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Pin1 promotes GR transactivation by enhancing recruitment to target genes.

Poolman TM, Farrow SN, Matthews L, Loudon AS, Ray DW - Nucleic Acids Res. (2013)

Bottom Line: The Pin1 effect required both its WW and catalytic domains, and GR recruitment to its GRE was Pin1-dependent.Therefore, Pin1 is a selective regulator of GR transactivation, acting through N-terminal phospho-serine residues to regulate GR recruitment to its target sites in the genome.As Pin1 is dysregulated in disease states, this interaction may contribute to altered GC action in inflammatory conditions.

View Article: PubMed Central - PubMed

Affiliation: Centre in Endocrinology and Diabetes, Institute of Human Development, University of Manchester, Manchester, M13 9PT, UK, Respiratory Therapy Area, GSK, Stevenage, SG1 2NY, UK, Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, UK and Manchester Academic Health Sciences Centre, Manchester M13 9NT, UK.

ABSTRACT
The glucocorticoid receptor (GR) is a ligand activated transcription factor, serving to regulate both energy metabolism and immune functions. Factors that influence cellular sensitivity to glucocorticoids (GC) are therefore of great interest. The N-terminal of the GR contains numerous potential proline-directed phosphorylation sites, some of which can regulate GR transactivation. Unrestricted proline isomerisation can be inhibited by adjacent serine phosphorylation and requires a prolyl isomerise, Pin1. Pin1 therefore determines the functional outcome of proline-directed kinases acting on the GR, as cis/trans isomers are distinct pools with different interacting proteins. We show that Pin1 mediates GR transactivation, but not GR trans-repression. Two N-terminal GR serines, S203 and S211, are targets for Pin1 potentiation of GR transactivation, establishing a direct link between Pin1 and the GR. We also demonstrate GC-activated co-recruitment of GR and Pin1 to the GILZ gene promoter. The Pin1 effect required both its WW and catalytic domains, and GR recruitment to its GRE was Pin1-dependent. Therefore, Pin1 is a selective regulator of GR transactivation, acting through N-terminal phospho-serine residues to regulate GR recruitment to its target sites in the genome. As Pin1 is dysregulated in disease states, this interaction may contribute to altered GC action in inflammatory conditions.

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The effect of Pin1 inhibition on GR phosphorylation, nuclear translocation and stability. (A) A549 cells were transfected with Pin1 or control siRNA for 48 h. Cells were then treated with 1 or 100 nM for 30 min. Immunoblots were probed for phospho- GR (S211), GR, Pin1 and tubulin. (B) Cells were pre-treated with juglone for 30 min before a 30-min treatment of 100 nM DEX. (C) A549 cells were pre-treated with juglone (30 µM) for 30 min before a 30-min treatment with 100 nM DEX. Cytosol and nuclear fractions were prepared, and subsequent immunoblots were probed for GR and Histone H1. (D) A549 cells were transfected with Pin1 or control siRNA for 48 h before being stimulated with DEX (100 nM) or 4 or 8 h, whole-cell extracts were probed for GR and Pin1. (E) A549 cells were transfected with Pin1 siRNA as described in (D), cycloheximide (50 µg/ml) was added to the cells for 4, 8 and 16 h, subsequent immunoblots were probed for GR, Pin1 and β-actin.
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gkt624-F4: The effect of Pin1 inhibition on GR phosphorylation, nuclear translocation and stability. (A) A549 cells were transfected with Pin1 or control siRNA for 48 h. Cells were then treated with 1 or 100 nM for 30 min. Immunoblots were probed for phospho- GR (S211), GR, Pin1 and tubulin. (B) Cells were pre-treated with juglone for 30 min before a 30-min treatment of 100 nM DEX. (C) A549 cells were pre-treated with juglone (30 µM) for 30 min before a 30-min treatment with 100 nM DEX. Cytosol and nuclear fractions were prepared, and subsequent immunoblots were probed for GR and Histone H1. (D) A549 cells were transfected with Pin1 or control siRNA for 48 h before being stimulated with DEX (100 nM) or 4 or 8 h, whole-cell extracts were probed for GR and Pin1. (E) A549 cells were transfected with Pin1 siRNA as described in (D), cycloheximide (50 µg/ml) was added to the cells for 4, 8 and 16 h, subsequent immunoblots were probed for GR, Pin1 and β-actin.

Mentions: We observed loss of the characteristic rapid GR-S211 phosphorylation in response to GC activation both with Pin1 siRNA, and also antagonism with juglone (Figure 4A and B), suggesting a role for Pin1 in acquiring or maintaining the phospho mark. Translocation of the GR to the nucleus, a critical regulatory step in gene regulation, was unaffected by Pin1 knockdown (Figure 4C), and there was no discernible effect on GR protein abundance, either under basal conditions or following ligand activation (Figure 4D). Attempts to discern differences in GR protein stability in the absence of Pin1, analogous to that seen for cyclin D1, did not reveal any significant differences (Figure 4E and Supplementary Figure S4). GR and Pin1 were also found in the same molecular complex as demonstrated using a co-immunoprecipitation immunoblot (Figure 5A and B). The strength of the interaction appeared to be enhanced by ligand activation, as demonstrated in the GR immunoprecipitation (Figure 5B).Figure 4.


Pin1 promotes GR transactivation by enhancing recruitment to target genes.

Poolman TM, Farrow SN, Matthews L, Loudon AS, Ray DW - Nucleic Acids Res. (2013)

The effect of Pin1 inhibition on GR phosphorylation, nuclear translocation and stability. (A) A549 cells were transfected with Pin1 or control siRNA for 48 h. Cells were then treated with 1 or 100 nM for 30 min. Immunoblots were probed for phospho- GR (S211), GR, Pin1 and tubulin. (B) Cells were pre-treated with juglone for 30 min before a 30-min treatment of 100 nM DEX. (C) A549 cells were pre-treated with juglone (30 µM) for 30 min before a 30-min treatment with 100 nM DEX. Cytosol and nuclear fractions were prepared, and subsequent immunoblots were probed for GR and Histone H1. (D) A549 cells were transfected with Pin1 or control siRNA for 48 h before being stimulated with DEX (100 nM) or 4 or 8 h, whole-cell extracts were probed for GR and Pin1. (E) A549 cells were transfected with Pin1 siRNA as described in (D), cycloheximide (50 µg/ml) was added to the cells for 4, 8 and 16 h, subsequent immunoblots were probed for GR, Pin1 and β-actin.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3794586&req=5

gkt624-F4: The effect of Pin1 inhibition on GR phosphorylation, nuclear translocation and stability. (A) A549 cells were transfected with Pin1 or control siRNA for 48 h. Cells were then treated with 1 or 100 nM for 30 min. Immunoblots were probed for phospho- GR (S211), GR, Pin1 and tubulin. (B) Cells were pre-treated with juglone for 30 min before a 30-min treatment of 100 nM DEX. (C) A549 cells were pre-treated with juglone (30 µM) for 30 min before a 30-min treatment with 100 nM DEX. Cytosol and nuclear fractions were prepared, and subsequent immunoblots were probed for GR and Histone H1. (D) A549 cells were transfected with Pin1 or control siRNA for 48 h before being stimulated with DEX (100 nM) or 4 or 8 h, whole-cell extracts were probed for GR and Pin1. (E) A549 cells were transfected with Pin1 siRNA as described in (D), cycloheximide (50 µg/ml) was added to the cells for 4, 8 and 16 h, subsequent immunoblots were probed for GR, Pin1 and β-actin.
Mentions: We observed loss of the characteristic rapid GR-S211 phosphorylation in response to GC activation both with Pin1 siRNA, and also antagonism with juglone (Figure 4A and B), suggesting a role for Pin1 in acquiring or maintaining the phospho mark. Translocation of the GR to the nucleus, a critical regulatory step in gene regulation, was unaffected by Pin1 knockdown (Figure 4C), and there was no discernible effect on GR protein abundance, either under basal conditions or following ligand activation (Figure 4D). Attempts to discern differences in GR protein stability in the absence of Pin1, analogous to that seen for cyclin D1, did not reveal any significant differences (Figure 4E and Supplementary Figure S4). GR and Pin1 were also found in the same molecular complex as demonstrated using a co-immunoprecipitation immunoblot (Figure 5A and B). The strength of the interaction appeared to be enhanced by ligand activation, as demonstrated in the GR immunoprecipitation (Figure 5B).Figure 4.

Bottom Line: The Pin1 effect required both its WW and catalytic domains, and GR recruitment to its GRE was Pin1-dependent.Therefore, Pin1 is a selective regulator of GR transactivation, acting through N-terminal phospho-serine residues to regulate GR recruitment to its target sites in the genome.As Pin1 is dysregulated in disease states, this interaction may contribute to altered GC action in inflammatory conditions.

View Article: PubMed Central - PubMed

Affiliation: Centre in Endocrinology and Diabetes, Institute of Human Development, University of Manchester, Manchester, M13 9PT, UK, Respiratory Therapy Area, GSK, Stevenage, SG1 2NY, UK, Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, UK and Manchester Academic Health Sciences Centre, Manchester M13 9NT, UK.

ABSTRACT
The glucocorticoid receptor (GR) is a ligand activated transcription factor, serving to regulate both energy metabolism and immune functions. Factors that influence cellular sensitivity to glucocorticoids (GC) are therefore of great interest. The N-terminal of the GR contains numerous potential proline-directed phosphorylation sites, some of which can regulate GR transactivation. Unrestricted proline isomerisation can be inhibited by adjacent serine phosphorylation and requires a prolyl isomerise, Pin1. Pin1 therefore determines the functional outcome of proline-directed kinases acting on the GR, as cis/trans isomers are distinct pools with different interacting proteins. We show that Pin1 mediates GR transactivation, but not GR trans-repression. Two N-terminal GR serines, S203 and S211, are targets for Pin1 potentiation of GR transactivation, establishing a direct link between Pin1 and the GR. We also demonstrate GC-activated co-recruitment of GR and Pin1 to the GILZ gene promoter. The Pin1 effect required both its WW and catalytic domains, and GR recruitment to its GRE was Pin1-dependent. Therefore, Pin1 is a selective regulator of GR transactivation, acting through N-terminal phospho-serine residues to regulate GR recruitment to its target sites in the genome. As Pin1 is dysregulated in disease states, this interaction may contribute to altered GC action in inflammatory conditions.

Show MeSH