The mitogen-activated protein kinase cascade is activated by B-Raf in response to nerve growth factor through interaction with p21ras.

Abstract

Nerve growth factor (NGF) activates the mitogen-activated protein (MAP) kinase cascade through a p21r'-dependent signal transduction pathway in PC12 cells. The linkage between p2l'as and MEKI 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-Rafl; however, the MEK-activating activity was found only in fractions containing B-Raf. c-Rafl-containing fractions did not exhibit a MEK-activating activity. Gel filtration analysis revealed that the B-Raf eluted with an apparent Mr 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-Rafl, directly interacted with activated p2l' immobilized on silica beads. NGF treatment of the cells had no effect on the ability of B-Raf or c-Rafl to bind to activated p2l'. 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 p2l' 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-Rafl activity was not found to function within this pathway.Nerve growth factor (NGF) is responsible for the survival and differentiation of distinct subsets of neurons both within the central nervous system and in the periphery. The clonal rat pheochromocytoma cells, PC12, have proven to be a valuable model in which to investigate NGF action (15). These cells respond to NGF by cessation of division and then differentiate into a sympathetic-like neuronal phenotype. NGF initiates its actions through interaction with the proto-oncogene trk (trkA) (5, 25). TrkA possesses an intrinsic protein tyrosine kinase activity that is activated upon NGF binding, resulting in receptor autophosphorylation and phosphorylation of other proteins which participate in the signal transduction process. The detailed mechanisms through which NGF elicits its specific biochemical effects have been elusive; however, it is clear that the transmission of signals from the cell surface involves the activation of the G protein p2lras. This is followed by the serial activation of other protein kinases, driving a cascade of protein phosphorylation that mediates the specific biochemical events characteristic of NGF action through the phosphorylation of cytosolic proteins and nuclear transcription factors (7,10,18,66 (8,14,33,38

Differential abilities of the Raf family of protein kinases to abrogate cytokine dependency and prevent apoptosis in murine hematopoietic cells by a MEK1-dependent mechanism

“…The levels of the ⌬Raf:ER protein often increases after ␤-estradiol treatment, this has been shown to be due to protein stabilization induced by ␤-estradiol. [39][40][41] Thus there was a dose-dependent activation of Raf and MEK activities in these cells which correlated with …”

Ha-ras and N-ras regulate MAPK activity by distinct mechanisms in vivo

“…Ras e ector molecules include the Raf kinases (Moodie et al, 1993Jaiswal et al, 1994;Van Aelst et al, 1993;Vojtek et al, 1993), MEKK (Russell et al, 1995), RalGDS family members (Hofer et al, 1994;Kikuchi et al, 1994;Spaargaren and Bischo , 1994;Wolthius et al, 1996), PI 3-OH kinase (PI3K) (Rodriguez-Viciana et al, 1994), Neuro®bromin (DiBattiste et al, 1993) and AF6 (Kuriyama et al, 1996). The mechanisms which determine and regulate the formation of speci®c Rase ector signalling complexes are not known.…”

Rheb is in a high activation state and inhibits B-Raf kinase in mammalian cells

“…Because B-Raf kinase is an important activator of Erk-1 and Erk-2 (Vossler et al, 1997;Jaiswal et al, 1994) and the transcription factor Elk-1 is a physiological target of these two MAP kinases (Johnson et al, 1996), we used a chimeric Elk-Gal4 construct to measure B-Raf kinase activation of a Gal-4-dependent reporter gene (Qiu et al, 2000). We performed these studies in two of the neuronal cell lines which have endogenous Rheb, i.e., NB2A and PC-12 cells, and in two fibroblastic cell lines, one with and one without endogenous Rheb, i.e., BHK and CHOK-1 cells.…”

“…One way, at least in part, to reconcile these seemingly disparate results would be if Rheb inhibits B-Raf kinase. Compared with Raf-1, BRaf has a higher basal kinase activity and interacts with their shared targets MEK-1 and MEK-2 with greater affinity; as a consequence, B-Raf appears to be the major Ras effector and MEK activator in several cell types including some strains of NIH3T3 cells Eychene et al, 1995;Papin et al, 1995Papin et al, , 1996Papin et al, , 1998Catling et al, 1994;Jaiswal et al, 1994;Pritchard et al, 1995;Reuter et al, 1995;Vossler et al, 1997). MEK is the immediate upstream activator of the mitogen-activated protein (MAP) kinases, ERK-1 and ERK-2, which are required for many of the growth-promoting effects of Ras (Campbell et al, 1998).…”