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Nerve growth factor-mediated activation of the mitogen-activated protein (MAP) kinase cascade involves a signaling
R K Jaiswal1, E Weissinger, W Kolch
1Alzheimer Research Laboratory, Department of Neurology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
Abstract:
The nerve growth factor (NGF)-mediated activation of the mitogen-activated protein (MAP) kinase cascade is an obligatory step in the morphological differentiation of PC12 cells. Signal transduction through the MAP kinase cascade is dependent upon activation of p21(ras) which binds directly to Raf family protein kinases, mediating their association with the membrane and activation. PC12 cells express two Raf isoforms, c-Raf and B-Raf. The activation of the MAP kinase cascade in response to NGF is due principally to the action of B-Raf. NGF treatment of PC12 cells resulted in the enhanced phosphorylation of B-Raf and c-Raf, and both exhibit reduced electrophoretic mobilities following stimulation of the cells. The NGF-stimulated phosphorylation of B-Raf was correlated with its enzymatic activation as measured by the phosphorylation of its substrate MEK. However, c-Raf does not exhibit significant levels of activity. B-Raf was present as a component of a high molecular mass complex, which included the molecular chaperone, heat shock protein 90 (HSP90). Importantly, c-Raf did not participate in the formation of such complexes. The B-Raf containing HSP90 complexes were normally present in PC12 cells, and their assembly was not dependent upon NGF stimulation. These data suggest that the ability of B-Raf to activate the MAP kinase cascade is due to its association with a large signaling complex, which is likely to impart signaling pathway specificity.
Insights
Nerve growth factor (NGF) activates the MAP kinase cascade in PC12 cells primarily through B-Raf. B-Raf
Area of Science:
- Cell signaling
- Molecular biology
- Neuroscience
Background:
- Nerve growth factor (NGF) triggers crucial cellular processes like differentiation in PC12 cells.
- The mitogen-activated protein (MAP) kinase cascade is central to NGF-mediated signaling.
- p21(ras) activation is key for MAP kinase cascade signal transduction.
Purpose of the Study:
- To investigate the roles of c-Raf and B-Raf isoforms in NGF-induced MAP kinase activation.
- To elucidate the mechanisms underlying B-Raf activation and its signaling complex formation.
Main Methods:
- PC12 cell culture and NGF stimulation.
- Western blotting to detect protein phosphorylation and electrophoretic mobility shifts.
- Enzymatic assays to measure Raf kinase activity against MEK substrate.
- Analysis of protein complex formation using co-immunoprecipitation and size exclusion chromatography.
Main Results:
- NGF stimulation enhanced phosphorylation and reduced electrophoretic mobility of both B-Raf and c-Raf.
- NGF-induced B-Raf phosphorylation correlated with increased enzymatic activity towards MEK.
- c-Raf showed minimal enzymatic activity, unlike B-Raf.
- B-Raf, but not c-Raf, was found in a pre-existing high molecular mass complex with heat shock protein 90 (HSP90).
Conclusions:
- B-Raf is the primary mediator of NGF-induced MAP kinase cascade activation in PC12 cells.
- Association with HSP90-containing complexes enables B-Raf's signaling capacity.
- These signaling complexes likely confer specificity to the MAP kinase pathway.