Influence of a GSK3β phosphorylation site within the proximal C-terminus of neurofilament-H on neurofilament dynamics

Abstract

Phosphorylation of the C-terminal tail of the heavy neurofilament subunit (NF-H) impacts NF axonal transport and residence within axons by fostering NF-NF associations that compete with transport. We tested the role of phosphorylation of a GSK-3β consensus site (S493) located in the proximal portion of the NF-H tail, in NF dynamics by transfection of NB2a/d1 cells with NF-H where S493 was mutated to aspartic acid (S493D) or to alanine (S493A) to mimic constitutive phosphorylation and non-phosphorylation. S493D underwent increased transport into axonal neurites, while S493A displayed increased perikaryal NF aggregates that were decorated by anti-kinesin. Increased levels of S493A co-precipitated with anti-kinesin indicating that reduced transport of S493A was not due to reduced kinesin association but due to premature NF-NF interactions within perikarya. S493D displayed increased phospho-immunoreactivity within axonal neurites at downstream C-terminal sites attributable to mitogen-activated protein kinase and cyclin-dependent kinase 5. However, S493D was more prone to proteolysis following kinase inhibition, suggesting that S493 phosphorylation is an early event that alters sidearm configuration in a manner that promotes appropriate NF distribution. We propose a novel model for sidearm configuration.