br Funding sources This work was supported in part
Funding sources This work was supported, in part, by NIH GM115388 to BGM.
Introduction Glucokinase (GCK) plays a central role in the regulation of glucose metabolism. Its activity is restricted to Mycophenolic acid synthesis with important roles in whole-body glucose homeostasis . In pancreatic beta-cells GCK acts as glucose sensor by integrating blood glucose levels and glucose metabolism with insulin secretion. In hepatocytes, where glucokinase is also expressed, its activity controls glycogen accumulation, glycolysis and gluconeogenesis rates. Moreover, a role of glucokinase has also been reported in the brain, pancreatic alpha cells and pituitary gonadotropes, for review see . GCK mutations can result in monogenic disorders characterized by hyper or hypoglycaemia. Heterozygous activating mutations cause hyperinsulinemic hypoglycaemia. Homozygous inactivating mutations cause permanent neonatal diabetes mellitus, whereas heterozygous inactivating mutations cause maturity-onset diabetes of the young type 2 (MODY2) . The pathophysiological mechanism of GCK associated disorders is a defect in glucose sensing that results in modification of the glucose threshold for beta-cell insulin secretion. Additionally, defects in liver glycogen storage and increased rate of gluconeogenesis have been demonstrated in MODY2 patients [4,5]. Glucokinase belongs to the hexokinase family (GCK is also named hexokinase IV), which converts glucose in glucose-6-phosphate with ATP as second substrate. This reaction is the first limiting step of glucose utilization in hepatocytes and beta-cells. The function of glucokinase is based on its particular kinetic characteristics, mainly low affinity and cooperativity for glucose. These properties are conferred by multiple conformational protein structure states. Briefly, in the absence of glucose, GCK has an inactive super-open conformation. Conversely, the glucose-bound enzyme adopts an active closed conformation [6,7]. In addition, glucokinase can be regulated by tissue-specific posttranscriptional mechanisms . In the liver, glucokinase is also regulated through protein-protein interactions by the glucokinase regulatory protein (GKRP), which inhibits the enzyme and also induces its nuclear retention in hepatocytes [8,9]. GCK-GKRP interaction is strengthened by fructose-6-phosphate (F6P) and counteracted by fructose-1-phosphate (F1P), which bind to the same site of GKRP [10,11]. During fasting, at low glucose concentrations, GKRP-F6P anchors the super-open GCK form, allowing its import into the nucleus. Mutational and structural studies of GKRP-bound GCK have shown that the GKRP-binding surface is located at the allosteric site in the hinge region of GCK, which is exposed in the super-open conformation [, , , , ]. Upon binding to GKRP, GCK translocates to the nucleus in hepatocytes or in heterologous cells co-transfected with GKRP and GCK, and the absence of GKRP results in exclusive cytoplasmic localization of this enzyme [9,16,17]. After feeding, when glucose (and fructose) concentrations rise, GCK adopts an active glucose-bound closed conformation and dissociates from GKRP-F1P. Substrate binding causes a structural rearrangement of GCK, which results in the dissociation of the complex . The dissociation of GCK from GKRP occurs prior to being exported from the nucleus to the cytoplasm [18,19]. The enzyme nuclear export is mediated by a leucine-rich nuclear export signal (NES) covering aminoacids 300 to 310 of glucokinase (GCK-NES: 300ELVRLVLLKLV310) . Due to the central role of glucokinase in the regulation of glucose homeostasis, this enzyme has been considered as a potential target for the development of new anti-diabetic drugs. However, tackling such a challenge requires a thorough knowledge of GCK regulatory mechanisms [20,21]. The biochemical characterization of GCK mutations associated to glycemic disorders has contributed significant insights into the regulatory mechanism of this enzyme . In this work, we carried out a systematic analysis of MODY2 mutations within the GCK-NES sequence.