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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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Mapping of the domains of interaction between nurr1 and LIMK1, ERK2, ERK5. (A) Schematic representation of the nurr1 deletion mutants used for GST-pulldown assays and summary of the quantification of obtained interactions with the kinases. Symbols represent quantification of binding interactions (n = 3–5). Intensity of binding was standardized to that of wt (100%); +, binding values ≤80%; ±, 79< × <60; −, ≤59%. (B) Representative GST-pulldown assays obtained by incubating 35S-labeled pCMX-nurr1 wt or deletion mutants (wt, ΔA-I, ΔAF2) with bacterially expressed GST alone (−) and GST-kinase fusion proteins (wt GST-LIM domain, -ERK2 or -ERK5).
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fig3: Mapping of the domains of interaction between nurr1 and LIMK1, ERK2, ERK5. (A) Schematic representation of the nurr1 deletion mutants used for GST-pulldown assays and summary of the quantification of obtained interactions with the kinases. Symbols represent quantification of binding interactions (n = 3–5). Intensity of binding was standardized to that of wt (100%); +, binding values ≤80%; ±, 79< × <60; −, ≤59%. (B) Representative GST-pulldown assays obtained by incubating 35S-labeled pCMX-nurr1 wt or deletion mutants (wt, ΔA-I, ΔAF2) with bacterially expressed GST alone (−) and GST-kinase fusion proteins (wt GST-LIM domain, -ERK2 or -ERK5).

Mentions: We further dissected the A/B and LBD regions of nurr1 to identify the specific binding domains implicated in the interaction with these protein kinases. Several minimal deletion mutants in the A/B (ΔA-D) and LBD (ΔF-I) domains of nurr1 were engineered (Figure 3A), in vitro radiolabeled and tested by GST-pulldown experiments with LIM domains of LIMK1, wt ERK2 and wt ERK5 proteins. As summarized in Figure 3A and shown in the representative experiments in Figure 3B, deletion of the region spanning from amino acid 1 to 52 of nurr1 did not affect its interaction with any of the kinases. Interestingly, deletion of the AF1 core domain (amino acid 52–84; ΔB) partially altered the physical binding of nurr1 with both MAPKs (ERK2 and ERK5), but not with LIMK1. Deletion of amino acid 166–180 (ΔD) severely impaired the binding of nurr1 to LIMK1 and partially to ERK2, but did not interfere with ERK5 interaction (Figure 3). On the other hand, the mutant ΔF (deleted from amino acid 368 to 420) was the only one to lose its ability to bind to ERK5, validating the previously observed binding of ERK5 to the LBD and not to the A/B domain of nurr1 (Figure 1C). The mutant ΔF also showed a lower binding to LIMK1, suggesting that this hinge region may play an important role in protein–protein interactions. Mutations in the C-terminal domain of nurr1 (ΔG-ΔAF2) mostly affected the interaction of nurr1 with LIMK1, in particular, as shown in Figure 3, more dramatic effects were obtained with mutants ΔH and ΔI. Loss of the AF2 domain (nurr1-ΔAF2) affected only the interaction with ERK5. Taken together, these data confirms that nurr1 interacts via distinct and separate binding sites to the different protein kinases identified by affinity chromatography, arguing for a specificity of the binding of Nurr1 to these novel partners.


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)

Mapping of the domains of interaction between nurr1 and LIMK1, ERK2, ERK5. (A) Schematic representation of the nurr1 deletion mutants used for GST-pulldown assays and summary of the quantification of obtained interactions with the kinases. Symbols represent quantification of binding interactions (n = 3–5). Intensity of binding was standardized to that of wt (100%); +, binding values ≤80%; ±, 79< × <60; −, ≤59%. (B) Representative GST-pulldown assays obtained by incubating 35S-labeled pCMX-nurr1 wt or deletion mutants (wt, ΔA-I, ΔAF2) with bacterially expressed GST alone (−) and GST-kinase fusion proteins (wt GST-LIM domain, -ERK2 or -ERK5).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1636490&req=5

fig3: Mapping of the domains of interaction between nurr1 and LIMK1, ERK2, ERK5. (A) Schematic representation of the nurr1 deletion mutants used for GST-pulldown assays and summary of the quantification of obtained interactions with the kinases. Symbols represent quantification of binding interactions (n = 3–5). Intensity of binding was standardized to that of wt (100%); +, binding values ≤80%; ±, 79< × <60; −, ≤59%. (B) Representative GST-pulldown assays obtained by incubating 35S-labeled pCMX-nurr1 wt or deletion mutants (wt, ΔA-I, ΔAF2) with bacterially expressed GST alone (−) and GST-kinase fusion proteins (wt GST-LIM domain, -ERK2 or -ERK5).
Mentions: We further dissected the A/B and LBD regions of nurr1 to identify the specific binding domains implicated in the interaction with these protein kinases. Several minimal deletion mutants in the A/B (ΔA-D) and LBD (ΔF-I) domains of nurr1 were engineered (Figure 3A), in vitro radiolabeled and tested by GST-pulldown experiments with LIM domains of LIMK1, wt ERK2 and wt ERK5 proteins. As summarized in Figure 3A and shown in the representative experiments in Figure 3B, deletion of the region spanning from amino acid 1 to 52 of nurr1 did not affect its interaction with any of the kinases. Interestingly, deletion of the AF1 core domain (amino acid 52–84; ΔB) partially altered the physical binding of nurr1 with both MAPKs (ERK2 and ERK5), but not with LIMK1. Deletion of amino acid 166–180 (ΔD) severely impaired the binding of nurr1 to LIMK1 and partially to ERK2, but did not interfere with ERK5 interaction (Figure 3). On the other hand, the mutant ΔF (deleted from amino acid 368 to 420) was the only one to lose its ability to bind to ERK5, validating the previously observed binding of ERK5 to the LBD and not to the A/B domain of nurr1 (Figure 1C). The mutant ΔF also showed a lower binding to LIMK1, suggesting that this hinge region may play an important role in protein–protein interactions. Mutations in the C-terminal domain of nurr1 (ΔG-ΔAF2) mostly affected the interaction of nurr1 with LIMK1, in particular, as shown in Figure 3, more dramatic effects were obtained with mutants ΔH and ΔI. Loss of the AF2 domain (nurr1-ΔAF2) affected only the interaction with ERK5. Taken together, these data confirms that nurr1 interacts via distinct and separate binding sites to the different protein kinases identified by affinity chromatography, arguing for a specificity of the binding of Nurr1 to these novel partners.

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