Regulation of nuclear import/export of carbohydrate response element-binding protein (ChREBP): interaction of an alpha-helix of ChREBP with the 14-3-3 proteins and regulation by phosphorylation.
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 alpha-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 alpha. Notably, 14-3-3 appears to compete with importin alpha for ChREBP binding. 14-3-3beta bound to a synthetic peptide spanning residues 125-144 and bearing a phosphate at Ser-140 with a dissociation constant of 1.1 microm, 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 14-3-3beta. These results suggest that interactions with importin alpha 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.
Sakiyama
Haruhiko
H
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
Wynn
R Max
RM
Lee
Wan-Ru
WR
Fukasawa
Masashi
M
Mizuguchi
Hiroyuki
H
Gardner
Kevin H
KH
Repa
Joyce J
JJ
Uyeda
Kosaku
K
D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism*; D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D005947:Glucose / Q000235:genetics / Q000378:metabolism; D006019:Glycolysis / Q000502:physiology; D006801:Humans; D028884:Karyopherins / Q000235:genetics / Q000378:metabolism; D050155:Lipogenesis / Q000502:physiology; D051379:Mice; D018345:Mice, Knockout; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding / Q000502:physiology; D017433:Protein Structure, Secondary; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D000097606:Exportin 1 Protein
D048948:14-3-3 Proteins; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D005947:Glucose; D028884:Karyopherins; D009687:Nuclear Proteins; D010455:Peptides; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D000097606:Exportin 1 Protein; C116385:MLXIPL protein, human; C506001:Mlxipl protein, mouse; C442825:Mlxipl protein, rat