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J Steroid Biochem Mol Biol. Author manuscript; available in PMC 2011 Jun 20.
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PMCID: PMC3118558



Upon ligand binding the 1α,25-dihydroxy vitamin D3 receptor (VDR) undergoes a conformational change that allows interaction with coactivator proteins including p160/SRC family members and the multimeric DRIP complex through the DRIP205 subunit. Casein kinase II (CKII) phosphorylates VDR both in vitro and in vivo at serine 208 within the hinge domain. This phosphorylation does not affect the ability of VDR to bind DNA, but increases its ability to transactivate target promoters. Here, we have analyzed whether phosphorylation of VDR by CKII modulates the ability of VDR to interact with coactivators in vitro. We find that both mutation of serine 208 to aspartic acid (VDRS208D) or phosphorylation of VDR by CKII enhance the interaction of VDR with DRIP205 in the presence of 1α,25-dihydroxy vitamin D3. We also find that the mutation VDRS208D neither affects the ability of this protein to bind DNA nor to interact with SRC-1 and RXRα. Together, our results indicate that phosphorylation of VDR at serine 208 contributes to modulate the affinity of VDR for the DRIP complex and therefore may have a role in vivo regulating VDR-mediated transcriptional enhancement.

Keywords: 1α, 25-dihydroxy vitamin D3 receptor, Coactivators, Transcription


1α,25-dihydroxy vitamin D3 modulates transcription after interacting with a specific cellular receptor (VDR), which is a member of the superfamily of nuclear receptors that recognize specific regulatory elements within the promoter region of target genes [1,2]. Upon ligand binding, VDR suffers a conformational change at the C-terminal ligand binding domain (LBD) that allows recognition of this domain by coactivators of the p160/SRC family, an event that is critical for transcriptional activation [1,2]. p160/SRC coactivators contain intrinsic histone acetyl transferase (HAT) activity. Therefore, protein complexes containing HAT activity are recruited to target gene promoters by nuclear receptors in a ligand-dependent manner. Once associated with these promoters the HAT activity can mediate the chromatin remodeling events that accompany steroid hormone-regulated transcriptional enhancement [3].

The multisubunit DRIP (VDR-Interacting Protein) complex also binds to VDR in response to 1α,25-dihydroxy vitamin D3 [1,2]. This interaction requires the LBD of VDR and occurs in much the same manner as for the p160/SRC coactivators, resulting also in transcriptional enhancement. DRIP interacts with nuclear receptors through a single subunit called DRIP205, which anchors the rest of the DRIP complex to the LBD of VDR. Several subunits found in the DRIP complex are also part of the Mediator complex [4], which interacts with the C-terminal domain (CTD) of the RNA polymerase II complex, therefore forming the holoenzyme complex. Hence, it has been proposed that DRIP205 may be functioning as a coactivator by directly connecting VDR and the basal transcriptional machinery [1].

Recent reports indicate that p160/SRC and DRIP205 coactivators are recruited to hormone-regulated promoters in a sequential and cyclical manner, exhibiting high exchange rates [5,6,7,8,9]. However, the molecular mechanisms that control this rapid and sequential recruitment have not been assessed. Previous reports have shown that phosphorylation reactions regulate the transactivation potential of VDR in osteoblastic cells [10,11]. Here, we evaluate whether phosphorylation of human VDR on serine 208 modulates the ability of this receptor to interact with the coactivators SRC-1 and DRIP205. We find that phosphorylation of this residue in vitro by CKII enhances the interaction between VDR and DRIP205 without affecting the interaction with SRC-1.

Materials and Methods

Production of GST fusion proteins and in vitro protein phosphorylation

The pGEX-VDRS208D vector coding for a mutated version of VDR in which serine 208 has been mutated to aspartic acid, was generated by site direct mutagenesis of the pCDNA-VDR plasmid [12] using the primers 5’-caatctggatctggatgaagaagattcag-3’ (forward) and 5’-ctgaatcttcttcatccagatccagattg-3’ (reverse). The mutated VDR gene was then cleaved with EcoRI and NotI and cloned into the pGEX5X3 vector (Pharmacia Biotech, Uppsala, Sweden). The fusion protein glutathione-S-transferase GST-VDR and GST-VDRS208D were obtained by expression in Escherichia coli BL21 as previously reported [12]. His tag-fused Casein Kinase II subunit alpha (CKIIα) was produced in bacteria by expressing pT7HX-His-CKIIα plasmid (kindly donated by Dr. Jorge Allende) and purified through Ni++ NTA affinity chromatography (Novagen, Darmstadt, Germany) under manufacture’s directions. 40 pmol of GST-fusion proteins were immobilized in 20 µl of glutathione sepharose resin (Pharmacia Biotech, Uppsala, Sweden) and phosphorylated in vitro with 2 pmol of purified CKIIα. The reaction was performed in 20 µl of Reaction Buffer (150 mM KCl; 0.5 mM DTT; 5 mM MgCl2; 20 mM Tris-HCl pH=7.4) supplemented with 100 µM ATP (Calbiochem, La Jolla, CA) for 30 min at 30°C. The resin was then washed several times with 1 mL of Reaction Buffer and the GST-fusion proteins eluted as described before [12].

GST-pull down assay

GST pull-down assays were carried out as describe before [13]. Co-precipitated VDR, RXRα, SRC-1 and DRIP205 proteins were detected by Western blotting using specific antibodies [C-20 for VDR, D-20 for RXRα̣, M-255 for DRIP205 (Santa Cruz Biotechnology, Santa Cruz, CA) and clone 1135 for SRC-1 (Upstate Biotechnology, Lake Placid, NY)].


Binding of GST-VDR and GST-VDRS208D to the osteocalcin (OC) VDRE was evaluated by EMSA as described before [14].

Results and Discussion

Previous reports indicated that phosphorylation reactions play an important role in the ability of VDR to upregulate transcription in a ligand-dependent manner [10,11]. It has been shown that human VDR can be phosphorylated in the serine residue 208 by the protein kinase CKII and that this modification increases the ability of this receptor to enhance transcription in response to 1α,25-dihydroxy vitamin D3 [15 and data not shown]. Therefore, we assessed whether phosphorylation in this serine residue 208 contributes to the cellular mechanisms that regulate the interaction of VDR with transcriptional coactivators.

We began our studies by evaluating the ability of bacterially produced VDR to bind coactivators in vitro that are present in nuclear extracts isolated from ROS 17/2.8 osteoblastic cells by GST-pull down assays. Recombinant full length VDR (GST-VDR, Figure 1A) or truncated forms of this protein where the C-terminal (GST-VDRΔ111, Figure 1A) or N-terminal (GST-VDRΔ1-111, Figure 1A) domains have been deleted were produced in bacteria as reported earlier [12]. Figure 1B shows that GST-VDR binds to RXRα, SRC-1, and DRIP205 proteins only in the presence of 1α,25-dihydroxy vitamin D3 (Figure 1B, compare lanes 2 and 3). As expected, these ligand-dependent interactions require an intact C-terminal LBD of VDR, as the GST-VDRΔ1-111 mutant receptor protein, which lacks the N-terminal region of VDR, was capable of recruiting SRC-1 and DRIP205 only in the presence of 1α,25-dihydroxy vitamin D3 (Figure 1B, lanes 6 and 7). Accordingly, the GST-VDRΔ111 mutant receptor, which lacks the LBD was unable to precipitate SRC-1 and DRIP205 in either the presence or absence of 1α,25-dihydroxy vitamin D3 (Figure 1B, lanes 2 and 3). Interestingly, we find that the GST-VDRΔ1-111 mutant receptor protein consistently binds to both coactivators with higher affinity than GST-VDR, which contains the full-length VDR protein (Figure 1B, compare lanes 3 and 7). This result indicates that the LBD domain of VDR can function as an independent domain and does not require the DNA binding domain or the short N-terminal AF-1 region to recruit coactivators. In addition, both truncated VDR forms bind poorly to RXRα (Figure 1B, compare lanes 3, 5 and 7), confirming previous reports indicating that at least two domains of VDR are required for efficient ligand-dependent association with RXRα [16].

Figure 1
Bacterially produced GST-VDR proteins bind SRC-1 and DRIP205 coactivators in a 1α,25-dihydroxivitamin D3-dependent manner

We next determined whether phosphorylation of VDR in vitro by CKII, changes the ability of this receptor to interact with coactivators SRC-1 and DRIP205 by GST-pull down assays. As shown in Figure 2B, GST-VDR interacts with both RXRα and DRIP205 in a 1α,25-dihydroxy vitamin D3-dependent manner (Figure 2B, compare lanes 3 and 4). Interestingly, when VDR is phosphorylated by CKII (data not shown) the interaction with DRIP205 is markedly increased (Figure 2B, compare lanes 4 and 6). This increased interaction is specific as the binding between phosphorylated VDR and its heterodimer partner RXRα remains similar to the level exhibited by unphosphorylated VDR (Figure 2B, middle panel, compare lanes 4 and 6). Similarly, this increased VDR-DRIP205 interaction was not due to the presence of the GST moiety, as GST neither interferes with VDR-coactivator interactions (Figure 2B, lane 2) nor is phosphorylated under our experimental conditions (data not shown).

Figure 2
Phosphorylation of VDR by CKII enhances interaction with coactivator DRIP205

In contrast to DRIP205, the 1α,25-dihydroxy vitamin D3-dependent association of VDR with SRC-1 is neither enhanced nor down-regulated after phosphorylation by CKII (Figure 2C, compare lanes 3 and 4). Together, these results indicate that phosphorylation of VDR by CKII specifically increases the affinity of this receptor for the coactivator DRIP205.

To further demonstrate that a phosphorylated serine 208 residue in VDR is required for an increased interaction with DRIP205, we mutated this residue to aspartic acid (GST-VDRS208D, see Figure 3A), therefore mimicking the presence of a negatively charged phosphate group in this position [17]. This aminoacid change does not affect the ability of the VDRS208D protein to recognize its specific DNA target sequence (Figure 3). Thus, EMSA analyses show that the bacterially-produced GST-VDRS208D protein binds to the rat osteocalcin (OC) VDRE with an affinity equivalent to that of wild-type GSTVDR (Figure 3B, compare lanes 2 and 3). Interestingly, GST-pull down analyses demonstrate that the VDRS208D protein interacts with the DRIP205 coactivator with markedly higher affinity (Figure 3C, upper panel, compare lanes 3 and 4). This increased interaction is specific as the 1α,25-dihydroxy vitamin D3-dependent association of VDRS208D protein with SRC-1 and RXRα remains unaffected (Figure 3B, compare lanes 3 and 4).

Figure 3
Replacing serine residue 208 of VDR by aspartic acid results in enhanced VDR-DRIP205 interaction

Taken together, our results indicate that phosphorylation on serine residue 208 can modulate the affinity of VDR for the DRIP complex. Therefore, this covalent modification may have a role in vivo by regulating 1α,25-dihydroxy vitamin D3-mediated transcriptional enhancement. The results are in agreement with previous reports indicating the relevance of phosphorylation reactions in 1α,25-dihydroxy vitamin D3-dependent transcriptional regulation [10,11]. Moreover, these results offer a molecular explanation for how phosphorylation may directly influence the ability of VDR to alternatively interact with p160/SRC and DRIP205 coactivators during transcriptional upregulation of 1α,25-dihydroxy vitamin D3-responsive genes.


The authors thank Dr. Fernando Cruzat for the interesting discussions and critical reading of this manuscript. We also thank Dr. Jorge Allende for generously providing the pT7HX-His-CKIIα plasmid. This work has been supported by grants from CONICYT-PBCT ACT-044 (to M.M) and NIH PO1 AR48818 (to G.S.S). G.A. was supported by fellowships from CONICYT and MECESUP-RED UCH0115. R.P. was supported by fellowships from CONICYT, MECESUP and FUNDACION ANDES.


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