Functional Role of the Interaction between Polysialic Acid and Myristoylated Alanine-rich C Kinase Substrate at the Plasma Membrane

• Published in: The Journal of biological chemistry Vol. 288; no. 9; pp. 6726 - 6742
• Main Authors: Theis, Thomas, Mishra, Bibhudatta, von der Ohe, Maren, Loers, Gabriele, Prondzynski, Maksymilian, Pless, Ole, Blackshear, Perry J., Schachner, Melitta, Kleene, Ralf
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.03.2013; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Cell Adhesion; Cell Membrane - genetics; Cell Membrane - metabolism; CHO Cells; Cricetinae; Cricetulus; Hippocampus - cytology; Hippocampus - metabolism; Intracellular Signaling Peptides and Proteins - genetics; Intracellular Signaling Peptides and Proteins - metabolism; Lipid Bilayers; MARCKS; Membrane Bilayer; Membrane Proteins - genetics; Membrane Proteins - metabolism; Mice; Mice, Knockout; Myristoylated Alanine-Rich C Kinase Substrate; NCAM; Nerve Tissue Proteins - genetics; Nerve Tissue Proteins - metabolism; Neural Cell Adhesion Molecule L1 - genetics; Neural Cell Adhesion Molecule L1 - metabolism; Neurite Outgrowth; Neurites - metabolism; Neurobiology; Peptides - genetics; Peptides - metabolism; Peptides - pharmacology; Plasma Membrane; Polysaccharide; Polysialic Acid; Sialic Acids - genetics; Sialic Acids - metabolism; Plasma Membrane; MARCKS; Polysialic Acid; NCAM; Membrane Bilayer; Neurite Outgrowth; Cell Adhesion; Polysaccharide
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.444034

Polysialic acid (PSA) is a homopolymeric glycan that plays crucial roles in the developing and adult nervous system. So far only a few PSA-binding proteins have been identified. Here, we identify myristoylated alanine-rich C kinase substrate (MARCKS) as novel PSA binding partner. Binding assays showed a direct interaction between PSA and a peptide comprising the effector domain of MARCKS (MARCKS-ED). Co-immunoprecipitation of PSA-carrying neural cell adhesion molecule (PSA-NCAM) with MARCKS and co-immunostaining of MARCKS and PSA at the cell membrane of hippocampal neurons confirm the interaction between PSA and MARCKS. Co-localization and an intimate interaction of PSA and MARCKS at the cell surface was seen by confocal microscopy and fluorescence resonance energy transfer (FRET) analysis after the addition of fluorescently labeled PSA or PSA-NCAM to live CHO cells or hippocampal neurons expressing MARCKS as a fusion protein with green fluorescent protein (GFP). Cross-linking experiments showed that extracellularly applied PSA or PSA-NCAM and intracellularly expressed MARCKS-GFP are in close contact, suggesting that PSA and MARCKS interact with each other at the plasma membrane from opposite sides. Insertion of PSA and MARCKS-ED peptide into lipid bilayers from opposite sides alters the electric properties of the bilayer confirming the notion that PSA and the effector domain of MARCKS interact at and/or within the plane of the membrane. The MARCKS-ED peptide abolished PSA-induced enhancement of neurite outgrowth from cultured hippocampal neurons indicating an important functional role for the interaction between MARCKS and PSA in the developing and adult nervous system. Background: Polysialic acid (PSA) plays important roles in the developing and adult nervous system. Results: The interaction of PSA with myristoylated alanine-rich C kinase substrate (MARCKS) at the plasma membrane regulates neurite outgrowth. Conclusion: The MARCKS/PSA interaction regulates PSA-triggered signal transduction. Significance: Study of the molecular mechanisms underlying PSA-induced cellular responses helps to understand the functions of PSA in the nervous system.