This work is designed to provide information on the mechanism of action of nerve growth factor. Nerve growth factor is a polypeptide required for the survival and development of the sympathetic and sensory nervous systems. Nerve growth factor controls the expression of specific genes in target neurons and, in that way, directs their development. The molecular mechanism by which the factor controls gene expression is not known. Our studies are focused on the intracellular events which follow the binding of nerve growth factor to its receptor. We have used PC12, a cell which differentiates in response to the factor. We have developed three cell-free phosphorylation systems, one cytoplasmic, one nuclear, and one ribosomal, which reflect the action of nerve growth factor on the cells. We have separated the components of the soluble system, kinase and substrate, and purified each. We have found that a decrease in kinase activity, rather than a decrease in substrate, is responsible for the overall decreases in phosphorylation. We have seen that the components, kinase and substrate, are present in many tissues, not just those that are nerve growth factor sensitive. We have purified the substrate from brain and are developing antibodies against it. We have explored the role of other, known kinases and have evidence that an early step in the action of nerve growth factor on phosphorylation involves protein kinase c. We are also exploring the nerve growth factor-induced changes in DNA. Our effort is to correlate these changes with changes in the phosphorylation of nuclear proteins, or with alterations in transcriptional activity and nuclear morphology. Finally, we are looking at the transcription of a specific gene, the gene for the epidermal growth factor recptor. The binding of epidermal growth factor is decreased in cells treated with nerve growth factor. We would like to know if this decrease occurs at the transcriptional level. If so, we want to clone the gene and study its functional state in nerve growth factor-treated cells.
The aim of these studies is to describe the actions of the factor at the molecular level.
