The study of oncogenes has provided an unexpected probe for neuronal development and function. Normal cellular pp60c-src, a protein of unknown function that is homologous to the transforming protein of Rous sarcoma virus, has been shown by this laboratory to be expressed at elevated levels in fully differentiated, functional neurons and in cells of the early embryo. This proposal will investigate a function for pp60c-src in transmembrane signalling in mature neurons and in undifferentiated cells of early embryos during commitment to the neural pathway. (a) pp60c-src will be localized within the neuronal plasma membrane at functionally distinct sites, where specific ion channels are located, by biochemical fractionation of neuronal membranes isolated from the chick neural retina and by immunoperoxidase staining of retinal neurons in culture. (b) Regulation of the tyrosine-specific protein kinase activity of pp60c-src by neural protein kinases will be studied in retinal neurons stimulated in vivo with B-adrenergic agonists (which activate cAMP-dependent protein kinase), phorbol esters (which activate protein kinase C) and selected neurotransmitters. Phosphorylation sites within the pp60c-src molecule will be identified by peptide mapping and phosphoamino acid analysis; and changes in the tyrosine kinase activity of pp60c-src measured using a synthetic peptide substrate. (c) A novel src-related tyrosine kinase (100K) that we have identified in the electric organ of the electric eel will be characterized by peptide mapping and cDNA cloning for future determination of its coding sequence and to provide probes for its study in higher animals. The eel src protein may be a new member of the src family of tyrosine kinases or have a unique domain that facilitates electrical signalling. (d) Finally, the expression of pp60c-src in cells of the early chick embryo that are the targets of neural inducing signals will be mapped by immunocytochemical staining. The proposed study of tyrosine kinases in the nervous system is a fundamentally new approach to the study of neuronal function and differentiation, and will also provide insight into the mechanism by which the very similar but mutant protein of Rous sarcoma virus transforms cells.
