Effect of oncogenic ras expression on activation of PI3kinase/AKT in myeloma cells.

(A) Wild-type control (C), mutant N-ras (N), or K-ras (K) cells were depleted of IL-6 as described in Figure 2A, and immunoblot was performed for total AKT (AKT-T) and phosphorylated AKT (AKT-P). Mean expression of phosphorylated AKT from 3 separate experiments is shown at the bottom of the panel, with control (C) cells set arbitrarily at 1. *Significantly different from control value,P < .05. (B) AKT in vitro kinase assay performed in separate experiments in which wild-type (WT) cells were compared with either mutant N-ras (N) or K-ras (K) cells after 48 hours of IL-6 depletion. Wild-type cells were also depleted of IL-6 (48 hours) and then treated with or without IL-6 (100 U/mL) for 15 minutes. Immunoblot assay shown for expression of total AKT in immunoprecipitates (AKT) and phosphorylated GSK-3 (P-GSK-3) used as AKT substrate. (C) Three cell lines depleted of IL-6 as in panel A, and immunoblot performed for total forkhead transcription factor (FKH-T) and phosphorylated FKH (FKH-P). Relative mean expression levels of phosphorylated FKH from 4 separate experiments shown at the bottom of the panel. *Significantly different from control (C) value,P < .05. (D) Upper panels: wild-type, mutant N-ras, and K-ras cells were depleted of IL-6 for 48 hours and then treated with or without wortmannin (wort) (0.1 μM) for 2 hours and with or without IL-6 (100 U/mL) for 15 minutes. Immunoblot was then performed for total AKT and phosphorylated AKT. In the lower panel, the same cells (wild-type [WT], mutant N-ras [N], or K-ras [K]) were either continuously cultured in IL-6 (+IL-6) or depleted of IL-6 (no IL-6) for 48 hours. They were then harvested, and immunoblot was performed for tyrosine-phosphorylated STAT-3 (expression of total STAT-3 was comparable in all 6 lanes; not shown).