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Multiple signaling pathways regulate the transcriptional activity of the orphan nuclear receptor NURR1.

Sacchetti P, Carpentier R, Ségard P, Olivé-Cren C, Lefebvre P - Nucleic Acids Res. (2006)

Bottom Line: Furthermore, nurr1 recruits ERK5 to a NBRE-containing promoter and is a potential substrate for this kinase.We have identified amino acids in the A/B domain of nurr1 important for mediating the ERK5 activating effects on nurr1 transcriptional activity.Our results suggest that nurr1 acts as a point of convergence for multiple signaling pathways that likely play a critical role in differentiation and phenotypic expression of dopaminergic (DAergic) neurons.

View Article: PubMed Central - PubMed

Affiliation: INSERM U459, Faculté de Médecine de Lille, 59045 Lille, France. paola.sacchetti@ki.se

ABSTRACT
The orphan nuclear receptor nurr1 (NR4A2) is an essential transcription factor for the acquisition and maintenance of the phenotype of dopamine (DA)-synthesizing neurons in the mesencephalon. Although structurally related to ligand-regulated nuclear receptors, nurr1 is functionally atypical due to its inability to bind a cognate ligand and to activate transcription following canonical nuclear receptor (NR) rules. Importantly, the physiological stimuli that activate this NR and the signaling proteins that regulate its transcriptional activity in mesencephalic neurons are unknown. We used an affinity chromatography approach and CSM14.1 cells of mesencephalic origin to isolate and identify several proteins that interact directly with nurr1 and regulate its transcriptional activity. Notably, we demonstrate that the mitogen-activated protein kinases, ERK2 and ERK5, elevate, whereas LIM Kinase 1 inhibits nurr1 transcriptional activity. Furthermore, nurr1 recruits ERK5 to a NBRE-containing promoter and is a potential substrate for this kinase. We have identified amino acids in the A/B domain of nurr1 important for mediating the ERK5 activating effects on nurr1 transcriptional activity. Our results suggest that nurr1 acts as a point of convergence for multiple signaling pathways that likely play a critical role in differentiation and phenotypic expression of dopaminergic (DAergic) neurons.

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Nurr1 transcriptional activity is regulated by the MEK5/ERK5 signaling pathway. (A) Dose-dependent induction by wt ERK5 of NBRE3xtkLuc activity in the presence of 25 ng of nurr1 in PC12 cells transfected with increasing doses of wt HA-ERK5. (B) Analysis of luciferase activity induced by MEK5 wt, ca and dn in the absence or presence of pCMX-nurr1 and wt HA-ERK5. (C) Comparison of transcriptional activity between wt and dn ERK5. PC12 cells were transfected with NBRE3xtkLuc, pCMX-nurr1, wt Flag-ERK5 or Flag-ERK5 AEF in the presence or absence of MEK5 ca. (D) Comparison of transcriptional activity induced by wt, AEF and kinase-dead K83M ERK5. Cells were transfected as in (A). Luciferase activity was normalized and results are expressed as described in Figure 4. Data are the means ± S.E. (bars) of 3–6 experiments.
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fig5: Nurr1 transcriptional activity is regulated by the MEK5/ERK5 signaling pathway. (A) Dose-dependent induction by wt ERK5 of NBRE3xtkLuc activity in the presence of 25 ng of nurr1 in PC12 cells transfected with increasing doses of wt HA-ERK5. (B) Analysis of luciferase activity induced by MEK5 wt, ca and dn in the absence or presence of pCMX-nurr1 and wt HA-ERK5. (C) Comparison of transcriptional activity between wt and dn ERK5. PC12 cells were transfected with NBRE3xtkLuc, pCMX-nurr1, wt Flag-ERK5 or Flag-ERK5 AEF in the presence or absence of MEK5 ca. (D) Comparison of transcriptional activity induced by wt, AEF and kinase-dead K83M ERK5. Cells were transfected as in (A). Luciferase activity was normalized and results are expressed as described in Figure 4. Data are the means ± S.E. (bars) of 3–6 experiments.

Mentions: Our screening also identified the MAPK/ERK kinase 5 (MEK5) as a potential modulator of nurr1 activity (Supplementary Table 1). Co-immunoprecipitation and GST-pulldown experiments established an interaction between nurr1 and ERK5 (Figures 2 and 3), the direct substrate of MEK5. We therefore tested whether overexpressing components of the MEK5/ERK5 signaling pathway altered nurr1 activity. Increasing doses of wt ERK5 did not affect the basal activity of the NBRE3xtkLuc construct in the absence of nurr1 (Figure 5A). Whereas nurr1-induced activation of the luciferase construct was significantly enhanced by wt ERK5 in a dose-dependent manner. We then tested the effects of MEK5 on the transcriptional activity of nurr1 using MEK5 wt, ca and dn forms (Figure 5B). As observed previously, none of the constructs activated non-specifically the NBRE3xtkLuc reporter, while nurr1 alone or in the presence of ERK5 increased luciferase activity. Cotransfection of nurr1, ERK5 and wt MEK5 as well as its ca form significantly enhanced luciferase levels, whereas the dn MEK5 had no significant effect (Figure 5B). To verify that ERK5 is indeed the conduit for transduction of MEK5 signal, we used a Flag-tagged wt ERK5 construct (Flag-ERK5 wt) as well as a ERK5 mutant lacking both activation sites for MEK5 (Flag-ERK5 AEF). In the presence of wt ERK5, constitutively active MEK5 was able to enhance nurr1-induced luciferase expression (Figure 5C). However, the level of luciferase expression due to the coexpression of nurr1 and the ERK5 AEF mutant did not change in the presence of MEK5 ca, suggesting that MEK5 could modulate nurr1 activity essentially through ERK5 activation. Surprisingly, the unphosphorylated AEF mutant was still capable of enhancing nurr1 activity, even in the absence of MEK5 contribution (Figure 5C). It has been previously suggested that, in non-activating conditions, ERK5 AEF could act as the wild-type form since it still retains the capacity to activate known substrates (26). Thus, to determine if basal ERK5 activity was sufficient to affect nurr1 transcriptional activity, we co-expressed nurr1 and different forms of ERK5 (HA-ERK5 wt, AEF and the kinase-dead ERK5 mutant K83M) and measured NBRE luciferase levels (Figure 5D). Wt ERK5 and the AEF mutant further enhanced nurr1-induced luciferase activity, with the mutant showing stronger activating effects on nurr1 activity (the HA-tagged AEF construct showed higher effects on nurr1 activity than the Flag-tagged construct; compare Figure 5C and D). Importantly, the kinase-dead mutant had no effects at any of the doses used. Hence, the basal activity of ERK5 seems sufficient to affect nurr1 function without excluding that, in particular conditions, activation of MEK5 could further contribute to the modulation of nurr1 transcriptional activity.


Multiple signaling pathways regulate the transcriptional activity of the orphan nuclear receptor NURR1.

Sacchetti P, Carpentier R, Ségard P, Olivé-Cren C, Lefebvre P - Nucleic Acids Res. (2006)

Nurr1 transcriptional activity is regulated by the MEK5/ERK5 signaling pathway. (A) Dose-dependent induction by wt ERK5 of NBRE3xtkLuc activity in the presence of 25 ng of nurr1 in PC12 cells transfected with increasing doses of wt HA-ERK5. (B) Analysis of luciferase activity induced by MEK5 wt, ca and dn in the absence or presence of pCMX-nurr1 and wt HA-ERK5. (C) Comparison of transcriptional activity between wt and dn ERK5. PC12 cells were transfected with NBRE3xtkLuc, pCMX-nurr1, wt Flag-ERK5 or Flag-ERK5 AEF in the presence or absence of MEK5 ca. (D) Comparison of transcriptional activity induced by wt, AEF and kinase-dead K83M ERK5. Cells were transfected as in (A). Luciferase activity was normalized and results are expressed as described in Figure 4. Data are the means ± S.E. (bars) of 3–6 experiments.
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Related In: Results  -  Collection

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fig5: Nurr1 transcriptional activity is regulated by the MEK5/ERK5 signaling pathway. (A) Dose-dependent induction by wt ERK5 of NBRE3xtkLuc activity in the presence of 25 ng of nurr1 in PC12 cells transfected with increasing doses of wt HA-ERK5. (B) Analysis of luciferase activity induced by MEK5 wt, ca and dn in the absence or presence of pCMX-nurr1 and wt HA-ERK5. (C) Comparison of transcriptional activity between wt and dn ERK5. PC12 cells were transfected with NBRE3xtkLuc, pCMX-nurr1, wt Flag-ERK5 or Flag-ERK5 AEF in the presence or absence of MEK5 ca. (D) Comparison of transcriptional activity induced by wt, AEF and kinase-dead K83M ERK5. Cells were transfected as in (A). Luciferase activity was normalized and results are expressed as described in Figure 4. Data are the means ± S.E. (bars) of 3–6 experiments.
Mentions: Our screening also identified the MAPK/ERK kinase 5 (MEK5) as a potential modulator of nurr1 activity (Supplementary Table 1). Co-immunoprecipitation and GST-pulldown experiments established an interaction between nurr1 and ERK5 (Figures 2 and 3), the direct substrate of MEK5. We therefore tested whether overexpressing components of the MEK5/ERK5 signaling pathway altered nurr1 activity. Increasing doses of wt ERK5 did not affect the basal activity of the NBRE3xtkLuc construct in the absence of nurr1 (Figure 5A). Whereas nurr1-induced activation of the luciferase construct was significantly enhanced by wt ERK5 in a dose-dependent manner. We then tested the effects of MEK5 on the transcriptional activity of nurr1 using MEK5 wt, ca and dn forms (Figure 5B). As observed previously, none of the constructs activated non-specifically the NBRE3xtkLuc reporter, while nurr1 alone or in the presence of ERK5 increased luciferase activity. Cotransfection of nurr1, ERK5 and wt MEK5 as well as its ca form significantly enhanced luciferase levels, whereas the dn MEK5 had no significant effect (Figure 5B). To verify that ERK5 is indeed the conduit for transduction of MEK5 signal, we used a Flag-tagged wt ERK5 construct (Flag-ERK5 wt) as well as a ERK5 mutant lacking both activation sites for MEK5 (Flag-ERK5 AEF). In the presence of wt ERK5, constitutively active MEK5 was able to enhance nurr1-induced luciferase expression (Figure 5C). However, the level of luciferase expression due to the coexpression of nurr1 and the ERK5 AEF mutant did not change in the presence of MEK5 ca, suggesting that MEK5 could modulate nurr1 activity essentially through ERK5 activation. Surprisingly, the unphosphorylated AEF mutant was still capable of enhancing nurr1 activity, even in the absence of MEK5 contribution (Figure 5C). It has been previously suggested that, in non-activating conditions, ERK5 AEF could act as the wild-type form since it still retains the capacity to activate known substrates (26). Thus, to determine if basal ERK5 activity was sufficient to affect nurr1 transcriptional activity, we co-expressed nurr1 and different forms of ERK5 (HA-ERK5 wt, AEF and the kinase-dead ERK5 mutant K83M) and measured NBRE luciferase levels (Figure 5D). Wt ERK5 and the AEF mutant further enhanced nurr1-induced luciferase activity, with the mutant showing stronger activating effects on nurr1 activity (the HA-tagged AEF construct showed higher effects on nurr1 activity than the Flag-tagged construct; compare Figure 5C and D). Importantly, the kinase-dead mutant had no effects at any of the doses used. Hence, the basal activity of ERK5 seems sufficient to affect nurr1 function without excluding that, in particular conditions, activation of MEK5 could further contribute to the modulation of nurr1 transcriptional activity.

Bottom Line: Furthermore, nurr1 recruits ERK5 to a NBRE-containing promoter and is a potential substrate for this kinase.We have identified amino acids in the A/B domain of nurr1 important for mediating the ERK5 activating effects on nurr1 transcriptional activity.Our results suggest that nurr1 acts as a point of convergence for multiple signaling pathways that likely play a critical role in differentiation and phenotypic expression of dopaminergic (DAergic) neurons.

View Article: PubMed Central - PubMed

Affiliation: INSERM U459, Faculté de Médecine de Lille, 59045 Lille, France. paola.sacchetti@ki.se

ABSTRACT
The orphan nuclear receptor nurr1 (NR4A2) is an essential transcription factor for the acquisition and maintenance of the phenotype of dopamine (DA)-synthesizing neurons in the mesencephalon. Although structurally related to ligand-regulated nuclear receptors, nurr1 is functionally atypical due to its inability to bind a cognate ligand and to activate transcription following canonical nuclear receptor (NR) rules. Importantly, the physiological stimuli that activate this NR and the signaling proteins that regulate its transcriptional activity in mesencephalic neurons are unknown. We used an affinity chromatography approach and CSM14.1 cells of mesencephalic origin to isolate and identify several proteins that interact directly with nurr1 and regulate its transcriptional activity. Notably, we demonstrate that the mitogen-activated protein kinases, ERK2 and ERK5, elevate, whereas LIM Kinase 1 inhibits nurr1 transcriptional activity. Furthermore, nurr1 recruits ERK5 to a NBRE-containing promoter and is a potential substrate for this kinase. We have identified amino acids in the A/B domain of nurr1 important for mediating the ERK5 activating effects on nurr1 transcriptional activity. Our results suggest that nurr1 acts as a point of convergence for multiple signaling pathways that likely play a critical role in differentiation and phenotypic expression of dopaminergic (DAergic) neurons.

Show MeSH
Related in: MedlinePlus