Regulation of Glucocorticoid Receptor Activity by 14–3-3-Dependent Intracellular Relocalization of the Corepressor RIP140

Johanna Zilliacus, Elin Holter, Hideki Wakui1, Hiroshi Tazawa, Eckardt Treuter and Jan-Åke Gustafsson

Department of Medical Nutrition (J.Z., H.W., H.T., J.-A.G.) and Department of Biosciences (E.H., E.T., J.-A.G.) Karolinska Institutet Novum, S-141 86 Huddinge, Sweden


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Proteins belonging to the 14–3-3 family interact with various regulatory proteins involved in cellular signaling, cell cycle regulation, or apoptosis. 14–3-3 proteins have been suggested to act by regulating the cytoplasmic/nuclear localization of their target proteins or by acting as molecular scaffolds or chaperones. We have previously shown that overexpression of 14–3-3 enhances the transcriptional activity of the glucocorticoid receptor (GR), which is a member of the nuclear receptor family. In this study, we show that 14–3-3 interacts with the nuclear receptor corepressor RIP140. In transfection assays, RIP140 antagonizes 14–3-3- enhanced GR transactivation. Using colocalization studies we demonstrate that 14–3-3 can export RIP140 out of the nucleus and, interestingly, can also change its intranuclear localization. Moreover, we also observed that 14–3-3 can bind various other nuclear receptors and cofactors. In summary, our findings suggest that 14–3-3-mediated intracellular relocalization of the GR corepressor RIP140 might be a novel mechanism to enhance glucocorticoid responsiveness of target genes. They furthermore indicate a more general role for 14–3-3 protein by influencing the nuclear availability of nuclear receptor-associated cofactors.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Proteins belonging to the 14–3-3 family have recently been shown to interact with many proteins involved in cellular signaling, such as Raf-1, protein kinase C, Bcr, MEK kinase, insulin-like growth factor I (IGF I) receptor, and IRS 1 (1, 2, 3). 14–3-3 proteins also bind proteins regulating the cell cycle (Cdk 25 and Wee1) and proteins involved in apoptosis (A20 and Bad) (1, 4, 5, 6). The functional role of the interactions has, in many cases, been unclear. In the case of Raf-1 it has been shown that 14–3-3 is permissive for recruitment of Raf-1 to the cell membrane and for Raf-1 activation (7). Interaction between 14–3-3 and Cdc25 retains Cdc25 in the cytoplasm, thus suppressing its ability to induce entry into mitosis (8, 9). Furthermore, 14–3-3 counteracts the apoptotic activity of BAD by disrupting the heterodimerization between BAD and Bcl2 (6). These effects could be explained by 14–3-3 stabilizing a specific structure of the heterodimerizing partner. 14–3-3 is a dimer and can also bind two different proteins simultaneously and thus function as a scaffold that connects two protein signals (10). Recently it has been demonstrated that 14–3-3 proteins can regulate the subcellular localization of a number of interacting proteins. Cdc25 has been shown to interact with 14–3-3 and be retained in the cytoplasm, either by a 14–3-3-mediated export from the nucleus due to a putative nuclear export signal present in 14–3-3 or by inhibition of nuclear import due to 14–3-3 binding next to a nuclear localization sequence on Cdc25 (8, 9, 11, 12, 13). 14–3-3 has also been shown to retain Forkhead transcription factor FKHRL1 (14), the yeast nutrient-regulated transcription factors MSN2 and MSN4 (15), protein kinase U-{alpha} (16), as well as histone deacetylase 4 and 5 (17, 18) in the cytoplasm. In contrast, 14–3-3 can enhance nuclear localization of telomerase (19) and the homeodomain Tlx-2 transcription factor (20). Many 14–3-3 interacting proteins contain the consensus 14–3-3 interacting sequence R(S)X1,2pSX(P) where pS is a phosphoserine (1). Other 14–3-3 interacting sequences have also been reported, and in many cases these include a serine residue (21).

We have recently demonstrated an interaction between the 14–3-3{eta} protein and the ligand-activated glucocorticoid receptor (GR) (22). Overexpression of the 14–3-3 protein enhances the transcriptional activity of GR in transient transfection experiments (22). GR mediates the effects of glucocorticoid hormones and is a member of the nuclear receptor family (23). The nuclear receptors have three major functional domains, an N-terminal transactivation domain, a central DNA-binding domain, and a C-terminal ligand-binding domain (LBD) (24). The LBD is, in addition, involved in transcriptional activation and interaction with several cofactor proteins (25, 26, 27). In the absence of ligand, GR is found in a cytoplasmic complex with heat shock protein 90 (hsp90) and other associated proteins (28). The hsp90 multiprotein complex stabilizes a structure of GR that is competent of ligand binding. The ligand-activated receptor regulates transcription either by binding to glucocorticoid response elements located close to target genes or by interacting with other transcription factors (23). The nuclear receptors interact with many transcriptional cofactors that modulate the activity of receptors, including RIP140, TRAP220, CBP, and members of the p160/SRC-1 coactivator family (e.g. SRC-1, TIF2, ACTR) (25, 26, 27). The p160/SRC-1 coactivators and CBP have been demonstrated to enhance the transcriptional activity of nuclear receptors, at least partly by modulating chromatin structure by histone acetylation (29), and TRAP220 has been suggested to function by connecting the receptor with the transcriptional machinery (26). RIP140 was originally identified as a coactivator protein for estrogen receptor (ER) that stimulated the transcriptional activity of the receptor (30), and it has also been shown to stimulate the activity of androgen receptor (AR) and aryl hydrocarbon receptor (AhR) (31, 32). In contrast, RIP140 does not act as a coactivator for other nuclear receptors tested such as peroxisome proliferator-activated receptor (PPAR), TR2, retinoic acid receptor (RAR), and thyroid hormone receptor (TR) (33, 34, 35). This led to the hypothesis that RIP140 might function in regulation of nuclear receptor activity by competition for nuclear receptor coactivators such as SRC-1 for binding to the receptor (34). Recently, the interaction between RIP140 and histone deacetylase 1 and 3 was reported, suggesting that RIP140 also may act as corepressor by negatively modulating chromatin structure and gene regulation (36). In the case of GR, RIP140 shows a strong inhibition of GR function both in transactivation and transrepression (37, 38), indicating that it generally inhibits glucocorticoid-dependent gene regulation.

To further elucidate the function of 14–3-3 proteins in the regulation of GR activity, we were interested in determining whether any nuclear receptor cofactors could directly interact with the 14–3-3 protein. In this paper we describe a functional interaction between 14–3-3 and RIP140 that could influence the transcriptional activity of GR.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Interaction of RIP140 with 14–3-3
To determine whether the GR interacting protein 14–3-3 could bind directly to any GR cofactor proteins, we performed glutathione S-transferase (GST) pull-down experiments. The results showed that the in vitro translated corepressor RIP140 did interact specifically with GST-14–3-3 (Fig. 1AGo, lane 4). The in vitro translated RIP140 protein also bound specifically to the GST-GR LBD in the presence of ligand (Fig. 1AGo, lane 3), in accordance with the previously described ligand-dependent interaction between RIP140 and GR (38). We next analyzed whether the same domains of RIP140 were involved in the interactions with 14–3-3 and GR. Analysis of the interaction of in vitro translated RIP140 domains with GST-14–3-3 and GST-GR LBD showed that N- and C-terminal domains of RIP140 bound both 14–3-3 and GR LBD (Fig. 1BGo, lanes 5, 6, 11, and 12), whereas the middle domain did not interact with either protein (Fig. 1BGo, lanes 8 and 9).



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Figure 1. Interaction of RIP140 with GR LBD and 14–3-3 and Mapping of the Binding Sites on RIP140: GST Pull-Down Assays

A, In vitro translated 35S-labeled RIP140 was incubated with GST (lane 2), GST-GR LBD (lane 3), or GST-14–3-3 (lane 4) bound to glutathione agarose beads in PDB with 0.01% Igepal CA-630. GST-GR LBD bound to glutathione agarose beads was incubated with 1 µM Dex for 2 h before mixing with translated protein. The eluted samples from washed beads were analyzed by SDS-PAGE and visualized by autoradiography. The input (lane 1) represents 15% of the amount of labeled protein incubated with GST-protein-bound beads. B, In vitro translated 35S-labeled RIP140 N (aa 1–472) (lanes 1 and 4–6), RIP140 M (aa 431–745) (lanes 2 and 7–9), and RIP140 C (aa 747-1,158) (lanes 3 and 10–12) were incubated with GST (lanes 4, 7, and 10), GST-GR LBD (lanes 5, 8, and 11), or GST-14–3-3 (lanes 6, 9, and 12) bound to glutathione agarose beads in PDB with 0.01% Igepal CA-630. GST-GR LBD bound to glutathione agarose beads was incubated with 1 µM Dex for 2 h before mixing with translated protein. The input (lanes 1–3) represents 15% of the amount of labeled protein incubated with GST-protein-bound beads.

 
Role of Protein Phosphorylation for 14–3-3 Interaction
14–3-3 has been shown in many cases to interact with phosphorylated forms of proteins (1), and phosphatase treatment of the 14–3-3 interacting protein can result in reduced binding (45, 46, 47). To test whether protein phosphorylation is involved in 14–3-3 binding to RIP140 we studied the binding of in vitro translated 14–3-3 with bacterially expressed GST-RIP140 domains, since proteins expressed in bacteria are not phosphorylated. The experiment showed that in vitro translated 14–3-3 did not bind GST-RIP140 N or C proteins (Fig. 2AGo, lanes 3 and 4). Control reactions showing interaction between in vitro translated GR and GST-RIP140 N and C (Fig. 2AGo, lanes 7 and 8) proved that there was no general defect in the bacterially expressed proteins. The role of RIP140 phosphorylation for 14–3-3 binding was further studied by analyzing the interaction of in vitro translated and alkaline phosphatase-treated RIP140 with GST-14–3-3. The results showed that alkaline phosphatase treatment of RIP140 decreased the interaction with 14–3-3 (Fig. 2BGo, compare lanes 3 and 6).



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Figure 2. Role of Protein Phosphorylation for 14–3-3 Interaction: GST Pull-Down Assays

A, In vitro translated 35S-labeled 14–3-3 (lanes 1–4) and GR (lanes 5–8) were incubated with GST (lanes 2 and 6), GST-RIP140 N (aa 1–281) (lanes 3 and 7), or GST-RIP140 C (aa 747-1,158) (lanes 4 and 8) bound to glutathione agarose beads in PDB with 0.01% Igepal CA-630. In vitro translated GR was diluted into PDB and incubated with 1 µM Dex for 2 h at 4 C before mixing with the GST protein. The eluted samples from washed beads were analyzed by SDS-PAGE and visualized by autoradiography. The input (lanes 1 and 5) represents 15% of the amount of labeled protein incubated with GST-protein-bound beads. B, In vitro translated 35S-labeled RIP140 was incubated in the presence or absence of alkaline phosphatase (AP) and mixed with GST (lanes 2 and 5) or GST-14–3-3 (lanes 3 and 6) bound to glutathione agarose beads in PDB with 0.03% Igepal CA-630. The input (lanes 1 and 4) represents 15% of the amount of labeled protein incubated with GST-protein-bound beads. The relative binding of alkaline phosphatase-treated RIP140 to GST-14–3-3 compared with untreated, was 0.22 ± 0.14 (mean ± SD, n = 4) as determined by PhosphoImager analysis.

 
RIP140 Antagonizes the 14–3-3-Enhanced GR Transactivation
To analyze the functional role of RIP140 interaction with 14–3-3, we performed transient transfection experiments in COS-7 cells. As expected, 14–3-3 enhanced the GR- induced transactivation (Fig. 3Go). The results of coexpression of RIP140 and 14–3-3 on GR-induced transactivation showed that RIP140-antagonized 14–3-3 enhanced GR transactivation (Fig. 3Go). In cells transfected with 5 ng of RIP140 expression plasmid, no 14–3-3-enhanced GR transactivation could be detected (Fig. 3Go). RIP140 also reduced the GR transactivation in the absence of coexpressed 14–3-3. RIP140 only worked via ligand- activated GR since no effect of RIP140 on basal transactivation levels in the absence of dexamethasone (Dex) was seen (Fig. 3Go).



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Figure 3. RIP140 Antagonizes the 14–3-3 Enhanced GR Transactivation

COS-7 cells were transiently transfected with 100 ng of p19-tk-luc and 5 ng of pCMV-ß gal reporter plasmids and 2.5 ng of pCMV4-hGR expression plasmid, as well as indicated amounts of pSG5-RIP140 and pBKCMV-14–3-3 expression plasmids or the corresponding empty plasmids. The total amount of each kind of plasmid was kept constant. The cells were treated with or without 1 µM Dex and harvested for luciferase and ß-galactosidase assays. The GR-regulated luciferase reporter gene activity was related to the control ß-galactosidase reporter gene activity. The results shown represent mean values and SD obtained from three independent experiments.

 
Noncompetitive Interactions between RIP140, 14–3-3, and GR
The negative effect of RIP140 on GR transactivation has previously been suggested to be due to RIP140 competing with positively acting coactivator proteins for binding to GR (34). We have previously described a ligand-dependent interaction between 14–3-3 and GR (22). To analyze whether the mechanism for the antagonizing effect of RIP140 on 14–3-3-enhanced GR transactivation was due to RIP140 competing with 14–3-3 for binding to GR, we performed GST pull-down competition experiments. Unlabeled in vitro translated RIP140 or bacterially expressed His-RIP140 C were added to binding reaction to determine the effect on interaction between GST-14–3-3 and in vitro translated GR in the presence of ligand. The results showed that neither of the RIP140 proteins could disrupt the binding of GR to 14–3-3 (Fig. 4AGo, compare lanes 3 and 5, and 8 and 10, respectively). To determine whether the positive effect of 14–3-3 on GR transactivation was due to 14–3-3 competing with RIP140 for binding to GR, unlabeled in vitro translated 14–3-3 was added to the binding reaction for GST-GR LBD and in vitro translated RIP140 and to the binding reaction for GST-RIP140 C and in vitro translated GR (Fig. 4BGo). In neither case did 14–3-3 inhibit the interaction between GR and RIP140 (Fig. 4BGo, compare lanes 3 and 5, and 8 and 10, respectively).



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Figure 4. Noncompetitive Interactions between RIP140, 14–3-3, and GR: GST Pull-Down Assays

A, RIP140 does not disrupt the interaction between GR and 14–3-3. In vitro translated 35S-labeled GR (7.5 µl) was incubated in the presence of 1 µM Dex with 10 µg of GST (lanes 2, 4, 7, and 9) or GST-14–3-3 (lanes 3, 5, 8, and 10) bound to glutathione agarose beads in the absence or presence of 30 µl of in vitro translated unlabeled RIP140 (lanes 2–5) or 10 µg of bacterially expressed His-RIP140 C (aa 747–1,158) (lanes 7–10) in PDB with 0.03% Igepal CA-630. The ratio of in vitro translated RIP140/GR was 3-fold and the ratio of His-RIP140/GR was more than 100-fold. GR was diluted into PDB and incubated with 1 µM Dex for 2 h at 4 C before mixing with the GST protein. The eluted samples from washed beads were analyzed by SDS-PAGE and visualized by autoradiography. The input (lanes 1 and 6) represents 10% of the amount of labeled protein incubated with GST-protein-bound beads. The relative binding of GR to GST-14–3-3 in the presence of added in vitro translated RIP140 or His-RIP140 C compared with in the absence, was 1.5 ± 0.7 (mean ± SD, n = 3) and 1.2 ± 0.5 (mean ± SD, n = 4), respectively, as determined by densitometric scanning of films. B, 14–3-3 does not disrupt the interaction between RIP140 and GR. Five microliters of in vitro translated 35S-labeled RIP140 (lanes 1–5) and GR (lanes 6–10) were incubated in the presence of 1 µM Dex with 10 µg of GST (lanes 2, 4, 7, and 9), GST-GR LBD (lanes 3 and 5), or GST-RIP140 C (aa 747–1,158) (lanes 8 and 10) bound to glutathione agarose beads in the absence or presence of 25 µl of in vitro translated unlabeled 14–3-3 in PDB with 0.01% Igepal CA-630. The ratio of in vitro translated 14–3-3/RIP140 and 14–3-3/GR was 5-fold. GR was diluted into PDB and incubated with 1 µM Dex for 2 h at 4 C before mixing with the GST protein. The input (lanes 1 and 6) represents 15% of the amount of labeled protein incubated with GST-protein bound beads. C, RIP140, 14–3-3, and GR can form a tertiary complex. Bacterially expressed His-RIP140 C was incubated in the presence of 1 µM Dex with GST (lanes 2 and 4) or GST-14–3-3 (lanes 3 and 5) bound to glutathione agarose beads in the absence or presence of in vitro translated unlabeled GR. GR was diluted into PDB and incubated with 1 µM Dex for 2 h at 4 C before mixing with the GST protein. The eluted samples from washed beads were analyzed by SDS-PAGE and visualized by Western blotting using a His tag-specific antibody.

 
We have demonstrated here that all three proteins, GR, RIP140, and 14–3-3, can interact pairwise with each other. To determine whether all three proteins could form a tertiary complex, we used the inability of bacterially expressed protein to bind to 14–3-3. The binding of bacterially expressed His-RIP140 C to GST-14–3-3 was analyzed in the presence or absence of in vitro translated GR (Fig. 4CGo). In the absence of GR, His-RIP140 C did not bind GST-14–3-3 (Fig. 4CGo, lane 3). However, in the presence of GR, His-RIP140 C interacted with GST-14–3-3 (Fig. 4CGo, lane 5), indicating that GR could function as a bridging protein between RIP140 and 14–3-3 and that all three proteins were present in the same complex.

14–3-3 Regulates the Subcellular Localization of RIP140
14–3-3 proteins have been shown to regulate the subcellular localization of some interacting proteins (8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). To determine whether this mechanism was involved in the case of 14–3-3 binding to RIP140 we studied the effect of 14–3-3 on the subcellular localization of green fluorescent protein (GFP)-fused RIP140 in transfected COS-7 cells. GFP-RIP140 was detected only in the nucleus, in a punctate pattern as previously described (Fig. 5AGo, panel a) (30). In contrast, in cells cotransfected with an expression plasmid for 14–3-3, the majority of GFP-RIP140 was relocalized into the cytoplasm (Fig. 5AGo, panel b). Interestingly, remaining nuclear GFP-RIP140 had lost its punctate expression pattern (Fig. 5AGo, panel b), suggesting a function for 14–3-3 in relocalization of RIP140 also within the nucleus. To study the expression of both 14–3-3 and RIP140 in the same cell, GFP-fused 14–3-3 and hemagglutinin (HA)-tagged RIP140 that could be detected using indirect immunofluorescence were used. In cells cotransfected with HA-RIP140 and GFP, HA-RIP140 was present only in the nucleus in a similar punctate pattern to GFP-RIP140 (Fig. 5BGo, panel a). As expected, the control GFP protein was detected both in the cytoplasm and the nucleus (Fig. 5BGo, panel b). In cells coexpressing HA-RIP140 and GFP-14–3-3, HA-RIP140 was found in the cytoplasm, confirming the 14–3-3-induced redistribution observed using GFP-RIP140 (Fig. 5BGo, panel c). In these cells GFP-14–3-3 could be detected both in the cytoplasm and the nucleus (Fig. 5BGo, panel d). In most cells, the coexpression of HA-RIP140 did not affect the localization of GFP-14–3-3 (data not shown). Apparently, the export function of 14–3-3 seems to dominate over the import function of RIP140.



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Figure 5. Relocalization of RIP140 from the Nucleus to the Cytoplasm by 14–3-3: Confocal Microscopy Analysis

A, COS-7 cells were transfected with 0.5 µg of pEGFP-C2-RIP140 and 0.5 µg of pcDNA3-FLAG (panel a) or with 0.5 µg of pEGFP-C2-RIP140 and 0.5 µg of pcDNA3-FLAG-14–3-3{eta} (panel b). The localization of GFP-RIP140 was visualized using a TCS SP Multiband Confocal Imaging System (Leica Corp.). B, COS-7 cells were transfected with 0.5 µg of pSG5-HA-hRIP140 and pEGFP-C2 (panels a and b) or with 0.5 µg of pSG5-HA-hRIP140 and pEGFP-C2–14-3–3{eta} (panels c and d). The localization of HA-RIP140 and GFP or GFP-14–3-3 in the same cells was visualized using indirect immunofluorescence and a Leica TCS SP Multiband Confocal Imaging System.

 
Interaction of 14–3-3 with Nuclear Receptors and Nuclear Receptor Cofactors
To determine whether 14–3-3 could interact with any other nuclear receptors, in addition to GR, we performed GST pull-down experiments. GST-14–3-3 interacted with in vitro translated GR, AR, ER{alpha}, ERß, and TR{alpha}, but not with retinoid X receptor {alpha} (RXR{alpha}) or PPAR{alpha} (Fig. 6AGo, lanes 5, 10, 15, 20, 25, 30, and 35). The interaction between GR and 14–3-3 was inhibited by molybdate, which stabilizes the GR-hsp90 complex that is formed in the absence of ligand (Fig. 6AGo, lane 3) (22). However, molybdate did not inhibit the interaction between 14–3-3 and the other steroid receptors, AR, ER{alpha}, or ERß (Fig. 6AGo, lanes 8, 13, and 18). The interaction between 14–3-3 and TR{alpha} was also ligand independent (Fig. 6AGo, lane 23).



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Figure 6. Interaction of 14–3-3 with Nuclear Receptors and Nuclear Receptor Cofactors: GST Pull-Down Assays

A, In vitro translated 35S-labeled GR (lanes 1–5), AR (lanes 6–10), ER{alpha} (lanes 11–15), ERß (lanes 16–20), TR{alpha} (lanes 21–25), RXR{alpha} (lanes 26–30), and PPAR{alpha} (lanes 31–35) were incubated with GST or GST-14–3-3 bound to glutathione agarose beads in PDB with 0.01% Igepal CA-630. The interaction of GR, AR, ER{alpha}, and ERß was studied in the presence of 20 mM molybdate (Mo) or 1 µM of the respective ligand, i.e. Dex, testosterone (Test), and 17ß-estradiol (E2) as indicated. The interaction of TR{alpha}, RXR{alpha}, and PPAR{alpha} was studied in the absence (-) or presence of 10 µM of their respective ligands, i.e. T3, 9-cis retinoic acid (9-cis), and WY-14,643 (WY) as indicated. The translated receptor proteins were diluted into PDB and incubated in the presence or absence of the corresponding ligand or molybdate for 2 h at 4 C before mixing with the GST protein. The eluted samples from washed beads were analyzed by SDS-PAGE and visualized by autoradiography. The input represents 10% of the amount of labeled protein incubated with GST-protein-bound beads. B, In vitro translated 35S-labeled TRAP220 (lanes 1 and 6–8), CBP (lanes 2 and 9–11), ACTR (lanes 3 and 12–14), SRC-1 (lanes 4 and 15–17), and TIF2 (lanes 5 and 18–20) were incubated with GST, GST-GR LBD, or GST-14–3-3 bound to glutathione agarose beads in PDB with 0.01% Igepal CA-630. GST-GR LBD bound to glutathione agarose beads was incubated with 1 µM Dex for 2 h before mixing with translated protein. The input (lanes 1–5) represents 15% of the amount of labeled protein incubated with GST-protein-bound beads.

 
Using the yeast two-hybrid method we tested whether nuclear receptor cofactors previously identified in a two-hybrid screening for PPAR{alpha} interacting proteins could also bind to GR LBD (34, 44, 48). The results showed that, in the presence of ligand, GR LBD interacted not only with RIP140 [amino acids (aa) 431–1,158], but also with TRAP220 (aa 503–754) and TIF2 (aa 320–1,119) (data not shown). To further study the interaction of GR LBD with cofactor proteins, we performed GST pull-down experiments. GST-GR LBD interacted in the presence of ligand with in vitro translated full-length TRAP220 and ACTR, and weakly with SRC-1 and TIF2 (Fig. 6BGo, lanes 7, 13, 16, and 19). No interaction was detected with in vitro translated full-length CBP (Fig. 6BGo, lane 10). To determine whether any of the GR cofactor proteins, in addition to RIP140, could interact with 14–3-3, we used GST-14–3-3 in the GST pull-down experiments. The results showed that the in vitro translated full-length TRAP220 and ACTR interacted specifically with GST-14–3-3 (Fig. 6BGo, lanes 8 and 14), whereas CBP, SRC-1, and TIF2 did not interact or interacted weakly (Fig. 6BGo, lanes 11, 17, and 20).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We show in this paper that the 14–3-3 protein can interact with the nuclear receptor corepressor RIP140 and change its subcellular localization. Therefore, we suggest that the positive effect of 14–3-3 on GR transactivation is due to 14–3-3 binding to RIP140 and removing the negatively acting RIP140 from the nucleus, allowing positively acting coactivator proteins to interact with the receptor. Recently it has been reported that 14–3-3 may act by regulating the subcellular localization of interacting proteins (8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). We show here that coexpression of 14–3-3 can relocalize RIP140 from the nucleus to the cytoplasm, as well as change the punctate pattern of remaining nuclear RIP140 to a homogeneous nuclear distribution. Therefore it is tempting to speculate that the 14–3-3- induced disappearance of the punctate pattern could be connected with functional changes of RIP140 within the nucleus. Further, it is interesting that agonist-, but not antagonist-, bound GR is also localized to intranuclear dot structures (49). 14–3-3 has been suggested to act by exporting proteins from the nucleus using a nuclear export sequence that is present in 14–3-3. Alternatively, 14–3-3 may inhibit nuclear import of interacting proteins by binding to a site on the interacting protein overlapping with a nuclear localization sequence. The mechanism for the redistribution of RIP140 into the cytoplasm by 14–3-3 is at present not known. However, the observed 14–3-3-induced intranuclear redistribution of RIP140 suggests that 14–3-3 is binding to RIP140 in the nucleus and not only acting by sequestering RIP140 in the cytoplasm.

RIP140 has previously been shown to compete with the coactivator TIF2 for binding to GR (37). We show here that overexpression of RIP140 inhibits the 14–3-3-enhanced GR transactivation, which can be due to an increased level of RIP140 that is not bound to 14–3-3 and can stay in the nucleus and compete with coactivators for binding to GR. Since RIP140 and 14–3-3 interact noncompetitively with GR, it is unlikely that the antagonistic effect of RIP140 on 14–3-3- enhanced GR transactivation is due to RIP140 disrupting the 14–3-3/GR complex or that the positive effect of 14–3-3 on GR transactivation is due to 14–3-3 disrupting the RIP140/GR. Both RIP140 and 14–3-3 interact with GR LBD. RIP140 has been shown to interact with the activation function 2 (AF2) domain in nuclear receptor LBDs (30) whereas the exact interaction surface for 14–3-3 on GR LBD has not been determined. The lack of binding competition between RIP140 and 14–3-3 suggests that 14–3-3 does not interact with the AF2 domain in GR LBD. It is possible that all three proteins can form a complex, but that this tertiary complex is inhibitory in contrast to the heterodimer between GR and 14–3-3.

Many 14–3-3 target proteins are phosphorylated on serine residues, and phosphatase treatment of the proteins has been shown to reduce the interaction with 14–3-3 (1, 45, 46, 47). RIP140 is a serine-rich protein, and we show here that phosphorylation of RIP140 may also be important for 14–3-3 recognition, since phosphatase-treated in vitro translated RIP140 or nonphosphorylated bacterially expressed RIP140 did not interact efficiently with 14–3-3. The effect of 14–3-3 of subcellular localization of interacting proteins has been demonstrated to be regulated by phosphorylation of the target protein and subsequent association with 14–3-3 (8, 14, 15). It would be interesting to determine whether a regulated serine phosphorylation of RIP140 contributes to the function of the protein and the subcellular localization.

Interestingly, the same domains of RIP140 are involved in both GR and 14–3-3 interaction. RIP140 contains many nuclear receptor boxes (NR boxes) (LXXLL motifs) that have been shown to be important for nuclear receptor interaction (34, 44, 50, 51, 52). The GR-interacting domains RIP140 N (aa 1–472) and RIP140 C (aa 747–1,158) contain five and three NR boxes, respectively. However, the middle domain RIP140 M (aa 431–745), which does not interact with GR, still contains two NR boxes. Possibly, sequences surrounding the LXXLL core of these NR boxes account for the low affinity to GR (44). The C-terminal part of RIP140 contains a consensus 14–3-3 binding motif (RTFSYP), whereas the N-terminal and middle parts of RIP140 only contain sequences with weak similarity to the consensus motif. However, in addition to the consensus motif, other less well characterized 14–3-3 binding sequences have been identified, and possibly such sequences are also involved in the 14–3-3/RIP140 interaction.

Our results indicate that in addition to RIP140, 14–3-3 can interact with the nuclear receptor coactivators TRAP220 and ACTR. Although the importance of these coactivators for GR signaling is not yet clear, 14–3-3 could be involved in the regulation of these cofactors as well. Nuclear receptor cofactors have been suggested to function within larger coactivator complexes such as the p160 complex and the TRAP220 complex (26). Since 14–3-3 has been suggested to act as a scaffold to bring together different proteins, it is interesting to speculate that 14–3-3, which potentially interacts with proteins in both complexes, may play a role in the formation or regulation of the coactivator complexes needed for receptor function. In addition, our results suggest that 14–3-3 can interact with other nuclear receptors. The binding of 14–3-3 to these receptors was ligand independent and not inhibited by molybdate, which is consistent with a weaker complex formation between these receptors and hsp90 (28). Since several nuclear receptors can interact with 14–3-3, the interaction between 14–3-3 and RIP140 that we describe here might be involved in the regulatory function of RIP140 on other receptors in addition to GR.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
GST fusion proteins were expressed from pGEX plasmids (Amersham Pharmacia Biotech, Arlington Heights, IL). The plasmids expressing GST-14–3-3{eta}, GST-RIP140 N (aa 1–281), and GST-RIP140 C (aa 747-1,158) have been described previously (22, 34). The plasmid expressing GST-LBD (aa 485–777) was constructed by subcloning of a BamHI fragment from pEG202/GR LBD into pGEX-5X-3 (22). The His-tagged RIP140 C (aa 747–1,158) was expressed from plasmid pET19 (Novagen Inc., Madison, WI) (34). pSG5-HA-hRIP140 expressing an HA-tagged human RIP140 (aa 1–1,158) has been described previously (34). GFP-RIP140 was made by subcloning the corresponding fragment from the pBK-CMV-based expression plasmid into pEGFP-C2 (CLONTECH Laboratories, Inc., Palo Alto, CA). GFP-14–3-3{eta} was generated by subcloning an EcoRI–XhoI fragment from pBK-CMV-14–3-3{eta} into pEGFP-C2 (CLONTECH Laboratories, Inc.). FLAG-14–3-3{eta} was made by cloning the same fragment into pcDNA3-FLAG (a gift from A. Ström). Plasmids for in vitro transcription/translation of hGR, 14–3-3{eta}, RIP140, RIP140 N (aa 1–472), RIP140 M (aa 431–745), RIP 140 C (aa 747-1,158), hAR, hER{alpha}, hERß, hTR{alpha}, rRXR{alpha}, rPPAR{alpha}, mTRAP220, hSRC1, mTIF2, hACTR, and mCBP were used (22, 34, 39, 40, 41, 42, 43, 44). Mammalian expression plasmids pCMV4-hGR and pBKCMV-14–3-3{eta} and reporter plasmids p19-tk-luc and pCMV-ß gal were described previously (22, 34).

Protein Expression and Purification
GST- and His-tagged proteins were expressed in the Escherichia coli strain BL21(DE3)pLysS. Bacteria were grown to an OD600 of 0.5, and the expression of fusion protein was induced for 2 h by adding isopropyl 1-thio-ß-D-galactopyranoside to a final concentration of 0.5 mM. Bacteria expressing GST and GST-14–3-3 were harvested by centrifugation and resuspended in 10 mM phosphate buffer, pH 7.4, 140 mM NaCl, 3 mM KCl, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml leupeptin, 0.1% Igepal CA-630. The suspension was treated with 0.1 mg/ml lysozyme, 20 µg/ml DNaseI, 10 µg/ml RNaseA and cleared by centrifugation. The resulting soluble protein lysate was incubated with glutathione-agarose beads (Sigma, St. Louis, MO) for 2 h at 4 C. The beads were washed with 10 mM phosphate buffer, pH 7.4, 140 mM NaCl, 3 mM KCl. Fusion proteins were eluted with 20 mM HEPES-KOH, pH 7.9, 10% (vol/vol) glycerol, 100 mM KCl, 5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 0.2 mM PMSF, 0.01% Igepal CA-630, 20 mM glutathione, and dialyzed using the same buffer without glutathione. GST-RIP140 proteins and GST-GR LBD, which were insoluble under normal purification conditions, were solubilized in sodium-N-lauroylsarcosinate, and the extracts were used directly in GST pull-down experiments. Bacteria expressing GST-RIP140 and GST-GR LBD were harvested by centrifugation and resuspended in 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 1 µg/ml leupeptin. The suspension was treated with 0.1 mg/ml lysozyme, 20 µg/ml DNaseI, 10 µg/ml RNaseA, and 1.5% sodium-N- lauroylsarcosinate and cleared by centrifugation. The cleared extract was used directly in GST pull-down experiments. Bacteria expressing His-RIP140 C were harvested by centrifugation and resuspended in 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM PMSF, 1 µg/ml leupeptin. The suspension was treated with 0.1 mg/ml lysozyme, 20 µg/ml DNaseI, 10 µg/ml RnaseA, and 0.9% sodium-N-lauroylsarcosinate and cleared by centrifugation. The resulting soluble protein lysate was incubated with a TALON affinity resin (CLONTECH Laboratories, Inc.) for 30 min at room temperature. The resin was washed with 20 mM Tris-HCl, pH 8.0, 100 mM NaCl. His-RIP140 C was eluted with 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM PMSF, 1 µg/ml leupeptin, 1% Tween 20, and 50 mM imidazole and dialyzed using the same buffer without Tween 20 or imidazole. [35S]Methionine-labeled proteins were synthesized in vitro using TnT-coupled reticulocyte lysate (Promega Corp., Madison, WI).

Alkaline Phosphatase Treatment
To dephosphorylate the in vitro translated RIP140 protein, 10 µl of translation reaction were incubated in the presence or absence of 5 U of alkaline phosphatase (Roche Molecular Biochemicals, Indianapolis, IN) in 50 mM Tris-HCl, pH 8.5, 0.1 mM EDTA at 37 C for 1 h. The reactions were subsequently used in GST pull-down experiments.

GST Pull-Down Experiments
GST-fusion protein (5 or 10 µg) bound to 30 µl glutathione beads was incubated with 5–10 µl of in vitro translated protein in 200 µl pull-down buffer (PDB) [20 mM HEPES-KOH, pH 7.9, 10% (vol/vol) glycerol, 100 mM KCl, 5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 0.2 mM PMSF, 1 mg/ml BSA and 0.01–0.03% Igepal CA-630] overnight at 4 C. The beads were recovered by centrifugation and washed four times with PDB without BSA, and bound proteins were eluted by SDS-PAGE sample buffer, analyzed on SDS-PAGE, and visualized by autoradiography. Dex, 1 µM, was used in all GST pull-down experiments using in vitro translated GR or GST-GR LBD. The translated receptor proteins were diluted into PDB and incubated in the presence or absence of the corresponding ligand or molybdate for 2 h at 4 C before mixing with the GST protein. In experiments using GST-GR LBD, the fusion protein bound to glutathione beads was incubated with ligand for 2 h before mixing with translated proteins. In competition GST pull-down experiments, His-RIP140 C or unlabeled in vitro translated protein was included in the binding reaction. For quantification, gels were analyzed using PhosphoImager (BAS-2000, Fuji Photo Film Co., Ltd., Tokyo, Japan) or films were analyzed using Image Scanner (Amersham Pharmacia Biotech, Arlington Heights, IL) and the relative amount of protein input was calculated by subtracting unspecific binding (GST) from specific binding (GST-fusion protein).

Western Blotting
Proteins were separated on SDS-PAGE and transferred to Hybond-C extra nitrocellulose filters (Amersham Pharmacia Biotech). Filters were blocked with 5% milk powder in PBS containing 0.05% Tween 20. For detection of His-RIP140 C the filter was incubated with a 1:500 dilution of a rabbit polyclonal antibody recognizing the His tag (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in PBS/Tween 20 for 2 h at room temperature. After washing, the filters were incubated with horseradish peroxidase-linked antirabbit antibody (Amersham Pharmacia Biotech) at a dilution of 1:8,000 in PBS/Tween 20 for 1 h at room temperature. After washing, the proteins were visualized using the Supersignal West Pico chemiluminescent substrate (Pierce Chemical Co.).

Reporter Gene Assays
COS-7 cells were seeded in 24-well plates and transfected with plasmids using the Fugene 6 transfection agent (Roche Molecular Biochemicals). After transfection, cells were maintained in the presence of 1 µM Dex or ethanol vehicle for 24 h and then harvested for luciferase and ß-galactosidase assay. The luciferase activity was determined using the Gen-Glow-1000 kit (Bioorbit, Turku, Finland) and ß-galactosidase using the Galacto-Light Plus kit (Tropix, Inc., Bedford, MA) in the luminometer Anthos Lucy I (Anthos Labtec-Instruments, Salzburg, Austria).

Confocal Microscopy
COS-7 cells were plated on glass cover slips in six-well plates and grown for 24 h. The cells were transfected for 8 h with 0.5 µg of each plasmid (total amount 1 µg) using Lipofectin (Life Technologies, Inc., Gaithersburg, MD). Then fresh medium was added, and the cells were further grown for 20 h before fixing with 3% paraformaldehyde in 5% sucrose/PBS, 20 min at room temperature. For indirect immunofluorescence, fixed cells were rinsed three times with PBS before being permeabilized with PBS/Tween (0.1 %), three times for 5 min at room temperature. The cells were blocked by 5% goat serum (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) in PBS/Tween (1 h at room temperature) before incubating with mouse monoclonal antibody, HA.1 (Babco, Richmond, CA) for 1 h (diluted 1:200 in PBS/Tween). After removal of the HA.1 antibody by washing three times 5 min with PBS/Tween, cells were treated with tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat antimouse IgG (H+L) (Jackson ImmunoResearch Laboratories, Inc.) for 1 h at room temperature (diluted 1:100 in PBS/Tween). Cells were washed five times for 5 min in PBS/Tween before being fixed to slides using antiphotobleaching fluorosave (Calbiochem, La Jolla, CA). Subcellular localization was determined using a TCS SP Multiband Confocal Imaging System. (Leica Corp., Deerfield, IL).


    ACKNOWLEDGMENTS
 
We thank Drs. R. M. Evans, G. Kuiper, J. Leers, B.W. O’Malley, M. G. Parker, K. Pettersson, A. Ström, and S. Nilsson for generously providing plasmids and Sanna Hulkko for technical assistance during the work.


    FOOTNOTES
 
Address requests for reprints to: Dr. Johanna Zilliacus, Department of Medical Nutrition, Karolinska Institutet, Novum, S-141 86 Huddinge, Sweden.

This work was supported by grants from The Swedish Medical Research Council (12557), Åke Wiberg Foundation, Lars Hierta Foundation, and Emil and Wera Cornell Foundation. Dr. Zilliacus was supported by a junior research position from the Swedish Medical Research Council. Dr. Holter was supported by a grant from Nordic Academy of Advanced Study (99.30.165–0).

1 Present address: Third Department of Internal Medicine, Akita University School of Medicine, 1–1-1 Hondo, Akita-shi, Akita 010-8543, Japan. Back

Received for publication May 22, 2000. Revision received December 19, 2000. Accepted for publication January 5, 2001.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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