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

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.

Figure 1

Identification of nurr1-interacting proteins in cells of mesencephalic origin. (A) Schematic representation of the N- and C-termini of nurr1 fused to GST and used as baits in GST-pulldown experiments. (B) Silver nitrate staining of pulldown samples obtained after incubation of immobilized GST–nurr1 fusion proteins with (+) or without (−) CSM14.1 nuclear extracts (1.5 mg). GST alone was used as control for non-specific interactions. Samples were separated on 6–15% gradient one-dimension (1D) gels before staining. Multiple identical experiments (n = 7) were performed and analyzed by 1D gel in strictly comparable conditions. Gels with similar electrophoretic profiles (n = 4) were used to excise colloidal blue stained protein bands which were further analyzed by mass spectroscopy after tryptic digestion. A representative silver stained gel is shown and arrows correspond to bands of interest excised from Colloidal blue stained gels for further analysis. (C) Detection of identified partners on 1D gels. After transfer to nitrocellulose membranes of GST-pulldown samples obtained using 300 μg of CSM14.1 nuclear extracts, western blots were performed using the specified antibodies (RXR and ERK) to detect newly identified interacting factors. Values on the left inside denote molecular weights.

Figure 2

Nurr1 interacts directly with multiple proteins identified by MS analysis. (A) ³⁵S-labeled LIMK1 or kinase domain of LIMK1 were incubated in the presence of bacterially expressed GST alone (−) and GST–nurr1 fusion proteins (LBD or A/BΔ domain) for 3 h. The first lane corresponds to 10% input. Protein interactions were analyzed by SDS–PAGE and assayed by autoradiography. (B) ³⁵S-labeled full-length nurr1 was incubated with ERK2, LIM domain, ERK5 fused to GST or GST alone (−) expressed, separated and assayed as in (A). (C–E) In vivo interactions between nurr1 and several signaling proteins. Co-immunoprecipitations with the anti-HA antibody of cellular extracts of CSM14.1 cells co-transfected with HA-tagged human nurr1 and empty vector (−) or expression plasmids of indicated proteins (+): (C) pcDNA3-LIMK1 and detected with anti-LIMK antibody, (D) pCMV-ERK2ca and visualized with pan-ERK antibody and (E) pFlag-ERK5 wt and detected with anti-BMK1/ERK5 antibody. Input represents levels of GST-LIMK, ERK2 and ERK5 in CMS14.1 cells.

Figure 3

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).

Figure 4

Modulation of nurr1-induced activation of NBRE3xtkLuc reporter gene by identified interacting proteins. (A) Analysis of luciferase activity in PC12 cells transfected with 25 ng of nurr1 and increasing amounts of wt ERK2 compared to identical doses of constitutively active ERK2 (ca) and kinase-dead (K52R). Normalized luciferase activity of nurr1 on NBRE3xtkLuc was expressed as Fold activation over NBRE3xtkLuc alone and set to 100%. All other values are compared to it. Data are the means ± S.E. (bars) of five experiments (two for insets) with n = 3 for each experiment (#, P < 0.05; *, P < 0.01; **, P < 0.001). (B) Luciferase activity of PC12 cells transfected with nurr1 and increasing amounts of wt full-length LIMK1 encoding plasmid. Results are expressed as described in (A).

Figure 5

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.

Figure 6

Nurr1 is phosphorylated by BMK1/ERK5 MAP kinase. (A) DNA immunoprecipitation of nurr1 and endogenous ERK5 recruited to NBREs in cells overexpressing HA-nurr1, but not to a promoter lacking nurr1 response elements, pGL3-tk-Luc. (B) Endogenous ERK5 immunoprecipitated from untransfected CSM14.1 cells was incubated with GST-A/BΔ nurr1 or GST fusion proteins and [γ-³²P]ATP for 30 min. Arrows indicate phosphorylated nurr1. For activation of MEK-ERK5 pathway, cells were stimulated for 15 min with 100 ng/ml of EGF. (C) Analysis of luciferase activity induced by coexpression of wt HA-ERK5 and wt pCMX-nurr1 or point mutations S89A, T168A and S177A on NBRE3xtkLuc. Normalized luciferase activity of wt nurr1 plus ERK5 was set to 100% and all other values were compared to it. Data are the means of ± S.E. (bars) of five independent experiments (n = 3 in each experiment).