The product of the proto-oncogene, c-src, is expressed at high levels in developing neural tissues and in cells of neural and non- neural origin which exhibit a specialized secretory functions. Examples of such cells include platelets, granulocytes, neurones and adrenal chromaffin cells Specific expression in these cells suggests a function for pp60c-src the normal homologue of the highly oncogenic protein, pp60v-src, i.e., participation in the exocytotic process. Studies of the neurally-derived bovine adrenal medullary chromaffin cells have provided three lines of evidence which support the notion that pp60c-src plays some role in the secretory function of these cells. First is the finding that membranes of the secretory vesicles of chromaffin cells contain high levels of pp60c-src specific tyrosine kinase activity. Second is the observation that a transient down-modulation of c-src enzyme activity and state of phosphorylation occurs concomitantly with release of the vesicle contents. And third is the identification of calpactin I, a purported substrate for the transforming protein, pp60v-src, as a cytosolic protein with Ca++ dependent chromaffin granule aggregating activity. This type of activity is requisite for the process of secretion. The experiments outlined in this grant are designed to investigate further the interaction between pp60c-src and its potential substrates and regulators in relation to the process f secretion. The goal of these studies is the elucidation of the function of pp60c-src in the chromaffin cell. The following specific aims are proposed: (a) Determine the reasons for the changes in pp60c-src activity and phosphate content in secretagogue stimulated cells by examining pp60c-src in subcellular model systems in detergent permeabilized cells and in intact cells for post-translational modifications or heterocomplex formation. In addition we will analyze mRNA purified from stimulated cells for expression of splice variants of c-src or of src related genes which may exhibit the altered properties we have observed. (b) Identify phosphotyrosine containing proteins of the chromaffin cell and investigate alterations in the phosphotyrosine composition of this group of proteins upon secretagogue stimulation. Approaches will include 32Pi labeling of intact detergent permeabilized cells, followed by gel electrophoresis and autoradiographic analysis of labeled proteins, and immunoblot analysis with anti-phosphotyrosine antibodies of whole cell extracts or subcellular fractions. Particular attention will be paid to those proteins that lose phosphotyrosine upon stimulation. (c) Assess the function of substrates identified in (b) first with respect to their activity in subcellular models for events occurring in exocytosis and second in cultured chromaffin cells. Particular emphasis will be put n the effects differential phosphorylation has on the tetrameric structure and on the chromaffin granule binding activity of calpactin I. These studies will be carried out with the ultimate goal of elucidating the function of the normal counterpart of the transforming protein, pp60v-src, to facilitate our understanding of the mechanism of oncogenesis by this agent.
Showing the most recent 10 out of 222 publications
