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The mitogen-activated protein kinase cascade is activated by B-Raf in response to nerve growth factor through
R K Jaiswal1, S A Moodie, A Wolfman
1Department of Neurology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
Abstract:
Nerve growth factor (NGF) activates the mitogen-activated protein (MAP) kinase cascade through a p21ras-dependent signal transduction pathway in PC12 cells. The linkage between p21ras 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 p21ras 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 p21ras. 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 p21ras 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.
Insights
Nerve growth factor (NGF) activates the MAP kinase cascade via B-Raf, not c-Raf1, in PC12 cells. This study identifies B-Raf as the key enzyme in this NGF-stimulated signaling pathway.
Area of Science:
- Cellular signaling pathways
- Molecular biology
- Signal transduction
Background:
- Nerve growth factor (NGF) triggers the mitogen-activated protein (MAP) kinase cascade through a p21ras-dependent pathway in PC12 cells.
- Understanding the specific components regulating MEK1 activity is crucial for elucidating this signaling cascade.
Purpose of the Study:
- To identify the specific MEK-activating activity stimulated by NGF in PC12 cells.
- To investigate the roles of B-Raf and c-Raf1 in the p21ras-dependent MAP kinase activation pathway.
Main Methods:
- Reconstitution of the MAP kinase cascade in vitro using a coupled assay.
- Screening for MEK activators.
- Gel filtration analysis to determine protein complex size.
- Immunoprecipitation using B-Raf-specific antisera.
- In vitro binding assays with activated p21ras.
Main Results:
- A single NGF-stimulated MEK-activating activity was identified as B-Raf.
- MEK-activating activity was exclusively found in B-Raf-containing fractions, not c-Raf1.
- B-Raf exists in a stable complex with an apparent molecular weight of 250,000–300,000.
- Both B-Raf and c-Raf1 bind to activated p21ras, independent of their activation state.
- NGF treatment increased MEK kinase association with p21ras.
Conclusions:
- NGF-stimulated B-Raf directly activates the MAP kinase cascade in PC12 cells.
- c-Raf1 does not appear to function in this specific NGF-induced MAP kinase activation pathway.
- B-Raf functions within a larger protein complex in this signaling pathway.