FIG. 3.

PMA treatment of cells prevents Ral-GDS activation by Ras. (A) PMA does not block Ras activation by EGF. COS-7 cells were pretreated with either buffer or PMA and then stimulated with EGF for the indicated time. The amount of active Ras-GTP in cell lysates was determined by affinity purification with the Ras binding domain of Raf followed by immunoblotting with anti-Ras antibodies. The data are representative of at least two independent experiments. (B) PMA blocks Ras activation of Ral-GDS in vivo. GST-Ral was transfected alone or together with Ral-GDS or with Ral-GDS plus constitutively activated Ras12V. After being incubated with ³²PO₄ for 4 h to label nucleotide pools, cells were incubated with buffer or PMA for 30 min. GST-Ral was then isolated from cell extracts with glutathione beads, and the ratio of GTP/(GTP plus GDP) bound to Ral was determined by TLC. The data represent the average of three experiments ± standard deviations. The levels of transfected Ral-GDS and Ras are shown at the bottom.