The Mitogen-Activated Protein Kinase Cascade Is Activated by B-Raf in Response to Nerve Growth Factor through Interaction with p21^ras

Abstract

Nerve growth factor (NGF) activates the mitogen-activated protein (MAP) kinase cascade through a p21^ras-dependent signal transduction pathway in PC12 cells. The linkage between p21^ras and MEK1 was investigated to identify those elements which participate in the regulation of MEK1 activity. We have screened for MEK activators using a coupled assay in which the MAP kinase cascade has been reconstituted in vitro. We report that we have detected a single NGF-stimulated MEK-activating activity which has been identified as B-Raf. PC12 cells express both B-Raf and c-Raf1; however, the MEK-activating activity was found only in fractions containing B-Raf. c-Raf1-containing fractions did not exhibit a MEK-activating activity. Gel filtration analysis revealed that the B-Raf eluted with an apparent M_r of 250,000 to 300,000, indicating that it is present within a stable complex with other unidentified proteins. Immunoprecipitation with B-Raf-specific antisera quantitatively precipitated all MEK activator activity from these fractions. We also demonstrate that B-Raf, as well as c-Raf1, directly interacted with activated p21^ras immobilized on silica beads. NGF treatment of the cells had no effect on the ability of B-Raf or c-Raf1 to bind to activated p21^ras. These data indicate that this interaction was not dependent upon the activation state of these enzymes; however, MEK kinase activity was found to be associated with p21^ras following incubation with NGF-treated samples at levels higher than those obtained from unstimulated cells. These data provide direct evidence that NGF-stimulated B-Raf is responsible for the activation of the MAP kinase cascade in PC12 cells, whereas c-Raf1 activity was not found to function within this pathway.