FIGURE 2.

FBP-induced oligomerization of PKM2 activates PKAR FRET. A, coimmunoprecipitation (IP) of PKAR2.3 with myc-PKAR2.3 immobilized on myc antibody-conjugated agarose beads in the absence or presence of 200 μm FBP or 10 mm serine. Binding (middle panel) is quantified as Citrine fluorescence excited at 500 nm, which reports on PKAR concentration independently of FRET. FRET efficiencies (right panel) were calculated as in Fig. 1. n ≥ 50 beads per treatment from 2 independent experiments. B, average PKAR FRET response to 11 mm glucose (Glc), glyceraldehyde (GA), pyruvate (Pyr), or the mitochondrial fuel ketoisocaproate (KIC) in Min6 cells. C, PKAR FRET efficiency in Min6 cells expressing PKAR2.1 (M2) or mutants in which PKM2 was replaced by the tetrameric PKM1 isoform (M1) or the dimeric PKM2 mutant R399E, shown ±25 mm Glc as indicated. n ≥ 50 cells per treatment from ≥3 independent experiments. Error bars in all panels represent the mean ± S.E., analysis of variance with Bonferroni post-test: ***, p < 0.0001.