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

• Main Authors: Rishel Brenna Vohnoutka, Edward F. Boumil, Yuguan Liu, Atsuko Uchida, Harish C. Pant, Thomas B. Shea
• Format: Article
• Language: English
• Published: The Company of Biologists 2017-10-01
• Series: Biology Open
• Subjects: Axonal cytoskeleton; Neurofilament; Phosphorylation; Protein conformation; Proteolysis
• Online Access: http://bio.biologists.org/content/6/10/1516

Phosphorylation of the C-terminal tail of the heavy neurofilament subunit (NF-H) impacts neurofilament (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.