Myristoylation-dependent and Electrostatic Interactions Exert Independent Effects on the Membrane Association of the Myristoylated Alanine-rich Protein Kinase C Substrate Protein in Intact Cells

Sharon L Swierczynski,Perry J Blackshear

doi:10.1074/jbc.271.38.23424

Abstract

The myristoylated alanine-rich protein kinase C substrate (MARCKS) is a widely expressed, prominent substrate for protein kinase C. MARCKS is largely associated with membranes in cells, and hydrophobic interactions involving the amino-terminal myristoyl moiety are thought to play a role in anchoring MARCKS to cellular membranes. In addition, experiments in cell-free systems have suggested that electrostatic interactions between the positively charged phosphorylation site/calmodulin binding domain (PSD) of MARCKS and negatively charged membrane lipids are also involved in this association. Although it has been inferred from phosphorylation experiments, the electrostatic nature of the interaction between the PSD and membranes has not been demonstrated directly in intact cells. We expressed human MARCKS mutated in the myristoylation site and the PSD in REF52 cells; the cells were then fractionated by ultracentrifugation. Both nonmyristoylatable MARCKS and MARCKS in which the four serines in the PSD were mutated to aspartic acids, mimicking phosphorylation, exhibited decreased membrane affinity when compared to the fully myristoylated, wild-type, tetra-Ser protein or a myristoylated, tetra-Asn mutant. A double mutant, nonmyristoylatable protein in which the four serines in the PSD were mutated to aspartic acids exhibited negligible membrane association. Similar results were obtained in 293 cells that stably expressed chicken MARCKS mutated in the same domains. The double mutant, nonmyristoylatable tetra-Asp chicken protein exhibited little membrane association as determined by both subcellular fractionation and immunoelectron microscopy. These results indicate that myristoylation and electrostatic interactions involving the PSD exert independent, essentially additive effects on the membrane association of MARCKS in intact cells.

Highlights

The myristoylated alanine-rich protein kinase C substrate (MARCKS) is a widely expressed, prominent substrate for protein kinase C
A double mutant, nonmyristoylatable protein in which the four serines in the phosphorylation site/calmodulin binding domain (PSD) were mutated to aspartic acids exhibited negligible membrane association
In five sections of cells expressing nonmyristoylatable, tetra-Asp MARCKS, 13 Ϯ 5% (S.D.) of the gold particles was associated with membranes. These results support the data obtained from the subcellular fractionation experiments in that tetraAsn MARCKS was associated predominantly with membrane structures, tetra-Asp MARCKS was distributed almost between membranes and cytosol, and nonmyristoylatable tetra-Asp MARCKS was almost exclusively cytosolic. These studies examined the effect of mutations in the myristoylation and PSD consensus sequences of MARCKS on the membrane association of the protein in intact cells

Summary

Introduction

The myristoylated alanine-rich protein kinase C substrate (MARCKS) is a widely expressed, prominent substrate for protein kinase C. Experiments in cellfree systems have suggested that electrostatic interactions between the positively charged phosphorylation site/calmodulin binding domain (PSD) of MARCKS and negatively charged membrane lipids are involved in this association. The double mutant, nonmyristoylatable tetra-Asp chicken protein exhibited little membrane association as determined by both subcellular fractionation and immunoelectron microscopy These results indicate that myristoylation and electrostatic interactions involving the PSD exert independent, essentially additive effects on the membrane association of MARCKS in intact cells. Several studies in cell-free systems have suggested that the positively charged PSD of MARCKS is involved in membrane association through electrostatic interactions [26,27,28,29,30] This interaction has been well characterized in vitro, the only evidence supporting the electrostatic nature of the association of the PSD with membranes in intact cells has been obtained from phosphorylation experiments. Because activation of PKC results in the phosphorylation of many intracellular substrates and affects numerous cellular processes, it is possible that the reduced affinity of MARCKS for membranes seen after PKC activation might not be due to electrostatic changes alone

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Conclusion