B-Raf inhibits programmed cell death downstream of cytochrome c release from mitochondria by activating the MEK Erk pathway

Growth factor-dependent kinases, such as phosphatidylinositol 3-kinase (PI 3-kinase) sind Raf kinases; have been implicated in the suppression of apop tosis. We have recently established Rat-1 fibroblast cell lines overexpress ing B-Raf, leading to activation of the MEK/Erk mitogen-activated protein k inase pathway. Overexpression of B-Raf confers resistance to apoptosis indu ced by growth factor withdrawal or PI 3-kinaae inhibition. This is accompan ied by constitutive activation of Erk without effects on the PI 3 kinase/Ak t pathway. The activity of MEK is essential for cell survival mediated by B -Raf overexpression, since either treatment with the specific MEK inhibitor PD98059 or expression of a dominant inhibitory MEK mutant blocks the antia poptotic activity of B-Raf. Activation of MEK is not only necessary but als o sufficient for cell survival because overexpression of constitutively act ivated MEK, Ras, or Raf-1, like B-Raf, prevents apoptosi's after growth fac tor deprivation. Overexpression of B-Raf did not interfere with the release of cytochrome c from mitochondria after growth factor deprivation. However , the addition of cytochrome c to cytosols: of cells overexpressing B-Raf f ailed to induce caspase activation. It thus appears that the B-Raf/MEK/Erk pathway confers protection against apoptosis at the level of cytosolic casp ase activation, downstream of the release of cytochrome c from mitochondria .