Fig. 3. Ser259 regulates coupling to Ras. (A) Myc-tagged wild-type Raf-1 (WT), RafS259A, and the double mutant RafR89L/S259A were expressed in COS-1 cells either alone or with RasV12 as indicated. Raf-1 immunoprecipitates were examined for kinase activity. (B) The mutation of Ser259 enhances the association with Ras. COS-1 cells were transfected with the indicated expression plasmids. Ras immunoprecipitates were immunoblotted for Ras and associated Raf proteins (upper panel). Crude lysates were immunoblotted to show that all Raf proteins were expressed at comparable levels (lower panel). (C) Cell fractionation. COS cells were transfected with the indicated expression plasmids, serum starved ‘–’ and treated with EGF (20 ng/ml) plus TPA (100 ng/ml), ‘E&T’, for 30 min. Cells were lysed in hypotonic, detergent-free buffer and fractionated into 100 000 g supernatant ‘S’ and pellet ‘P’ containing membranes. The fractions were extracted with 1% Triton X-100, and Raf-1 immunoprecipitates were examined for kinase activity, Ser259 phosphorylation and Raf-1 distribution. Kinase activity was quantified by a phosphoimager and is given below the lanes in arbitrary units. (D) COS cells were transfected with myc-Raf-1 along with RasV12 or vector. The cells were fractionated as described above and equal amounts of whole-cell lysate from each fraction were blotted for Ser259, Raf-1 and Ras levels using the respective antibodies.