Investigation into the mechanism regulating MRP localization.
The major PKC substrates MARCKS and MacMARCKS (MRP) are membrane-binding proteins implicated in cell spreading, integrin activation and exocytosis. According to the myristoyl-electrostatic switch model the co-operation between the myristoyl moiety and the positively charged effector domain (ED) is an essential mechanism by which proteins bind to membranes. Loss of the electrostatic interaction between the ED and phospholipids, such as Ptdins(4,5)P2, results in the translocation of such proteins to the cytoplasm. While this model has been extensively tested for the binding of MARCKS far less is known about the mechanisms regulating MRP localization. We demonstrate that after phosphorylation, MRP is relocated to the intracellular membranes of late endosomes and lysosomes. MRP binds to all membranes via its myristoyl moiety, but for its localization at the plasma membrane the ED is also required. Although the ED of MRP can bind to Ptdins(4,5)P2 in vitro, this binding is not essential for its retention at or targeting to the plasma membrane. We conclude that the co-operation between the myristoyl moiety and the ED is not required for the binding to membranes in general but that it is essential for the targeting of MRP to the plasma membrane in a Ptdins(4,5)P2-independent manner.
van den Bout
Iman
I
Division of Cell Biology, Netherlands Cancer Institute, 121 Plesmanlaan, 1066 CX Amsterdam, The Netherlands.
van Rheenen
Jacco
J
van Angelen
Annelies A
AA
de Rooij
Johan
J
Wilhelmsen
Kevin
K
Jalink
Kees
K
Divecha
Nullin
N
Sonnenberg
Arnoud
A
D000818:Animals; D001665:Binding Sites; D002148:Calmodulin-Binding Proteins; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004705:Endocytosis; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008840:Microfilament Proteins; D009154:Mutation; D009226:Myristates / Q000378:metabolism; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010766:Phosphorylation; D021381:Protein Transport
D002148:Calmodulin-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; C000619710:MARCKS protein, human; C000619711:Marcks protein, mouse; C512945:Marcksl1 protein, mouse; D008565:Membrane Proteins; D008840:Microfilament Proteins; D009226:Myristates; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D000076250:Myristoylated Alanine-Rich C Kinase Substrate