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J. Biol. Chem., Vol. 275, Issue 48, 38032-38039, December 1, 2000

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Thyroxine Promotes Association of Mitogen-activated Protein Kinase and Nuclear Thyroid Hormone Receptor (TR) and Causes Serine Phosphorylation of TR^*

Paul J. Davis^§¶, Ai Shih, Hung-Yun Lin, Leon J. Martino^§, and Faith B. Davis^§

From the  Samuel S. Stratton Veterans Affairs Medical Center and the ^§ Molecular and Cellular Medicine Program, Department of Medicine and the Center for Cell Biology and Cancer Research, Albany Medical College, Albany, New York 12208

Received for publication, March 27, 2000, and in revised form, September 7, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Activated nongenomically by L-thyroxine (T₄), mitogen-activated protein kinase (MAPK) complexed in 10-20 min with endogenous nuclear thyroid hormone receptor (TR1 or TR) in nuclear fractions of 293T cells, resulting in serine phosphorylation of TR. Treatment of cells with the MAPK kinase inhibitor, PD 98059, prevented both T₄-induced nuclear MAPK-TR co-immunoprecipitation and serine phosphorylation of TR. T₄ treatment caused dissociation of TR and SMRT (silencing mediator of retinoid and thyroid hormone receptor), an effect also inhibited by PD 98059 and presumptively a result of association of nuclear MAPK with TR. Transfection into CV-1 cells of TR gene constructs in which one or both zinc fingers in the TR DNA-binding domain were replaced with those from the glucocorticoid receptor localized the site of TR phosphorylation by T₄-activated MAPK to a serine in the second zinc finger of the TR DNA-binding domain. In an in vitro cell- and hormone-free system, purified activated MAPK phosphorylated recombinant human TR1 (). Thus, T₄ activates MAPK and causes MAPK-mediated serine phosphorylation of TR1 and dissociation of TR and the co-repressor SMRT.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have demonstrated in cultured cells that L-thyroxine (T₄)¹ can nongenomically activate signal transduction proteins such as mitogen-activated protein kinase (MAPK) (1) and, through serine phosphorylation by MAPK, can enhance the activity of several nuclear transactivator proteins. Among the latter are the signal transducer and activator of transcription (STAT) proteins that mediate growth factor (2) and cytokine (1, 3) signals. This T₄ effect is initiated by a G protein-coupled receptor in the plasma membrane (1) and has been observed in cells that contain endogenous nuclear thyroid hormone receptor (TR), such as 293T cells and human skin fibroblasts (BG-9), and in cells which are devoid of functional TR (CV-1 and HeLa (4)).

In contrast, genomic actions of thyroid hormone require binding of the hormone, predominantly 3,5,3'-triiodo-L-thyronine (T₃), by specific receptors in the cell nucleus. T₃-liganded TR may bind as a monomer or a homo- or heterodimer with retinoid X receptor to thyroid hormone response elements in the regulatory upstream regions of specific hormone-responsive genes (5-7). In the absence of T₃, TR exists in the transcriptionally inactive (repressed) state. This state is imposed by the binding to unliganded TR of the co-repressor proteins, SMRT (silencing mediator of retinoid and thyroid hormone receptors) and NCoR (nuclear co-repressor) (8). SMRT binding to TR has been localized to the hinge region of the receptor (amino acids 211-240) (9). Binding of T₃ to TR results in dissociation of co-repressor proteins from TR and the recruitment of activator proteins that facilitate enhanced transcriptional activity of the receptor (10).

Serine phosphorylation of TR isoforms has been described by several laboratories (11-15). In such studies phosphorylation has been inferred from stimulation of the activity of cellular cAMP-dependent protein kinase (PKA) (11, 12), a serine/threonine kinase, or from serine kinase inhibition or phosphatase inhibition in treated cells (14, 16). There have been several results of such phosphorylation in these model systems. For example, serine phosphorylation of TR1 has been shown to decrease TR monomer binding to DNA (11). TR1 is selectively stabilized against protease degradation by serine phosphorylation and transcriptional activity of the receptor is significantly increased (14, 15). Leitman et al. (12) have also shown increased transcriptional activity of TR1 in response to phosphorylation. Comparing phosphorylatable and nonphosphorylatable forms of TR2, Katz et al. (17) concluded that serine phosphorylation of the receptor isoform decreased its ability to heterodimerize with retinoid X receptor at a thyroid hormone response element site. Using a serine/threonine kinase inhibitor, H7, Jones et al. (16) showed a reduction in T₃-induced transcriptional activity of both TR1 and TR1. In some of these studies (12), the results were highly cell line-specific.

The mechanism by which serine phosphorylation of TR1 is achieved is unclear, aside from the possible involvement of PKA (11, 12). In the study reported here, we describe a signal transduction mechanism by which T₄, acting at the cell surface, promotes MAPK-dependent serine phosphorylation of TR1 and resultant dissociation of TR and SMRT. We also identify a region of TR1 that is required for MAPK binding to the receptor.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- L-T₄, L-T₃, D-T₄, D-T₃, 3,3',5'-triiodothyronine (rT₃), tetraiodothyroacetic acid (tetrac), 3,5,3'-triiodothyroacetic acid (triac), 8-bromo-cyclic-AMP (8-Br-cAMP), myelin basic protein (MBP), 6-n-propyl-2-thiouracil (PTU), T₄-agarose, and protein A-agarose were obtained from Sigma. Stock solutions of thyroid hormone and analogues were prepared in 0.04 N KOH, 4% propylene glycol, and dilutions were made to final analogue concentrations as indicated. In all experiments in which T₄ was added to cultured cells, the total and free T₄ concentrations were 10⁷ and 10¹⁰ M, respectively, and total and free T₃ concentrations were below the limits of detection. PD 98059 was obtained from Calbiochem (La Jolla, CA), and geldanamycin was from the Drug Synthesis and Chemistry Branch of the National Cancer Institute (Bethesda, MD). Stock solutions of these inhibitors were prepared in 100% Me₂SO, so that a final concentration of 0.1% Me₂SO was achieved in cell cultures and had no effect on experimental results. KT5720 was obtained from Kamiya Biomedical Co. (Thousand Oaks, CA). LipofectAMINE Plus was obtained from Life Technologies, Inc.

TR nucleotides, expressed in pcDNA1/Amp and including full-length hTR1, rTR-N (containing DBDs and LBDs), and rTR-LBD (LBD only), were generously provided by Dr. Paul M. Yen (NIDDK, NIH, Bethesda, MD), as were a TGT hybrid construct of TR1 with the DBD of the glucocorticoid receptor (GR) substituted for that of TR (provided with permission of Dr. Ronald Evans, Salk Institute, La Jolla, CA) and two TR hybrid mutants in which one or the other zinc finger of the TR DBD is replaced with the corresponding zinc finger of GR (T-TG-T or T-GT-T; obtained with permission of Dr. J. Larry Jameson, Northwestern University, Chicago, IL). Recombinant TR1 (residues 102-461) was generously provided by Dr. Brian L. West (Metabolic Research Unit, University of California-San Francisco, CA).

Cell Cultures, Treatment, and Preparation of Nuclear Fractions-- TR-replete 293T cells were obtained from Dr. K. Pumiglia (Albany Medical College, Albany, NY); CV-1 cells, which lack TR, were on hand in the laboratory (1). All cells were maintained and grown in Dulbecco's modified essential medium supplemented with 10% fetal bovine serum. Almost confluent cells were placed for 2 days in medium containing 0.25% serum that had been previously depleted of thyroid hormone by the method of Samuels et al. (18), as modified by Weinstein et al. (19), and then in serum-free medium for 2 h. Cells were then treated with T₄ or other analogues for the times indicated. In selected experiments the inhibitors PD 98059 and geldanamycin were added to cells for 5 h, and T₄ was added for the last 30 min; KT5720 was added for 70 min, and 8-Br-cAMP and T₄ were added for the last 30 min of KT5720 incubation.

After cell treatment, cells were harvested, and nuclear extracts were prepared as follows: the cells were washed twice with ice-cold phosphate-buffered saline and lysed in hypotonic buffer as described previously (1-3). After sample centrifugation at 4 °C and 13,000 rpm for 1 min, supernatants were collected as cytoplasmic extracts. Nuclear extracts were prepared according to the method of Wen et al. (20) by resuspension of the crude nuclei in high salt buffer (hypotonic buffer with 20% glycerol and 420 mM NaCl) at 4 °C with rocking for 30 min. The supernatants were collected after centrifugation at 4 °C and 14,000 × g for 10 min.

Immunoprecipitation and Immunoblotting-- Following normalization of sample protein content, immunoprecipitation was performed using polyclonal anti-phosphotyrosine (Transduction Laboratories, Lexington, KY), monoclonal antibody to the amino-terminal or carboxyl-terminal halves of the amino-terminal (AB) domain (amino acids 1-93 of TR1 (Santa Cruz, Santa Cruz, CA), polyclonal antibody to amino acids 73-93 of rat TR1 (generously provided by Dr. William W. Chin, Indianapolis, IN), monoclonal antibody to amino acids 235-414 of human TR1 (C3 antibody, Affinity Bioreagents, Inc., Golden, CO), monoclonal antibody to MAPK (Transduction), polyclonal anti-phosphoserine (Research Diagnostics, Flanders, NJ), or polyclonal antibody to tyrosine/threonine-phosphorylated MAPK (New England Biolabs, Beverly, MA). After overnight incubation of samples at 4 °C with rocking, protein A-agarose was added, and samples were rocked for 1 h at 4 °C. After two washes with hypotonic buffer containing 0.2% Nonidet P-40, immunoprecipitates were eluted with 2× sample buffer and proteins (10 µg of protein/sample) were resolved on discontinuous SDS-PAGE gel (7.5-9.0%). Proteins were transferred to Immobilon membranes (Millipore, Bedford, MA) by electroblotting. After blocking with 5% milk in Tris-buffered saline containing 0.1% Tween, membranes were immunoblotted with one of several antibodies including monoclonal anti-MAPK or -TR1, polyclonal anti-tyrosine/threonine-phosphorylated MAPK, anti-phosphoserine, anti-phosphothreonine (Research Diagnostics), anti-TR1, or anti-SMRT (Santa Cruz). The secondary antibodies were rabbit anti-mouse, goat anti-rabbit, or donkey anti-goat IgG (1:1000, DAKO Corporation, Carpenteria, CA). Immunoblots were visualized by chemiluminescence (ECL; Amersham Pharmacia Biotech) and quantitated by digital imaging (BioImage, Millipore). The possibility of nonspecific immunoprecipitation was ruled out by use of anti-HLA-DR for immunoprecipitation, followed by MAPK immunoblot of the immunoprecipitated proteins; no MAPK was detectable. Supernatants from TR1 immunoprecipitation contained no TR1, indicating that the immunoprecipitation was complete. Immunoblots shown in the figures are representative of three or more experiments.

Transfection of TR1 and Mutants of TR1-- The DNA samples for TR1, two truncated mutants (TR-N and TR-LBD), and three hybrid constructs (TGT, T-GT-T, and T-TG-T), in the pcDNA expression vector were transformed into competent DH5 cells and plated on ampicillin-treated agar plates. Single colonies were chosen and grown overnight in Circle Grow culture broth (Bio101, Carlsbad, CA) containing ampicillin (10 mg/ml). Plasmid DNA was isolated and purified by a modified alkaline lysis protocol (RPM4G kit, Bio101). CV-1 cells were transfected with TR1 or mutants in pcDNA or with pcDNA alone using LipofectAMINE Plus according to supplier's instructions. Cells grown to 60% confluence in serum-free, antibiotic-free medium were replenished with 5 ml of fresh medium, and LipofectAMINE/DNA mixtures previously prepared were then added, followed by incubation for 6 h at 37 °C. An equal volume of medium containing 20% T₄-depleted serum was then added to the DNA-LipofectAMINE Plus mixture, and the cells were incubated for an additional 6-8 h. The medium was replaced with Dulbecco's modified essential medium containing 10³ M PTU and 0.25% T₄-depleted serum, and the cells were incubated for 16 h and then treated with T₄ (10⁷ M) or diluent for 30 min and harvested for preparation of nuclei as described above. Nuclear samples were immunoblotted with a TR1 antibody that detects TR1 and all mutants or were immunoprecipitated with the same antibody, and precipitated proteins were then separated by PAGE and immunoblotted with antibodies to MAPK, phosphoserine, or phosphothreonine.

In Vitro Serine Phosphorylation of Recombinant TR1 by Activated MAPK-- 5 µg of recombinant TR1 (amino acids 102-461) were immunoprecipitated with C3 antibody, and the immunoprecipitated protein eluted from protein A-agarose. The immunoprecipitate was incubated for 30 min at 30 °C with 5 units of activated MAPK (New England Biolabs) in 50 mM Tris-HCl (pH 8.0), 0.5 mM EDTA, 25 mM MgCl₂, 1 mM dithiothreitol, 10% glycerol, and 20 µM ATP containing 0.05 µCi of [-³²P]ATP (PerkinElmer Life Sciences), following the method of Kato et al. (21). Immunoprecipitated TR1 from 293T cells was also exposed to activated MAPK. Control samples contained 1 µg of MBP instead of TR1. After incubation proteins were solubilized, separated by PAGE, and radioautographed.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

T₄ Induces Activation of MAPK and Nuclear Translocation of Co-immunoprecipitated MAPK and TR1-- Human embryonic kidney (293T) cells contain endogenous TR1. To study co-immunoprecipitation, or complexing, of TR1 and MAPK, nuclear fractions were prepared from 293T cell lysates of control samples and of cells treated with T₄ (10⁷ M) for 10-90 min and were immunoprecipitated with antibody to the carboxyl-terminal half of the AB domain of TR1. The immunoprecipitated proteins were eluted, separated by SDS-polyacrylamide gel electrophoresis, and transferred to membranes for immunoblotting with antibody to MAPK. Shown in Fig. 1A is a representative immunoblot. There is an increase in nuclear MAPK complexed with TR1 immunoprecipitates that reaches a peak in 40 min (22-fold increase over control in the figure, decreasing to 12-fold in 90 min). In nine experiments, the fold increases in nuclear MAPK associated with TR1 in 30 and 40 min were 6.7 ± 1.5 (mean ± S.E.) and 8.6 ± 2.5, respectively, as shown in Fig. 1B. These changes parallel the timing of T₄-induced increases in tyrosine phosphorylation and nuclear translocation of MAPK that we have previously reported (1).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Accumulation in 293T cell nuclei of co-immunoprecipitated TR1 and MAPK. A, nuclear fractions of 293T cells treated with 107 M T4 for 10-90 min or with T4 diluent were immunoprecipitated with antibody to the carboxyl-terminal half of the AB domain of TR1, and the immunoprecipitates were immunoblotted with anti-MAPK. In all studies 10 µg of protein from each sample was used for gel electrophoresis. The MAPK antibody reacts principally with ERK2 (42 kDa). There was time-dependent association of TR1 and MAPK in from 10-90 min, with a maximal 22-fold effect at 40 min. B, graphic results of nine experiments performed as in A are shown as mean ± S.E. of the fold increase in band integrated optical density (I.O.D.) of each sample compared with the untreated samples shown at left (value = 1). Increases (mean ± S.E.) of 6.7 ± 1.5-fold (p < 0.0025; one-way analysis of variance) and 8.6 ± 2.5-fold (p < 0.01), respectively, in 30 and 40 min were seen in nine experiments. Significance is shown as follows: ***, p < 0.001; **, p < 0.025; and *, p < 0.01. C, an experiment conducted as described for A utilized antibody to tyrosine/threonine-phosphorylated MAPK for immunoblotting of TR1 immunoprecipitates and shows maximal complexing with TR1 at 30-40 min. This antibody detects both ERK1 and ERK2. In four experiments, there were significant fold increases in activated MAPK in 10 min (10.3 ± 3.8, p < 0.05), 30 min (13.1 ± 3.2, p < 0.01), 40 min (24.1 ± 8.6, p < 0.05), and 90 min (4.4 ± 1.0, p < 0.025).

In studies using a selective antibody to tyrosine/threonine-phosphorylated, or activated, MAPK for immunoblotting of TR1 immunoprecipitates, we observed 21- and 24-fold increases in activated MAPK in 30 and 40 min, respectively (Fig. 1C). In additional experiments, nuclear fractions were immunoprecipitated with antibody to phosphorylated MAPK, and the resulting proteins were immunoblotted with anti-TR1, thus reversing the antibody order. Again, complexing of TR1 and MAPK was found, with 1.4- and 7.8-fold increases in TR1 bands in 30 and 40 min of T₄ treatment, respectively (not shown). We have therefore demonstrated that T₄ in a physiologic concentration can transiently 1) activate MAPK causing its translocation to the nucleus and 2) promote the association of activated MAPK with TR1 in an immunoprecipitable complex. This effect of T₄ occurs in as early as 10 min and persists for up to 90 min. Studies utilizing antibody to the amino-terminal half of the AB domain of TR1 demonstrated similar results, as did studies with a polyclonal antibody to amino acids 73-93 of the rat TR1 AB domain (not shown).

In time course experiments similar to the study shown in Fig. 1A, nuclear extracts of 293T cells treated with T₄ were immunoblotted with TR1 antibody without prior immunoprecipitation; Western blots demonstrated 2-5-fold increases in nuclear TR1 in 10-60 min, respectively, as shown in Fig. 2A. Nuclear samples were also immunoprecipitated with monoclonal TR1 antibody, and the precipitates were subjected to PAGE and immunoblotted with polyclonal anti-TR1 (Fig. 2B). Again, progressive nuclear accumulation of the receptor during T₄ treatment is seen.

View larger version (44K): [in this window] [in a new window]   Fig. 2.   Effect of T4 on nuclear accumulation of TR1 and evidence of cytosolic TR1 in 293T cells. A, 293T cells were treated with 107 M T4 for 10-90 min as in experiments shown in Fig. 1. Nuclear proteins were immunoblotted with antibody to the carboxyl-terminal half of the AB domain of TR1. Compared with untreated cells, there was a 2-5-fold increase in nuclear TR1 in 10-60 min in the study shown, and in seven studies there was a 2.1 ± 0.3-fold (p < 0.01) increase in 30 min. B, nuclear samples of the 293T cells shown in A were immunoprecipitated with monoclonal TR1 antibody. The immunoprecipitated proteins were separated by PAGE and immunoblotted with polyclonal antibody to amino acids 62-82 in the AB domain of TR1. A time-dependent increase in nuclear TR1 with T4 treatment is again seen. C, 293T cells treated with T4 for 30 min display increased TR1 in the nuclear fraction compared with that of cells not treated with hormone. In four experiments the increase with T4 was 2.0 ± 0.6-fold. However, cytosolic TR1 is less concentrated and does not appear to change with T4.

We determined in 293T cells whether T₄ treatment for 30 min altered the abundance of TR1 per unit of nuclear fraction protein (10 µg of protein/lane). A representative blot is shown in Fig. 2C. It is assumed that the increase in nuclear TR1 originated from the pool of cytosolic TR1 also shown in this figure. We have also found cytosolic TR1 in another cell line (NIH3T3 cells; not shown), and others have reported the presence of TR in cytoplasm (22). Extranuclear TR1 concentration in our studies was 4.5 ± 2.5% of nuclear receptor concentration, where relative concentration was expressed as band integrated optical density per 10 µg of lane protein. The pool of cytosolic TR1 may be larger than Fig. 2C implies, however, given the 2-fold greater concentration of total protein in cytoplasm than in nuclear fractions and the relative volumes of nucleus and cytosol in intact cells.

Iodothyronine Analogues and Formation of TR1-MAPK Complexes-- We have previously reported that physiologic concentrations of T₄ are more effective in nongenomic models of hormone action than physiologic concentrations of T₃ (1-3) and that triac and tetrac may block the action of T₄ at the plasma membrane (1, 3). We therefore studied whether D-T₄, D-T₃, rT₃, tetrac, and triac caused complexing of TR1 and MAPK in 293T cell nuclei. In Fig. 3A T₄ is shown to enhance co-immunoprecipitation of TR1 and MAPK by 11.6-fold. In contrast, L-T₃ in concentrations of 10¹⁰ and 10⁷ M was ineffective in promoting this complex in five studies. D-T₄, D-T₃, and rT₃ were similarly inactive in the same studies compared with control cells. The ability of both tetrac and triac (10⁷ M) to block the effect of T₄ is seen in Fig. 3B. In this study T₄ enhanced the amount of MAPK in the TR1 immunoprecipitate 10.4-fold. Tetrac and triac had negligible effects when present alone, but both analogues almost completely blocked T₄ action.

View larger version (36K): [in this window] [in a new window]   Fig. 3.   Effect of thyroid hormone analogues on complexing of TR1 and MAPK in 293T cells. A, 293T cells were treated with 107 M L-T4, D-T4, L-T3, D-T3, rT3, or tetrac or 1010 M L-T3 for 30 min, after which immunoprecipitation of nuclear extracts with anti-TR1 and immunoblotting with anti-MAPK were carried out. This blot shows an 11.6-fold increase in MAPK content of TR1 immunoprecipitates with L-T4 (lane 2 compared with lane 1). In five experiments the fold increase with T4 was 5.2 ± 1.8 (p < 0.05), whereas none of the other analogues, including T3 (lanes 4 and 5), had any consistent effect. B, this figure shows a 10-fold increase in TR1/MAPK complexing (lane 2), and in eight experiments the fold increase with 107 M T4 at 30 min was 3.3 ± 1.1 (p < 0.05). Tetrac and triac (107 M) were ineffective as agonists (lanes 3 and 5) but were effective as antagonists of the T4 effect (lanes 4 and 6). Shown in lane 7 is a positive control sample for MAPK. con, control.

T₄-induced Nuclear Complexing of TR1 and MAPK and Serine Phosphorylation of TR1 Are Blocked by a MEK Inhibitor-- To study the contribution of the T₄-activated MAPK pathway to the complexing of MAPK and TR1, the MEK inhibitor PD 98059 was used. MEK is a dual tyrosine-threonine kinase that activates MAPK, a serine or threonine kinase (23). We have previously shown that PD 98059 (30 µM) blocks T₄-induced tyrosine phosphorylation and nuclear translocation of MAPK (1) and the complexing of MAPK with both STAT1 and STAT3 (2). 293T cells were pretreated with PD 98059 (30 µM) for 5 h and with T₄ (10⁷ M) for the last 30 min. Fig. 4A demonstrates T₄-induced co-immunoprecipitation of TR1 and tyrosine-phosphorylated MAPK, with a 2.4-fold increase in activated MAPK in T₄-treated cells (comparing lanes 1 and 2). PD 98059 inhibited the T₄ effect by 88% in the study shown (lane 2 versus lane 3). Additional studies examined the effect of PD 98059 on serine phosphorylation of TR1. Samples were immunoprecipitated with anti-TR1, and precipitates were immunoblotted with anti-phosphoserine. In the experiment shown in Fig. 4B, there is 2.9-fold enhancement of TR1 serine phosphorylation by T₄ (lanes 1 and 2) and 97% inhibition of serine phosphorylation by PD 98059 (lanes 2 and 3).

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Inhibition by a MEK inhibitor of the co-immunoprecipitation of TR1 and activated MAPK and of serine phosphorylation of TR1 in 293T cells. A, T4-induced association in 293T cells of tyrosine-phosphorylated MAPK and TR1 (lane 2, 2.4-fold increase in this experiment; 4.2 ± 1.8-fold increase, n = 4) was inhibited by 30 µM PD 98059 (PD) (88% inhibition, lane 3; 62 ± 14% inhibition, n = 3). B, T4 stimulated serine phosphorylation of TR1 3-fold comparing lanes 1 and 2 (3 ± 0.4-fold stimulation, n = 4), an effect that was inhibited by PD 98059 (lane 3; 69 ± 11% inhibition, n = 4). Shown at right is a TR1 immunoblot of the sample shown in lane 1, supporting the identity of the serine-phosphorylated protein as TR1.

T₄ Effect on TR1/MAPK Complex Formation Is PKA-mediated-- Because a role for PKA in the serine phosphorylation of TR1 has been suggested (11, 12), we examined the effect of KT5720 and 8-Br-cAMP, respectively, an inhibitor and an agonist of PKA (24). In the study shown in Fig. 5A, T₄ enhanced 293T cell nuclear uptake of TR1 complexed with MAPK 7.7-fold (lane 2). KT5720 inhibited T₄-induced complexing of TR1 and MAPK (lanes 3 and 4, 33 and 61% inhibition, respectively). 8-Br-cAMP added to the effect of T₄ (lane 8) and was itself agonistic (lane 5). The effect of 8-Br-cAMP was also partially blocked by KT5720 (lanes 6 and 7).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Contribution of PKA activity to T4-induced formation of nuclear TR1-MAPK complexes in 293T cells. A, T4 caused association of TR1 and MAPK (lane 2, 6.8 ± 2.5-fold in three experiments) in 293T cells, an effect inhibited by KT5720 (lanes 3 and 4; KT5720 (KT) 1 or 100 nM, 61 ± 16% and 76 ± 12% inhibition, respectively, n = 3). 8-Bromo-cAMP (8-Br, 1 mM) also enhanced TR1-MAPK association (lane 5; 9.3-fold in the figure, 7.5 ± 3.9-fold, n = 3), an effect reduced by KT5720 (lanes 6 and 7; 32 and 37% inhibition, respectively, n = 3). The effects of T4 and 8-Br-cAMP are additive (lane 8). B, T4 enhancement of the co-immunoprecipitation of nuclear TR1 and MAPK (7.8-fold, lane 2; 4.4 ± 1.8-fold, n = 3) was inhibited 65% by PD 98059 (PD, 30 µM, lane 3; 64 ± 6% inhibition compared with lane 2, n = 3). Similarly, 8-Br-cAMP (1 mM) enhanced co-immunoprecipitation (4.7-fold, lane 4; 8 ± 4-fold increase, n = 3), and this effect was inhibited by PD 98059 (lane 5; 50 ± 11% inhibition compared with lane 4, n = 3). C, in 293T cell samples immunoprecipitated with anti-phosphoserine and the immunoprecipitates then immunoblotted with anti-TR1, T4 enhanced serine phosphorylation of TR1 (lane 2; 14 ± 3-fold increase, n = 3), an effect inhibited by PD 98059 (lane 3; 82 ± 4% inhibition, n = 3). The findings in lanes 1-3 of this study were similar to those in lanes 1-3 of Fig. 4B, even though the antibody order for immunoprecipitation and immunoblotting was reversed. Similarly, 8-Br-cAMP (lane 4) stimulated receptor serine phosphorylation (8 ± 4-fold, n = 3), which was also inhibited by PD 98059 (lane 5; 65 ± 15% inhibition, n = 3). Shown at right is a sample of 293T cell nuclear extract immunoblotted with anti-TR1, without immunoprecipitation.

The PKA-mediated T₄ Effect on Nuclear TR1/MAPK Complex Formation Depends on MEK Activity-- To further study whether the effect of 8-Br-cAMP on nuclear complexing of TR1 and MAPK was direct via PKA or mediated through the MAPK pathway, we examined the effects of T₄ and 8-Br-cAMP in 293T cells in the absence and presence of PD 98059. In the experiment shown in Fig. 5B, T₄ enhanced complexing of TR1 and MAPK (lane 2), as did 8-Br-cAMP (lane 4). PD 98059 inhibited the effect of T₄ by 65% (lane 3) and the effect of 8-Br-cAMP by 62% (lane 5), indicating that the action of PKA on nuclear TR1/MAPK association takes place at, or upstream of, MEK in the MAPK signal transduction pathway.

We then examined the effect of T₄, 8-Br-cAMP, and PD 98059 on the appearance of TR1 in anti-phosphoserine immunoprecipitates. Fig. 5C demonstrates 15-fold nuclear accumulation of serine-phosphorylated TR1 in nuclei of T₄-treated 293T cells (lane 2) and 6-fold accumulation with 8-Br-cAMP (lane 4). Inhibition of both T₄ and 8-Br-cAMP effects by PD 98059 was evident (lanes 3 and 5, respectively). These studies suggest that PKA, whether activated by T₄ or by 8-Br-cAMP, requires MEK action on MAPK to bring about serine phosphorylation of TR1.

T₄-induced Nuclear Complexing of MAPK with Transfected TR1 and Mutants of TR1 in CV-1 Cells-- Having established that T₄ induces TR1/MAPK complexing and serine phosphorylation of the endogenous receptor in 293T cells, we studied CV-1 cells transfected with TR1 and found that T₄ produced the same effects. Preliminary studies with two truncated mutants of TR1, rTR-N (amino acids 94-461, containing both DBD and LBD) and rTR-LBD (amino acids 174-461, without DBD) showed that the DBD must be present for T₄-induced complexing of MAPK and receptor to occur (results not shown). Based on this finding, we developed a strategy to localize the site in the TR DBD of MAPK-receptor interaction. CV-1 cells were transfected with three hybrid constructs, substituting human GR sequences for corresponding segments of human TR1: T-TG-T, containing the second zinc finger of GR instead of that of TR; T-GT-T, with the first zinc finger of GR replacing that of TR; and TGT, which contains the entire DBD of GR in lieu of the TR DBD (Fig. 6A). Results in Fig. 6B confirm transfection of the probes. These cells were treated with PTU and T₄ as described above under "Experimental Procedures," and examined for TR1/MAPK complexing. T₄-stimulated co-immunoprecipitation of TR1 and MAPK is seen only in cells with intact TR1 (Fig. 6C, upper panel, lane 2) or with the T-GT-T construct, in which the second zinc finger of TR is intact (lane 6). In contrast, in cells with the second zinc finger of TR replaced with that of GR or with the entire DBD replaced with that of GR, no T₄-induced complex formation of TR and MAPK is seen (lanes 4 and 8, respectively). The TGT construct substitutes GR (), containing neither serine nor threonine, for TR () which has two serines (142, 144) and three threonines. However, only one of these amino acids in TR (serine 142) is located next to a proline, a basic requirement for MAPK substrates (25). Anti-phosphothreonine immunoblots of TR immunoprecipitates revealed no receptor threonine phosphorylation (not shown). These results support an essential role for the second zinc finger of TR1 in T₄-induced receptor complexing with activated MAPK and suggest that serine 142 is the phosphorylation site.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Contribution of the two zinc fingers of TR1 to T4-induced interaction of MAPK and the receptor. A, diagram illustrating the structures of TR1 and three hybrid mutants containing one or both zinc fingers of the GR in place of corresponding segments of TR. AB, amino acids 1-93. B, CV-1 cells were transfected with one of the four structures in A, treated with T4 (107 M) for 30 min in the presence of PTU as described under "Experimental Procedures," and nuclear samples were immunoblotted with TR1 AB domain antibody, to show that adequate levels of receptor were transfected. C, nuclear samples were immunoprecipitated with TR1 antibody, and the resulting proteins were immunoblotted with antibodies to MAPK or phosphoserine. Evidence of T4-induced TR1/MAPK complexing (MAPK, upper panel), and serine phosphorylation of TR (pSer, lower panel) is seen in lanes 2 and 6 but is missing in lanes representing cells with either the entire GR DBD (TGT, lane 8) or the second zinc finger of GR (T-TG-T, lane 4). Anti-phosphothreonine immunoblots of TR immunoprecipitates demonstrated no evidence of threonine phosphorylation (results not shown).

Effect of Activated MAPK on Serine Phosphorylation of Recombinant TR1-- Kato et al. (21) have demonstrated that activated MAPK can cause serine phosphorylation of the estrogen receptor in vitro. To determine whether MAPK can directly serine phosphorylate TR1, in vitro studies of thyroid hormone receptor phosphorylation were performed with activated MAPK and TR1. Recombinant TR1 (amino acids 102-461, 5 µg), was further purified by immunoprecipitation with the C3 antibody and then incubated for 30 min at 30 °C with activated MAPK (5 units/µl), and 20 µM ATP including 0.05 µCi of [-³²P]ATP in the buffer medium described by Kato et al. (21). A control sample contained 1.0 µg of MBP and activated MAPK. Phosphorylation of proteins was evaluated by PAGE and radioautography, and results are shown in Fig. 7. Phosphorylation of MBP by activated MAPK is seen in lane 1. In the lane 2 sample MAPK was absent, and no phosphorylation of recombinant TR1 is seen. The lane 3 sample contained activated MAPK and immunoprecipitated recombinant TR1, and a 43-kDa labeled band is seen, consistent with the size of the TR1 fragment. A sample of immunoprecipitated endogenous TR1 from 293T cells was also exposed to activated MAPK and radiolabeled ATP, and in lane 4 a band is seen at a molecular mass of 56 kDa, consistent with that of intact TR1.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Effect of activated purified MAPK on phosphorylation of recombinant TR1. Purified recombinant TR1 (amino acids 102-461) was immunoprecipitated with the C3 TR antibody, and the precipitate was incubated for 30 min at 30 °C with activated MAPK (5 units/ml) and 20 µM ATP containing 0.05 µCi of [-32P]ATP. A positive control sample contained 1.0 µg of MBP as substrate and is shown in lane 1, demonstrating phosphorylation of MBP by the activated MAPK. In lane 2 no MAPK is present, and consequently no phosphorylation of TR1 is seen. Lane 3 demonstrates phosphorylation of immunoprecipitated recombinant TR1 by the activated MAPK; the molecular mass of this receptor fragment is 43 kDa. Immunoprecipitated TR1 from 293T cells was also phosphorylated by activated MAPK, and a clear band of 56 kDa is seen in lane 4. The figure shown is representative of three experiments.

Effect of T₄-induced Nuclear Complexing of Activated MAPK and TR1 on Binding of SMRT to TR1-- 293T cells were treated with T₄ and/or tetrac (10⁷ M) for 30 min, and selected samples were pretreated for 1 h with 30 µM PD 98059 or 10 µM geldanamycin. The latter causes depletion of cellular Raf-1, a serine/threonine kinase responsible for activation of MEK (1). Shown in Fig. 8A is the presence of SMRT in the TR1 immunoprecipitate of control cells (lane 1) and a marked reduction in SMRT binding to TR1 when cells were treated with T₄ (lane 2). Geldanamycin and PD 98059 both inhibited the T₄ effect on SMRT binding to TR1 (lanes 3 and 4, respectively). Tetrac alone had no effect on SMRT binding to TR1 (lane 5) but completely blocked T₄-induced displacement of SMRT from the receptor (lane 6). From prior studies we know that tetrac inhibits T₄ binding to plasma membranes (26) and blocks T₄-induced activation of MAPK (1), STAT1 (3), and STAT3.² In additional studies, T₄-agarose (10⁷ M T₄) inhibited SMRT binding to TR1 by 50% (not shown). These findings support a role for T₄ in the release of SMRT from TR1 in a manner that is dependent on 1) an intact MAPK pathway and 2) tetrac-inhibitable binding of T₄ to a plasma membrane receptor.

View larger version (30K): [in this window] [in a new window]   Fig. 8.   Effect of T4, tetrac, and MAPK pathway inhibitors on co-immunoprecipitation of TR1 and SMRT in 293T cells. A, treatment of 293T cells with T4 (107 M) for 30 min was followed by preparation of nuclear fractions, immunoprecipitation with TR1 antibody, and immunoblotting of the precipitate with antibody to the corepressor, SMRT. T4 caused a 52% reduction in SMRT complexed with TR1 (lane 2; 64 ± 12% reduction, n = 3). The T4 effect was blocked by pretreatment of cells for 16 h with geldanamycin (Gel, 10 µM) or for 5 h with PD 98059 (PD, 30 µM). In three studies, 100 and 75% inhibition of the T4 effect, respectively, were found with geldanamycin and PD 98059. Tetrac was ineffective alone (lane 5) but completely blocked the action of T4 in this study (lane 6; 44% reduction in three studies). B, 293T cells were treated with T4 or T3 in the concentrations shown for 30 min and then studied as described in A, above. T4 (108 and 107 M) reduced SMRT binding to TR1 by 34 and 87%, respectively, in the study shown, and by 38 ± 9 and 80 ± 6% in four experiments. T3 (1010 and 107 M) reduced SMRT binding to TR1 by 49 ± 18 and 70 ± 9% in four experiments.

The effect of T₃ on SMRT binding to TR1 was compared with that of T₄. In four experiments, levels of SMRT complexed with TR1 were reduced by 38 ± 9 and 80 ± 6% of control levels in cells treated with 10⁸ and 10⁷ M T₄, respectively, and the SMRT levels were reduced by 49 ± 18 and 70 ± 9% of control in cells treated with T₃ (10¹⁰ and 10⁷ M, respectively). A representative blot is shown in Fig. 8B. Similar findings were obtained in studies with NCoR antibody (not shown). Thus, a physiologic concentration of T₄ (10⁷ M) is more effective than a physiologic concentration of T₃ (10¹⁰ M) in displacing SMRT from endogenous TR1 in 293T cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A number of hormones have recently been reported to influence activity of kinase cascades, including the MAPK pathway. Activation of steps in this pathway by gonadotropin-releasing hormone (27), norepinephrine (28), insulin (29), estradiol (21, 30), and T₄ (1) are several examples of such hormones. MAPK has been shown to serine phosphorylate the nuclear estrogen receptor, and overexpression of MEK or Ras in Cos-1 cells potentiates estrogen-induced transcriptional activity of the estrogen receptor (21). Modulation of glucocorticoid receptor activity by MAPK has also been described (31). Chick oviduct nuclear progesterone receptor is another phosphoprotein whose hormone-induced activation is serine protein kinase-dependent (32). A variety of changes in the activity and stabilization of TR have been ascribed to serine phosphorylation (11, 13, 15, 16, 33, 34), and PKA activity has been implicated in the process (11, 12), but no further mechanism of control of serine phosphorylation of TR has been suggested.

We have found that T₄ nongenomically promotes the phosphorylation and nuclear uptake of MAPK and the co-immunoprecipitation of nuclear MAPK with STAT1 and STAT3 (1, 2). We have shown that this effect results in phosphorylation of serine 727 of STAT1 (1). We therefore examined the possibility that iodothyronines, particularly T₄, might promote serine phosphorylation of TR1 by activation of MAPK. Were this to be the case, thyroid hormone in the form of T₄ could modulate the activity of the nuclear receptor by a mechanism distinct from the binding of T₃ directly to TR1. That is, without competing for the T₃-binding site on TR1, T₄ might affect the transcriptional activity of the liganded nuclear T₃ receptor by promoting serine phosphorylation of the protein.

In the present studies we demonstrated in 293T cells that treatment with physiologic concentrations of T₄ caused endogenous nuclear TR1 to increase and promoted MAPK and TR1 co-immunoprecipitation in nuclear fractions. The time course of these effects was relatively rapid, being apparent in 10 min and maximal by 30-40 min. The association of TR1 and MAPK was evident whether we immunoprecipitated TR1 first and then reprobed with anti-MAPK antibody or immunoprecipitated MAPK and then identified TR1 in that precipitate.

The structure-activity relationships of the iodothyronine analogues studied in this MAPK-TR model were identical to those we have previously reported in the nongenomic activation by thyroid hormone of MAPK and its association with STAT proteins (1, 2). L-T₄ was more active at physiologic concentrations than L-T₃, and T₄-agarose was as active as T₄. The fact that deaminated analogues (tetrac and triac) were not thyroid hormone agonists but were capable of blocking the action of T₄ is consistent with the existence of a cell surface receptor for thyroid hormone that we have previously described (26, 35, 36), which is pertussis toxin- and GTPS-sensitive (37). We have previously demonstrated that tetrac and triac block T₄ potentiation of the antiviral (38) and immunomodulatory (3) actions of interferon-, even though these analogues themselves have no effect on interferon- action.

We have detected TR1 in cytosol of 293T cells (Fig. 2B). TR1 is generally regarded to be restricted to the nucleus but has been reported recently by Zhu et al. (22) to be present in cytosol. In our studies of 293T cells, we found TR1 in cytosol in much lower concentrations than those in corresponding nuclear fractions. The role of cytosolic TR1 is not clear; it may be nascent in an inactivated state or may be undergoing degradation. Although nuclear estrogen receptor and plasma membrane estrogen receptor apparently represent transcripts of the same gene (39), the TR1 detected in cytosol in our studies is not currently thought by us to represent trafficking of TR1 to the cell surface. We base this conclusion on the structure-activity relationships of iodothyronine analogues in their actions at the cell surface (1, 3, 26), compared with those at the nuclear TR (40, 41).

In the present experiments, we showed that MEK activity was required to cause nuclear translocation of phosphorylated MAPK and association of MAPK with TR1 in cell nuclei. PD 98059, an inhibitor of MEK, prevented activation of MAPK and the nuclear association of phosphorylated MAPK and TR1 and inhibited T₄-induced serine phosphorylation of TR1. Thus, the MEK-MAPK cascade that has been implicated in serine phosphorylation of estrogen receptor (21, 30) is also operative in T₄-treated cells in which TR1 is serine-phosphorylated. In studies not presented here, we have found that T₄ can also promote the nuclear association of estrogen receptor and MAPK.²

The fact that TR1 and MAPK were found to be associated in the nucleus did not prove that the receptor was a substrate for MAPK. However, using a phosphoserine antibody we documented that endogenous nuclear TR1 complexed with MAPK was serine-phosphorylated in T₄-treated cells and that this serine phosphorylation was inhibited by PD 98059. In contrast, we found no evidence for threonine phosphorylation of TR.

Studies with two truncated mutants of TR1 suggested that to demonstrate T₄-induced co-immunoprecipitation of MAPK and the receptor, the DBD must be present. With the use of hybrid constructs of TR1 and GR in which the zinc fingers of the DBDs were exchanged, we identified the second zinc finger of the TR DBD as a necessary participant in MAPK action. In cells with either this portion of TR or the entire TR DBD replaced with that of GR, T₄ treatment did not result in co-immunoprecipitation of TR and MAPK or serine phosphorylation of the TR/GR hybrid. The second zinc finger of the DBD has also been found to be important for homodimerization of TR and transcriptional activation (42).

We also demonstrated that constitutively activated MAPK phosphorylates purified recombinant human TR1 in vitro. The receptor fragment we used (residues 102-461) contains 2 serines in the DBD and 14 in the LBD. From our studies described above, we concluded that MAPK binds to the DBD, and serine phosphorylates the DBD. None of the serines in TR are in environments that qualify as optimum consensus phosphorylation sites, namely, PX_n(S/T)P, where X is a neutral or basic amino acid and n = 1 or 2, or as the minimum sequence site ((S/T)P; Ref. 21). However, MAPK substrates may lack consensus sequences, and the enzyme may phosphorylate a PS sequence (25) similar to that which occurs in the TR DBD at residues 141-142. Another PS sequence occurs at amino acids 98-99 of TR, but this segment was not present in the recombinant TR1 used in our studies.

It is the hinge region of TR (amino acids 211-240), located in the amino-terminal portion of the LBD, that binds the co-repressor proteins SMRT and NCoR (9), which contribute to basal repression of transcriptional activation. Our studies demonstrate that T₄ alone, in the absence of T₃, is effective in displacing SMRT and NCoR from the receptor. This action of T₄ has characteristics similar to T₄-induced activation of MAPK and TR1/MAPK complexing, including dependence on MEK activity, effectiveness of T₄-agarose (1), and inhibition by tetrac and triac (1). We therefore postulate that T₄ treatment causes serine phosphorylation in the DBD of TR1, leading to allosteric changes in the proximal region of the LBD resulting in dissociation of SMRT from TR1. Such a change in the state of the receptor would result, even in the absence of T₃ and binding of T₃ by TR1, in derepression of the receptor without ligand-induced transcriptional activation. That this may be the case has recently been shown in a preliminary study, where T₄ in the absence of T₃ caused return of TR1 from a state of transcriptional repression to the basal state but did not cause transcriptional activation of the receptor.²

We have previously shown that thyroid hormone can potentiate the action of interferon- by a signal transduction pathway that involves both protein kinase C and PKA (24). We therefore studied the possibility that T₄ might also promote the serine phosphorylation of TR1 by a PKA-dependent mechanism and found that the MEK-MAPK pathway by which T₄ acts to phosphorylate TR1 is indeed subject to activation by 8-Br-cAMP and inhibition by KT5720. Our observations suggest that the involvement of PKA in TR1 phosphorylation reported by others depends upon MAPK activation and that T₄-stimulated PKA contributes to activation and nuclear translocation of MAPK.

	FOOTNOTES

^* This work was supported in part by funds from the Office of Research Development, Medical Research Service, Department of Veterans Affairs (to P. J. D.) and by a grant from the Candace King Weir Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom all correspondence should be addressed: Dept. of Medicine, MC-16, Albany Medical College, Albany, NY 12208. Tel.: 518-262-6138; Fax: 518-262-5008; E-mail: pjdavis@albany.net.

Published, JBC Papers in Press, September 11, 2000, DOI 10.1074/jbc.M002560200

² H.-Y. Lin, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: T₄, L-thyroxine; T₃, 3,5,3'-triiodo-L-thyronine; tetrac, tetraiodothyroacetic acid; triac, triiodothyroacetic acid; rT₃, 3,3',5'-triiodo-L-thyronine; TR, thyroid hormone receptor 1; DBD, DNA-binding domain; LBD, ligand-binding domain; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; PD, PD 98059; PTU, 6-n-propyl-2-thiouracil; MBP, myelin basic protein; STAT, signal transducer and activator of transcription; PKA, cyclic AMP-dependent protein kinase; 8-Br-cAMP, 8-bromo-cyclic AMP; GR, glucocorticoid receptor; PAGE, polyacrylamide gel electrophoresis; GTPS, guanosine 5'-O-(3-thio)triphosphate.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES