The localization of the protein tyrosine kinase pp60c-src to the plasma membrane and to the membrane of secretory vesicles in neurally derived bovine chromaffin cells has suggested that tyrosine phosphorylations may be associated with the process of secretion. In the present study we have identified two cytosolic proteins of approximately 42 and 45 kD that become phosphorylated on tyrosine in response to secretagogue treatment. Phosphorylation of these proteins reached a maximum (3 min after stimulation) before maximum catecholamine release was observed (5-10 min after stimulation). Both secretion and tyrosine phosphorylation of p42 and p45 required extracellular Ca2+. Tyrosine-phosphorylated proteins of similar Mr have previously been identified in 3T3-L1 adipocytes stimulated with insulin (MAP kinase; Ray, L. B., and T. W. Sturgill. 1987. Proc. Natl. Acad. Sci. USA. 84:1502-1506) and in avian and rodent fibroblasts stimulated with a variety of mitogenic agents (Cooper, J. A., D. F. Bowen-Pope, E. Raines, R. Ross, and T. Hunter. 1982. Cell. 31:263-273; Nakamura, K. D., R. Martinez, and M. J. Weber. 1983. Mol. Cell. Biol. 3:380-390). Comparisons of the secretion-associated 42-kD protein of chromaffin cells with the 42-kD protein of Swiss 3T3 fibroblasts and 3T3-L1 adipocytes provide evidence that these three proteins are highly related. This evidence includes comigration during one-dimensional SDS-PAGE, cochromatography using ion exchange and hydrophobic matrices, similar isoelectric points, identical cyanogen-bromide peptide maps, and cochromatography of MAP kinase activity with the tyrosine-phosphorylated form of pp42. This protein(s), which appears to be activated in a variety of cell types, may serve a common function, perhaps in signal transduction involving a cascade of kinases.

The derivation and characterization of 22 hybridoma clones producing monoclonal antibodies (Mabs) specific for the transforming protein of Rous sarcoma virus, pp6Osrc, are described. All Mabs reacted with pp6Ov-src encoded by Prague, Schmidt-Ruppin, and Bratislava 77 strains of Rous sarcoma virus. Of these Mabs, 10 efficiently immunoprecipitated pp60Ocsrc from chicken embryo cells. Of these 10 Mabs, 2 (GD11 and EB8) readily detected pp6O-src from a variety of rodent and human cultured cells and from rat brain tissue in an in vitro immune complex kinase assay. Mapping experiments have tentatively localized the determinant(s) recognized by GD11 and EB8 to a region of the src protein bounded by amino acid residues 82 to 169, whereas the remaining Mabs appeared to recognize determinants residing within residues 1 to 82 or 169 to 173. Most of the Mabs complexed denatured pp6O-src in a Western immunoblot, and several were used to localize pp6Ov-src in Rous sarcoma virus-transformed chicken embryo cells by indirect immunofluorescence microscopy. * Corresponding author. MATERIALS AND METHODS Cells, viruses, and plasmids. Primary cultures of CE cells were prepared from 10-day-old gram-negative, chf-negative, Marek's disease-negative embryos (SPAFAS, Norwich, Conn.) and maintained in culture as described previously (30). Secondary cultures of CE cells were infected with the following strains of RSV: PrA-RSV, SRD-RSV, B77C-RSV, or mutants of PrA-RSV, tsCHdlll9, CHdl121, or CHdl300 (6; J.

Nicotine‐induced catecholamine secretion in bovine adrenomedullary chromaffin cells is accompanied by rapid tyrosine phosphorylation of multiple cellular proteins, most notably the mitogen‐activated protein kinases (MAPKs). The requirement for activation of tyrosine kinases and MAPKs in chromaffin cell exocytosis was investigated using a panel of tyrosine kinase inhibitors. Genistein and tyrphostin 23, two compounds that inhibit tyrosine kinases by distinct mechanisms, were found to inhibit secretion by >90% in cells stimulated by nicotine, 55 mM KCI, or the Ca2+ ionophore A23187. Inhibition of secretion induced by all three secretagogues correlated with a block in both protein tyrosine phosphorylation and activation of the MAPKs and their activators (MEKs) in situ. However, neither genistein nor tyrphostin 23 inhibited the activities of the MAPKs or MEKs in vitro. These results indicate that the target(s) of inhibition lie down‐stream of Ca2+ influx and upstream of MEK activation. This Ca2+‐activated tyrosine kinase activity could not be accounted for entirely by c‐Src or Fyn (two nonreceptor tyrosine kinases that are expressed abundantly in chromaffin cells), because their in vitro kinase activities were not inhibited by tyrphostin 23 and only partially inhibited by genistein. These results demonstrate that an unidentified Ca2+‐activated tyrosine kinase(s) is required for MAPK activation and exocytosis in chromaffin cells and suggest that MAPK participates in the regulation of secretion.

Adrenal medullary chromaffin cells derive from the neural crest during embryogenesis and differentiate into dedicated secretory cells that release catecholamines in response to acetylcholine in vivo or nicotinic agonists in vitro. Previous studies have indicated that tyrosine kinases participate in early secretagogue‐induced events in these cells and are required for exocytosis. Abundant levels of the cytoplasmic tyrosine kinases, c‐Src and c‐Yes, have been detected in chromaffin cells, thereby implicating them as kinases relevant to these events. However, c‐Src has been found to undergo a decrease in activity following secretagogue‐stimulation, and c‐Yes appears to exist in a constitutively low activity state, suggesting that other tyrosine kinases are involved. Furthermore, other members of the Src family of tyrosine kinases have been implicated as playing roles in secretion in a variety of cell types. Therefore, we sought to determine if other Src family members were present in chromaffin cells, and if so, to examine them for subcellular localization and changes in activity following treatment with nicotinic agonists. To this end, antibodies for Fyn, Lck, Lyn, and Fgr were assembled and used in immunoprecipitation, in vitro autokinase, and Western immunoblotting assays. Of these four kinases, only Fyn was found to be expressed at detectable levels. Differential centrifugation studies revealed that Fyn resides predominantly (>95%) in the crude plasma membrane fraction and undergoes nicotinic‐ and carbachol‐induced activation. This activation is reduced by the nicotinic antagonist, mecamylamine, is not elicited by muscarine, and is dependent upon the presence of extracellular Ca2+. These results suggest that Fyn is involved in signalling through the nicotinic receptor and may be one of the relevant kinases responsible for at least some of the tyrosine phosphorylations detected after stimulation. © 1996 Wiley‐Liss, Inc.

Transformation of cells by Ro,us sarcoma virus is mediated by the product of the viral src gene, pp6OSrc. A hybridoma cell line producing an immunoglobulin G3 antibody to pp6Osrc was isolated after lymph node cells from immune mice were fused with mouse myeloma cells (P3-NS1-1). Mice were immunized with p6Osrc purified from Escherichia coli cells expressing the src gene product. The monoclonal antibody immunoprecipitated pp60src from Rous sarcoma virustransformed cells and recognized an antigenic determinant located in the aminoterminal third of the pp6Osrc protein. Cellular transformation by Rous sarcoma virus (RSV) requires the functional expression of the RSV src gene (19, 40). Brugge and coworkers first identified the RSV src gene product, showing that sera from rabbits bearing RSV-induced tumors (TBR sera) immunoprecipitated a 60,000-molecular-weight phosphoprotein (pp6Osrc) from RSV-transformed cells but not from normal cells or from cells infected with transformation-defective RSV (1, 2, 31). The RSV src gene product is a protein kinase (7, 24). pp60Src isolated as an immune complex with TBR sera exhibits a unique phosphotransferase activity, catalyzing the phosphorylation of tyrosine in the immunoglobulin heavy chain (7-9, 11, 21, 25, 37, 38). This activity appears to be an intrinsic property of pp6src, since purified preparations of it readily phosphorylate tyrosine in a variety of substrates (11, 25, 30). In RSV-transformed cells, the cellular expression of pp6Osrc results in the phosphorylation of tyrosine in several specific cellular proteins (10, 12, 32, 36). These unique phosphorylation events, coupled with the intrinsic kinase activity of pp6Osrc, suggest that phosphorylation of defined cellular target proteins plays a critical role in cellular transformation.

Secretion of catecholamines by adrenal chromaffin cells is a highly regulated process that involves serine/threonine and tyrosine phosphorylations. The nonreceptor tyrosine kinase pp60c‐sre is expressed at high levels and localized to plasma membranes and secretory vesicle membranes in these cells, suggesting an interaction of this enzyme with components of the secretory process. To test the hypothesis that pp60c‐sic is involved in exocytosis, we transiently expressed exogenous c‐src cDNA using a vaccinia virus vector in primary cultures of bovine adrenomedullary chromaffin cells. Chromaffin cells infected with a c‐src recombinant virus restored the diminished secretory activity accompanying infection by wild type virus alone or a control recombinant virus. The level of enhanced catecholamine release correlated directly with the time and level of exogenous c‐src expression. These results could not be attributed to differences in cytopathic effects of wild type versus recombinant viruses as assessed by cell viability assays, nor to differences in norepinephrine uptake or basal release, suggesting that pp60c‐src is involved in stimulus‐secretion coupling in infected cells. Surprisingly, exogenous expression of an enzymatically inactive mutant c‐src also restored catecholamine release, indicating that regions of the introduced c‐src protein other than the kinase domain may affect catecholamine release. Secretory activity was elevated by both forms of c‐src in response to either nicotine or carbachol (which activate the nicotinic and the nicotinic/muscarinic receptors, respectively). In contrast, release of catecholamines upon membrane depolarization (as elicited by 55 mM K+) or by treatment with the calcium ionophore A23187 was unaffected by either vaccinia infection or increased levels of pp60c‐src. These results suggest that pp60c‐src affects secretory processes in vaccinia‐infected cells that are activated through ligand‐gated, but not voltage‐gated, ion channels.

The major component of the core structure of avian sarcoma leukosis viruses is a 27 kD molecular weight polypeptide, p27. Spleen cells from mice immunized with the Schmidt-Ruppin strain of Rous sarcoma virus (RSV) were fused with mouse myeloma cells (SP2/0), and hybridoma cell lines producing monoclonal antibodies to p27 were isolated. The monoclonal antibodies were all of the IgG1 subclass with kappa light chains. These antibodies immunoprecipitated p27 and its precursor proteins from extracts of RSV-transformed cells. Reciprocal competitive binding experiments defined five nonoverlapping antigenic determinants within p27. The monoclonal antibodies also immunoprecipitated the transforming protein, p110gag-myc, from avian myelocytomatosis virus transformed cells. Their usefulness in studies of virion maturation and viral oncogenesis is discussed.

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