Effect of oncogenic ras expression on activation of ERK in myeloma cells.

(A) Empty vector–transfected control cells (C) or mutant ras–transfected cells (mutant N-ras [N] or K-ras [K]) were depleted of IL-6 for 0 hours (day 0), 48 hours (day +2), or 72 hours (day +3), and immunoblot was performed for total ERK (ERK-T) and phosphorylated ERK (ERK-P). Mean expression of phosphorylated ERK from 3 separate experiments is shown at the bottom of the panel, with control cells set arbitrarily at 1. *Significantly different from control value, P < .05. (B) ERK in vitro kinase assay performed in which wild-type (WT) cells were compared with mutant N-ras (N) or K-ras (K) cells after 48 or 72 hours of IL-6 depletion. Immunoblot assay shown for expression of phosphorylated ELK-1 (ELK-P) used as an ERK substrate. (C) In left panels, mutant N-ras or K-ras cells were depleted of IL-6 for 48 hours and then treated with or without PD98059 for 2 hours at 10 μM. In the right panel, wild-type cells (empty vector transfected) were depleted of IL-6 for 48 hours and then treated without IL-6, with IL-6 alone (100 U/mL for 15 minutes), or with IL-6, preceeded by 2 hours of PD98059 (10 μM).