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A nuclear action of the eukaryotic cochaperone RAP46 in downregulation of glucocorticoid receptor activity.

Schneikert J, Hübner S, Martin E, Cato AC - J. Cell Biol. (1999)

Bottom Line: We demonstrate a specific cytoplasmic-nuclear recruitment of RAP46 by the liganded GR that results in inhibition of the transactivation function of the receptor.BAG-1, a shorter isoform with only a duplication of the [EEX(4)] sequence, does not inhibit GR activity.The nuclear effects of RAP46 and BAG-1L are specific since GR-mediated inhibition of AP-1 activity was not affected.

View Article: PubMed Central - PubMed

Affiliation: Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics, D-76021 Karlsruhe, Germany.

ABSTRACT
RAP46 is a eukaryotic cochaperone that associates with several proteins, including the heat shock protein hsp70/hsc70 and the glucocorticoid receptor (GR). Here we show a downregulation of GR-mediated transactivation by RAP46 via a mechanism independent of a cytoplasmic action of this cochaperone. We demonstrate a specific cytoplasmic-nuclear recruitment of RAP46 by the liganded GR that results in inhibition of the transactivation function of the receptor. A repeated sequence motif [EEX(4)](8) at the NH(2) terminus of RAP46 or BAG-1L, a larger isoform of RAP46, is responsible for this downregulation of GR activity. BAG-1, a shorter isoform with only a duplication of the [EEX(4)] sequence, does not inhibit GR activity. The [EEX(4)](8) motif, when linked to an otherwise unrelated protein, abrogated the inhibitory action of endogenous RAP46 on GR-mediated transactivation. The nuclear effects of RAP46 and BAG-1L are specific since GR-mediated inhibition of AP-1 activity was not affected. These studies identify the [EEX(4)](8) sequence as a signature motif for inhibition of GR-mediated transactivation and demonstrate a specific nuclear action of a eukaryotic cochaperone in the regulation of GR activity.

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Effect of RAP46 and BAG-1L on DNA-binding, transactivation, and transrepression by the GR. COS-7 cells were transiently transfected with either a control vector, a plasmid encoding the GR, RAP46, or BAG-1L. After transfection, the cells were treated with 0.1 μM dexamethasone for 36 h and whole cell extracts were prepared for EMSA. In addition, the effect of RAP46 and BAG-1L on transactivation at the MMTV promoter and GR-mediated repression at the human collagenase I promoter were analyzed. MCF-7 cells were transiently transfected with a control vector or a plasmid encoding RAP46 or BAG-1L, in addition to the MMTV firefly luciferase indicator or collagenase luciferase constructs and a plasmid encoding the renilla luciferase as an internal control. The activity of the collagenase promoter was induced with 80 ng/ml TPA and the cells were treated immediately after transfection with vehicle alone (0.1% ethanol; −) or 0.1 μM dexamethasone (+) for 36 h. The bar chart shows the normalized luciferase activity (firefly/renilla) of the MMTV and collagenase gene constructs. The results represent the mean value (± SD) of three independent experiments.
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Figure 7: Effect of RAP46 and BAG-1L on DNA-binding, transactivation, and transrepression by the GR. COS-7 cells were transiently transfected with either a control vector, a plasmid encoding the GR, RAP46, or BAG-1L. After transfection, the cells were treated with 0.1 μM dexamethasone for 36 h and whole cell extracts were prepared for EMSA. In addition, the effect of RAP46 and BAG-1L on transactivation at the MMTV promoter and GR-mediated repression at the human collagenase I promoter were analyzed. MCF-7 cells were transiently transfected with a control vector or a plasmid encoding RAP46 or BAG-1L, in addition to the MMTV firefly luciferase indicator or collagenase luciferase constructs and a plasmid encoding the renilla luciferase as an internal control. The activity of the collagenase promoter was induced with 80 ng/ml TPA and the cells were treated immediately after transfection with vehicle alone (0.1% ethanol; −) or 0.1 μM dexamethasone (+) for 36 h. The bar chart shows the normalized luciferase activity (firefly/renilla) of the MMTV and collagenase gene constructs. The results represent the mean value (± SD) of three independent experiments.

Mentions: In the liganded state, when both the GR and MR are in the nucleus, RAP46 downregulated the transactivation function of only the GR, but not that of the MR (Fig. 3a and Fig. B; note that GGG and MMM refer to GR and MR). This repression was independent of whether dexamethasone, cortisol, or aldosterone was the activating ligand (Fig. 3 B). The lack of RAP46 effect on transactivation by the MR is possibly due to the different cellular localizations of these two proteins in the presence of ligand (Fig. 1D 2; note nuclear and cytoplasmic localization of MR and RAP46). We therefore repeated the transfection experiments with the MR and the RAP46 isoform BAG-1L, which is constitutively localized in the nucleus (Froesch et al. 1998; our unpublished results). In this study, transactivation by the MR was not repressed by BAG-1L, although the response of the GR was inhibited (results not shown; see Fig. 7). This indicates a fundamental difference in the action of RAP46 towards the MR and the GR.


A nuclear action of the eukaryotic cochaperone RAP46 in downregulation of glucocorticoid receptor activity.

Schneikert J, Hübner S, Martin E, Cato AC - J. Cell Biol. (1999)

Effect of RAP46 and BAG-1L on DNA-binding, transactivation, and transrepression by the GR. COS-7 cells were transiently transfected with either a control vector, a plasmid encoding the GR, RAP46, or BAG-1L. After transfection, the cells were treated with 0.1 μM dexamethasone for 36 h and whole cell extracts were prepared for EMSA. In addition, the effect of RAP46 and BAG-1L on transactivation at the MMTV promoter and GR-mediated repression at the human collagenase I promoter were analyzed. MCF-7 cells were transiently transfected with a control vector or a plasmid encoding RAP46 or BAG-1L, in addition to the MMTV firefly luciferase indicator or collagenase luciferase constructs and a plasmid encoding the renilla luciferase as an internal control. The activity of the collagenase promoter was induced with 80 ng/ml TPA and the cells were treated immediately after transfection with vehicle alone (0.1% ethanol; −) or 0.1 μM dexamethasone (+) for 36 h. The bar chart shows the normalized luciferase activity (firefly/renilla) of the MMTV and collagenase gene constructs. The results represent the mean value (± SD) of three independent experiments.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169481&req=5

Figure 7: Effect of RAP46 and BAG-1L on DNA-binding, transactivation, and transrepression by the GR. COS-7 cells were transiently transfected with either a control vector, a plasmid encoding the GR, RAP46, or BAG-1L. After transfection, the cells were treated with 0.1 μM dexamethasone for 36 h and whole cell extracts were prepared for EMSA. In addition, the effect of RAP46 and BAG-1L on transactivation at the MMTV promoter and GR-mediated repression at the human collagenase I promoter were analyzed. MCF-7 cells were transiently transfected with a control vector or a plasmid encoding RAP46 or BAG-1L, in addition to the MMTV firefly luciferase indicator or collagenase luciferase constructs and a plasmid encoding the renilla luciferase as an internal control. The activity of the collagenase promoter was induced with 80 ng/ml TPA and the cells were treated immediately after transfection with vehicle alone (0.1% ethanol; −) or 0.1 μM dexamethasone (+) for 36 h. The bar chart shows the normalized luciferase activity (firefly/renilla) of the MMTV and collagenase gene constructs. The results represent the mean value (± SD) of three independent experiments.
Mentions: In the liganded state, when both the GR and MR are in the nucleus, RAP46 downregulated the transactivation function of only the GR, but not that of the MR (Fig. 3a and Fig. B; note that GGG and MMM refer to GR and MR). This repression was independent of whether dexamethasone, cortisol, or aldosterone was the activating ligand (Fig. 3 B). The lack of RAP46 effect on transactivation by the MR is possibly due to the different cellular localizations of these two proteins in the presence of ligand (Fig. 1D 2; note nuclear and cytoplasmic localization of MR and RAP46). We therefore repeated the transfection experiments with the MR and the RAP46 isoform BAG-1L, which is constitutively localized in the nucleus (Froesch et al. 1998; our unpublished results). In this study, transactivation by the MR was not repressed by BAG-1L, although the response of the GR was inhibited (results not shown; see Fig. 7). This indicates a fundamental difference in the action of RAP46 towards the MR and the GR.

Bottom Line: We demonstrate a specific cytoplasmic-nuclear recruitment of RAP46 by the liganded GR that results in inhibition of the transactivation function of the receptor.BAG-1, a shorter isoform with only a duplication of the [EEX(4)] sequence, does not inhibit GR activity.The nuclear effects of RAP46 and BAG-1L are specific since GR-mediated inhibition of AP-1 activity was not affected.

View Article: PubMed Central - PubMed

Affiliation: Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics, D-76021 Karlsruhe, Germany.

ABSTRACT
RAP46 is a eukaryotic cochaperone that associates with several proteins, including the heat shock protein hsp70/hsc70 and the glucocorticoid receptor (GR). Here we show a downregulation of GR-mediated transactivation by RAP46 via a mechanism independent of a cytoplasmic action of this cochaperone. We demonstrate a specific cytoplasmic-nuclear recruitment of RAP46 by the liganded GR that results in inhibition of the transactivation function of the receptor. A repeated sequence motif [EEX(4)](8) at the NH(2) terminus of RAP46 or BAG-1L, a larger isoform of RAP46, is responsible for this downregulation of GR activity. BAG-1, a shorter isoform with only a duplication of the [EEX(4)] sequence, does not inhibit GR activity. The [EEX(4)](8) motif, when linked to an otherwise unrelated protein, abrogated the inhibitory action of endogenous RAP46 on GR-mediated transactivation. The nuclear effects of RAP46 and BAG-1L are specific since GR-mediated inhibition of AP-1 activity was not affected. These studies identify the [EEX(4)](8) sequence as a signature motif for inhibition of GR-mediated transactivation and demonstrate a specific nuclear action of a eukaryotic cochaperone in the regulation of GR activity.

Show MeSH
Related in: MedlinePlus