THE JOURNAL OF BIOLOGICAL CHEMISTRY 8 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 17, Issue of June 15, pp. 10025-10029. 1990 Prmted in U.S. A.

Phosphorylation of the 1,25-Dihydroxyvitamin D3 Receptor A PRIMARY EVENT IN 1,2SDIHYDROXYVITAMIN Ds ACTION*

(Received for publication, December 18, 1989)

Thomas A. Brown and Hector F. DeLucaS From the DeDartment of Biochemistrv. Universitv of Wisconsin, college of Agricultural and Life Sciences, Madison, Wkonsin 53iOS

.,I I .

1,25-Dihydroxyvitamin Ds is believed to mediate the regulation of calcium metabolism by a steroid hormone receptor mechanism. We now demonstrate that phos- phorylation of the 1,25-dihydroxyvitamin DS receptor occurs rapidly in embryonic chick duodenal organ cul- ture in response to 1,25-dihydroxyvitamin DB. Phos- phorylation of the receptor is strongly induced within 1 h by 1,25-dihydroxyvitamin Da and occurs prior to the initiation of calcium uptake or induction of cal- cium-binding protein in this system. Phosphorylation is not affected by inhibition of protein synthesis by cycloheximide. Since chick duodenal organ culture is a vitamin D-responsive system that closely parallels in viva rachitic chicken intestine, the occurrence of receptor phosphorylation in response to 1,25-dihy- droxyvitamin D3 strongly suggests that it is a physio- logically relevant event. In addition, since it occurs prior to the other responses to 1,25-dihydroxyvitamin D3, it appears possible that phosphorylation may play a significant role in the 1,25-dihydroxyvitamin DB regulation of transcription in the intestine.

The 1,2Sdihydroxyvitamin DB (1,25-(OH),D,)’ receptor mediates the regulation of calcium metabolism by 1,25- (OH)*D3 (1). This receptor is a member of the steroid/thyroid hormone receptor gene family (2, 3) and is thought to act through a classic steroid hormone mechanism (4). According to the classic steroid hormone model (5), 1,25-(OH)zD3 dif- fuses into the nucleus of target cells and interacts with its receptor. The receptor-1,25-(OH)2D3 complex then binds to specific DNA sequences in or near target genes and modulates their transcription. In a major target organ of 1,25-(OH)zD3, the intestine, the modulation of gene expression results in transport of calcium and phosphorus from the lumen to the blood, contributing to a rise in serum calcium and phosphorus (1).

In mouse fibroblast 3T6 cells, 1,25-(OH)zD3 stimulates phosphorylation of the 1,25-(OH)*D3 receptor (6). Other ste- roid hormone receptors are also phosphorylated in a number of systems (7). Phosphorylation is an important mechanism by which biological systems are controlled (8). Therefore,

* This work was supported by Program Project Grant DK-14881 from the National Institutes of Health and by the Harry Steenbock Research Fund of the Wisconsin Alumni Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed: Dept. of Biochem- istry, University of Wisconsin, 420 Henry Mall, Madison, WI 53706.

1 The abbreviation used is: 1,25-(OH)& 1,25-dihydroxyvitamin D3.

there is much interest in characterizing phosphorylation of steroid receptors and understanding its role, if any, in receptor function. In most instances, phosphorylation of steroid recep- tors is induced by their respective ligands (7), including a recent report (9) that the number of phosphates per glucocor- ticoid receptor is increased by agonist. The induction of receptor phosphorylation by agonists supports the idea that phosphorylation is involved in the function of receptors. Many functional roles for steroid receptor phosphorylation have been suggested, including the regulation of steroid bind- ing, DNA binding, specificity of DNA binding, nuclear local- ization, association with heat shock proteins, and association with auxiliary transcription factors (7). It is also possible that phosphorylation may play a role in receptor turnover rate and recycling (7). Unfortunately, a role for receptor phosphoryla- tion in any of these events has not yet been demonstrated.

The phosphorylation sites of several steroid receptors have been localized to discrete domains. Phosphorylation sites have been identified on serine residues in the amino-terminal do- main (A/B domain) of the glucocorticoid (10, ll), progester- one (12,13), and thyroid hormone (14) receptors. In addition, there is a report (10) of glucocorticoid receptor phosphoryla- tion in the DNA-binding domain (domain C). However, a conflicting report (11) showed phosphorylation of the gluco- corticoid receptor in the A/B domain and in the steroid- binding domain (domain E), but not in the DNA-binding domain. Using a variety of systems, Auricchio (7) has reported phosphorylation of the estrogen receptor on tyrosine. Auric- chio and co-workers (15) have shown that one site of tyrosine phosphorylation lies in the steroid-binding domain.

The goal of this study is the demonstration of 1,25-(OH)2D3 receptor phosphorylation in a major target organ of 1,25- (OH)zD3, the intestine. The intestine is experimentally acces- sible for these studies since embryonic chick duodenal organ culture is a well-characterized system that closely resembles the in uivo rachitic chick intestine (16, 17). In addition, 1,25- (OH)*DB action on the intestine is not complicated by the requirement for parathyroid hormone as is action at the kidney and bone (1). Cultured duodena have several well- characterized responses to 1,25-(OH)zD.1 including increases in calcium uptake and the synthesis of vitamin D-dependent calcium-binding protein (16, 17).

In this paper, we report the immunoprecipitation of the [35S]methionine- and 32P0,-labeled 1,25-(OH)2D3 receptor from cultured embryonic chick duodena. Receptor phos- phorylation is markedly increased by addition of 1,25- (OH)2D3 within 1 h and prior to induction of vitamin D- dependent calcium uptake and calcium-binding protein.

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10026 1,25-Dihydroxyvitamin D3

MATERIALS AND METHODS’

RESULTS AND DISCUSSION

The 1,25-(0H)~D~ receptor was isolated from chick duo- denal organ cultures. Extracts prepared from duodena that were cultured for 24 h in complete Waymouth’s medium in the absence of 1,25-(OH)zD3 contained -150 fmol of receptor/ mg of protein as determined by a hydroxylapatite binding assay. To demonstrate specific immunoprecipitation of the 1,25-(OH)2D3 receptor from these cultures, the proteins were labeled by incubation of the cultures in [35S]methionine- containing medium for 2 h. The specific activity of the ex- tracted protein was -10,000 cpm/pg of protein. The 1,25- (OH)ZDB receptor was immunoprecipitated from these ex- tracts using monoclonal antibodies coupled to Sepharose (Fig. 1). Monoclonal antibody IVG8Cll-Sepharose (lane I) im- munoprecipitated several proteins. Immunoprecipitation with IVG8Cll uersus control antibodies MOPCBl (an antibody which does not recognize the 1,25-(OH)*D3 receptor; lane 2) and XVIElOBGA5 (a monoclonal antibody which recognizes only the pig 1,25-(OH)zD3 receptor; lane 3) revealed two proteins of -60,000 and -58,000 Da that are the 1,25-(OH)zD3 receptors. A slightly smaller species of 56,000 Da was also specifically immunoprecipitated by IVG8Cll. To demonstrate that the 1,25-(OH)*D3 receptor was still present in the extracts precipitated with the control resins, the supernatants after precipitation with the two control antibodies were subjected to immunoprecipitation with IVG8Cll. As shown in lanes 4 and 5, the receptor was present. As a further control to demonstrate the specificity of immunoprecipitation, excess non-Sepharose-bound antibody was added in solution to the extract prior to immunoprecipitation. Addition of excess MOPC21 (lane 6) did not affect the immunoprecipitation of the three receptor protein bands. Excess added IVG8Cll (lane 7) effectively competed with IVG8Cll-Sepharose for the receptor, and the three proteins at 60, 58, and 56 kDa were not precipitated. Addition of excess IVG8Cll did not elimi- nate the receptor from solution since the receptor was im- munoprecipitated with antibody VDBFlS-Sepharose (an an- tibody recognizing a different epitope of the 1,25-(OH)2D3 receptor; lane 8) from the supernatants containing excess IVG8Cll. A 41-kDa protein was present in the control pre- cipitations (lanes 2 and 3) and was not affected by addition of excess IVG8Cll (lane 7). This protein was therefore non- specific background in the assay and was not related to the 1,25-(OH)2D3 receptor. The two major receptor forms are analogous to the 60,300- and 58,600-Da receptors purified from chicken (26) and immunoprecipitated from in vitro translatedchicken intestinalpolyadenylated RNA (27). Based on their observations, Pike and co-workers (26,27) suggested that the 58-kDa form was a proteolytic product of the com- plete 60-kDa receptor. Since we always detected both forms in approximately equal amounts, we cannot draw conclusions as to the origin of the two forms. The faint 56-kDa receptor species we observed with [“‘Slmethionine may be a proteolytic product.

The phosphorylated 1,25-(OH)*D3 receptor was detected by immunoprecipitation from duodenal organ cultures that were labeled in low methionine medium in which the only phos- phate present was from HZ3*P04. Cultures were labeled for 4 h with 0.2 mCi of H~‘2P04/ml of medium in the presence of

’ Portions of this paper (including “Materials and Methods” and Figs. l-4 and 6) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

75 nM 1,25-(OH)*D3, and a specific activity of -600 cpm/pg of protein was obtained. Comparison of the immunoprecipi- tation of 32P04-labeled extract with IVG8Cll-Sepharose (Fig. 2, lanes 3 and 4) to the control precipitations with MOPCBl- Sepharose (lane I) or XVIElOBGA5-Sepharose (lane 2) showed two labeled proteins of 60 and 58 kDa that were specifically recognized by receptor-specific antibody. The im- munoprecipitation of these two proteins was eliminated by addition of excess IVG8Cll (lane 5), whereas it was unaffected by addition of MOPC21 (lane 6). The 1,25-(OH)*D3 receptor could be immunoprecipitated from the supernatant from the sample of lane 5 with VDBFlB-Sepharose (lane 7). Because of the low amount of 32P04 incorporated into protein and the diffuse nature of the autoradiography, we could not determine if the smaller protein band at 56 kDa was phosphorylated.

To ensure that the response to 1,25-(OH)*D3 is not changed by the use of low methionine/zero phosphate medium, we tested the time course of 1,25-(OH)2D3-dependent calcium uptake under these conditions (Fig. 3). No significant increase in 1,25-(OH)PDs-dependent calcium uptake was seen at 1 h after 1,25-(OH)*D3 addition. At 6 and 12 h after 1,25-(OH)zD3 addition, there was a significant increase in calcium uptake. Also, the induction of calcium-binding protein by 1,25- (OH)zD3 was measured by immunoprecipitation from [3”S] methionine-labeled extracts. As shown in Fig. 4, 35S-labeled calcium-binding protein, evident at the expected apparent molecular mass of 28 kDa, was induced dramatically at 6 and 12 h after addition of 1,25-(OH)zD3 to these cultures. The induction of calcium uptake and calcium-binding protein ob- served is consistent with previous reports in complete medium (17, 19). The -fold induction of calcium uptake was greater in the zero phosphate medium than previously seen with com- plete medium (17, 19).

The time course of 32P04 incorporation into the 1,25- (OH)*D3 receptor after 1,25-(OH)*D3 addition was monitored (Fig. 5). In some experiments, the increase of 32P04 incorpo- ration was evident at 15 min after 1,25-(OH)2D3 addition (lane 2), but it was consistently increased at 1 h (lane 3). The incorporation continued to increase at longer times after 1,25- (OH)zD3 addition. Addition of 1,25-(OH)tDa dramatically in- creased the amount of 32P0., incorporated, although a low level of the phosphorylated receptor was detectable in the absence of added 1,25-(OH)2D3. The low level of receptor phosphorylation observed in the absence of added 1,25- (OH)zD3 was most likely a basal level of receptor phosphoryl- ation which is independent of ligand binding. However, this may have been caused by the small amounts of 1,25-(OH)pD3 present in these vitamin D-sufficient embryos.

1,25-Dihydroxyvitamin D3 caused rapid phosphorylation of its receptor. A clear signal was observed consistently at 1 h after addition of 1,25-(OH)zD3. This was prior to the detection of an increase in 1,25-(OH),Ds-induced calcium uptake and calcium-binding protein. Since different assays with different sensitivities are being compared, we cannot determine with certainty the temporal order of the 1,25-(OH)zDa responses. In fact, calcium-binding protein was previously shown to increase within 1 h when samples were assayed by computer analysis of two-dimensional electrophoresis (20). However, certainly, receptor phosphorylation was detected early enough to suggest a functional role of receptor phosphorylation in transcription induction by 1,25-(OH)gDz.

At a concentration of 50 pM cycloheximide, shown previ- ously to block calcium transport and calcium-binding protein responses (20, 25), the incorporation of [35S]methionine into total protein was diminished by 85%. However, cycloheximide had no effect on the increased 32P04 incorporation into the

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FIG. 5. Time course of 1,25-(OH)zD3 receptor phosphoryl- ation in chick organ culture. Chick duodena were cultured for 12 h in Waymouth’s medium and then for 12 h in low phosphate medium containing 0.125 mCi of Hs”‘P04/ml. The receptor was precipitated from extracts as described under “Materials and Methods” using antibody IVG8Cll. Lanes l-6 represent addition of 150 nM 1,25- (OH)zDs 0, 0.25, 1, 4, 6, and 10 h, respectively, prior to the end of the culture period. The positions of molecular mass standards (in kilo- daltons) are shown.

1,25-(OH)?Ds receptor (Fig. 6). It is therefore clear that recep- tor phosphorylation is a primary action of 1,25-(OH)2D3, not requiring the synthesis of new proteins. Since it has been previously shown that cycloheximide eliminates vitamin D- dependent calcium uptake in this system (25), the possibility that receptor phosphorylation is an indirect consequence of calcium uptake is eliminated. Furthermore, phosphorylation does not occur as a direct or indirect effect of a change in the concentration of specific proteins by a regulation of transcrip- tion.

Although in other systems, 1,25-(OH)2D3 has been shown to up-regulate its receptor (27), the 1,25-(OH)nD3 receptor phosphorylation observed here is not due to an increase in the amount of the receptor isolated from tissue. The immu- noprecipitation of the [3”S]methionine-labeled receptor at various times after 1,25-(OH)*D3 addition indicated no signif- icant increase in the labeled receptor detected at 6 and 12 h (data not shown). Estimate of the immunoprecipitated recep- tor by immunoblotting also did not indicate an increase in total receptor protein (data not shown). The phosphorylation observed here cannot be explained by an increase in receptor synthesis rate since phosphorylation was unaffected by block- ing protein synthesis with cycloheximide.

The regulation of proteins by protein kinases and phospha- tases is important in the control of cellular processes (8). Phosphorylation has been found to be necessary for transcrip- tional activation by the transcription factor CREB (28). How- ever, although the phosphorylation of many members of the steroid/thyroid hormone receptor gene family has been re- ported, its function remains unclear in each case. Recently, an effect of phosphorylation on progesterone receptor-specific DNA binding (29) or stimulation of transcription (30) could not be demonstrated. Also, no difference in phosphorylation state between the untransformed and transformed glucocor- ticoid receptors was detected (31). On the other hand, tyrosine phosphorylation of the estrogen receptor has been reported to activate steroid binding activity (15).

Although this report is suggestive of an important role of phosphorylation of the 1,25-(OH)2D:j receptor, such a conclu- sion is not yet warranted. However, our results are sufficiently compelling to encourage a determination of the sites of phos- phorylation, the nature of the kinase carrying out the phos- phorylation, and a determination if phosphorylation is re- quired for 1,25-(OH)pD:1 to exert its activity through its recep- tor.

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T A Brown and H F DeLuca1,25-dihydroxyvitamin D3 action.

Phosphorylation of the 1,25-dihydroxyvitamin D3 receptor. A primary event in

1990, 265:10025-10029.J. Biol. Chem. 

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