PKC-dependent RasGRP3 phosphorylation on Thr133 occurs in B cells after stimulation and is correlated with Ras-Erk signaling. (A) Ramos B cells were left untreated or stimulated with PMA for 10 minutes or anti-IgM for 5 minutes, and the levels of Thr133-phosphorylated RasGRP3 (pRasGRP3), immunoglobulin G (IgG), RasGRP3, phosphorylated Erk, Ras-GTP, and total Ras (tRas) were determined. (B) Ramos B cells were treated as in panel A. Phosphorylated RasGRP3 was immune-precipitated with anti-3/pThr133 antibodies and precipitated RasGRP3 was then detected by immunoblotting. Levels of phospho-Erk and total Ras in lysates were also determined. (C) Ramos B cells were pretreated with inhibitors followed by stimulation with anti-IgM antibodies, as indicated, and lysates were probed for Thr133-phosphorylated RasGRP3, total RasGRP3, and phosphorylated Erk. PMA-treated cells served as a positive control. Ro indicates Ro318220; Go, Go6976. (D) Ramos B cells were pretreated as in panel C, and then stimulated with PMA for 10 minutes followed by analysis of RasGRP3 (top) and other signaling molecules (bottom). Numbers are quantification of band intensity. (E) Mouse B cells were stimulated with anti-IgM antibodies for 5 minutes or PMA for 10 minutes. RasGRP3 phosphorylation and RasGRP3 recovery (top) were determined as in panel A, as were the levels of phospho-Erk, Ras-GTP, and total Ras in lysates (bottom). Results are representative of at least 2 independent experiments.