SUMOylation of Pancreatic Glucokinase Regulates Its Cellular Stability and Activity* (original) (raw)
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Molecular and Cellular Endocrinology, 2017
The localization of glucokinase in pancreatic beta-cell nuclei is a controversial issue. Although previous reports suggest such a localization, the mechanism for its import has so far not been identified. Using immunofluorescence, subcellular fractionation and mass spectrometry, we here present evidence in support of glucokinase localization in beta-cell nuclei of human and mouse pancreatic sections, as well as in human and mouse isolated islets, and murine MIN6 cells. We have identified a conserved, seven-residue nuclear localization signal (30 LKKVMRR 36) in the human enzyme. Substituting the residues KK 31,32 and RR 35,36 with AA led to a loss of its nuclear localization in transfected cells. Furthermore, our data indicates that SUMOylation of glucokinase modulates its nuclear import, while high glucose concentrations do not significantly alter the enzyme nuclear/cytosolic ratio. Thus, for the first time, we provide data in support of a nuclear import of glucokinase mediated by a redundant mechanism, involving a nuclear localization signal, and which is modulated by its SUMOylation. These findings add new knowledge to the functional role of glucokinase in the pancreatic beta-cell.
SUMOylation regulates Kv2.1 and modulates pancreatic β-cell excitability
Journal of Cell Science, 2009
The covalent attachment of small ubiquitin-like modifier (SUMO) proteins regulates protein localization and function. SUMOylation has recently been shown to modulate ion-channel function; however, the extent to which this affects native currents and cellular excitability remains to be determined. The voltage-dependent K+ (Kv) channel Kv2.1 regulates pancreatic β-cell excitability and insulin secretion. We found that YFP-tagged SUMO1 (SUMO1-YFP) can be immunoprecipitated with Kv2.1 when these two proteins are coexpressed in HEK 293 cells. Furthermore, direct infusion of recombinant SUMO1 peptide or coexpression of SUMO1-YFP inhibited current through cloned Kv2.1 by 80% and 48%, respectively. Insulin-secreting cells express SUMO variants 1 and 3, and expression of the SUMO1-YFP in human β-cells or insulinoma cells inhibited native Kv currents (by 49% and 33%, respectively). Inhibition of the channel resulted from an acceleration of channel inactivation and an inhibition of recovery fr...
Sumoylation Regulates the Transcriptional Activity of MafA in Pancreatic Cells
Journal of Biological Chemistry, 2009
MafA is a transcriptional regulator expressed primarily in pancreatic  cells. It binds to the RIPE3b/C1-binding site within the ins gene promoter, which plays a critical role in regulating ins gene expression in response to glucose. Here, we show that MafA is post-translationally modified by the small ubiquitinrelated modifiers SUMO-1 and -2. Mutation of a single site in MafA, Lys 32 , blocks its sumoylation in  cells. Incubation of  cells in low glucose (2 mM) or exposure to hydrogen peroxide increases sumoylation of endogenous MafA. Forced sumoylation of MafA results in reduced transcriptional activity toward the ins gene promoter and increased suppression of the CHOP-10 gene promoter. Sumoylation of MafA has no apparent effect on either its nuclear localization in  cells or its ubiquitindependent degradation. This study suggests that modification of MafA by SUMO modulates gene transcription and thereby  cell function.
Journal of Biological Chemistry, 1999
Glucokinase (GK) gene mutations cause diabetes mellitus in both humans and mouse models, but the pathophysiological basis is only partially defined. We have used cre-loxP technology in combination with gene targeting to perform global,  cell-, and hepatocyte-specific gene knockouts of this enzyme in mice. Gene targeting was used to create a triple-loxed gk allele, which was converted by partial or total Cre-mediated recombination to a conditional allele lacking neomycin resistance, or to a null allele, respectively.  cell-and hepatocytespecific expression of Cre was achieved using transgenes that contain either insulin or albumin promoter/ enhancer sequences. By intercrossing the transgenic mice that express Cre in a cell-specific manner with mice containing a conditional gk allele, we obtained animals with either a  cell or hepatocyte-specific knockout of GK. Animals either globally deficient in GK, or lacking GK just in  cells, die within a few days of birth from severe diabetes. Mice that are heterozygous null for GK, either globally or just in the  cell, survive but are moderately hyperglycemic. Mice that lack GK only in the liver are only mildly hyperglycemic but display pronounced defects in both glycogen synthesis and glucose turnover rates during a hyperglycemic clamp. Interestingly, hepatic GK knockout mice also have impaired insulin secretion in response to glucose. These studies indicate that deficiencies in both  cell and hepatic GK contribute to the hyperglycemia of MODY-2.
Diabetes, 1998
There remains a wide gap between theoretical concepts and experimental realities in the enzyme kinetics and biochemical genetics of the pancreatic P-cell glucokinase-glucose sensor. It is the goal of present efforts in many laboratories to bridge this gap. This perspective intends to provide a timely review of this crucial aspect of research in glucose homeostasis. It deals briefly with some fundamentals of glucokinase enzyme kinetics, offers some pertinent biochemical genetic considerations, takes stock of the current experimental database of the field by emphasizing human studies and referring to recent mouse studies, and ventures a few extrapolations into the future of this endeavor.
SUMO downregulates GLP-1-stimulated cAMP generation and insulin secretion
American Journal of Physiology-Endocrinology and Metabolism, 2012
Glucagon-like peptide-1 (GLP-1)-based incretin therapy is becoming central to the treatment of type 2 diabetes. Activation of incretin hormone receptors results in rapid elevation of cAMP followed by enhanced insulin secretion. However, the incretin effect may be significantly impaired in diabetes. The objective of this study is to investigate downregulation of GLP-1 signaling by small ubiquitin-related modifier protein (SUMO). Mouse islets exposed to high glucose showed increased expression of endogenous SUMO transcripts and its conjugating enzyme Ubc-9. Overexpression of SUMO-1 in mouse insulinoma 6 (MIN6) cells and primary mouse β-cells resulted in reduced static and real-time estimates of intracellular cAMP upon receptor stimulation with exendin-4, a GLP-1 receptor (GLP-1R) agonist. GLP1-R was covalently modified by SUMO. Overexpression of SUMO-1 attenuated cell surface trafficking of GLP-1R, which resulted in significantly reduced insulin secretion when stimulated by exendin-4....
Site-specific characterization of endogenous SUMOylation across species and organs
Nature communications, 2018
Small ubiquitin-like modifiers (SUMOs) are post-translational modifications that play crucial roles in most cellular processes. While methods exist to study exogenous SUMOylation, large-scale characterization of endogenous SUMO2/3 has remained technically daunting. Here, we describe a proteomics approach facilitating system-wide and in vivo identification of lysines modified by endogenous and native SUMO2. Using a peptide-level immunoprecipitation enrichment strategy, we identify 14,869 endogenous SUMO2/3 sites in human cells during heat stress and proteasomal inhibition, and quantitatively map 1963 SUMO sites across eight mouse tissues. Characterization of the SUMO equilibrium highlights striking differences in SUMO metabolism between cultured cancer cells and normal tissues. Targeting preferences of SUMO2/3 vary across different organ types, coinciding with markedly differential SUMOylation states of all enzymes involved in the SUMO conjugation cascade. Collectively, our systemic ...
Biochemical Journal, 2008
Human GKβ (pancreatic β-cell glucokinase) is the main glucose-phosphorylating enzyme in pancreatic β-cells. It shares several structural, catalytic and regulatory properties with Hxk2 (hexokinase 2) from Saccharomyces cerevisiae. In fact, it has been previously described that expression of GKβ in yeast could replace Hxk2 in the glucose signalling pathway of S. cerevisiae. In the present study we report that GKβ exerts its regulatory role by association with the yeast transcriptional repressor Mig1 (multicopy inhibitor of GAL gene expression 1); the presence of Mig1 allows GKβ to bind to the SUC2 (sucrose fermentation 2) promoter, helping in this way in the maintenance of the repression of the SUC2 gene under high-glucose conditions. Since a similar mechanism has been described for the yeast Hxk2, the findings of the present study suggest that the function of the regulatory domain present in these two proteins has been conserved throughout evolution. In addition, we report that GKβ i...
Glucokinase and glucose homeostasis: proven concepts and new ideas
2005
The enzyme GK (glucokinase), which phosphorylates glucose to form glucose 6-phosphate, serves as the glucose sensor of insulin-producing β-cells. GK has thermodynamic, kinetic, regulatory and molecular genetic characteristics that are ideal for its glucose sensor function and allow it to control glycolytic flux of the β-cells as indicated by control-, elasticity-and response-coefficients close to or larger than 1.0. GK operates in tandem with the K + and Ca 2+ channels of the β-cell membrane, resulting in a threshold for glucose-stimulated insulin release of approx. 5 mM, which is the set point of glucose homoeostasis for most laboratory animals and humans. Point mutations of GK cause 'glucokinase disease' in humans, which includes hypo-and hyper-glycaemia syndromes resulting from activating or inactivating mutations respectively. GK is allosterically activated by pharmacological agents (called GK activators), which lower blood glucose in normal animals and animal models of T2DM. On the basis of crystallographic studies that identified a ligand-free 'super-open' and a liganded closed structure of GK [Grimsby, Sarabu, Corbett and others (2003) Science 301, 370-373; Kamata, Mitsuya, Nishimura, Eiki and Nagata (2004) Structure 12, 429-438], on thermostability studies using glucose or mannoheptulose as ligands and studies showing that mannoheptulose alone or combined with GK activators induces expression of GK in pancreatic islets and partially preserves insulin secretory competency, a new hypothesis was developed that GK may function as a metabolic switch per se without involvement of enhanced glucose metabolism. Current research has the goal to find molecular targets of this putative 'GK-switch'. The case of GK research illustrates how basic science may culminate in therapeutic advances of human medicine. Determinants of the glucose homeostatic set point Glucose homeostasis in man and many animals is maintained by feedback designed to keep the blood glucose (G) close to a set point characteristic for each species. This set point is close to 5 mM. Critical elements of the loop include: (1) G-sensitive insulin and glucagon-producing βand α-cells, respectively; (2) the hormones insulin and glucagon; (3) hormone-sensitive tissues that consume, store or produce glucose, i.e. liver, skeletal and heart muscle and adipose tissue; and (4) the regulated parameter G. The G set point is determined by the G thresholds for stimulating insulin or glucagon secretion. When G exceeds 5 mM insulin secretion increases above basal and when glucose falls below this level glucagon release is augmented above basal. Note that stimulation of glucagon release by lowering glucose is actually due to reducing the glucose inhibition of secretion stimulated by other agonists (e.g. amino acids or adrenaline). Importantly, the G set point is close to the cross-over point of the glucose dependency curves for hormone secretion rates of these two opposing endocrine cell types.