A 42-kD tyrosine kinase substrate linked to chromaffin cell secretion exhibits an associated MAP kinase activity and is highly related to a 42-kD mitogen-stimulated protein in fibroblasts.

Abstract

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.

Inhibition of MAP kinase kinase (MEK) blocks endothelial PGI2 release but has no effect on von Willebrand factor secretion or E‐selectin expression

“…Growing evidence from a number of cell types, including terminally differentiated cells, additionally implicates p42 m~pk in the control of acute responses to receptor stimulation such as exocytosis and arachidonic acid release [14,15]. In endothelial cells p42 mapk can be activated by growth factors [16], GPCA [17,18], shear stress [19,20] or inflammatory cytokines (this study) but the functional relevance of stimulus-induced changes in p42 mapk remains to be determined.…”

Activated 3′,5′-Cyclic AMP-dependent Protein Kinase Is Sufficient to Induce Neuroendocrine-like Differentiation of the LNCaP Prostate Tumor Cell Line

“…The dominant negative RI␣ subunit, dnRI␣, was provided in a murine metallothionine I promoter-driven expression vector (pMTdnRI␣; Ref. Immunoblotting-After treatment, cells were washed with phosphate-buffered saline (50 mM sodium phosphate, 150 mM NaCl, pH 7.4), lysed in HO buffer (50 mM HEPES, 100 mM NaCl, 1% Noniodet P-40, 2 mM EDTA, 1 g/ml leupeptin, 2 g/ml aprotinin, 0.5 mM sodium vanadate, 40 mM p-nitrophenyl phosphate, 2 M microcystin, pH 7.2) on ice, and processed for SDS-polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose (Schleicher and Schuell), as described (29). Immunoblotting for FLAG-tagged kinases, NSE, and PKA was performed using the anti-FLAG epitope antibody, M5 (Eastman Kodak Co.), a polyclonal anti-NSE antiserum (DAKO, Carpinteria, CA), and a polyclonal anti-PKA CI␣ catalytic subunit antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), respectively.…”

Regulation of the Neuronal Nicotinic Acetylcholine Receptor by Src Family Tyrosine Kinases

“…Cells-Chromaffin cells were cultured from freshly harvested bovine adrenal glands as described previously (42) and modified (43). Cells were maintained in serum-free N2 medium (Invitrogen) at 37°C in a humidified, 5% CO 2 environment.…”