Fig. 3: HK2 directly binds GSK3β and R1a in vitro to form complexes that are disrupted by G6P.

a Nickel-coated 96-well plates were incubated overnight at 4 °C with His-PRKAR1a (50 nM) and then incubated with Myc-HK2 (0.25–64 nM) in blocking buffer or with blocking buffer without HK2. HK2 binding was detected and quantified with anti-Myc-HRP-conjugated antibody, and an HRP-catalyzed reaction with a chromogenic substrate solution. Left panel shows generated binding curves. Curves were fitted based on a one-site-binding model in GraphPad Prism followed by a comparison of fits with p < 0.0001. Right panel: binding curve after increasing concentrations of G6P. Binding was conducted as above except that Myc-HK2 (32 nM) was incubated for 2 h. Thirty minutes before the end of Myc-HK2 incubation, increasing concentrations of G6P were added to the wells and detection was carried out as above. Results are the mean ± SEM of three independent experiments. p < 0.0001 one-way ANOVA. b Glutathione-coated 96-well plates were incubated overnight at 4 °C with GST-GSK3β (50 nM) and then incubated with or without Myc-HK2 (0.25–64 nM). Binding was quantified in a. Left panel: binding curves were generated as in a (comparison of fits with p < 0.0001). Right panel: binding curve after increasing concentrations of G6P as described in a. Results are the mean ± SEM of three independent experiments. p < 0.0001 one-way ANOVA. c Nickel-coated 96-well plates were incubated overnight at 4 °C with His-PRKAR1a (50 nM) or with blocking buffer in the absence of PRKAR1a, and then incubated with Myc-HK2 (32 nM) in blocking buffer or with blocking buffer without HK2 for 2 h. GST-GSK3β or blocking buffer without GSK3β was then added for 2 h. GSK3β binding was detected with anti-GST-HRP-conjugated antibody, and an HRP-catalyzed reaction with a chromogenic substrate solution. Left panel: binding curves were generated as in a (comparison of fits with p < 0.0001). Right panel: binding curve after increasing concentrations of G6P as described in a. Nickel-coated plates were successively incubated with His-PRKAR1a (50 nM) overnight, HK2-Myc (32 nM) for 2 h and then GST-GSK3β (32 nM) for 2 h. Right panel: results are the mean  ±  SEM of four independent experiments. panel one-way anova p < 0.0001.