Demyristoylation of the major substrate of protein kinase C (MARCKS) by the cytoplasmic fraction of brain synaptosomes.

Myristoylated alanine-rich C kinase substrate (MARCKS) is a major substrate of protein kinase C, whose interaction with the plasma membrane is dependent on its phosphorylation by protein kinase C and on its N-terminal myristoylation. We describe a hitherto undescribed demyristoylation activity of the protein in cytoplasmic fraction of synaptosomes from bovine brain. The activity is dependent on ATP but independent from calcium. The formation of the demyristoylated form, characterized by an increased mobility on SDS gel (70 kDa instead of 85 kDa), was confirmed by mass spectrometry and amino acid sequencing. The molecular mass of the demyristoylated protein as well as the incorporation of radioactive phosphate from [gamma-32P]ATP indicated that one phosphoryl group was incorporated during the demyristoylation process. Calmodulin, which binds to the protein kinase C phosphorylation domain of MARCKS, inhibited the reaction in a calcium-dependent manner. These data suggest that the demyristoylation is regulated by the signal transduction pathways and that the two conserved domains of the protein, namely the N-terminal myristoylated region and the phosphorylation site domain, are functionally interdependent. The localization of MARCKS in the cells may be regulated not only by its phosphorylation with protein kinase C but also by a reversible myristoylation of the protein in situ.