RasGRP3 Thr133Ala expressed in rat2 cells is defective at PMA-induced Ras-Erk signaling and PMA-induced growth. (A) Rat2 cells expressing empty vector (Puro), RasGRP3, or RasGRP3Thr133Ala (T133A) were either left unstimulated or stimulated with PMA for 10 minutes. Lysates were assayed for Thr133-phosphorylated RasGRP3, RasGRP3, phospho-Erk, Ras-GTP, and total Ras by immunoblotting. Numbers are quantification of band intensity. (B) Rat2 cells similar to those in panel A were assayed for Rap-GTP and total Rap (tRap). Numbers are quantification of band intensity. (C, left) Rat2 cells similar to those in panel A were plated in complete medium. One set of plates was harvested later the same day (□), while 2 sets were incubated for 3 days in medium containing 0.5% FBS alone (▦) or supplemented with 100 nM PMA (▪) before performing cell counts. Values represent the means ± standard deviations of triplicates within a single experiment (P < .01). (Right) RasGRP3 expression levels in these cells were estimated using anti-RasGRP3 antibodies. Results are representative of at least 2 independent experiments.