Regulation of nuclear import/export of carbohydrate response element-binding protein (ChREBP)

Interaction of anα-helix of ChREBP with the 14-3-3 proteins and regulation by phosphorylation

Haruhiko Sakiyama, R. Max Wynn, Wan Ru Lee, Masashi Fukasawa, Hiroyuki Mizuguchi, Kevin H. Gardner, Joyce J. Repa, Kosaku Uyeda

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Abstract

Carbohydrate response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in the glucose-mediated induction of gene products involved in hepatic glycolysis and lipogenesis. Glucose affects the activity of ChREBP largely through post-translational mechanisms involving phosphorylation-dependent cellular localization. In this work we show that the N-terminal region of ChREBP (residues 1-251) regulates its subcellular localization via an interaction with 14-3-3. 14-3-3 binds an α-helix in this region (residues 125-135) to retain ChREBP in the cytosol, and binding of 14-3-3 is facilitated by phosphorylation of nearby Ser-140 and Ser-196. Phosphorylation of ChREBP at these sites was essential for its interaction with CRM1 for export to the cytosol, whereas nuclear import of ChREBP requires dephosphorylated ChREBP to interact with importin α. Notably, 14-3-3 appears to compete with importin α for ChREBP binding. 14-3-3β bound to a synthetic peptide spanning residues 125-144 and bearing a phosphate at Ser-140 with a dissociation constant of 1.1 μM, as determined by isothermal calorimetry. The interaction caused a shift in the fluorescence maximum of the tryptophan residues of the peptide. The corresponding unphosphorylated peptide failed to bind 14-3-3β. These results suggest that interactions with importin α and 14-3-3 regulate movement of ChREBP into and out of the nucleus, respectively, and that these interactions are regulated by the ChREBP phosphorylation status.

Original languageEnglish (US)
Pages (from-to)24899-24908
Number of pages10
JournalJournal of Biological Chemistry
Volume283
Issue number36
DOIs
StatePublished - Sep 5 2008

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14-3-3 Proteins
Phosphorylation
Cell Nucleus Active Transport
Response Elements
Carrier Proteins
Carbohydrates
Karyopherins
Glucose
Cytosol
Peptides
Bearings (structural)
Lipogenesis
Calorimetry
Glycolysis
Protein Binding
Tryptophan
Transcription Factors
Genes
Fluorescence
Phosphates

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

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title = "Regulation of nuclear import/export of carbohydrate response element-binding protein (ChREBP): Interaction of anα-helix of ChREBP with the 14-3-3 proteins and regulation by phosphorylation",
abstract = "Carbohydrate response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in the glucose-mediated induction of gene products involved in hepatic glycolysis and lipogenesis. Glucose affects the activity of ChREBP largely through post-translational mechanisms involving phosphorylation-dependent cellular localization. In this work we show that the N-terminal region of ChREBP (residues 1-251) regulates its subcellular localization via an interaction with 14-3-3. 14-3-3 binds an α-helix in this region (residues 125-135) to retain ChREBP in the cytosol, and binding of 14-3-3 is facilitated by phosphorylation of nearby Ser-140 and Ser-196. Phosphorylation of ChREBP at these sites was essential for its interaction with CRM1 for export to the cytosol, whereas nuclear import of ChREBP requires dephosphorylated ChREBP to interact with importin α. Notably, 14-3-3 appears to compete with importin α for ChREBP binding. 14-3-3β bound to a synthetic peptide spanning residues 125-144 and bearing a phosphate at Ser-140 with a dissociation constant of 1.1 μM, as determined by isothermal calorimetry. The interaction caused a shift in the fluorescence maximum of the tryptophan residues of the peptide. The corresponding unphosphorylated peptide failed to bind 14-3-3β. These results suggest that interactions with importin α and 14-3-3 regulate movement of ChREBP into and out of the nucleus, respectively, and that these interactions are regulated by the ChREBP phosphorylation status.",
author = "Haruhiko Sakiyama and Wynn, {R. Max} and Lee, {Wan Ru} and Masashi Fukasawa and Hiroyuki Mizuguchi and Gardner, {Kevin H.} and Repa, {Joyce J.} and Kosaku Uyeda",
year = "2008",
month = "9",
day = "5",
doi = "10.1074/jbc.M804308200",
language = "English (US)",
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pages = "24899--24908",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
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TY - JOUR

T1 - Regulation of nuclear import/export of carbohydrate response element-binding protein (ChREBP)

T2 - Interaction of anα-helix of ChREBP with the 14-3-3 proteins and regulation by phosphorylation

AU - Sakiyama, Haruhiko

AU - Wynn, R. Max

AU - Lee, Wan Ru

AU - Fukasawa, Masashi

AU - Mizuguchi, Hiroyuki

AU - Gardner, Kevin H.

AU - Repa, Joyce J.

AU - Uyeda, Kosaku

PY - 2008/9/5

Y1 - 2008/9/5

N2 - Carbohydrate response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in the glucose-mediated induction of gene products involved in hepatic glycolysis and lipogenesis. Glucose affects the activity of ChREBP largely through post-translational mechanisms involving phosphorylation-dependent cellular localization. In this work we show that the N-terminal region of ChREBP (residues 1-251) regulates its subcellular localization via an interaction with 14-3-3. 14-3-3 binds an α-helix in this region (residues 125-135) to retain ChREBP in the cytosol, and binding of 14-3-3 is facilitated by phosphorylation of nearby Ser-140 and Ser-196. Phosphorylation of ChREBP at these sites was essential for its interaction with CRM1 for export to the cytosol, whereas nuclear import of ChREBP requires dephosphorylated ChREBP to interact with importin α. Notably, 14-3-3 appears to compete with importin α for ChREBP binding. 14-3-3β bound to a synthetic peptide spanning residues 125-144 and bearing a phosphate at Ser-140 with a dissociation constant of 1.1 μM, as determined by isothermal calorimetry. The interaction caused a shift in the fluorescence maximum of the tryptophan residues of the peptide. The corresponding unphosphorylated peptide failed to bind 14-3-3β. These results suggest that interactions with importin α and 14-3-3 regulate movement of ChREBP into and out of the nucleus, respectively, and that these interactions are regulated by the ChREBP phosphorylation status.

AB - Carbohydrate response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in the glucose-mediated induction of gene products involved in hepatic glycolysis and lipogenesis. Glucose affects the activity of ChREBP largely through post-translational mechanisms involving phosphorylation-dependent cellular localization. In this work we show that the N-terminal region of ChREBP (residues 1-251) regulates its subcellular localization via an interaction with 14-3-3. 14-3-3 binds an α-helix in this region (residues 125-135) to retain ChREBP in the cytosol, and binding of 14-3-3 is facilitated by phosphorylation of nearby Ser-140 and Ser-196. Phosphorylation of ChREBP at these sites was essential for its interaction with CRM1 for export to the cytosol, whereas nuclear import of ChREBP requires dephosphorylated ChREBP to interact with importin α. Notably, 14-3-3 appears to compete with importin α for ChREBP binding. 14-3-3β bound to a synthetic peptide spanning residues 125-144 and bearing a phosphate at Ser-140 with a dissociation constant of 1.1 μM, as determined by isothermal calorimetry. The interaction caused a shift in the fluorescence maximum of the tryptophan residues of the peptide. The corresponding unphosphorylated peptide failed to bind 14-3-3β. These results suggest that interactions with importin α and 14-3-3 regulate movement of ChREBP into and out of the nucleus, respectively, and that these interactions are regulated by the ChREBP phosphorylation status.

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U2 - 10.1074/jbc.M804308200

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