Myristoylation-dependent N-terminal cleavage of the myristoylated alanine-rich C kinase substrate (MARCKS) by cellular extracts

The myristoylated alanine-rich C kinase substrate (MARCKS) has been propose d to regulate the plasticity of the actin cytoskeleton at its site of attac hment to membranes. In macrophages, MARCKS is implicated in various cellula r events including motility, adhesion and phagocytosis. In this report we s how that macrophage extracts contain a protease which specifically cleaves human MARCKS, expressed in a cell-free system or in E. coli, between Lys-6 and Thr-7. Cleavage of MARCKS decreases its affinity for macrophage membran es by ca. one order of magnitude, highlighting the contribution of the myri stoyl moiety of MARCKS to membrane binding. Importantly, cleavage requires myristoylation of MARCKS. Furthermore, MARCKS-related protein (MRP), the se cond member of the MARCKS family, is not digested. Since Thr-Ti is lacking in MRP this suggests that Thr-7 at the P1 position is important for the rec ognition of lipid-modified substrates. A different product is observed when MARCKS is incubated with a calf brain cytosolic extract. This product can be remyristoylated in the presence of myristoyl-Coa and N-myristoyl transfe rase, demonstrating that cycles of myristoylation/demyristoylation of MARCK S can be achieved in vitro. Although the physiological relevance of these e nzymes still needs to be demonstrated, our results reveal the presence of a new class of cleaving enzymes recognizing lipid-modified protein substrate s. (C) 2000 Societe francaise de biochimie et biologie moleculaire / Editio ns scientifiques et medicales Elsevier SAS.