The functional specificity of external cues such as trophic stimulation or synaptic activity is dependent upon the regulation of intracellular signaling pathways. Subcellular localization of MAPK is one means of regulating its specificity by controlling its pool of effector molecules.
Specific aim 1 outlines a series of experiments designed to test the hypothesis that MAPK is translocated from the cytoplasm to the nucleus in neuronal cell types. PC-12 cells and hippocampal neuron cultures will be examined upon stimulation by trophic factors and depolarization. Endogenous MAPK will be examined using immunocytochemistry and cell fractionation. Transiently expressed GFP-labeled MAPK-localization will be monitored using time-lapse epifluorescent microscopy.
Specific aim 2 will test the hypothesis that stimulation of a novel MAPK-activating pathway is similarly sufficient to cause the nuclear import of MAPK. This pathway is stimulated by PKA and utilizes Rap1 and B-Raf to activate MAPK in neurons. In PC-12 cells, NGF, but not EGF, activates this pathway. The sufficiency of this PKA-dependent pathway (PKA/Rap1/B-Raf) to cause MAPK translocation will be examined using this model. In hippocampal neurons, the ability of this pathway to stimulate Erk movement will be tested. Finally, the necessity of this pathway in the cyto-nuclear translocation caused by trophic factors and electrical activity will be examined using dominant negative and constitutively active mutant proteins of Rap1.
