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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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p35 interacts with NIF-1.(A) Mouse NIF-1 encodes a 1,291-amino acid protein and contains 6 zinc finger domains, an LXXLL domain, and a leucine zipper-like motif. (B & C) Mapping of interaction domains between NIF-1 and p35. Yeast was co-transformed with different domains of p35 and NIF-1. +, strong interaction; −, absence of interaction. (B) The C-terminal region of NIF-1 (amino acids 1,066–1,291) containing the 6th zinc finger domain, leucine zipper-like motif, and short C-terminus was sufficient to interact with p35. (C) The N-terminal region of p35 (corresponding to the p10 fragment) was required for the interaction between p35 and NIF-1. p10 comprises the 98 N-terminal amino acids of p35, while p25 contains the C-terminal region of p35. (D & E) Direct interaction between NIF-1 and p35. Recombinant GST fusion proteins encoding different regions of NIF-1 were incubated with lysate prepared from p35-overexpressing COS-7 cells (D) or recombinant p35 protein (E). The bound proteins were pulled down by glutathione-Sepharose and analyzed by western blot analysis (Lysate, as an input control). Bottom panel: Coomassie-stained gel. (F) Association of p35 with NIF-1 in mammalian cells. COS-7 cells were transiently transfected with NIF-1 and p35. Cell lysate was immunoprecipitated (IP) with p35 or NIF-1 antibody as indicated and subjected to western blot analysis. Rabbit normal IgG (IgG) was used as a negative control.
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pone.0110584-g001: p35 interacts with NIF-1.(A) Mouse NIF-1 encodes a 1,291-amino acid protein and contains 6 zinc finger domains, an LXXLL domain, and a leucine zipper-like motif. (B & C) Mapping of interaction domains between NIF-1 and p35. Yeast was co-transformed with different domains of p35 and NIF-1. +, strong interaction; −, absence of interaction. (B) The C-terminal region of NIF-1 (amino acids 1,066–1,291) containing the 6th zinc finger domain, leucine zipper-like motif, and short C-terminus was sufficient to interact with p35. (C) The N-terminal region of p35 (corresponding to the p10 fragment) was required for the interaction between p35 and NIF-1. p10 comprises the 98 N-terminal amino acids of p35, while p25 contains the C-terminal region of p35. (D & E) Direct interaction between NIF-1 and p35. Recombinant GST fusion proteins encoding different regions of NIF-1 were incubated with lysate prepared from p35-overexpressing COS-7 cells (D) or recombinant p35 protein (E). The bound proteins were pulled down by glutathione-Sepharose and analyzed by western blot analysis (Lysate, as an input control). Bottom panel: Coomassie-stained gel. (F) Association of p35 with NIF-1 in mammalian cells. COS-7 cells were transiently transfected with NIF-1 and p35. Cell lysate was immunoprecipitated (IP) with p35 or NIF-1 antibody as indicated and subjected to western blot analysis. Rabbit normal IgG (IgG) was used as a negative control.

Mentions: As a first step to identify p35-interacting protein(s), full-length p35 was used as a bait to screen a postnatal day 14 mouse muscle cDNA library using a yeast two-hybrid screen [15]. The results revealed a positive clone encoding NIF-1 interacting with p35. Mouse NIF-1 encodes a 1,291-amino acid protein that contains 6 Cys-2/His-2 zinc fingers, an N-terminal stretch of acidic amino acids, and a C-terminal leucine zipper-like motif (Fig. 1A). The NIF-1 fragment that interacts with p35 spans the C-terminal half region of NIF-1 (amino acids 772–1,291; Fig. 1A & B). We further characterized the interaction between NIF-1 and p35 by expressing different fragments of them in yeast. The region of NIF-1 flanking the 6th zinc finger and leucine zipper-like motif (amino acids 1,066–1,291) were sufficient for binding to p35 (Fig. 1B). On the other hand, the N-terminal region of p35, corresponding to the p10 fragment, is responsible for its interaction with NIF-1 (Fig. 1C).


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)

p35 interacts with NIF-1.(A) Mouse NIF-1 encodes a 1,291-amino acid protein and contains 6 zinc finger domains, an LXXLL domain, and a leucine zipper-like motif. (B & C) Mapping of interaction domains between NIF-1 and p35. Yeast was co-transformed with different domains of p35 and NIF-1. +, strong interaction; −, absence of interaction. (B) The C-terminal region of NIF-1 (amino acids 1,066–1,291) containing the 6th zinc finger domain, leucine zipper-like motif, and short C-terminus was sufficient to interact with p35. (C) The N-terminal region of p35 (corresponding to the p10 fragment) was required for the interaction between p35 and NIF-1. p10 comprises the 98 N-terminal amino acids of p35, while p25 contains the C-terminal region of p35. (D & E) Direct interaction between NIF-1 and p35. Recombinant GST fusion proteins encoding different regions of NIF-1 were incubated with lysate prepared from p35-overexpressing COS-7 cells (D) or recombinant p35 protein (E). The bound proteins were pulled down by glutathione-Sepharose and analyzed by western blot analysis (Lysate, as an input control). Bottom panel: Coomassie-stained gel. (F) Association of p35 with NIF-1 in mammalian cells. COS-7 cells were transiently transfected with NIF-1 and p35. Cell lysate was immunoprecipitated (IP) with p35 or NIF-1 antibody as indicated and subjected to western blot analysis. Rabbit normal IgG (IgG) was used as a negative control.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0110584-g001: p35 interacts with NIF-1.(A) Mouse NIF-1 encodes a 1,291-amino acid protein and contains 6 zinc finger domains, an LXXLL domain, and a leucine zipper-like motif. (B & C) Mapping of interaction domains between NIF-1 and p35. Yeast was co-transformed with different domains of p35 and NIF-1. +, strong interaction; −, absence of interaction. (B) The C-terminal region of NIF-1 (amino acids 1,066–1,291) containing the 6th zinc finger domain, leucine zipper-like motif, and short C-terminus was sufficient to interact with p35. (C) The N-terminal region of p35 (corresponding to the p10 fragment) was required for the interaction between p35 and NIF-1. p10 comprises the 98 N-terminal amino acids of p35, while p25 contains the C-terminal region of p35. (D & E) Direct interaction between NIF-1 and p35. Recombinant GST fusion proteins encoding different regions of NIF-1 were incubated with lysate prepared from p35-overexpressing COS-7 cells (D) or recombinant p35 protein (E). The bound proteins were pulled down by glutathione-Sepharose and analyzed by western blot analysis (Lysate, as an input control). Bottom panel: Coomassie-stained gel. (F) Association of p35 with NIF-1 in mammalian cells. COS-7 cells were transiently transfected with NIF-1 and p35. Cell lysate was immunoprecipitated (IP) with p35 or NIF-1 antibody as indicated and subjected to western blot analysis. Rabbit normal IgG (IgG) was used as a negative control.
Mentions: As a first step to identify p35-interacting protein(s), full-length p35 was used as a bait to screen a postnatal day 14 mouse muscle cDNA library using a yeast two-hybrid screen [15]. The results revealed a positive clone encoding NIF-1 interacting with p35. Mouse NIF-1 encodes a 1,291-amino acid protein that contains 6 Cys-2/His-2 zinc fingers, an N-terminal stretch of acidic amino acids, and a C-terminal leucine zipper-like motif (Fig. 1A). The NIF-1 fragment that interacts with p35 spans the C-terminal half region of NIF-1 (amino acids 772–1,291; Fig. 1A & B). We further characterized the interaction between NIF-1 and p35 by expressing different fragments of them in yeast. The region of NIF-1 flanking the 6th zinc finger and leucine zipper-like motif (amino acids 1,066–1,291) were sufficient for binding to p35 (Fig. 1B). On the other hand, the N-terminal region of p35, corresponding to the p10 fragment, is responsible for its interaction with NIF-1 (Fig. 1C).

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