FIG. 13.

Effects of G-kinase on the activities of mutant Raf(S43-A) and B-Raf kinase. G-kinase-expressing C11 cells were transiently transfected as described for Fig. 11; cells were serum starved and treated with 8-pCPT-cGMP and/or EGF as described for Fig. 5. Immunoprecipitates of c-Raf kinase (A) or B-Raf kinase (B) were incubated with MEK-1 and [γ-³²PO₄]ATP for 20 min as described in Materials and Methods. Autoradiographs of phosphorylated MEK-1 are shown in the upper panels; the immunoprecipitates were also analyzed by immunoblotting with c-Raf or B-Raf kinase antibody as indicated in the lower panels. (A) C 11 cells were transfected with empty vector (control vector) or with expression vectors encoding either wild-type c-Raf kinase [Raf (WT)] or Raf(S43-A). c-Raf kinase activity was determined as described above. Lane 10, MEK-1 incubated in the absence of immunoprecipitates. (B) C11 cells were transfected with control vector or with an expression vector for B-Raf kinase; B-Raf kinase activity and the amount of B-Raf in the immunoprecipitates were determined as described above. The amount of MEK-1 phosphorylation seen with immunoprecipitates from cells transfected with empty vector was little above the background MEK-1 phosphorylation observed with control immunoprecipitates obtained with nonimmune rabbit serum (lane 9).