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p35 regulates the CRM1-dependent nucleocytoplasmic shuttling of nuclear hormone receptor coregulator-interacting factor 1 (NIF-1).

Zhao XS, Fu WY, Chien WW, Li Z, Fu AK, Ip NY - PLoS ONE (2014)

Bottom Line: Our results show that p35 interacts with NIF-1 and regulates its nucleocytoplasmic trafficking via the nuclear export pathway.Furthermore, we identified a nuclear export signal on p35; mutation of this site or blockade of the CRM1/exportin-dependent nuclear export pathway resulted in the nuclear accumulation of p35.These findings reveal a new p35-dependent mechanism in transcriptional regulation that involves the nucleocytoplasmic shuttling of transcription regulators.

View Article: PubMed Central - PubMed

Affiliation: Division of Life Science, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China; Molecular Neuroscience Center, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China; State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China.

ABSTRACT
Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase, which plays critical roles in a wide spectrum of neuronal functions including neuronal survival, neurite outgrowth, and synapse development and plasticity. Cdk5 activity is controlled by its specific activators: p35 or p39. While knockout studies reveal that Cdk5/p35 is critical for neuronal migration during early brain development, functions of Cdk5/p35 have been unraveled through the identification of the interacting proteins of p35, most of which are Cdk5/p35 substrates. However, it remains unclear whether p35 can regulate neuronal functions independent of Cdk5 activity. Here, we report that a nuclear protein, nuclear hormone receptor coregulator (NRC)-interacting factor 1 (NIF-1), is a new interacting partner of p35. Interestingly, p35 regulates the functions of NIF-1 independent of Cdk5 activity. NIF-1 was initially discovered as a transcriptional regulator that enhances the transcriptional activity of nuclear hormone receptors. Our results show that p35 interacts with NIF-1 and regulates its nucleocytoplasmic trafficking via the nuclear export pathway. Furthermore, we identified a nuclear export signal on p35; mutation of this site or blockade of the CRM1/exportin-dependent nuclear export pathway resulted in the nuclear accumulation of p35. Intriguingly, blocking the nuclear export of p35 attenuated the nuclear accumulation of NIF-1. These findings reveal a new p35-dependent mechanism in transcriptional regulation that involves the nucleocytoplasmic shuttling of transcription regulators.

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Mediation of p35-triggered NIF-1 nuclear export via a CRM1-dependent pathway.(A & B) COS-7 cells were transfected with p35 and HA-tagged NIF-1. Twenty-four hours after transfection, the cells were treated with LMB (5 or 10 ng/mL) for 4–8 h, stained with HA antibody, and examined by fluorescence microscopy. Representative fluorescence images (A) and quantitative analysis of the cells showing exclusive nuclear accumulation of NIF-1 (B). At least 100 cells were scored for each condition in each trial. Results represent the mean ± SEM of 3 replicates (***p <0.05, one-way ANOVA followed by the Student–Newman–Keuls test). (C–E) p35-stimulated redistribution of NIF-1 from the nucleus to cytoplasm is independent of Cdk5 activity. (C) Active Cdk5 phosphorylated recombinant NIF-1 protein. Recombinant GST-NIF-1-C protein was subjected to phosphorylation assay by Cdk5 (GST and histone H1 were used as negative and positive controls, respectively). (D) COS-7 cells were transfected with the HA-tagged NIF-1 and p35, and subsequently treated with roscovitine (Ros, 10 or 25 µM) for 4 or 8 h. The cells with exclusive nuclear accumulation of NIF-1 were quantified. (E) Inhibition of Cdk5 activity by dnCdk5 expression did not affect the p35-stimulated nuclear export of NIF-1. COS-7 cells were transfected with NIF-1, p35, and dnCdk5 constructs as indicated. Quantitative analysis as described in (D). Results represent the mean ± SEM of 3 replicates (***p <0.05, one-way ANOVA followed by the Student–Newman–Keuls test).
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pone.0110584-g003: Mediation of p35-triggered NIF-1 nuclear export via a CRM1-dependent pathway.(A & B) COS-7 cells were transfected with p35 and HA-tagged NIF-1. Twenty-four hours after transfection, the cells were treated with LMB (5 or 10 ng/mL) for 4–8 h, stained with HA antibody, and examined by fluorescence microscopy. Representative fluorescence images (A) and quantitative analysis of the cells showing exclusive nuclear accumulation of NIF-1 (B). At least 100 cells were scored for each condition in each trial. Results represent the mean ± SEM of 3 replicates (***p <0.05, one-way ANOVA followed by the Student–Newman–Keuls test). (C–E) p35-stimulated redistribution of NIF-1 from the nucleus to cytoplasm is independent of Cdk5 activity. (C) Active Cdk5 phosphorylated recombinant NIF-1 protein. Recombinant GST-NIF-1-C protein was subjected to phosphorylation assay by Cdk5 (GST and histone H1 were used as negative and positive controls, respectively). (D) COS-7 cells were transfected with the HA-tagged NIF-1 and p35, and subsequently treated with roscovitine (Ros, 10 or 25 µM) for 4 or 8 h. The cells with exclusive nuclear accumulation of NIF-1 were quantified. (E) Inhibition of Cdk5 activity by dnCdk5 expression did not affect the p35-stimulated nuclear export of NIF-1. COS-7 cells were transfected with NIF-1, p35, and dnCdk5 constructs as indicated. Quantitative analysis as described in (D). Results represent the mean ± SEM of 3 replicates (***p <0.05, one-way ANOVA followed by the Student–Newman–Keuls test).

Mentions: As p35 promoted the redistribution of NIF-1 from the nucleus to the cytoplasm in COS-7 cells, we examined whether p35 triggers NIF-1 nuclear export. The CRM1-dependent nuclear export pathway is a major pathway for transporting proteins out of the nucleus [25]. Treatment with LMB, a specific inhibitor of CRM1-mediated export [26], abolished the p35-dependent export of NIF-1 from the nucleus to the cytoplasm, resulting in the nuclear accumulation of NIF-1 (Fig. 3A & B). LMB treatment increased the restricted nuclear localization of NIF-1 in COS-7 cells expressing both NIF-1 and p35 (from ∼30% to>60%) similar to that observed in the cells expressing NIF-1 alone (Fig. 2D & 3B). These findings suggest that p35 triggers NIF-1 nuclear export via a CRM1-dependent nuclear export pathway.


p35 regulates the CRM1-dependent nucleocytoplasmic shuttling of nuclear hormone receptor coregulator-interacting factor 1 (NIF-1).

Zhao XS, Fu WY, Chien WW, Li Z, Fu AK, Ip NY - PLoS ONE (2014)

Mediation of p35-triggered NIF-1 nuclear export via a CRM1-dependent pathway.(A & B) COS-7 cells were transfected with p35 and HA-tagged NIF-1. Twenty-four hours after transfection, the cells were treated with LMB (5 or 10 ng/mL) for 4–8 h, stained with HA antibody, and examined by fluorescence microscopy. Representative fluorescence images (A) and quantitative analysis of the cells showing exclusive nuclear accumulation of NIF-1 (B). At least 100 cells were scored for each condition in each trial. Results represent the mean ± SEM of 3 replicates (***p <0.05, one-way ANOVA followed by the Student–Newman–Keuls test). (C–E) p35-stimulated redistribution of NIF-1 from the nucleus to cytoplasm is independent of Cdk5 activity. (C) Active Cdk5 phosphorylated recombinant NIF-1 protein. Recombinant GST-NIF-1-C protein was subjected to phosphorylation assay by Cdk5 (GST and histone H1 were used as negative and positive controls, respectively). (D) COS-7 cells were transfected with the HA-tagged NIF-1 and p35, and subsequently treated with roscovitine (Ros, 10 or 25 µM) for 4 or 8 h. The cells with exclusive nuclear accumulation of NIF-1 were quantified. (E) Inhibition of Cdk5 activity by dnCdk5 expression did not affect the p35-stimulated nuclear export of NIF-1. COS-7 cells were transfected with NIF-1, p35, and dnCdk5 constructs as indicated. Quantitative analysis as described in (D). Results represent the mean ± SEM of 3 replicates (***p <0.05, one-way ANOVA followed by the Student–Newman–Keuls test).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4199748&req=5

pone.0110584-g003: Mediation of p35-triggered NIF-1 nuclear export via a CRM1-dependent pathway.(A & B) COS-7 cells were transfected with p35 and HA-tagged NIF-1. Twenty-four hours after transfection, the cells were treated with LMB (5 or 10 ng/mL) for 4–8 h, stained with HA antibody, and examined by fluorescence microscopy. Representative fluorescence images (A) and quantitative analysis of the cells showing exclusive nuclear accumulation of NIF-1 (B). At least 100 cells were scored for each condition in each trial. Results represent the mean ± SEM of 3 replicates (***p <0.05, one-way ANOVA followed by the Student–Newman–Keuls test). (C–E) p35-stimulated redistribution of NIF-1 from the nucleus to cytoplasm is independent of Cdk5 activity. (C) Active Cdk5 phosphorylated recombinant NIF-1 protein. Recombinant GST-NIF-1-C protein was subjected to phosphorylation assay by Cdk5 (GST and histone H1 were used as negative and positive controls, respectively). (D) COS-7 cells were transfected with the HA-tagged NIF-1 and p35, and subsequently treated with roscovitine (Ros, 10 or 25 µM) for 4 or 8 h. The cells with exclusive nuclear accumulation of NIF-1 were quantified. (E) Inhibition of Cdk5 activity by dnCdk5 expression did not affect the p35-stimulated nuclear export of NIF-1. COS-7 cells were transfected with NIF-1, p35, and dnCdk5 constructs as indicated. Quantitative analysis as described in (D). Results represent the mean ± SEM of 3 replicates (***p <0.05, one-way ANOVA followed by the Student–Newman–Keuls test).
Mentions: As p35 promoted the redistribution of NIF-1 from the nucleus to the cytoplasm in COS-7 cells, we examined whether p35 triggers NIF-1 nuclear export. The CRM1-dependent nuclear export pathway is a major pathway for transporting proteins out of the nucleus [25]. Treatment with LMB, a specific inhibitor of CRM1-mediated export [26], abolished the p35-dependent export of NIF-1 from the nucleus to the cytoplasm, resulting in the nuclear accumulation of NIF-1 (Fig. 3A & B). LMB treatment increased the restricted nuclear localization of NIF-1 in COS-7 cells expressing both NIF-1 and p35 (from ∼30% to>60%) similar to that observed in the cells expressing NIF-1 alone (Fig. 2D & 3B). These findings suggest that p35 triggers NIF-1 nuclear export via a CRM1-dependent nuclear export pathway.

Bottom Line: Our results show that p35 interacts with NIF-1 and regulates its nucleocytoplasmic trafficking via the nuclear export pathway.Furthermore, we identified a nuclear export signal on p35; mutation of this site or blockade of the CRM1/exportin-dependent nuclear export pathway resulted in the nuclear accumulation of p35.These findings reveal a new p35-dependent mechanism in transcriptional regulation that involves the nucleocytoplasmic shuttling of transcription regulators.

View Article: PubMed Central - PubMed

Affiliation: Division of Life Science, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China; Molecular Neuroscience Center, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China; State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China.

ABSTRACT
Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase, which plays critical roles in a wide spectrum of neuronal functions including neuronal survival, neurite outgrowth, and synapse development and plasticity. Cdk5 activity is controlled by its specific activators: p35 or p39. While knockout studies reveal that Cdk5/p35 is critical for neuronal migration during early brain development, functions of Cdk5/p35 have been unraveled through the identification of the interacting proteins of p35, most of which are Cdk5/p35 substrates. However, it remains unclear whether p35 can regulate neuronal functions independent of Cdk5 activity. Here, we report that a nuclear protein, nuclear hormone receptor coregulator (NRC)-interacting factor 1 (NIF-1), is a new interacting partner of p35. Interestingly, p35 regulates the functions of NIF-1 independent of Cdk5 activity. NIF-1 was initially discovered as a transcriptional regulator that enhances the transcriptional activity of nuclear hormone receptors. Our results show that p35 interacts with NIF-1 and regulates its nucleocytoplasmic trafficking via the nuclear export pathway. Furthermore, we identified a nuclear export signal on p35; mutation of this site or blockade of the CRM1/exportin-dependent nuclear export pathway resulted in the nuclear accumulation of p35. Intriguingly, blocking the nuclear export of p35 attenuated the nuclear accumulation of NIF-1. These findings reveal a new p35-dependent mechanism in transcriptional regulation that involves the nucleocytoplasmic shuttling of transcription regulators.

Show MeSH
Related in: MedlinePlus