Phosphorylation and dephosphorylation of chromaffin cell proteins in response to stimulation

Abstract

Stimulation of bovine chromaffin cell in culture changed (increased or decreased) the phosphorylation state of several proteins as examined by 32P incorporation. Enhanced phosphorylation of 22 protein bands as well as increased dephosphorylation of a 20.4 kilodaltons protein band was observed when extracts of cultured chromaffin cells stimulated by either acetylcholine or high K + were subjected to mono-dimensional gel electrophoresis. For several protein bands, the degree of phosphorylation was larger in cells stimulated by acetylcholine than in those challenged by a depolarizing concentration of K +. The most affected phosphoproteins have apparent molecular weights of 14,800, 29,000, 33,000, 57,000 (tubulin subunit), 63,000 (tyrosine hydroxylase subunit) and 94,000. The presence of a low extracellular calcium concentration (0.5 mM Ca 2+ plus 15 mM Mg 2+) in the incubation medium inhibited (38–100%) the acetylcholine-evoked increases in protein phosphorylation observed previously for 18 protein bands. Trifluoperazine at the concentration required for 50% inhibition of acetylcholine-induced catecholamine release decreases (33–100%) the stimulation-induced phosphorylation in all polypeptides, with the exception of the 14.8 kilodaltons and the dephosphorylated 20.4 kilodaltons components which were not affected. Two-dimensional gel electrophoresis analysis revealed that exposure of chromaffin cells to acetylcholine produced two types of effect on protein phosphorylation: (1) activation of protein kinase activities affecting about 30 polypeptides; (2) activation of protein phosphatase activities resulting in the dephosphorylation of about 40 polypeptides, most of them appearing as minor phosphoproteins, with the exception of the α-subunit of pyruvate dehydrogenase and the 20.4 kilodaltons polypeptide. On the basis of their molecular properties (molecular weight and pI) and their abundance in chromaffin cells, the 80 kilodaltons phosphoprotein which focused at pI 4.8 and the 117.5 kilodaltons phosphoprotein which focused at pI 5.0 were identified as chromogranins A and B, respectively. The relationship between acetylcholine-induced protein phosphorylation (or dephosphorylation) and catecholamine secretion was also investigated. The time course of protein phosphorylation (or dephosphorylation) paralleled or preceded [ 3H]noradrenaline release for 16 phosphoproteins. This was also true for the 20.4 kilodaltons polypeptide that was dephosphorylated. However, the involvement of the 20.4 kilodaltons component as well as the 184, 210, 249 and 313 kilodaltons phosphoproteins in the secretory process is unlikely considering that their phosphorylation (or dephosphorylation) appeared to be independent of extracellular calcium. The present findings demonstrate that protein phosphorylation and dephosphorylation are stimulated during catecholamine secretion from chromaffin cells and that the phosphorylation state of these proteins is affected by calcium deprivation and a calmodulin antagonist. However, the relationship between the level of protein phosphorylation and the events involved in the secretory process remain to be established.