Membrane Association of the Myristoylated Alanine-rich C Kinase Substrate (MARCKS) Protein.: MUTATIONAL ANALYSIS PROVIDES EVIDENCE FOR COMPLEX INTERACTIONS

Abstract

The myristoylated alanine-rich C kinase substrate (MARCKS) protein, a prominent cellular substrate for protein kinase C, is associated with membranes in various cell types. MARCKS is myristoylated at its amino terminus; this modification is thought to play the major role in anchoring MARCKS to cellular membranes. Recent studies have suggested that the protein's basic phosphorylation site/calmodulin binding domain may also be involved in the membrane association of MARCKS through electrostatic interactions. The present studies used mutations in the primary structure of the protein to investigate the nature of the association between MARCKS and cell membranes. In chick embryo fibroblasts, activation of protein kinase C led to a decrease in MARCKS membrane association as determined by cell fractionation techniques. Cell-free assays revealed that nonmyristoylated MARCKS exhibited almost no affinity for fibroblast membranes, despite readily demonstrable binding of the wild-type protein. Similar experiments in which the four serines in the phosphorylation site domain were mutated to aspartic acids, mimicking phosphorylation, decreased, but did not eliminate, membrane binding when compared to either the wild-type protein or a comparable tetra-asparagine mutant. Addition of calmodulin in the presence of Ca2+ also inhibited binding of the wild-type protein to membranes, presumably by neutralizing the phosphorylation site domain, or by physically interfering with its membrane association. Surprisingly, expression of a nonmyristoylatable mutant form of MARCKS in intact cells led to only a 46% decrease in its plasma membrane association, as determined by cell fractionation and immunoelectron microscopy. These results are consistent with a complex model of the interaction of MARCKS with cellular membranes, in which the myristoyl moiety, the positively charged phosphorylation site domain, and possibly other domains make independent contributions to membrane binding in intact cells.