Chromaffin cell F-actin disassembly and potentiation of catecholamine release in response to protein kinase C activation by phorbol esters is mediated through myristoylated alanine-rich C kinase substrate phosphorylation

The large majority of chromaffin vesicles are excluded from the plasma memb rane by a cortical F-actin network. Treatment of chromaffin cells with phor bol 12-myristate 13-acetate produces disassembly of cortical F-actin, incre asing the number of vesicles at release sites (Vitale, M. L., Seward, E. P. , and Trifaro, J. M. (1995) Neuron 14, 353-363). Here, we provide evidence for involvement of myristoylated alanine-rich protein kinase C substrate (M ARCKS), a protein kinase C substrate, in chromaffin cell secretion. MARCKS binds and crosslinks F-actin, the latter is inhibited by protein kinase C-i nduced MARCKS phosphorylation. MARCKS was found in chromaffin cells by immu noblotting. MARCKS was also detected by immunoprecipitation. In intact or p ermeabilized cells MARCKS phosphorylation increased upon stimulation with 1 0(-7) M phorbol 12-myristate 13-acetate. This was accompanied by cortical F -actin disassembly and potentiation of secretion. MARCKS phosphorylation, c ortical F-actin disassembly, and potentiation of Ca2+-evoked secretion were inhibited by a peptide (MARCKS phosphorylation site domain sequence (MPSD) ) with amino acid sequence corresponding to MARCKS phosphorylation site. MP SD was phosphorylated in the process. A similar peptide (alanine-substitute d phosphorylated site domain) with four serine residues of MPSD substituted by alanines was ineffective. These results provide the first evidence for MARCKS involvement in chromaffin cell secretion and suggest that regulation of cortical F-actin crosslinking might be involved in this process.