Fig. 1
K-Ras(V12) interacts with PI3-Kα and activates Akt. A, B Interaction of K-Ras(V12) with PI3-K. A PANC-1 cells, transiently expressing TAP-EGFP, TAP-EGFP-K-Ras(V12), or TAP-EGFP-K-Ras(N17), and non-transfected control cells were lysed 24 h after transfection. TAP-containing proteins were precipitated from 5 mg of cell lysate (1 mg for TAP-EGFP) by binding to IgG sepharose beads and eluted by cleavage of the TAP tag with TEV protease. Lysates and eluates were analyzed by SDS PAGE and Western blotting using anti-p85α PI3-K or anti-GFP antibody and ECL. The left panel [lysate] shows the expression of the TAP-EGFP proteins and endogenous PI3-K p85α subunit in 8 μg (first lane) or 40 μg of lysate. The right panel [eluate] shows the precipitated, TEV-cleaved (cl.) TAP-proteins and demonstrates that PI3-K co-precipitated only with TAP-EGFP-K-Ras(V12). One representative assay (n ≥ 3) is shown. B Purified, post-translationally modified HA-Ras proteins [HA-H-Ras(V12), HA-K-Ras(V12), HA-N-Ras(V12), 500 ng each], were immobilized on anti-HA μMacs beads and incubated with PANC-1 cell lysate [lanes 1–3]. As controls, anti-HA μMacs beads incubated with PANC-1 lysate [PANC-1 lysate] or purified HA-K-Ras(V12) [HA-K-Ras(V12)], and to determine endogenous PI3-K p85α an aliquot of PANC-1 lysate (50 µg) [Control] were used. The Western blot in the upper panel demonstrates the detection of the co-precipitated PI3-K whereas the lower panel shows a silver-stained gel with one third of the precipitated HA-Ras and IgG (light chain) proteins (n = 3). C-F Activation of Akt by EGFP-K-Ras(V12). C PANC-1 cells and PANC-1 cell clones stably expressing EGFP, EGFP-K-Ras(V12) (clone 4.1 and 4.4), EGFP-N-Ras(V12), EGFP-H-Ras(G12), or EGFP-K-Ras(N17) were analyzed for expression of EGFP, EGFP-Ras proteins, and Akt by Western blotting. D The amount of pAktSer473 (upper blot) and total Akt (lower blot) was detected by Western blotting in serum starved EGFP and EGFP-Ras cell clones. pAktSer473 is increased in K-Ras(V12)-expressing PANC-1 cells (n > 3). E Serum-starved PANC-1 cell clones were incubated with 10 μM LY294002 for 20 min, 30 ng/ml EGF for 10 min, or with EGF after preincubation with LY294002. Cell lysates were subjected to Western blotting for detection of pAktSer473 and Akt (n > 3). Inhibition of PI3-K reduced basal as well as EGF-induced Akt phosphorylation. F Lysates of HEK293 and Colo-699 cells stably expressing EGFP or EGFP-K-Ras(V12) were subjected to Western blotting. The amount of pAktSer473, Akt, EGFP-Ras, and Ras was detected by using appropriate antibodies demonstrating that pAkt is increased in K-Ras(V12)-expressing cells