Figure 1.
Protein-protein interaction between NQO2 and AKT.
(A) GRAMM-X molecular modeling analysis was used to examine the possible interaction of NQO2 (PDB: 1SG0) and AKT (PDB: 3O96). The output data from the GRAMM-X program showed the potential interaction between these two proteins. H-bonds formed between proteins are shown as blue solid lines and the distance of H-bonds between amino acid residues are presented as angstrom (Å). (B) Gel filtration (technical advice provided by Dr. Z. Zhang) was performed to examine complex formation between NQO2 and AKT. CWR22Rv1 cell lysates were applied to a Superdex 200 column and eluted fractions were analyzed by western blotting. (C) Wild-type NQO2 and AKT recombinant proteins were immunoprecipitated with anti-NQO2 or anti-AKT antibodies and probed with either anti-AKT or anti-NQO2 antibody. AKT signals were quantified by densitometry analysis and dose-dependent increase of AKT signal was detected in NQO2 immunoprecipitates. (D) Analysis of co-localization of NQO2 and AKT in PC-3 cells. Cells were incubated with anti-NQO2 and anti-AKT antibodies, and counter-stained with DAPI for identification of location of cell nucleus. The presence of NQO2 and AKT was identified and visualized by FITC-conjugated or rhodamine-conjugated secondary antibodies, respectively, as described in Materials and Methods. The images were examined by immunofluorescence microscopy. The co-localization of NQO2 (green) with AKT (red) appeared as a yellow color in the merged images.
Figure 2.
Effects of NQO2 on resveratrol mediated AKT control.
(A) Mapping of the sites of NQO2 and AKT interaction using full-length and truncated deletion constructs of NQO2 and AKT, respectively. (B) Determination of the interactions between NQO2 and AKT by pull-down assays. His-AKT, His-AKT-dPH or His-AKT-PH (up to 2.5 µg) was used to pull down purified GST-NQO2 or GST-N161H NQO2. The bound proteins were subjected to 10% SDS-PAGE and then immunoblotted using monoclonal antibody against AKT, NQO2 or His. Conditions for C (control) and R (resveratrol) consisted of GST-tagged-NQO2 pre-incubated with or without 1 mM resveratrol added prior to performance of the pull down assays.
Table 1.
Determination of the binding of AKT activators, Ins(1,3,4,5)P4 (IP4), PtdIns(3,4,5)P3 (PIP3) or phosphatidylserine (PS) to AKT using small molecule docking.
Figure 3.
Binding of resveratrol to AKT.
(A) Docking of resveratrol to AKT (top panel) using DOCK 6.3 program. The grid score (GS) represented the sum of the van der Waals (VDW) and electrostatic scores (ES); compounds showing low GS are likely to have a high affinity towards the receptor (targeting protein). The affinity of resveratrol and ATP to AKT was compared and shown in lower panel. (B) Binding of specific proteins from K562 cell extracts to immobilized resveratrol affinity columns and isolation of resveratrol target proteins. Cell lysates fractionated on control and resveratrol affinity columns were eluted with NaCl, ATP and resveratrol. Silver stained protein profiles of the ATP and resveratrol eluted fractions without (left side of the gel) and with (right side of the gel) competition by resveratrol were shown. (C) Western blot analysis examined the elution profiles and demonstrated the presence of AKT and NQO2 in resveratrol eluted fractions in column separation performed without prior competition of cell lysates with resveratrol. (D) Effect of resveratrol on interaction and complex formation between NQO2 and AKT was evaluated by PatchDock and scored by FireDock. (E) Changes on AKT protein expression by resveratrol were examined in control shRNA08 and NQO2 knockdown shRNA25 cells. Cells were treated with increasing doses of resveratrol (0, 2.5, 10 and 25 µM) for 72 h and changes on AKT were analyzed by western blot analysis. The blots were also probed for actin which served as loading control.
Figure 4.
Analysis of stability of AKT protein and mRNA in relation to knockdown status of NQO2.
(A) Turnover/degradation of AKT in shRNA08 or shRNA25 cells. Cells were treated with 30 µg/ml CHX to inhibit new protein synthesis for the times indicated. Changes in AKT was analyzed by western blot analysis, with Hsp70 expression used as loading control. AKT expression was quantified by densitometry analysis and the expression was presented as fold relative to time zero. (B) Effect of knockdown NQO2 on AKT mRNA stability. Both shRNA08 and shRNA25 cells were treated with transcription inhibitor actinomycin D (5 µg/ml). Cells were harvested at 0, 1, 2 and 4 h after treatment. Total cellular RNA was isolated and residual mRNAs were determined by RT-PCR, using GAPDH mRNA expression as internal control. AKT expression was quantified by densitometric analysis and the expression was presented as fold relative to time zero. (C) Effect of resveratrol on AKT mRNA. Cells (shRNA08 and shRNA25 cells) were treated with increasing doses of resveratrol (0, 2.5 and 10 µM) for 72 h and changes on AKT mRNA levels were assayed by RT-PCR, quantified by densitometry, and expressed as fold differences by normalization against GAPDH.
Figure 5.
A proposed mechanism on the role of NQO2 in regulating AKT and AKT-mediated signaling events.
(A) Hypotheses: NQO2 acts as a physiologic partner and modulator of AKT by disruption of its activation. NQO2/AKT interaction may (i) disrupt or limit the access of PIP3 for membrane translocation by AKT or (ii) inhibit the catalytic activity of AKT. (B) NQO2 regulates GSK-3β-mediated cyclin D1 degradation by binding AKT and/or inhibiting its phosphorylation-dependent activation. This sequence of events is effectively attenuated in NQO2 knockdown cells or when cells are exposed to resveratrol, which acts by effectively binding and sequestrating NQO2, rendering it incapable of forming a complex with AKT.