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    • Yeast two hybrid screening studies have provided the

    2022-01-20

    Yeast two-hybrid screening studies have provided the foundation for countless investigations of protein-protein interactions [[141], [142], [143]]. To identify additional hepatic proteins similar to GKRP that are implicated in GCK regulation, a group of researchers conducted a two-hybrid study using rat liver GCK cDNA [144]. Their screen revealed both GKRP and a 37 kDa tyrosine phosphatase as interaction partners. A BLAST search of the coding sequence identifies this protein as dual specificity protein phosphatase 12, which shares 83% sequence identity to the homologous human isoform. In vitro assays revealed that this protein was able to dephosphorylate GCK and increase GCK's activity in a dose-dependent manner and at different stoichiometries [144]. These results, while being the only published report of this interaction, offer unique insights into the regulatory mechanisms that govern cellular GCK. As the interplay between phosphorylation and proteolysis has been well described [145], it seems likely that the phosphorylation state of GCK might contribute to its stability and intracellular concentration. The physiological consequences of direct interaction with this tyrosine phosphatase, and those of the dephosphorylation it catalyzes, are still poorly understood. Another yeast two-hybrid screen revealed interaction between propionyl-CoA carboxylase β subunit (pβPCCase) and both the liver and pancreatic isoforms of GCK [146]. The interaction was confirmed in an immobilized binding assay and the authors observed an increase in GCK's Vmax and an improved (±)-CPSI 1306 K0.5 value in a pβPCCase concentration-dependent manner [146]. Interestingly, the product of pβPCCase, (S)-methylmalonyl CoA, is a precursor for gluconeogeneic intermediates [147], indicating a seemingly contradictory role between GCK and pβPCCase. It is unclear how these seemingly subsidiary interactions of GCK cooperate with the more established regulatory mechanisms within the broader context of the cell. However, continued investigation surrounding the aforementioned GCK interaction partners and explorations aimed at discovering latent GCK regulatory schemes are critical to future therapeutic strategies.
    Concluding remarks Biochemical, biophysical and cellular investigations performed over the last decade have resulted in tremendous advances in our molecular understanding of GCK regulation. These studies have yielded a semi-quantitative model for the enzyme's kinetic cooperativity and have uncovered two distinct mechanisms by which the enzyme is activated in hyperinsulinemia-associated variants. Molecular and cellular studies have also identified a variety of interaction partners and post-translational events capable of regulating GCK activity. Taken together, these data support the view that GCK regulation is best represented as a network of processes that operate in close coordination with the metabolic state of the cell. Several perspective and review articles have integrated known cellular GCK regulatory strategies into a holistic understanding of pancreatic β-cell and liver hepatocyte function [6,46,148]. The next major step in understanding global GCK regulation is to incorporate our understanding of molecular and cellular regulatory mechanisms into a unified model for GCK regulation in vivo. Several considerations, outlined below, suggest that undertaking such a task is both feasible and close at hand. GCK's conformational heterogeneity also appears correlated with subcellular localization of the enzyme, a characteristic that is conferred by GCK interaction partners. Nuclear localization of GCK in hepatocytes requires binding to GKRP and only occurs when the enzyme adopts the inactive, super-open state. Conversely, current evidence suggests that recognition by PFK-2/FBPase-2 involves the glucose-bound state, an interaction that is believed to be predominantly cytosolic in nature. Moreover, PFK-2/FBPase-2-mediated regulation of GCK is more favorable in pancreatic β-cells than in liver hepatocytes because capillary bed is not hindered by the presence of the liver-specific GKRP. The third known site of GCK localization, the mitochondria, is likely to involve interactions with multiple components of the 5-membered BAD-containing complex. No information is available regarding the conformation(s) involved in tethering GCK at the mitochondria, emphasizing the need for further study. Combining recent advances in super-resolution microscopy with the ability to engineer conformationally restricted variants of GCK could provide new insights into the link between cellular localization and enzyme conformation. Live-cell imaging studies could also establish the extent to which GCK can participate in higher order multienzyme complexes similar to the recently described purinosome and glucosome [149,150]. Clearly, more investigations of GCK structure and dynamics within the complex cellular environment are warranted, and the information gleaned from such investigations promises to drive the field of GCK regulation into new, unexplored avenues.