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.
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.
Swierczynski
S L
SL
Howard Hughes Medical Institute, Division of Endocrinology, Metabolism, and Nutrition, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
Blackshear
P J
PJ
D000215:Acylation; D000818:Animals; D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites; D002642:Chick Embryo; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D016296:Mutagenesis; D019814:Myristic Acid; D009227:Myristic Acids / Q000737:chemistry / Q000378:metabolism*; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000235:genetics; D013045:Species Specificity; D055672:Static Electricity; D013347:Subcellular Fractions
D047908:Intracellular Signaling Peptides and Proteins; C000619710:MARCKS protein, human; D008565:Membrane Proteins; D009227:Myristic Acids; D011506:Proteins; D011994:Recombinant Proteins; D019814:Myristic Acid; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D001224:Aspartic Acid; D012694:Serine; D001216:Asparagine