Identification of the ubiquitin-like domain of midnolin as a new glucokinase interaction partner.

Glucokinase acts as a glucose sensor in pancreatic beta cells. Its posttranslational regulation is important but not yet fully understood. Therefore, a pancreatic islet yeast two-hybrid library was produced and searched for glucokinase-binding proteins. A protein sequence containing a full-length ubiquitin-like domain was identified to interact with glucokinase. Mammalian two-hybrid and fluorescence resonance energy transfer analyses confirmed the interaction between glucokinase and the ubiquitin-like domain in insulin-secreting MIN6 cells and revealed the highest binding affinity at low glucose. Overexpression of parkin, an ubiquitin E3 ligase exhibiting an ubiquitin-like domain with high homology to the identified, diminished insulin secretion in MIN6 cells but had only some effect on glucokinase activity. Overexpression of the elucidated ubiquitin-like domain or midnolin, containing exactly this ubiquitin-like domain, significantly reduced both intrinsic glucokinase activity and glucose-induced insulin secretion. Midnolin has been to date classified as a nucleolar protein regulating mouse development. However, we could not confirm localization of midnolin in nucleoli. Fluorescence microscopy analyses revealed localization of midnolin in nucleus and cytoplasm and co-localization with glucokinase in pancreatic beta cells. In addition we could show that midnolin gene expression in pancreatic islets is up-regulated at low glucose and that the midnolin protein is highly expressed in pancreatic beta cells and also in liver, muscle, and brain of the adult mouse and cell lines of human and rat origin. Thus, the results of our study suggest that midnolin plays a role in cellular signaling of adult tissues and regulates glucokinase enzyme activity in pancreatic beta cells.

The ubiquitin-proteasome system regulates the stability and activity of the glucose sensor glucokinase in pancreatic ß-cells.

The ubiquitin-proteasome system is important to maintain pancreatic ß-cell function. Inhibition of the proteasome significantly reduced glucose-induced insulin secretion. Key regulators of the stimulus/secretion cascade seem to be affected by protein misfolding if the proteasome is down-regulated as recently reported in humans with Type 2 diabetes. It remains unknown, however, whether the glucose sensor enzyme glucokinase is involved in this process. A direct interaction between glucokinase and ubiquitin could be shown in vivo by FRET, suggesting regulation of glucokinase by the proteasome. After proteasome inhibition glucokinase activity was significantly reduced in MIN6 cells, whereas the protein content was increased, indicating protein misfolding. Enhancing the availability of chaperones by cyclohexamide could induce refolding and restored glucokinase activity. Glucokinase aggregation due to proteasome blocking with MG132, bortezomib, epoxomicin or lactacystin could be detected in MIN6 cells, primary ß-cells and hepatocytes using fluorescence-based assays. Glucokinase aggresome formation proceeded microtubule-assisted and was avoided by cyclohexamide. Thus the results of the present study provide support for glucokinase misfolding and aggregation in case of a diminished capacity of the ubiquitin-proteasome system in pancreatic ß-cells. In the Type 2 diabetic situation this could contribute to reduced glucose-induced insulin secretion.

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase and small chemical activators affect enzyme activity of activating glucokinase mutants by distinct mechanisms.

Glucokinase (GK), a monomeric glucose-phosphorylating enzyme characterised by high structural flexibility, acts as a glucose sensor in pancreatic beta cells and liver. Pharmaceutical efforts to control the enzyme are hampered by an incomplete understanding of GK regulation. We investigated GK characteristics of wild-type and activating S64Y and G68V mutant proteins in the presence of various combinations of the synthetic activators RO-28-1675 and compound A, the endogenous activator fructose-2,6-bisphosphatase (FBPase-2), and the inhibitor mannoheptulose. S64Y impedes formation of a turn structure that is characteristic for the inactive enzyme conformation, and complex formation with compound A induces collision with the large domain. G68V evokes close contact of connecting region I and helix α13 with RO-28-1675 and compound A. Both mutants showed higher activity than the wild-type at low glucose and were susceptible to further activation by FBPase-2 and RO-28-1675, alone and additively. G68V was less active than S64Y, but was activatable by compound A. In contrast, compound A inhibited S64Y, and this effect was even more pronounced in combination with mannoheptulose. Mutant and wild-type GK showed comparable thermal stability and intracellular lifetimes. A GK-6-phosphofructo-2-kinase (PFK-2)/FBPase-2 complex predicted by in silico protein-protein docking demonstrated possible binding of the FBPase-2 domain near the active site of GK. In summary, activating mutations within the allosteric site of GK do not preclude binding of chemical activators (GKAs), but can alter their action into inhibition. Our postulated GK-PFK-2/FBPase-2 complex represents the endogenous principle of activation by substrate channelling which permits binding of other small molecules and proteins.