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.

FIGURE 1.

Modulation of ChREBP importin-α binding, transcriptional activity, and nuclear localization by mutation of putative bipartite NLS lysine residues. A, residues 101–200 of ChREBP indicating locations of the “classical” NLS site, α2 and α3 helices, Ser¹⁴⁰ and Ser¹⁹⁶ phosphorylation sites, and lysine residues of the putative bipartite NLS. B, interaction of importin-α with ChREBP. WT FLAG-ChREBP and the indicated FLAG-ChREBP lysine mutants expressed in HEK293T cells were immunoprecipitated from cell lysates with anti-FLAG beads; GST-importin-α (0.05 μm) was added to reaction mixtures containing the pellets and incubated for 1.5 h at 4 °C. The beads were washed, and bound proteins were analyzed by SDS-PAGE and Western blotting using GST and FLAG antibodies. These experiments were repeated three times, and a representative Western blot is shown. The bar diagrams were generated by densitometric scanning to quantitate importin-α binding to ChREBP. *, p < 0.01; **, p < 0.05. C, transcriptional activity of FLAG-ChREBP lysine, Ser¹⁴⁰, and Ser¹⁹⁶ mutants. DNA constructs expressing the indicated FLAG-ChREBP proteins, firefly luciferase under control of LPK promoter, and Renilla luciferase (an internal control) were co-transfected into primary cultured rat hepatocytes during a 4-h incubation in DMEM containing 5.5 mm glucose (Glc). Fresh medium containing either 5.5 (open bars) or 27.5 mm (filled bars) glucose was added, and the cells were incubated for an additional 20 h. One set of cultures received cAMP (0.1 mm) in addition. Luciferase activities were measured and expressed as firefly luciferase activity relative to Renilla luciferase activity. The values presented are the mean ± S.D. of the results of all five independent experiments. *, p < 0.05; **, p < 0.01. D, nuclear localization of ChREBP in primary hepatocytes. Primary cultured rat hepatocytes were transfected with DNA constructs expressing WT GFP-ChREBP or indicated GFP-ChREBP lysine mutants during a 4-h incubation in DMEM with 5.5 mm glucose. Fresh medium containing either 5.5 (open bars) or 27.5 mm (filled bars) glucose was added, and the cells were incubated an additional 20 h. The values presented are the mean ± S.D. of three sets of ∼100 fluorescent cells. *, p < 0.05.

FIGURE 2.

Measurements for binding of two distinct NLS-containing peptides from ChREBP to importin-α by isothermal titration calorimetry. For measuring binding of the 156–176-residue peptide (A) and the residue 158–190 peptide (B) to importin-α, the solution containing 600 μm of each peptide in the syringe was injected in 8-μl increments into the reaction well containing 1.4 ml of 48.6 μm importin-α (based on monomer concentration) at 20 °C in a VP-ITC microcalorimeter.

FIGURE 3.

Effects of α2 helix mutations on 14-3-3 binding to ChREBP. Anti-FLAG beads were used to isolate WT and the α2 helix A129P/Y131P substitution and Δ126–134 deletion mutants of full-length (FL) FLAG-ChREBP (left) or FLAG-ChREBP N-terminal peptide (1–250, right) with bound endogenous (end) 14-3-3 from transfected HEK293T cell lysates. Exogenous 14-3-3β(exo), 0.4 μm, was added to reaction mixtures and incubated as described in the legend to Fig. 1. The beads were washed, and bound proteins were analyzed by SDS-PAGE and Western blotting using FLAG and pan 14-3-3 antibodies.

FIGURE 4.

14-3-3 proteins bind to the two binding sites of ChREBP with similar affinity. Anti-FLAG beads were used to isolate wild-type FLAG-ChREBP with bound endogenous (end) 14-3-3 from HEK293 cells. Variable concentrations of exogenous 14-3-3β (exo) protein were added to reaction mixtures containing the pellets and incubated as described in the legend to Fig. 1. Bound proteins were analyzed by SDS-PAGE and Western blotting and quantified by densitometric scanning. The plot on the left shows exogenous 14-3-3β (exo) bound to ChREBP with increasing concentrations of exogenous 14-3-3β added.

FIGURE 5.

Interactions of 14-3-3β with the α2 and the α3 helices of ChREBP. FLAG-tagged ChREBP, WT, the α3 helix mutant V179P/Y187P, and the double α2/α3 helix A129P/Y131P-V179P/Y187P mutant, were expressed in HEK293 cells and immunoprecipitated with anti-FLAG beads. Exogenous 14-3-3β (0.14 μm) was added to the reaction mixtures and incubated as described in the legend to Fig. 1. ChREBP-bound14-3-3β was eluted and analyzed by SDS-PAGE and Western blotting with pan 14-3-3 antibody.

FIGURE 6.

Importin-α and 14-3-3 proteins compete for binding to wild-type ChREBP and the α2 helix mutant A129P/Y131P. Anti-FLAG beads were used to isolate WT FLAG-ChREBP or the A129P/Y131P mutant with bound endogenous (end) 14-3-3 from lysates of transfected HEK293 cells. Varying concentrations of purified importin-α were added to reaction mixtures in the presence of purified 14-3-3β (0.18 μm). Following incubation and washing as described in the legend to Fig. 1, bound proteins were analyzed by SDS-PAGE and Western blotting with FLAG, GST, and pan 14-3-3 antibodies. These experiments were repeated twice with similar results.

FIGURE 7.

Residues 1–200 of ChREBP show locations of motifs and phosphorylation sites key for glucose-regulated nuclear import and export of ChREBP. The α1, α2, and α3 helices, the bipartite lysine importin-α binding NLS site, NES1 and -2, CRM1 (exportin) binding sites, and Ser¹⁴⁰ and Ser¹⁹⁶ phosphorylation sites are indicated. ChREBP of rat (24), mouse (3), and human (9) are compared.

FIGURE 8.

Proposed model of the regulation of ChREBP subcellular localization by 14-3-3 and importin-α binding in response to glucose. In response to high glucose, phosphorylated (inactive) ChREBP, complexed with 14-3-3 in the cytosol, is first dephosphorylated (not shown) and releases 14-3-3 and CRM1. Dephosphorylated (active) ChREBP forms a complex with importin-α and -β to translocate into nucleus. It is possible that 14-3-3 may remain bound to ChREBP during import. A decrease in glucose results in inactivation of ChREBP by phosphorylation by PKA that can occur within the nucleus and complex formation with 14-3-3 and CRM1 followed by translocation to the cytosol. Note that for clarity, 14-3-3 proteins (27 kDa) are not presented to scale.