FIG. 3.

PI3K and Akt activate NF-κB by upregulating the transactivation potential of the p65 subunit. (A) Activated H-Ras(V12) and MEKK stimulate cellular pathways which result in nuclear translocation and increased DNA binding of NF-κB. Human 293T cells were transiently transfected with the vector control (VC) or activated forms of MEKK, H-Ras, Akt, or PI3K (PI3K*) (3 μg each). Nuclear extracts were prepared and EMSAs were performed to assess the presence of NF-κB DNA binding activity. Nuclear extracts isolated from TNF-stimulated 293T cells (15 ng/ml for 15 min) served as a positive control for NF-κB DNA binding activity. (Bottom) Nuclear extracts analyzed for NF-κB DNA binding activity were reanalyzed using a ³²P-labeled Oct-1-specific double-stranded probe to confirm the quality of the nuclear proteins. Note that the Oct-1-specific DNA-protein complex but not the free probe is shown. No Add, no addition. (B) IKKβ or H-Ras(V12) expression stimulates IκBα degradation. NIH 3T3 cells were transfected with plasmids bearing the genes encoding the Flag-tagged IκBα and His-tagged LacZ (2 μg each). Additionally, cells were transfected with the vector control (VC) or with plasmids bearing genes encoding wild-type IKKβ or activated forms of H-Ras, MEKK, PI3K, or Akt (1 μg each). Forty-eight hours posttransfection, cells were lysed and proteins (80 μg per lane) were resolved by PAGE. Transfected IκBα protein was detected using a Flag-specific antibody (M2; Sigma). Protein loading and transfection efficiencies were controlled by performing immunoblot analysis for His-tagged LacZ expression within each experimental group (data not shown). (C) Activated PI3K and Akt stimulate the transactivation domain of NF-κB. NIH 3T3 cells were transiently transfected with the Gal4-luciferase reporter (100 ng) and with plasmids bearing the genes encoding either Gal4-p65 or Gal4–Elk-1 (100 ng each). Cells were also transfected with the empty vector control (VC) or activated H-Ras(V12), PI3K, or Akt. Luciferase levels were measured and expressed as multiples of the level of activation of the empty vector control. Data presented are the means ± standard deviations of results of three independent experiments.