Pp60 (c-src), the normal cellular homologue of the viral transforming protein, pp60 (v-src), is a 60 kDa nonreceptor tyrosine kinase that is found in highest concentrations in post-mitotic cells specialized for exocytosis. Examples of such cells include platelets, chromaffin cells and cells of the myeloid lineage. Other pp60 (c-src)-related kinases have also been in secretory cells, e.g.,pp55(c-fgr) in neutrophils and pp59(fyn) in platelets. These findings suggest that pp60(c-src) and its related kinases participate in the process of regulated secretion. Neurally-derived, bovine adrenal chromaffin cells provide an ideal system in which to study the molecular mechanisms of secretion, due to the development of conditions for in vitro and of subcellular model systems representing several distinct stages of the secretory process. In the search for secretion-sensitive tyrosine kinase substrates in this model system, we detected two proteins of 42 and 45 kDa, whose tyrosyl phosphorylations increased 15-30 fold upon stimulation with nicotine, carbachol or 55 mMK(+). Further characterization of these proteins revealed that p42, in particular, exhibited properties nearly identical to those of the mitogen-sensitive, transformation-sensitive p42 of fibroblasts and the insulin-sensitive microtubule-associated protein (MAP) kinase of adipocytes. MAP kinase is a serine/threonine kinase that phosphorylates MAP 2 and S6 kinase in vitro an requires both tyrosine and threonine phosphorylations for activation. Little, however, is known about the in vivo functions of either MAP kinase or the p42/p45 proteins in any cell system. Recently, rat cDNA sequences (ERK1) thought a portion of an insulin-sensitive MAP kinase have been cloned. Biochemical and genetic evidence suggest that MAP kinase and the ERK1 product are highly related. We propose to utilize the rat ERK1 sequences to molecularly clone and sequence the bovine ERK1 cDNA and other related bovine cDNAs, to construct prokaryotic and eukaryotic expression systems for these cDNAs, and produce immunological reagents specific for each of the related gene products. These reagents will then be used in biochemical studies of the bovine chromaffin cell to identify enzymes that regulate ERK1 and related kinases, to define cellular substrates of ERK1 and related kinases, and to determine their subcellular localizations. Biological studies are proposed which exploit the unique in vitro model systems of the chromaffin cell as well as permeabilized and intact cells to determine the possible function(s) of ERK1 and related kinases in secretion. The overall goal of these studies is to assess the role of this family of serine/threonine kinases in the secretory with particular emphasis on their roles as """"""""switch kinases"""""""", those kinases that catalyze some of the many serine/threonine phosphorylations known to occur after secretagogue stimulation, but whose activities appear to be regulated by tyrosine/threonine phosphorylation.
