Abstract
The glycolytic enzyme glucokinase (GCK) and the pro-apoptotic protein BAD reportedly reside within a five-membered complex that localizes to the mitochondria of mammalian hepatocytes and pancreatic β-cells. Photochemical crosslinking studies using a synthetic analog of BAD’s BH3 domain and in vitro transcription/translation experiments support a direct interaction between BAD and GCK. To investigate the biochemical and biophysical consequences of the BAD:GCK interaction, we developed a method for the production of recombinant human BAD. Consistent with published reports, recombinant BAD displays high affinity for Bcl-xL (KD = 7 nM), and phosphorylation of BAD at S118, within the BH3 domain, abolishes this interaction. Unexpectedly, we do not detect association of recombinant, full-length BAD with recombinant human pancreatic GCK over a range of protein concentrations using various biochemical methods including size-exclusion chromatography, chemical cross-linking, analytical ultracentrifugation, and isothermal titration calorimetry. Furthermore, fluorescence polarization assays and isothermal titration calorimetry detect no direct interaction between GCK and BAD BH3 peptides. Kinetic characterization of GCK in the presence of high concentrations of recombinant BAD show modest (<15%) increases in GCK activity, observable only at glucose concentrations well below the K0.5 value. GCK activity is unaffected by BAD BH3 peptides. These results raise questions as to the mechanism of action of stapled peptide analogs modeled after the BAD BH3 domain, which reportedly enhance the Vmax value of GCK and stimulate insulin release in BAD-deficient islets. Based on our results, we postulate that the BAD:GCK interaction, and any resultant regulatory effect(s) upon GCK activity, requires the participation of additional members of the mitochondrial complex.
Highlights
Glucokinase (GCK) catalyzes the rate-limiting step of glycolysis in β-cells and hepatocytes, where it regulates glucose-stimulated insulin secretion (GSIS) and glycogen synthesis, respectively [1,2]
The discovery of a complex that contains both BAD and GCK, along with the observation that BAD-deficient mice display glucose homeostatic dysfunction, led to speculation that glycolytic metabolism and apoptosis might be coordinated by BAD in the pancreas and liver [7]
Hinds et al report that recombinant BAD produced in Escherichia coli is highly susceptible to proteolysis [30]
Summary
Glucokinase (GCK) catalyzes the rate-limiting step of glycolysis in β-cells and hepatocytes, where it regulates glucose-stimulated insulin secretion (GSIS) and glycogen synthesis, respectively [1,2]. Genetic deficiencies in GCK are associated with maturity onset diabetes of the young type 2 (MODY-II) and permanent neonatal diabetes mellitus [3,4]. GCK was found to co-localize with the pro-apoptotic protein BAD in a 232 kDa complex at the mitochondrial membrane of hepatocytes and pancreatic β-cells [7]. The discovery of a complex that contains both BAD and GCK, along with the observation that BAD-deficient mice display glucose homeostatic dysfunction, led to speculation that glycolytic metabolism and apoptosis might be coordinated by BAD in the pancreas and liver [7]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
