Fig. 1. Activation of B-Raf by oncogenic Ras requires both Thr598 and Ser601 phosphorylation. (A) Sequence alignment of Raf kinase activation loop (residues 593–622 for human B-Raf). Residues subjected to site-directed mutagenesis are indicated by arrows. An asterisk denotes the protein kinase C phosphorylation sites. (B) Oncogenic Ras-induced kinase activation of B-Raf and its mutants. Fifty nanograms of HA-tagged pcDNA3 (vector control, lane 1), HA-tagged B-Raf (lanes 2--4), B-RafT598A (lane 5 and 6), B-RafS601A (lanes 7 and 8), B-RafS613A (lanes 9 and 10), B-RafAA (lanes 11 and 12) or B-RafED (lanes 13 and 14) were transiently transfected in COS cells alone (open bars) or with 100 ng of oncogenic Ras (HRasV12, solid bars), as indicated at the top of the panel. The kinase was immunoprecipitated and its activity, reflected by phosphorylation of GST–Elk1 (pElk1), was measured using the coupled assay (see Materials and methods). The upper panel shows autoradiographs of pElk1 that are representative of five independent experiments. The intensity of pElk1 bands was quantified by PhosphorImager (lower panel); results were subtracted from the reading of lane 1 (background) and expressed as fold increase with respect to the cells transfected with wild-type B-Raf without stimulation (lane 2). Lane 4* denotes that GST–MEK was omitted in the assay as a control. Results are mean ± SD from three independent experiments. Western blot of the immunoprecipitated kinase is shown in the middle panel, indicating equivalent protein loadings. (C) Activation of B-RafED by oncogenic Ras. COS cells were transfected with vector (lane 1), 50 ng of wild-type B-Raf (lanes 2 and 3), 5 ng of B-RafED (lanes 4 and 5), 25 ng of B-RafED (lanes 6 and 7) or 50 ng of B-RafED (lanes 8 and 9) in the presence or absence of HRasV12, as indicated at the top of the panel. Results of kinase assay and western blotting of B-Raf using α-HA were representative of two separate experiments. (D) Carbachol-induced kinase activation of B-Raf and its mutants. Cells were co-transfected with the above DNA constructs and hM3. After 24 h, cells were starved in FBS-free medium for 5 h followed by stimulation with carbachol for 5 min. Kinase immunoprecipitation, kinase assay and quantitation of results are as described in (B).