Rk. Jaiswal et al., THE MITOGEN-ACTIVATED PROTEIN-KINASE CASCADE IS ACTIVATED BY B-RAF INRESPONSE TO NERVE GROWTH-FACTOR THROUGH INTERACTION WITH P21(RAS), Molecular and cellular biology, 14(10), 1994, pp. 6944-6953
Nerve growth factor (NGF) activates the mitogen-activated protein (MAP
) kinase cascade through a p21(ras)-dependent signal transduction path
way in PC12 cells. The linkage between p21(ras) and MEK1 was investiga
ted to identify those elements which participate in the regulation of
MEK1 activity. We have screened for MEK activators using a coupled ass
ay in which the MAP kinase cascade has been reconstituted in vitro. We
report that we have detected a single NGF-stimulated MEK-activating a
ctivity 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 exhib
it a MEK-activating activity. Gel filtration analysis revealed that th
e 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 pr
oteins. Immunoprecipitation with B-Raf-specific antisera quantitativel
y precipitated all MEK activator activity from these fractions. We als
o demonstrate that B-Raf, as well as c-Raf1, directly interacted with
activated p21(ras) immobilized on silica beads. NGF treatment of the c
ells had no effect on the ability of B-Raf or c-Raf1 to bind to activa
ted p21(ras). These data indicate that this interaction was not depend
ent upon the activation state of these enzymes; however, MEK kinase ac
tivity was found to be associated with p21(ras) following incubation w
ith NGF-treated samples at levels higher than those obtained from unst
imulated 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 t
his pathway.