Importin-α Protein Binding to a Nuclear Localization Signal of Carbohydrate Response Element-Binding Protein (ChREBP)

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 alpha 2 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-alpha; this heterodimer then forms a complex with importin-beta to interact with the nuclear pore complex. In this work, we recharacterized the importin-alpha 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-alpha binding, glucose-stimulated transcriptional activity and nuclear localization. Asecondary 14-3-3 protein binding site also was identified, the alpha 3 helix (residues 170-190) phospho-Ser(196) domain. Importin-alpha and 14-3-3 were found to bind competitively to this secondary site. These results suggest an important mechanism by which importin-alpha 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.