title: Importin-alpha protein binding to a nuclear localization signal of carbohydrate response element-binding protein (ChREBP).
abstract: Carbohydrate response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in the glucose-mediated induction of genes involved in hepatic glycolysis and lipogenesis. Circulating blood glucose levels affect ChREBP activity in hepatocytes largely by post-translational mechanisms that include phosphorylation-dependent subcellular localization. Previously, we showed that ChREBP is retained in the cytosol by phosphorylation-dependent binding to 14-3-3 protein dimers and identified the α2 helix (residues 125-135) phospho-Ser(140) domain as the primary 14-3-3 binding site (Sakiyama, H., Wynn, R. M., Lee, W. R., Fukasawa, M., Mizuguchi, H., Gardner, K. H., Repa, J. J., and Uyeda, K. (2008) J. Biol. Chem. 283, 24899-24908). To enter the nucleus in response to high glucose, ChREBP must bind importin-α; this heterodimer then forms a complex with importin-β to interact with the nuclear pore complex. In this work, we recharacterized the importin-α binding nuclear localization signal (NLS) of rat ChREBP, identifying it as an extended classical bipartite NLS encompassing minimally residues 158-190. Replacing Lys(159)/Lys(190) residues of ChREBP with alanine resulted in loss of importin-α binding, glucose-stimulated transcriptional activity and nuclear localization. A secondary 14-3-3 protein binding site also was identified, the α3 helix (residues 170-190) phospho-Ser(196) domain. Importin-α and 14-3-3 were found to bind competitively to this secondary site. These results suggest an important mechanism by which importin-α and 14-3-3 control movement of ChREBP in and out of the nucleus in response to changes in glucose levels in liver and thus further suggest that the extended NLS of ChREBP is a critical glucose-sensing, glucose-responsive site.
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390, USA.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Ge
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Nakagawa
authors.forename: R Max
authors.initials: RM
authors.lastname: Wynn
authors.forename: Yuh Min
authors.initials: YM
authors.lastname: Chook
authors.forename: Bonnie C
authors.initials: BC
authors.lastname: Miller
authors.forename: Kosaku
authors.initials: K
authors.lastname: Uyeda
mesh_terms: D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D001786:Blood Glucose / Q000378:metabolism; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D051379:Mice; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D011485:Protein Binding / Q000502:physiology; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D051381:Rats; D017207:Rats, Sprague-Dawley; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D028901:alpha Karyopherins / Q000235:genetics / Q000378:metabolism*
chemical_list: D048948:14-3-3 Proteins; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001786:Blood Glucose; C116385:MLXIPL protein, human; C506001:Mlxipl protein, mouse; C442825:Mlxipl protein, rat; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D014157:Transcription Factors; D028901:alpha Karyopherins