Fig. 5.

AKT2 is a kinase for CAD. (A–I) Immunoblotting. (A) β-Catenin^Δ(ex3)/+ MEFs were treated with DMSO or CCT for 6 h. β-Catenin^Δ(ex3)/+ MEFs (B) or HepG2 cells (C) were transfected with scramble or AKT2 siRNAs. (D) After S6K siRNAs transfection, Huh7 cells were treated with DMSO or CCT (5 μM) for 6 h. (E) Control or AKT2-OE Huh7 cells were transfected with S6K siRNAs. (F) Tsc2^−/− MEFs were transfected with scramble or Akt2-targeted siRNAs. (G) Coimmunoprecipitation (Co-IP) assays performed in 293 cells. (H) The 293 cells were cotransfected with vector (EV) or wild-type (WT), S1859A, or S1406A of FLAG-HA-CAD along with HIS-HA-AKT2 vector for 48 h. Anti-HIS antibody-protein A/G magnetic beads were used for Co-IP. (I) In vitro kinase assays were performed with FLAG-HA-CAD proteins and AKT2 proteins with or without 1 μM CCT. (J) Docking analysis of AKT2 and RRLSSFV (containing S1406) or HRASDPG (containing S1859) of CAD protein. RRLSSFV and HRASDPG are in yellow. AKT2 is in slate. Mn²⁺ are shown as magenta sphere. ANP is in cyan. Red dashes represent hydrogen bond interaction, blue dashes represent salt bridge, and marine dashes represent metal contacts.