Neurotrophins are a growing family of protein factors involved in the development, sculpting and maintenance of the nervous system. Nerve growth factor (NGF) is the first-isolated and most thoroughly characterized mem- ber of the neurotrophin family. Among the most significant biologic effects of NGF are promotion of the survival and neuronal differentiation of distinct elements of the nervous system. These include peripheral sympathetic and sensory neurons as well as central cholinergic neurons of the basal forebrain, a region prominently involved, patho logically, in neural degeneration associated with Alzheimer's Disease. Understanding the mechanisms of NGF action could lead to treatments for a variety of neurodegenerative conditions. The principle model used to investigate NGF signaling is the NGF- responsive, PC12 cell culture line. PC12 cells expresses the high affinity NGF receptor, p140trkA, which is a transmembrane protein with tyrosine kinase activity. Upon addition of NGF, the tyrosine kinase activity of p140 trkA is stimulated and summarily, previously round, replicating cells cease to divide and become, phenotypically, sympathetic neurons replete with electrically excitable neuritic processes. The alkaloid, K-252a, an inhibitor of protein kinase activity, blocks selectively all the actions of NGF in PC12 cells including stimulation of p140 trkA tyrosine kinase activity indicating the import- ance of protein phosphorylation with regard to NGF signal initiation and propagation. Investigative studies con- ducted in this laboratory have examined 1) the biochemical characteristics of NGF-stimulated phospholipase C- gamma (PLC) phosphorylation and 2) the constitution of an NGF signalling pathway in previously unresponsive cells by expressing p140trkA in C6-2B glioma cells. NGF stimulates the phosphorylation of an important signal transducing molecule, PLC, on both tyrosine and serine amino acid residues. The tyrosine phosphorylation is mediated directly by NGF-stimulated, p140 trkA tyrosine kinase activity, whereas, PLC serine phosphorylation appears to occur secondarily. Efforts are ongoing to identify the responsible serine kinase(s). Based upon pharmacologic studies involving inhibitors selective for specific classes of serine kinases it appears that both protein kinase A and protein kinase C may be involved demonstrating the capacity for NGF to activity multiple protein kinase cascades. Transfection of C6-2B glioma cells with the gene for p140 trkA resulted in the functional expression of high affinity NGF receptors demonstrated by receptor-ligand crosslinking studies, detection of NGF-stimulated p140 trkA autophosphorylation, and induction of the transcription factor, c-fos.
