Functional characterization of B-Raf mutants identified in CFC and Cancer. Kinase activities of B-Raf missense CFC mutants (black bars) are compared with those of B-Raf mutations found in cancer (gray bars). Empty vector, wild type B-Raf (WTB-Raf), or the indicated B-Raf point mutants were transfected in 293T cells and B-Raf activity was measured on Flag- immunoprecipitates.
CFC- Cardio-facio-cutaneous.[Germline Mutations in Genes Within the MAPK Pathway Cause Cardio-facio-cutaneous Syndrome]

BRAF and ERK activation. a, BRAF kinase activity. Myc-epitope-tagged versions of BRAF and the various mutants were expressed in COS cells alone as indicated. The activity of the BRAF proteins in cell extracts was examined using the Raf kinase cascade assay. Each sample was assayed in triplicate and error bars are used to indicate the standard deviations from the mean. Absence of error bars indicates less than 3% error. Similar results were obtained in two independent transfections.
[Mutations of the BRAF gene in human cancer]

All four mutations had elevated basal kinase activity compared with wild-type BRAF (WTBRAF); however, the basal activity of G468ABRAF and V600EBRAF was substantially higher (about 12.5- and 10.7-fold that of WTBRAF, respectively), whereas the activation of G464VBRAF and L597VBRAF was more modest (about 2- and 5.7-fold, respectively). Consistent with their in vitro activities, G469ABRAF and V600EBRAF stimulated phosphorylation of endogenous ERK1/2 more strongly than G464VBRAF or L597VBRAF. The data demonstrate that these mutants are active in vitro and stimulate the activity of the pathway in vivo to different degrees.