recent achievements of molecular endocrynology will certainly contribute greatly to the study of the role of receptor phosphorylation. Several steroid receptors can now be synthesized in vitro from their cDNA and comparison of their properties with those of in vioo synthesized receptors will suggest or exclude the possibility that post-translational modifications are necessary to confer function(s) to the receptors. For instance the fact that only a small percentage of the synthetic human E2R binds hormone [72] suggests that a post-translational modification is necessary to confer the maximal function to this receptor. Therefore synthetic receptors can be exploited as substrates of endogenous kinases. The use of receptor mutants adds further interest to this approach by offering the possibility to localize the residues whose phosphorylation controls a receptor function, CONCLUSIONS

Phosphorylation of steroid receptors has been observed in cells, tissues and animals under a variety of conditions (see Table 1). Although such a phosphorylation has been frequently implicated in the regulation of receptor functions, conclusive evidence of this is very limited. Studies on cell-free systems shows that hormone binding of uterus E,R is regulated by receptor tyrosine phosphorylation. The possibility that phosphorylation modulates hormone binding to other steroid receptors is supported by considerable indirect data. Recently some reports have attempted to elucidate whether receptor phosphorylation causes the hormone induced ability of receptor to interact with DNA and nuclei. These studies gave negative results. Nevertheless further investigation is required to reach a conclusion on this point. The use of in oitro synthesized receptors and their mutants, antiphosphotyrosine antibody and endogenous kinases and phosphatases acting on receptors, promise a rapid extension of our knowledge of the regulatory function of steroid receptor phosphorylation. Acknowledgements-The critical reading of the manuscript by Drs A. Migliaccio and B. Moncharmont is gratefully acknowledged together with the excellent editorial work 01 Mr Gian Michele La Placa. This work was supported by grants from the Associazione Italiana per la Ricerca sul

620

FE~INA~

Cancro; from the Italian National Research Council, Special Project Oncology, Contract no. 87.1167.44 and from the Minister0 Pubblica Istruzione, Italy.

Aurt~ccmo

15.

REFERENCES 1. Munck A. and Brinck-Johnsen T.: Specific and non specific physicochemical interactions of glucocorticoids related steroids with rat thymus cells in vitro. J. biol. Chem. 243 (1968) 55565565. 2. Ushiro H. and Cohen S.: Identification of phosphotyrosine as a product of epidermal growth factor-activated protein kinase in A 431 cell membranes. J. &of. Chetn. 255 (1980) 83638365. 3. Kasuga M., Zick Y., Blithe D. L., Crettaz and Kahn C. R.: Insulin stimulates tvrosine uhosohorvlation of the insulin receptor in cell&e sysiem. -Nat&e 298 (1982) 667669. 4. Ek B., Westermark B., Wasteson A. and Heldin C. H.: Stimulation of tyrosine-specific phosphorylation by pfatelet-derived growth factor. Nature 295 (1982) 419-420. 5. Nishimura J., Huang J. S. and Denel T. F.: Plateletderived growth factor stimulates tyrosine-specific protein kinase activity in Swiss mouse 3T3 cell membranes.

16.

17.

**.

19. 20.

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