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. 2001 Aug 14;98(17):9713-8.
doi: 10.1073/pnas.171184698. Epub 2001 Jul 31.

Regulation of the transcriptional coactivator PGC-1 via MAPK-sensitive interaction with a repressor

D Knutti  1 , D Kressler, A Kralli
Affiliations

Regulation of the transcriptional coactivator PGC-1 via MAPK-sensitive interaction with a repressor

D Knutti et al. Proc Natl Acad Sci U S A. .

Abstract

Mechanisms and signals that regulate transcriptional coactivators are still largely unknown. Here we provide genetic evidence for a repressor that interacts with and regulates the nuclear receptor coactivator PGC-1. Association with the repressor requires a PGC-1 protein interface that is similar to the one used by nuclear receptors. Removal of the repressor enhances PGC-1 coactivation of steroid hormone responses. We also provide evidence that interaction of the repressor with PGC-1 is regulated by mitogen-activated protein kinase (MAPK) signaling. Activation of the MAPK p38 enhances the activity of wild-type PGC-1 but not of a PGC-1 variant that no longer interacts with the repressor. Finally, p38 activation enhances steroid hormone response in a PGC-1-dependent manner. Our data suggest a model where the repressor and nuclear receptors compete for recruiting PGC-1 to an inactive and active state, respectively. Extracellular signals such as nuclear receptor ligands or activators of the MAPK p38 can shift the equilibrium between the two states.

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Figures

Figure 1
Figure 1
PGC-1 interacts with GR via Leu-motifs L2 and L3. (A) PGC-1 has three Leu-motifs, L1 in the transcriptional activation domain (shaded dark gray), and L2 and L3 in the nuclear receptor interaction domains (NID1 and NID2) (6). The double leucines that were substituted by double alanines are underlined, and their residue numbers are indicated. (B) Interaction of PGC-1 variants with the LBD of GR, using a two-hybrid assay in yeast. Cells expressing Gal4-GR.LBD and either the Gal4 AD alone (AD) or the indicated PGC-1 variants fused to the AD were grown in the presence of 25 μM corticosterone and assayed for β-gal activity. Data are normalized to wild-type PGC-1 (wt) activity being equal to 100. (C) Enhancement of hormone response by PGC-1 variants. COS7 cells transfected with the GR expression plasmid p6RGR, the GR-responsive luciferase reporter pTAT3Luc, and either pcDNA3 vector or pcDNA3/HA-PGC-1 variants, were treated for 20 h with 50 nM corticosterone and assayed for luciferase activity. PGC-1 had no effect in the absence of hormone. Data are expressed as fold-enhancement of GR activity by PGC-1 in the presence of hormone.
Figure 2
Figure 2
The Leu-motifs regulate the transcriptional activity of PGC-1. Disruption of motifs L2 and L3 increases PGC-1 activity in COS7 (A) and HeLa cells (B). Cells were transfected with expression plasmids for Gal4–PGC-1 variants and the Gal4-responsive luciferase reporter pGK-1, and assayed for luciferase activity. The activity of wild-type Gal4–PGC-1 (wt) was normalized to 1 within each experiment, and represents an average activation of 200-fold in COS7 and 50-fold in HeLa cells, compared with Gal4 DBD alone. (C) Disruption of motifs L2 and L3 does not affect PGC-1 activity in yeast. Yeast expressing the indicated Gal4–PGC-1 variants were assayed for activity from a Gal4-responsive β-gal reporter. Transcriptional activity in yeast expressing just the Gal4 DBD was set equal to 1.
Figure 3
Figure 3
Coexpression of a fragment containing sites L2 and L3 enhances PGC-1 activity. HeLa cells were transfected with Gal4–PGC-1 [wild type (wt) or mutant (L2/3A)], together with 1 μg of vector alone or vector expressing amino acid 91–408 of PGC-1 bearing the L2/3 sites [wild type (wt) or mutant (L2/3A)], and the Gal4-responsive luciferase reporter pGK-1. Data are normalized to the luciferase activity of wild-type Gal4–PGC-1 in the presence of vector alone being equal to 1.
Figure 4
Figure 4
Competition of the repressor increases the ability of PGC-1 to enhance the hormone response. (A) HeLa cells expressing PGC-1 stably, under the control of a tetracycline-regulatable promoter, were transfected with 1 μg of vector alone or vector expressing either the L2A or the L2/3A mutant 91–408 PGC-1 fragment, together with the GR expression plasmid p6RGR and the GR-responsive luciferase reporter pTAT3Luc. Cells were cultured in the presence of doxycycline (0.5 μg/ml) to repress PGC-1 expression (no PGC-1) or absence of doxycycline (+ PGC-1), treated for 24 h with 5 nM corticosterone, and assayed for luciferase activity. (B) Protein levels of the stably expressed full-length PGC-1 and the transiently expressed 91–408 PGC-1 fragments. Extracts were from cells grown in the absence of doxycycline (+ PGC-1); no PGC-1 was detected in cells grown in the presence of doxycycline. Proteins were detected with an anti-protein A antibody for full-length PGC-1 (Upper) and an anti-HA antibody for the 91–408 fragments (Lower).
Figure 5
Figure 5
Activation of the MAPK p38 increases Gal4–PGC-1 activity in a repressor-dependent manner. (A) Expression of the constitutively active MKK6 (MKK6bE) activates the wild-type PGC-1 (Gal4–PGC-1 wt), but not the L2/3A mutant (Gal4–PGC-1 L2/3A). Data are expressed as activation by coexpression of the indicated MKK6 variant. (B) The p38 inhibitor SB203580 inhibits the enhancement by MKK6bE and reduces Gal4–PGC-1 activity. SB203580 (30 μM) or vehicle DMSO were added to cells 12 h after transfection. Data are expressed as activation by coexpression of the indicated MKK6 variant, in the absence or presence of SB203580 (A and B). HeLa cells were transfected with the Gal4-reporter pGK-1 and plasmids expressing either Gal4 DBD, Gal4–PGC-1 wild-type (wt), or Gal4–PGC-1 mutant (L2/3A). Empty vector, pcDNA3-MKK6bAA (inactive kinase mutant), or pcDNA3-MKK6bE (constitutively active variant) was cotransfected as indicated. Activity in the presence of vector alone and the absence of SB203580 drug was set equal to 1 for all Gal4 constructs.
Figure 6
Figure 6
Activation of the MKK6/p38 pathway enhances PGC-1-mediated activation of glucocorticoid response. Expression vectors for GR (p6RGR), constitutively active or inactive MKK6 (MKK6bE or MKK6bAA), and the GR reporter pTAT3Luc were transfected into HeLa cells together with either just vector (Left) or PGC-1 expression plasmid pcDNA3/HA-PGC-1 (Right). SB203580 (30 μM) or DMSO vehicle were added 15 h after transfection, and corticosterone (5 nM) 5 h later. Luciferase activity was assayed 16 h after hormone addition. Data are from one experiment performed in duplicate and are representative of four independent experiments.
Figure 7
Figure 7
Model for the regulation of the coactivator PGC-1. PGC-1 associates with a repressor (R), which maintains the coactivator in an inactive state. Hormone-activated GR interacts with PGC-1, displaces the repressor and recruits the coactivator to sites of transcription. A conformational change in PGC-1 upon receptor binding, as seen by Puigserver et al. (15), is symbolized by the different shape of PGC-1 and may stabilize the active state. Phosphorylation by the MAPK p38 favors the release of the repressor, thereby enhancing PGC-1 activity and glucocorticoid responses.
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