Functional characterization of MODY2 mutations in the nuclear export signal of glucokinase

Abstract

Glucokinase (GCK) plays a key role in glucose homeostasis. Heterozygous inactivating mutations in the GCK gene cause the familial, mild fasting hyperglycaemia named MODY2. Besides its particular kinetic characteristics, glucokinase is regulated by subcellular compartmentation in hepatocytes. Glucokinase regulatory protein (GKRP) binds to GCK, leading to enzyme inhibition and import into the nucleus at fasting. When glucose concentration increases, GCK-GKRP dissociates and GCK is exported to the cytosol due to a nuclear export signal (NES). With the aim to characterize the GCK-NES, we have functionally analysed nine MODY2 mutations located within the NES sequence. Recombinant GCK mutants showed reduced catalytic activity and, in most cases, protein instability. Most of the mutants interact normally with GKRP, although mutations L306R and L309P impair GCK nuclear import in cotransfected cells. We demonstrated that GCK-NES function depends on exportin 1. We further showed that none of the mutations fully inactivate the NES, with the exception of mutation L304P, which likely destabilizes its α-helicoidal structure. Finally, we found that residue Glu300 negatively modulates the NES activity, whereas other residues have the opposite effect, thus suggesting that some of the NES spacer residues contribute to the low affinity of the NES for exportin 1, which is required for its proper functioning. In conclusion, our results have provided functional and structural insights regarding the GCK-NES and contributed to a better knowledge of the molecular mechanisms involved in the nucleo-cytoplasmic shuttling of glucokinase. Impairment of this regulatory mechanism by some MODY2 mutations might contribute to the hyperglycaemia in the patients.

A novel reverse two-hybrid method for the identification of missense mutations that disrupt protein–protein binding

“…We identified ten mutated residues in GK that are associated with a complete loss of GKRP binding. Our results are consistent with previous studies showing that three of these mutations (S64P, G72E and C252Y) led to a loss of inhibition of GK activity by GKRP in vitro 19,25 and we previously found that the L306R mutation disrupt the two-hybrid interaction with GKRP 22 . Two other mutated residues (T60A and A201V) are located at the GK-GKRP binding interface and we further showed that these mutations result in a complete loss of inhibition of GK activity by GKRP in vitro and the inability of GKRP to promote GK nuclear retention in cell cultures.…”

“…HepG2 cells were co-transfected with plasmids expressing GFP-GK and GKRP-mCherry fusion proteins and analyzed by confocal microscopy. In agreement with previous work 22 , wild type GFP-GK accumulates in the nucleus together with GKRP-mCherry (Fig. 4A).…”

“…Leu306 is the only mutated residue located far from the GK-GKRP binding region. This residue is part of the GK nuclear export sequence (NES) and we previously reported that the L306R substitution identified in our screen prevents GKRP binding, most likely due to protein folding defects 22 . In line with this, we found that the protein expression level of this mutant is significantly decreased compared to wild type and other mutants.…”

“…2 µm high-copy-number and integrative plasmids encoding LexA-Tsg101 were constructed by cloning the Tsg101 coding sequence in the BamH1 site of LexA(1-202)+PL 35 or a pRS402 36 derivative containing the LexA sequence under the control of the ADH1 promotor and terminator. pACT2-GK and pGBKT7-GKRP encoding GAD-GK and GBD-GKRP have been described previously 37 , as well as plasmids expressing GST-GK, GFP-GK, GKRP-Flag and GKRP-mCherry 22 . Single mutations in pACT2-GK were introduced by site-directed mutagenesis.…”

“…GK enzymatic assays and structural analysis. Recombinant wild-type and mutant GST-GK, and Flagtagged human GKRP were bacterially expressed and purified as described previously 21,22 . Determination of GK kinetic parameters and GKRP inhibition assays were performed as reported earlier 22 .…”