The Role of Phosphorylation on Mouse Neurofilament Medium Protein (NF-M) Sidearms

Abstract

Neurofilaments (NF) are important cytoskeletal filaments that are assembled from three distinct molecular weight proteins - neurofilament light (NF-L), medium (NF-M), and heavy (NF-H). The three proteins are bound to each other laterally forming 10-nm filamentous rods along with sidearm extensions that belong to the C-terminal tails of the proteins. These tails vary in number and sequences of their amino acid residues, and are abundant with charges. Additionally, the sidearm polypeptides attain negative charges through serine phosphorylation of the Lys-Ser-Pro (KSP) repeat motifs that are particularly found in NF-H and NF-M sidearms. NF protrusions mediate the interaction between neighboring filaments, and maintain axonal diameter. However, the role of individual NF proteins and their phosphorylations in regulating interfilament distances, and hence axonal diameter, is not fully understood. A number of studies have implicated NF-M proteins as critical in regulating axonal caliber. However, the conventional viewpoint that NF-M phosphorylation increases axonal caliber has been challenged by recent experimental study that disputes the effect of NF-M phosphorylation in modifying axonal caliber (Garcia et al. J. Neurosci. (2009) 29: 1277-1284). By employing gene replacement technique, the authors deduced that phosphorylation of NF-M KSP repeat is not required for myelin-dependent radial axonal growth. To better understand the effect of NF-M phosphorylation, we investigated the structural organization of mouse NF under phosphorylated and dephosphorylated conditions. We employed the 3D sequence-based coarse-grained model of NF brush (Chang et. al., J. Mol. Biol. (2009) 391:648-660) to perform Monte Carlo simulations of mouse NF by using the sequence and the stoichiometry of mouse NF proteins. Our result shows that the phosphorylation of mouse NF-M does not change the radial extension of NF-M, supporting the notion that NF-M phosphorylation has no effect on axonal diameter of mouse.