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    • A majority of investigations of the GCK

    2021-12-29

    A majority of investigations of the GCK-PFK-2/FBPase-2 interaction have taken place within cells. Immunofluorescence studies in several fixed cell lines indicate that GCK colocalizes with PFK-2/FBPase-2 in the cytoplasm following overexpression [[68], [69], [70]]. Live cell imaging conducted on ML 239 australia overexpressing fluorescent fusion proteins support colocalization of GCK and PFK-2/FBPase in a glucose-dependent manner [71]. In these studies, colocalization was most notable following treatment with elevated glucose levels (10–25 mM), whereas the interaction was less apparent in cells exposed to 3 mM glucose. Together with the observation that PFK-2/FBPase-2 stimulates the Vmax value of GCK, these observations support a model in which PFK-2/FBPase interacts preferentially with the closed, glucose-bound conformation of GCK [71]. This stands in contrast to the interaction of GCK with GKRP, which is specific to the super-open conformation. Association with PFK-2/FBPase-2 produces modest increases of GCK activity in insulin-producing cells following adenoviral overexpression [64]. Interestingly, this activation was shown to be sensitive to forskolin, a cAMP stimulator, when using the liver PFK-2/FBPase-2 isozyme, but not the islet PFK-2/FBPase-2 isozyme [64]. This observation is consistent with a role for protein kinase A mediated phosphorylation in regulating the GCK-PFK-2/FBPase-2 interaction. The activity of liver PFK-2/FBPase-2 is regulated by phosphorylation of Ser32, a modification that is stimulated by treatment with glucagon. Phosphorylation of Ser32 is also stimulated by adenosine monophosphate (AMP), via the action of AMP-activated protein kinase (AMPK) [72]. Glucagon inhibits glucose-stimulated translocation of GCK from the nucleus to the cytoplasm in liver cells and a phosphorylation inactive variant of liver PFK-2/FPBase-2 reverses this effect [73]. These results suggest that unphosphorylated PFK-2/FBPase-2 interacts with GCK to stimulate the active closed form of the enzyme and promote cytoplasmic localization. Glucagon stimulates protein kinase A activity and AMP stimulates AMPK activity, both of which promote PFK-2/FBPase-2 phosphorylation, impairing the interaction with GCK and facilitating GKRP-dependent nuclear sequestration of GCK [70]. It is important to note, however, that one study failed to detect a quantitative difference in the nuclear-to-cytoplasmic ratio of GCK that is dependent upon the phosphorylation state of PFK-2/FBPase-2 [74]. Several important aspects of the GCK-PFK-2/FBPase-2 interaction remain unresolved. Several lines of evidence suggest that while the GCK-PFK-2/FBPase-2 interaction is significantly weaker than that of GCK-GKRP interaction, it still represents the strongest interaction of GCK with a protein activator [63,71]. Notably, reconstitution of the complex using purified proteins has yet to be achieved [63,71]. Thus, the regions of GCK responsible for the PFK-2/FBPase-2 interaction are unknown and the extent to which these might overlap with sites of naturally occurring GCK activating mutations is unclear. Uncertainly also centers around whether the interaction involves other, yet-to-be determined factors that could enhance the strength of the complex [63]. Finally, there is incomplete knowledge regarding how this interaction is modulated by metabolic cues beyond glucose, AMP and glucagon, and how the interaction is coordinated with other potential binding partners at other locations with the cell. Despite these questions, there is strong evidence from a number of independent investigations that the GCK-PFK-2/FBPase-2 interaction plays an important role in liver and pancreatic β-cell function [[68], [69], [70]] and further studies are clearly warranted.
    Regulation of GCK via transient interaction partners
    Subordinate interaction partners GCK has been reported to colocalize with actin filaments [136]. Immunofluorescence staining revealed a mesh-like distribution of endogenous GCK in the cytoplasm of rat hepatocytes, which appeared to coincide with actin staining. Additionally, COS-7 cells, which are adherent and behave like fibroblasts, were transiently transfected with either liver or pancreatic GCK. In these dissimilar cells, colocalization with actin, independent of glucose concentrations, was observed in both cases [136]. This pattern was not observed in MIN6 β-cells, however. Tissue-specific promoters result in GCK expression levels that are significantly lower in pancreatic β-cells than hepatocytes [137], suggesting that an improved detection limit might be required to observe GCK-actin colocalization in these cells. Although the initial report of GCK-actin colocalization by Murata and coworkers represents the only evidence of this phenomenon, the interaction remains intriguing for two reasons: (1) Arabadopsis hexokinase associates with actin to modulate actin polymerization rate and glucose signaling [[138], [139], [140]] and (2) GCK has been reported to colocalize with other motor proteins including α-tubulin, β-tubulin and kinesin [68]. Perhaps GCK associates with motor proteins to facilitate rapid movement in response to metabolic stimuli and/or to alter its localization to discrete sites within the cell.