20(S)-Hydroxycholesterol GKRP binds to the inactive super op

GKRP binds to the inactive, super-open conformation of GCK, acting as a competitive inhibitor of glucose association with the enzyme (Fig. 3) [45]. Upon formation of the inhibitory complex with GKRP, GCK is sequestered into the hepatocyte nucleus [48]. The detailed mechanism by which GKRP mediates translocation of GCK into the nucleus is not fully undertood. GKRP is predominantly localized to the hepatocyte nucleus under a variety of conditions [48,49], but the regulatory protein has also been detected in the cytosol and in a mitochondrial complex with GCK [50]. GKRP appears to recruit GCK to the nucleus via a “piggy-back” mechanism [48], however direct nuclear localization of GCK via a signaling sequence remains possible, since a nuclear localization signal (NLS) has been identified in pancreatic GCK and this region is conserved in the liver isoform [48,51]. Nevertheless, numerous studies have indicated that nuclear localization of liver GCK is fully dependent upon a functional GKRP. GKRP-mediated inhibition is modulated by several phosphorylated 20(S)-Hydroxycholesterol that bind to the protein's ancestral etherase active site. Fructose 6-phosphate and sorbitol 6-phosphate promote interaction of GKRP with GCK, whereas fructose 1-phosphate weakens the interaction [42,52,53]. The GCK-GKRP interaction is also disrupted by synthetic drugs that bind to a distinct site on GKRP that is located near the carbohydrate binding site [54]. Recent kinetic and mutational work suggest that the differential effects of these ligands upon complex stability are dictated by the conformational state of the flexible N-terminus [55,56]. Ligands that favor an extended N-terminus poise GKRP for optimal coulombic interactions with residues located near the hinge region of GCK. Conversely, ligands that favor a more compact N-terminus disrupt these stabilizing interactions and weaken the protein-protein interaction. GKRP-mediated inhibition of GCK is also modulated by several post-translational processes in the liver. GKRP promoted nuclear translocation of GCK may be impaired by GCK SUMOylation, a modification that also serves to stabilize and activate the enzyme [51]. GKRP itself is acetylated near the N-terminus by the p300 acetyltansferase, which prolongs the protein's lifespan and enhances its inhibitory potential [57]. Sirtuin 2, an NAD + dependent deacetylase reverses this modification and also deacetylates Lys126, which reportedly promotes hepatic glucose uptake [58]. The existence of several layers of control over the GCK-GKRP complex demonstrates the physiological importance of regulating GCK activity via this protein-protein interaction. Although much is known about the GCK-GKRP interaction, several questions remain. The known, natural carbohydrate ligands of GKRP bind with rather low affinity, suggesting that they may not be physiologically relevant. Indeed, one of the main unresolved issues is whether other natural ligands might be important for governing the GCK-GKRP interaction in cells. Another area of uncertainty centers around potential additional, yet-to-be described functions of GKRP that could be independent of its GCK inhibitory function. Although GKRP does not retain detectable enzymatic activity characteristic of its ancestor [59], it could interact with other proteins and/or fulfill additional functions within the hepatocyte nucleus. Genome-wide association studies have linked mutations in the gkrp locus to hyperlipidemia and cardiovascular disease, however the link between these diseases and the GCK inhibitory activity of GKRP is murky [60,61]. Finally, as mentioned above, GKRP has been observed to be mitochondrially associated in primary hepatocytes, as role in localizing GCK to this (or other) cellular locations is unclear [50].
Activation of GCK by phosphofructokinase-2/fructose bisphosphatase-2 Phosphofructokinase-2/fructose bisphosphatase-2 (PFK-2/FBPase-2) is an enzyme responsible for the synthesis and degradation of fructose-2,6-bisphosphate, an important allosteric regulator of glycolysis and gluconeogenesis. Phage display of a peptide library identified a GCK binding epitope of sequence SLKVWT, which matches a conserved stretch within the FBPase-2 domain of the bifunctional liver enzyme [62]. Subsequent studies demonstrated that the phosphatase domain alone is sufficient for interaction with GCK [62,63]. As PFK-2/FBPase-2 is expressed in both liver hepatocytes and pancreatic β-cells, it contributes to GCK regulation in both organs. This chapter, therefore, will discuss the interaction in the context of both organs. In vitro studies using recombinant human pancreatic GCK and rat liver PFK-2/FPBase-2 indicate the proteins can interact directly, albeit weakly, with a 1:1 stoichiometry (Fig. 4) [63]. The Vmax value of GCK is modestly enhanced by exposure to 5–10 fold molar excess of recombinant PFK-2/FBPase-2, in a manner that is independent of activation by known synthetic allosteric activators [63,64]. Notably, interaction with PFK-2/FBPase-2 does not alter GCK's responsiveness to glucose, as reflected by the enzyme's K0.5 value [64], however one report suggests that the interaction perturbs GCK's hallmark cooperativity [65]. In addition to direct activation of GCK, PFK-2/FBPase-2 appears to stabilize GCK, in part by protecting the protein against oxidative damage [66]. GCK contains 13 reduced cysteine residues that are essential for proper activity and glucose responsesiveness [67]. Thus, an interaction that provides protection against reactive oxygen species likely serves a significant physiological role.