Fig 2: A schematic model of Aldob-mediated negative regulation of Akt activation.In normal cells, Aldob interacts with Akt in an Akt kinase activity-dependent manner, which promotes phosphatase PP2A binding to p-Akt and results in p-Akt dephosphorylation. Aldob is involved in the intracellular homeostasis of Akt phosphorylation through modulating substrate/phosphatase interactions. In Aldob-deficient cancer cells, down-regulation of Aldob releases its negative regulation of Akt activity, leading to constitutive activation of oncogenic Akt signaling, which facilitates cell cycle progression and increases glucose consumption and metabolic flux to glycolysis and TCA cycle for cancer cell proliferation and tumorigenesis. Targeting hyperactive Akt through directly inhibiting Akt activity or reactivating PP2A phosphatase activity may be a viable therapeutic approach to treat Aldob-deficient human cancers. Bold arrows represent up-regulation of indicated signaling events in response to the absence of Aldob. Aldob, aldolase B; DHAP, dihydroxyacetone phosphate; GSK3ß, glycogen synthase kinase 3ß; HK, hexokinase; mTORC2, mechanistic target of rapamycin complex 2; p-Akt, phosphorylated Akt; PDK1, phosphoinositide-dependent kinase 1; PP2A, protein phosphatase 2A; SMAP, small-molecule activator of PP2A; TCA cycle, tricarboxylic acid cycle.

Fig 3: Aldob regulates Akt activity through protein phosphatase PP2A.(A and B) IB analysis of anti-Flag IP and WCL derived from Huh7 cells transfected with the indicated constructs. (C) GST pull-down assays were performed with WCL derived from Flag-PP2A-C-transfected Huh7 cells and recombinant proteins GST-Akt1 and WT-Aldob or Aldob-R304A mutant. GST was used as the negative control. (D) In vitro PP2A dephosphorylation assays were performed with phosphorylated Myc-Akt1 and active PP2A in the presence or absence of recombinant WT-Aldob or Aldob-R304A mutant (top). The relative signal intensities of pT308-Akt and pS473-Akt were quantified (bottom). (E) IB analysis of WCL derived from Huh7 cells transfected with the indicated constructs. Resulting cells were treated for 4 hours with OA at 100 nM. (F) IB analysis of endogenous anti-Aldob and anti-Akt1 IP and WCL derived from 2 pairs of human HCC samples. IgG was used as the negative control. The data underlying this figure can be found in S1 Data. Aldob, aldolase B; IB, immunoblot; IP, immunoprecipitation; GST, glutathione S-transferase; IgG, immunoglobulin G; OA, okadaic acid; PP2A, protein phosphatase 2A; WCL, whole cell lysate; WT, wild-type.