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

Thomas Theis,Bibhudatta Mishra,Maren Von Der Ohe,Gabriele Loers,Maksymilian Prondzynski,Ole Pless,Perry J Blackshear,Melitta Schachner,Ralf Kleene

doi:10.1074/jbc.m112.444034

Abstract

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.

Highlights

Polysialic acid (PSA) plays important roles in the developing and adult nervous system
Binding of fluorescent-labeled PSA-NCAM to cells expressing mutated myristoylated alanine-rich C kinase substrate (MARCKS)-green fluorescent protein (GFP) was observed, but fluorescence resonance energy transfer (FRET) analysis showed either no FRET signals or a low number of FRET signals for distances of less than 30 nm between the labeled PSA-NCAM and the mutated MARCKS-GFP (Fig. 7B). These results indicate that soluble PSA-NCAM interacts with the effector domain of MARCKS and that mutation of the effector domain leads to disruption or disturbance of this interaction
These results indicate that the effect of soluble PSA on neurite outgrowth is mostly independent of cellular NCAM and PSA-carrying NCAM but is mainly mediated via the effector domain of MARCKS

Summary

Background

Polysialic acid (PSA) plays important roles in the developing and adult nervous system. MARCKS is a rod-shaped, acidic protein that possesses three highly conserved regions: 1) the N terminus, which contains a consensus sequence for myristoylation that is involved in membrane binding of MARCKS [17], 2) the MH2 domain, which resembles the cytoplasmic tail of the cation-independent mannose-6-phosphate receptor, and 3) the phosphorylation site domain, which contains the PKC phosphorylation sites and the effector domain [18, 19]. The effector domain is highly basic, in contrast to the rest of the highly acidic protein, and has been shown to be crucial for the function of MARCKS, whereas myristoylation of MARCKS is not required for many of the in vivo functions of MARCKS as indicated by studies using expression of non-myristoylatable MARCKS in MARCKS-null mice [19]. We provide evidence that MARCKS and PSA interact at the cell membrane of hippocampal neurons and that the interaction between MARCKS and PSA modulates the neuritogenesis of hippocampal neurons

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION