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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Mol Cell Biochem. Author manuscript; available in PMC Mar 14, 2009.
Published in final edited form as:
Mol Cell Biochem. Apr 2001; 220(1-2): 1–13.
PMCID: PMC2655343
NIHMSID: NIHMS92278

The SMRT corepressor is a target of phosphorylation by protein kinase CK2 (casein kinase II)

Abstract

The Silencing-Mediator for Retinoid/Thyroid hormone receptors (SMRT) interacts with, and mediates transcriptional repression by, a variety of eukaryotic transcription factors, including the nuclear hormone receptors. The ability of SMRT to function as a transcriptional ‘corepressor’ is regulated by a variety of signal transduction pathways. We report here that SMRT is a phosphoprotein in vivo, and is also phosphorylated in vitro by unfractionated cell extracts. A major site of phosphorylation of SMRT is a protein kinase CK2 motif centered on serine 1492, and located within a C-terminal SMRT domain that mediates interaction of the corepressor with the nuclear hormone receptors. Phosphorylation of SMRT by CK2 stabilizes the ability of the SMRT protein to interact with nuclear hormone receptors. Our results indicate that SMRT is a member of an expanding family of transcriptional regulators that are modified, and potentially regulated, in response to protein kinase CK2.

Keywords: corepressor, nuclear hormone receptors, protein kinase, CK2, casein kinase 2

Introduction

Nuclear hormone receptors, such as the steroid, retinoid, and thyroid hormone receptors, are hormone-regulated transcription factors that play many critical roles in vertebrate differentiation, reproduction, and homeostasis [1-5]. Nuclear hormone receptors function as ligand-regulated transcription factors, binding to the promoters of specific target genes and regulating their transcription in response to cognate hormone. Intriguingly, many nuclear hormone receptors possess bipolar transcriptional properties, and can either repress or activate expression of a given target promoter depending on the hormone status, the nature of the DNA binding site, and the impact of other signal transduction pathways operating in the cell [6-9].

The ability of nuclear hormone receptors to function in transcription in such a diverse manner is due to the ability of these receptors to recruit an array of auxiliary proteins, denoted corepressors and coactivators, that mediate the actual molecular events that lead to transcriptional regulation [10-14]. Thyroid hormone receptors (T3Rs), for example, typically bind to the SMRT (silencing mediator for retinoid/thyroid hormone receptors) and N-CoR (nuclear hormone receptor-corepressor) family of corepressor proteins in the absence of hormone, leading to repression of target gene transcription [15-19]. Conversely, the binding of hormone by T3R leads to a physical release of the corepressors from the nuclear receptor and the acquisition of novel coactivator proteins, such as SRC-1 (Steroid Receptor Coactivator-1), p300, TRAPs (T3R-Associated Proteins), or DRIPs (vitamin D3-Receptor Interacting Proteins), which convert the T3R into a transcriptional activator [10, 20-23]. The precise mechanisms by which these coactivators and corepressors regulate transcription remain incompletely understood, but appear to include both modifications of the chromatin template and interactions with components of the general transcriptional machinery [12, 14, 20-22, 24-31].

Cognate ligand plays a important role in regulating the transcriptional properties of the nuclear hormone receptors by regulating the interaction of these receptors with the SMRT/N-CoR corepressors [15-17, 19, 32-35]. However, the ability of nuclear hormone receptors to repress or activate transcription is also under tight regulation by many nonligand signal transduction pathways operating in the cell. For example, activation of the epidermal growth factor receptor, a membrane-associated tyrosine kinase, strongly interferes with transcriptional repression by T3R, apparently by interfering with the ability of corepressor to interact with the nuclear hormone receptor [36, 37]. Similarly, corepressor function has also been reported to be regulated in response to cyclic AMP signaling [38]. In many cases the nuclear hormone receptor is not itself detectably modified in response to these signaling events, suggesting that it is the corepressor that is the actual target of the regulatory pathway [36, 39]. We therefore have investigated if the SMRT corepressor is post-translationally modified in cells, and if these post-translational modifications can alter SMRT function. We report here that indeed SMRT is phosphorylated when expressed in mammalian cells in culture, and that SMRT can also be phosphorylated in vitro when incubated with nuclear extracts of these cells. A major site of SMRT phosphorylation, in vitro and in vivo, is a protein kinase CK2 phosphorylation motif located at serine 1492 within the C-terminal receptor-interaction domain of the SMRT corepressor. Phosphorylation of this site by CK2 (formally known as casein kinase II) resulted in a modest, but reproducible stabilization of the ability of SMRT to interact with thyroid hormone receptors. We conclude that SMRT is a member of the expanding class of transcription factors that are modified by protein kinase CK2, and for which this modification may play a role in function.

Materials and methods

Plasmid constructs

The pSG5-T3Rα, pGEX-SMRT, pCMV-SMRT, and green fluorescent protein (GFP)-SMRT derivatives were constructed as previously described [17, 39, 40]. Base substitution mutations, designed to abolish potential phosphorylation sites within the SMRT sequence, were created by oligonucleotide-mediated mutagenesis using polymerase chain reaction and standard recombinant DNA methodologies [41]. Deletion mutations within the C-terminus of the pGEX-KG SMRT (1291−1495) molecular clone were created by an exonuclease III/S1 nuclease protocol, using a double-stranded nested deletion kit (Amersham Pharmacia, Piscataway, NJ, USA). The mammalian expression vector, pCMV5-CK2α, was obtained from G.J. Rosman and A. Dusty Miller (Fred Hutchison Cancer Research Center).

In vitro kinase assays

CV-1 cells were maintained in Dulbecco-modified Eagles medium (Gibco/BRL Life Technologies, Rockville, MD, USA) supplemented with 10% fetal bovine serum and penicillin/streptomycin. Cells were harvested by mechanical scrapping and collected by centrifugation. CV-1 cell lysates were prepared by sonication of harvested cells in 25 mM HEPES pH 7.5, 300 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1% Triton X-100, 0.1 mM dithiothreitol, and COMPLETE protease inhibitor cocktail (Berhinger-Mannheim, Germany). The GST-SMRT fusion proteins were expressed in recombinant Escherichia coli, immobilized on glutathioneagarose, and repeatedly washed, as previously described [36]. The immobilized GST-SMRT proteins were then incubated at 30°C for 20 min with 30 μl of CV-1 cell lysate in 400 μl of 20 mM HEPES, pH 7.7, 20 mM MgCl2, 20 mM β-glycerol phosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, and 20 μM ATP containing 5 μCi [γ-32P] ATP. Alternatively, the immobilized GST-SMRT proteins were incubated with 500 units of purified, recombinant CK2 isolated from E. coli (New England Biolabs, Beverly, MA, USA) in 200 μl of 20 mM Tris-Cl, pH 7.5, 50 mM KCl, 10 mM MgCl2, and 100 μM ATP containing 5 μCi [γ-32P] ATP. The kinase reactions were terminated by washing the samples twice and resuspending the samples in SDS-sample buffer. The samples were then boiled for 5 min and the proteins were resolved by SDS-polyacrylamide gel electrophoresis (PAGE). Phosphorylated proteins were visualized and quantified by PhosphoImager analysis using a Storm 820 or 840 System (Molecular Dynamics, Sunnyvale, CA, USA).

Phosphorylation-dephosphorylation assays

One microgram of the pSG5-SMRT (1291−1495) expression vector was introduced into 1.5 × 105 CV-1 cells in the presence or absence of 500 ng of the pCMV5-CK2α expression vector, using a Lipofectin protocol (Gibco/BRL Life Technologies, Rockville, MD, USA). The cells were harvested 48 h later by mechanical scraping into 1.2 ml of Tris-Buffered Saline buffer (10 mM Tris-Cl, pH 8.0, 150 mM NaCl) and concentrated by centrifugation. For the phosphorylation assay, the cells were resuspended in 100 μl of 20 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1 mM Na3VO4, 10 mM NaF, 1 mM dithiothreitol, and COMPLETE protease inhibitor cocktail, and were lysed by sonication. Cell lysates were then analyzed by SDS-PAGE electrophoresis followed by an immunoblotting procedure using SMRT-directed antibodies [39]. Two electrophoresis systems were employed to analyze the electrophoretic shift associated with phosphorylation of the SMRT protein: a high cross-linker system (12% polyacrylamide, 0.5% bis-acrylamide) or an SDS/urea, low cross-linker system (12% polyacrylamide, 0.32% bis-acrylamide) [42]. For the dephosphorylation assay, the transfected cells were resuspended in 50 mM Tris-Cl, pH 7.9, 100 mM NaCl, 10 mM MgCl2, and COMPLETE protease inhibitor. The cell lysates were then incubated in the presence or absence of 10 units of calf intestinal alkaline phosphatase (New England Biolabs, Beverly, MA, USA) for 10 min at 37°C. The reactions were terminated by mixing the samples with SDS-Urea sample buffer [42], and the samples were subjected to electrophoresis and immunoblotting as for the phosphorylation assay.

Thyroid hormone receptor-corepressor binding assay in vitro

The GST-SMRT (1291−1495) fusion protein was expressed in E. coli, was immobilized on glutathione-agarose, and was purified as described above. The immobilized GST-SMRT protein was then incubated at 30°C for 40 min with 500 units of purified, recombinant CK2 isolated from E. coli (New England Biolabs, Beverly, MA, USA) in 100 μl of 20 mM Tris-Cl, pH 7.5, 50 mM KCl, 10 mM MgCl2, and 200 μM ATP, or was mock-treated in the same buffer without kinase. 35S-radiolabled T3Rα protein was synthesized by a coupled in vitro transcription-translation system (Promega, Madison, WI, USA), and was incubated with the immobilized GST-SMRT in HEMG buffer, as described before [39]. After repeated washing, any radiolabeled T3R remaining bound to the GST-SMRT matrix was eluted, resolved by SDS-PAGE, and visualized/quantified by PhosphoImager analysis.

Laser scanning confocal microscopy

The subcellular localization of GFP-fusions of the wild-type and S1492A mutant forms of the GFP-SMRT protein was determined as previously described [39].

Transient transfections

Approximately 7 × 104 CV-1 cells per assay were transfected using the Lipofectin protocol with 100 ng of pCMV5-CK2α vector, 20 ng of pSG5-T3Rα vector, 100 ng of a thymidine kinase promoter-luciferase reporter containing a DR-4 hormone response element, and 50 ng of a pCMV-lacZ reporter (used as an internal control) [36]. Equal quantities of the empty vectors were substituted as appropriate to maintain the total amount of DNA the same in all transfections. Five hours after the transfection procedure, the cells were transferred to medium containing 100 nM of T3-thyronine hormone, or an equivalent amount of ethanol carrier, and the cells were maintained at 37°C for an additional 43 h. Luciferase and β-galactosidase assays were performed as previously described [36].

Protein stability assay

Approximately 1.5 × 105 CV-1 cells per assay were transfected with 1 μg of pSG5-SMRT expression vector and 500 ng of pCMV5-CK2α (or an equivalent amount of an empty expression vector). Thirty-six hours after transfection, the cells were transferred to serum-free medium containing 20 μg/ml of cycloheximide to inhibit further protein synthesis, and samples were harvested at different time points thereafter. For each time point, cells were washed, suspended in 100 μl of 10 mM Tris-Cl, pH 8.0, 150 mM NaCl, 1 mM DTT and COMPLETE protease inhibitor cocktail, lysed by sonication, and subjected to SDS-PAGE and immunoblotting.

Results

The SMRT corepressor is phosphorylated in vivo

We first investigated if the SMRT corepressor protein was subject to post-translational modifications in cells, either constitutively or in response to EGF-receptor signaling, that might be reflected as changes in its electrophoretic mobility. We employed SDS-polyacylamide gel electrophoresis and Western blot analysis to detect the SMRT corepressor. Notably, the SMRT protein displayed a heterogeneous electrophoretic mobility in CV-1 cells in the absence of treatment, with a further decrease in apparent mobility observed when the cells were treated with tumor growth factor (TGF)α, an inducer of EGF-receptor signaling (Fig. 1A). The heterogeneous mobility of the corepressor, and its change in response to EGF-receptor signaling, was also observed when employing constructs restricted to the C-terminus of SMRT (Fig. 1B, and data not shown), indicating that this putative modification(s) mapped, at least in part, to within the receptor-interaction domain of the corepressor. Changes in the electrophoretic mobility of many proteins can be attributed to phosphorylation; we therefore tested the ability of alkaline phosphatase to reverse the heterogeneous mobility of the SMRT protein. Treatment with purified preparations of calf alkaline phosphatase converted the slower migrating SMRT species to a faster migrating form (Fig. 1B). We conclude that a basal level of phosphorylation occurs within the C-terminal domain of the SMRT corepressor that is augmented by EGF-signaling and reversed by phosphatase treatment.

Fig. 1
Electrophoretic mobility/phosphorylation of SMRT in CV-1 cells. (A) Heterogeneity in the electrophoretic mobility of SMRT in CV-1 cells, and its alteration in response to TGFα treatment. CV-1 cells were transfected by a pSG5-SMRT expression vector ...

The SMRT protein is also a substrate for phosphorylation in vitro

To identify the sites of, and the kinases responsible for, the phosphorylation of SMRT described above, we tested the ability of CV-1 cell lysates to phosphorylate in vitro various subdomains of SMRT (Fig. 2A). Using this method, we observed that a GST-fusion representing the C-terminal 204 amino acids of SMRT (codons 1291−1495) was strongly phosphorylated in our in vitro kinase assay, whereas GST-fusions representing other domains of SMRT, or the non-recombinant GST polypeptide itself, were relatively ineffective as substrates (Fig. 2B). Theses results indicated that the same C-terminal region of SMRT modified in vivo could also be phosphorylated in vitro.

Fig. 2
Phosphorylation in vitro of different SMRT subdomains by CV-1 cell lysates. (A) Schematic representation of the SMRT corepressor. A representation of the SMRT protein is presented from N- to C- terminus. The regions involved in transcriptional silencing ...

To further delineate the site(s) of phosphorylation within SMRT, we generated a series of small deletions within the 3′ end of the SMRT open reading frame, expressed the corresponding proteins as GST fusions, and tested these fusions for the ability to serve as substrates in the our in vitro phosphorylation assay (Fig. 2C). A SMRT 3′ deletion that removed only non-coding regions of the mRNA had no effect on SMRT phosphorylation, whereas a slightly larger deletion, removing fourteen C-terminal amino acids from the corepressor (Δ1482), severely impaired the ability of the corresponding GST-SMRT fusion polypeptide to be phosphorylated by the CV-1 cell lysates (Fig. 2C). GST-fusions representing more extensive SMRT deletions were similarly unable to be phosphorylated in the kinase assay (e.g. Δ1480 and data not shown). The deletions did not significantly alter the level of expression or the stability of the resulting GST-SMRT fusion proteins, and all constructs were tested at comparable protein concentrations in the in vitro assay (data not shown).

Our results therefore indicated that either the actual site of phosphorylation itself, or an important recognition or conformational motif essential for kinase recognition, lay within the C-terminal 14 amino acids of the corepressor. There are five amino acids in the SMRT sequence within this region that conceivably might serve as sites of acid-stable phosphorylation, represented by three serines, one threonine, and one tyrosine (Fig. 3A, underlined). We therefore individually substituted each of these amino acids within the SMRT C-terminal domain to alanine, and tested the ability of the resulting GST-fusions to serve as substrates in the in vitro kinase assay (Fig. 3B). The GST-SMRT fusion with the S1492A substitution was significantly impaired in the ability to be phosphorylated in our in vitro assay, whereas the other four amino acid substitutions displayed wild-type, or near wild-type, substrate activity (Fig. 3B, and quantified in Fig. 3C). We conclude that the retention of serine 1492 is important for the ability of wild-type SMRT to be phosphorylated in vitro, and therefore probably represents the predominant site of phosphorylation by CV-1 cell extracts in our assay.

Fig. 3
Phosphorylation in vitro of SMRT mutants by CV-1 cell lysates. (A) C-terminal amino acid sequence of SMRT. The amino acid sequence of SMRT is presented, using the numbering system derived from the 1495 amino acid version of SMRT as initially described ...

Serine 1492 of SMRT is phosphorylated by protein kinase CK2

Inspection of the SMRT amino acid sequence revealed that serine 1492, identified above, lies within a protein kinase CK2 recognition motif (S-X-X-D/E; [43]). To determine if serine 1492 was indeed a substrate for CK2, we next tested the ability of purified CK2 to phosphorylate the SMRT protein in vitro. The GST-SMRT-RID-2 fusion polypeptide was readily phosphorylated by CK2 under these conditions (Fig. 4A). In contrast no phosphorylation was detected employing equal or greater amounts of non-recombinant GST polypeptide, or of GST-SMRT constructs representing more N-terminal domains of corepressor (compare the phosphorylation in Fig. 4A with the Coomassie blue staining pattern in Fig. 4B). The phosphorylation of the GST-SMRT C-terminus was dependent on addition of CK2, indicating that this modification was not due to a hypothetical autophosphorylation of the GST-SMRT polypeptide or due to a contaminating bacterial kinase (Fig. 4A). Estimates based on specific activity considerations suggest that approximately one mole of phosphate could be incorporated per mole of GST-SMRT fusion protein (data not shown).

Fig. 4
Phosphorylation in vitro of different SMRT subdomains by purified CK2. (A) 32P-incorporation. GST fusion proteins representing different portions of SMRT, as indicated below the Fig., were synthesized in E. coli, were immobilized on glutathione-agarose, ...

To test if serine 1492 of SMRT was the actual site of phosphorylation under these conditions, we compared the ability of our different alanine substitution mutants to serve as substrates for purified CK2 (Fig. 5). Consistent with the identification of serine 1492 as a likely CK2 recognition site by sequence analysis, the S1492A mutation severely impaired the ability of SMRT to be phosphorylated by CK2 in vitro. In contrast, the four other amino acid substitution mutants tested exhibited virtually wild-type levels of phosphorylation by CK2.

Fig. 5
Phosphorylation in vitro of SMRT mutants by purified CK2. (A) Effects of alanine substitutions on phosphorylation of SMRT by purified CK2. The different alanine substitutions of GST-SMRT (1291−1495), shown in Fig. 3A, were tested for the ability ...

To determine if SMRT could be phosphorylated by CK2 in vivo, as well as in vitro, we tested the ability of an exogenously-introduced molecular clone of the CK2α catalytic subunit to further enhance the basal levels of SMRT phosphorylation observed in transfected CV-1 cells. Co-expression of CK2α increased the amount of SMRT displaying a retarded electrophoretic mobility relative to SMRT isolated from mock-treated cells, and both this CK2-mediated modification, and the basal modification observed in the absence of CK2, were reversed by calf-intestine alkaline phosphatase (Fig. 6A, left panel). Similarly, a retardation in the electrophoretic mobility of SMRT could also be observed when a purified GST-SMRT construct, isolated from E. coli, was incubated with purified CK2 in vitro (Fig. 6A, right panel). Although these changes in mobility in response to the introduction of CK2 could be observed in several different electrophoretic systems, it was most clearly detected in an urea/SDS/polyacrylamide gel system in which phosphorylation results in an increased, rather than a decreased, SMRT mobility (Fig. 6B). Consistent with our interpretation that the faster mobility of SMRT in the urea gel system represents a change in phosphorylation state of serine 1492, the increased mobility associated with CK2 coexpression was reversed on alkaline phosphatase treatment and was not observed with the SMRT S1492A mutant (Figs 6C and 6D). Taken as a whole, our experiments indicate that serine 1492 is a major site of phosphorylation within the SMRT corepressor, and that this amino acid is modified by either by CK2, or by a kinase possessing a CK2 recognition specificity.

Fig. 6
Effect of CK2α over-expression on the electrophoretic mobility and phosphorylation state of SMRT in transfected cells. (A) Effect of overexpression of CK2α on the electrophoretic mobility of SMRT using SDS-PAGE. Left panel: CV-1 cells ...

As noted previously, the basal phosphorylation of the SMRT protein in CV-1 cells was further altered in response to EGF-receptor signaling. This change in SMRT mobility in response to EGF-receptor signaling was of a greater magnitude than that observed in response to CK2, suggesting that SMRT might be modified at multiple sites, and that not all of these modifications were due to CK2 phosphorylation of serine 1492 (compare Fig. 1 to Fig. 6A). Confirming this hypothesis, although the S1492A SMRT mutant could not be phosphorylated by CK2, this mutant protein was nonetheless modified in response to EGF-receptor signaling (data not shown). In separate work, we have determined that at these additional modifications of SMRT in response to EGF-receptor signaling represent phosphorylation by components of a MAP kinase kinase kinase (MAPKKK) pathway [39].

Phosphorylation by CK2 stabilizes the ability of SMRT to interact with thyroid hormone receptor in vitro

We next wished to determine if phosphorylation of SMRT by protein kinase CK2 had a detectable effect on corepressor function. Given that the location of the CK2 site is within a region of SMRT involved in the interaction of corepressor with nuclear hormone receptors, we first tested if phosphorylation of this site in vitro altered the ability of the corepressor to bind to T3R. We employed an ‘GST-pull-down’ protocol to measure the ability of a GST-SMRT fusion polypeptide, synthesized in E. coli and immobilized on glutathione agarose, to bind to 35S-radiolabeled T3R, synthesized by in vitro transcription and translation (Fig. 7). In the absence of CK2 treatment, the GST-SMRT fusion protein bound the 35S-tagged T3R, whereas little or no binding of T3R was observed to a non-recombinant GST construct employed as a negative control (Fig. 7A). Pre-treatment of the GST-SMRT construct with CK2 led to a modest, although reproducible, enhancement of the ability of the SMRT construct to interact with T3R in this assay. This enhancement of the SMRT/T3R interaction was most clearly observed when the GST-binding assay was performed at moderate concentrations of thyroid hormone (Fig. 7A) or at high salt concentrations (Fig. 7B). High salt and thyroid hormone are both able to disrupt the SMRT/T3R interaction, and our results suggest that CK2 phosphorylation partially counteracts these destabilizing effects. It should be noted that the GST-SMRT preparation was efficiently phosphorylated in our experiments, with virtually the entire GST-SMRT polypeptide being converted by CK2 to the SDS-PAGE mobility characteristic of the phosphorylated form (Fig. 6A and data not shown).

Fig. 7
Effects of CK2 phosphorylation on the interaction of SMRT with T3Rα. (A) Increased binding of phosphorylated GST-SMRT to T3Rα at low T3 concentrations. The GST-SMRT(1291−1495) fusion was synthesized in E. coli, immobilized on glutathione-agarose, ...

We next examined if other properties of SMRT were detectably altered by CK2 modification. CK2 modification has been noted to alter the stability of many proteins (e.g. [44-47]). We therefore tested the turnover rate of SMRT in cells in the absence or presence of exogeneously introduced CK2α, comparing wild-type SMRT and the S1492A mutant. Notably, no significant difference in the turnover or half-life of SMRT, could be detected in response to CK2 under these conditions (Fig. 8A). Phosphorylation by CK2 has also been reported to alter the subcellular localization of many protein substrates (e.g. [48-51]). We therefore next employed GFP-fusions of SMRT to characterize the subcellular distribution of the corepressor in the absence or presence of a CK2 phosphorylation site. As reported previously (e.g. [39, 52]), wild-type SMRT, either native or expressed as a GFP-fusion, exhibits a primarily nuclear localization (Fig. 8B); within the resolution of this technique, the GFP-SMRT S1492A mutant displayed the same nuclear localization as did the GFP-SMRT wild-type (Fig. 8B).

Fig. 8
Effects of CK2 on SMRT stability, subcellular localization, and T3R-mediated gene regulation. (A) Effect of CK2α over-expression on the stability of SMRT protein. CV-1 cells were transfected with the wild type or S1492A mutant SMRT expression ...

The ability of nuclear hormone receptors to recruit corepressors or coactivators is reflected in their ability to repress or activate target gene expression in cells [9]. We therefore tested if the stabilization of the interaction of SMRT with T3R by CK2 phosphorylation, observed in vitro, was also manifested as a change in the repression/activation properties of the T3R when assayed in transfected cells. We employed transient transfections of CV-1 cells, introducing T3Rα, a thymidine kinase promoter-luciferase reporter gene construct containing a binding site for T3R, and either the CK2α expression vector, or equivalent amounts of empty vector (Fig. 8C). As anticipated, T3Rα repressed the expression of the luciferase reporter gene in the absence of hormone. Addition of T3 hormone resulted in a dose-dependent reversal of this repression and an increased activation of reporter gene expression (Fig. 8C). Notably, however, this ability of T3Rα to respond to T3 hormone by reversing repression, and by inducing expression of the reporter gene was impaired by overexpression of CK2α (Fig. 8C, compare the solid and the dashed lines). In contrast, the expression of the reporter gene itself (i.e. in the absence of T3Rα) was not influenced by CK2 expression (Fig. 8C and data not shown). Although the effect of CK2 in this assay was modest, it was reproducible, and paralleled the stabilization of the T3R/SMRT interaction we observe in vitro at intermediate hormone or salt concentrations. These results are consistent with the hypothesis that, by stabilizing the corepressor/nuclear receptor interaction, CK2 phosphorylation of SMRT shifts the equilibrium between corepressor and coactivator binding by T3R, resulting in enhanced corepressor occupancy at intermediate hormone concentrations, and impaired activation.

Discussion

SMRT is phosphorylated by a protein kinase CK2 activity in vitro and in vivo

We wish to better understand the mechanisms by which the transcriptional properties of nuclear hormone receptors are regulated in cells. One major aspect of this regulation is mediated by the binding of cognate hormone to the nuclear hormone receptor, which can convert the receptor from a corepressor-bound transcriptional silencer to a coactivator-bound transcriptional activator [32-35]. However, nuclear hormone receptors serve as a nexus for many different signal transduction pathways in the cell, and non-ligand-based signaling also can have profound effects on transcriptional regulation by the nuclear receptors. For example, many nuclear hormone receptors can be phosphorylated, and these phosphorylation events can mimic, enhance, or oppose the actions of the hormone ligand itself (reviewed in [53-57]). In addition, a number of the auxiliary proteins that modulate or mediate nuclear hormone receptor function appear to themselves be targets of protein kinase signal transducers [39, 58]. We and others have noted previously that the ability of SMRT and N-CoR corepressors to interact with, and mediate repression by, T3Rs and retinoic acid receptors are subject to regulation by a variety of protein kinase signaling pathways in the cell, including growth factor and cyclic AMP-responsive pathways [36-38]. Aspects of this regulation suggested that the SMRT protein, not the nuclear hormone receptor, might serve as the actual target of these protein kinase-mediated regulatory pathways. In the current manuscript, we have shown that the SMRT protein is indeed phosphorylated in cells, and that at least one component of this phosphorylation is mediated by CK2 or by a closely-related activity.

This phosphorylation of SMRT maps to the C-terminal receptor-interaction domain of the corepressor, and can be observed in vitro using a bacterially-synthesized SMRT-derived polypeptide as substrate and either unfractionated CV-1 cell extracts or a purified CK2, as the source of kinase. Deletion of even a small region of the SMRT C-terminus abolished the ability of the corepressor to serve as a substrate for phosphorylation, indicating that the likely site of phosphorylation was very near the corepressor C-terminus. Higher definition genetic mapping, using single amino acid substitutions, demonstrated that the integrity of serine 1492 of SMRT was crucial for phosphorylation in vitro by either CV-1 cells lysates or by purified CK2. Serine 1492 is embedded within a S-X-X-E amino acid motif characteristic of CK2 phosphorylation substrates, consistent with Ser 1492 representing the actual site of phosphorylation in these experiments.

The phosphorylation of SMRT at amino acid 1492 was the most prominent phosphorylation carried out in vitro by CV-1 cells lysates, and was also observed in transfected cells in response to the introduction of the catalytic a subunit of CK2. Nonetheless, serine 1492 is clearly not the only site of phosphorylation of SMRT either in vitro or in vivo. At least 10% of the phosphorylation of the SMRT C-terminus by CV-1 cell lysates in vitro was retained by the S1492A mutant, indicating that other phosphorylation sites must exist elsewhere in this protein. Similarly, the S1492A substitution altered, but did not abolish the ability of SMRT to display an altered mobility in response to EGF-receptor signaling in transfected cells; at least several of these additional modifications to SMRT represent sites of phosphorylation by the components of a MAP kinase kinase kinase cascade [39].

The studies reported here have been confined to the SMRT corepressor. N-CoR is a second member of the corepressor family, and is approximately 50% related to SMRT at the amino acid level [16, 59, 60]. Intriguingly, a CK2 consensus site is located in N-CoR at the equivalent position as that in SMRT (N-CoR amino acid 2449), suggesting that this post-translational modification may also be operative in N-CoR.

Phosphorylation of SMRT by CK2 alters the ability of corepressor to bind to T3R in vitro

We sought to determine what effect, if any, the phosphorylation of serine 1492 might have on SMRT function in vitro or in vivo. SMRT contains two adjacent, but independent protein domains, RID-1 and RID-2, that mediate the ability of the corepressor to interact with its nuclear hormone receptor partners [17-19, 32, 34, 35, 61-67]. The different RID regions display distinct affinities for different nuclear receptors: retinoic acid receptors interact primarily with RID-1, retinoid X receptors interact primarily with RID-2, and the T3Rs are able to interact with both the RID-1 and RID-2 domains of SMRT. Serine 1492 lies within the RID-2 domain; we therefore examined if phosphorylation of SMRT by CK2 could alter the ability of the corepressor to interact with nuclear hormone receptors. CK2 phosphorylation resulted in a modest, but reproducible enhancement in the interaction between GST-SMRT-RID-2 and T3R; this enhanced interaction was manifested primarily as a stabilization of the SMRT/T3R interaction against the disruptive effects of T3 hormone or high salt. Notably, the stabilizing effects of CK2 were observed when SMRT was pre-treated with CK2 and extensively washed prior to the introduction of the T3R, indicating that these effects are due to phosphorylation of SMRT, and not of T3R. Although mapping within the broadly defined RID-2 domain, Ser 1492 lies outside of an I/L-X-X-I-I motif that can function as the minimal contact site between SMRT and T3Rs [32, 34, 35]. The effects of Ser 1492 phosphorylation on the SMRT/T3R interaction may be mediated through hypothetical additional contacts between the nuclear receptors and SMRT RID-2 that map outside of the I/L-X-X-I-I motif itself, or conceivably, phosphorylation of Ser 1492 may alter the overall conformation of the SMRT RID-2 domain in a manner that stabilizes the I/L-X-X-I-I contact site.

Paralleling the stabilization of the SMRT/T3R interaction we observe in vitro, overexpression of CK2α in transfected cells resulted in a diminution of transcriptional activation by T3R under conditions of low to intermediate hormone concentration. We suggest that by enhancing the affinity of SMRT for T3R, CK2 phosphorylation impairs the release of corepressor from the receptor in response to hormone ligand, thereby modifying the equilibrium between T3R molecules in the population bound to corepressor, and the T3R molecules in the population bound to coactivator. This enhanced corepressor association would manifest as an attenuation of reporter gene activation (e.g. [9]), as is indeed observed. Of course, given the multiple roles of CK2 in cells, we cannot rule out that the effects of CK2 on T3R-mediated transcription regulation are mediated, at least in part, by indirect mechanisms not involving the corepressor. However, we note that the basal levels of reporter gene expression in the absence of T3R were not influenced by CK2 overexpression. Similarly, the levels of expression of a pCMV-β-galactosidase reporter, employed as an internal normalization control, were not impaired by overexpression of CK2, nor was the level of expression, subcellular distribution, or stability of SMRT detectably altered by introduction of the CK2 construct. Taken as a whole, therefore, our data suggests that CK2 phosphorylation can stabilize the physical and structural interaction of SMRT with its nuclear receptor partners.

Protein kinase CK2, corepressors, and the control of transcription

Protein kinase CK2 activity has been implicated as playing important roles in transcriptional regulation, cell cycle progression, and growth control in a variety of organisms (reviewed in [43, 68-72]). CK2 is widely expressed in many different cell types, and has been reported to phosphorylate hundreds of different protein substrates. Many of these targets of CK2 phosphorylation are transcriptional regulators; for example, c-Myc, c-Myb, c-Jun, p53, serum response factor, retinoblastoma protein, and RNA polymerase I and II are all phosphorylated by CK2 [43, 68-72]. In addition to these transcription factors, a variety of additional regulatory proteins, such as protein kinase A, DNA ligase, DNA topoisomerase, SV-40 large T-antigen, and several eukaryotic translational initiation factors, are also targets of protein kinase II modification [43, 68-72]. Phosphorylation by CK2 has been shown to alter the properties of a number of these substrate proteins; for example, CK2 phosphorylation inhibits the ability of c-Jun and of c-Myb to bind to DNA, but enhances the nuclear uptake of SV-40 T-antigen [48, 49, 73-75]. Consistent with a role for casein kinase as a global regulator of cell metabolism, CK2 appears to be essential for viability in Saccharomyces, Schizosaccharomyces, and Dictyostelium [76-79]. However, little is known about how CK2 activity itself is regulated in vivo. CK2 does not appear to respond to known secondary messengers, although CK2 levels are elevated in proliferating cells and some studies have suggested that CK2 activity can be stimulated by peptide growth factors and other mitogenic stimuli [43, 68, 71, 72]. In this context, it is intriguing that SMRT, an important transcriptional cofactor for the nuclear hormone receptors and for certain non-receptor transcription factors, such as PLZF and BCL-6 oncoproteins (e.g. [10]), is also a target of CK2 modification. We have shown here that modification of SMRT by CK2 may influence the ability of SMRT to interact with its nuclear receptor partners. It will require additional work to determine precisely how CK2 phosphorylation alters SMRT activity in the intact organism, and how this modification relates to the mechanisms by which growth stimuli and other cellular regulatory pathways modulate nuclear hormone receptor function under physiological conditions.

Acknowledgements

We thank G.J. Rosman and A.D. Miller for generously providing the CK2α molecular clone employed in this research. We also thank Valentina Taryanik for dedicated technical assistance. This work was supported by Public Health Services/NIH Grants R37 CA-53394 and R01 DK-53528.

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