Inhibition of p70 S6 kinase (A) and suppression of cell proliferation (B) by Rap in Kit225 cells entering into the cell cycling in response to IL-2. Kit225 cells deprived of IL-2 for 48 hours were cultured with or without Rap in the presence of 2 nmol/L IL-2. (A) A total of 10 nmol/L Rap was added 0 (▪), 6 (•), 18 (▵), or 36 hours (○) after the initiation of the culture with IL-2. As a control, Kit225 cells were cultured with IL-2 only (□). The cell lysates prepared from 4 × 10⁶ cells harvested at 0, 1, 6, 7, 18, 19, 36, and 37 hours of the culture were subjected to immunoprecipitation with anti-p70 S6 kinase antibody. The kinase activity of the immunoprecipitates was assayed with S6 peptide as the substrate and was shown as [γ-³²P]ATP incorporation into S6 peptide. Each point represents the average of duplicate samples. (B) IL-2–depleted Kit225 cells were cultured with 10 nmol/L Rap (columns 2 to 4) or without Rap (column 1) in the presence of 2 nmol/L IL-2 for 48 hours. Column 2, Rap was added concomitantly with IL-2; column 3, at 6 hours; column 4, at 18 hours after the initiation of the culture. The exponentially growing Kit225 cells were cultured in the absence of Rap (column 5) or the presence of 10 nmol/L Rap (column 6) for 48 hours. The incorporation of [³H]thymidine by cultured cells for the last 6 hours was measured. Each column represents the means ± standard deviation (SD) of the triplicate cultures.