TEL/PDGFβR stimulates phosphorylation of Akt and p70S6 kinase through a LY294002-responsive pathway.

(A) Ba/F3 (lanes 1-3) were harvested in log phase growth (lane 1) or deprived of IL-3 for 4 hours and then restimulated with media containing no IL-3 (lane 2) or 50 ng/mL IL-3 for 15 minutes. Ba/F3/TEL/PDGFβR cells were either harvested in log phase growth (lane 4) or treated with the indicated compound for 16 hours. Cells were lysed in 1% Triton lysis buffer, proteins were separated by SDS-PAGE and blotted for phospho-Akt. Blot was stripped and reprobed for expression of total Akt. Similar inhibition of Akt was seen in Ba/F3 TEL/PDGFβR cells treated with LY294002 (25 μM; lane 6) or STI571 (1 μM; lane 7). (B) In separate experiments, phosphorylation of p70S6 kinase was examined. Ba/F3 cells were either analyzed during log phase growth with 0.5 ng/mL IL-3 (lane 1) or deprived of IL-3 for 4 hours and then restimulated with media containing 50 ng/mL IL-3 for 15 minutes (lane 2) or no IL-3 (lane 3). Ba/F3-TEL/PDGFβR cells were harvested in log phase growth (lane 4) in the absence of IL-3. (C) Ba/F3 and Ba/F3-TEL/PDGFβR cells were harvested in log phase growth or treated with indicated inhibitors for 4 hours. D indicates DMSO; LY, LY294002 25 μM; STI, STI571 1 μM; R, rapamycin; and U, U0126 20 μM. Cells were lysed in 1% Triton, proteins were separated by SDS-PAGE and blotted by using the indicated antibodies. Phospho-p70S6 kinase antibody cross-reacts with a splice variant of p70, p85 S6 kinase, which is not known to be regulated by cytokines.