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

Edward F Boumil,Yuguan Liu,Atsuko Uchida,Thomas B Shea,Harish C Pant,Rishel Brenna Vohnoutka

doi:10.1242/bio.028522

Abstract

ABSTRACTPhosphorylation 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.

Highlights

The cytoskeleton provides structural support to axons allowing for axonal outgrowth and for maintenance of synaptic connections formed by outgrowing axons (Luo 2002, Barnes & Polleux 2009, Kapitein & Hoogenraad 2011)
Following knockdown of endogenous NF-H and expression of NF constructs, a significant increase in S493 was mutated to aspartic acid (S493D) was observed within axonal neurites versus that of wild type NF-H (wtH) or S493A, while reduced levels of S493A were observed within axonal neurites verus wtH (Fig. 1A)
To examine the potential effect of S493 phosphorylation on downstream NF-H tail domain phosphorylation, immunoblots were probed with monoclonal antibody RT97, which reacts with a conformationally-dependent phospho-epitope located within the NF-H tail downstream of S493 (Veeranna et al, 2008)

Summary

Introduction

The cytoskeleton provides structural support to axons allowing for axonal outgrowth and for maintenance of synaptic connections formed by outgrowing axons (Luo 2002, Barnes & Polleux 2009, Kapitein & Hoogenraad 2011). C-terminal phosphorylation precludes NF proteolysis and increases NF residence time within axons (Lee et al 2014, Pant & Veeranna 1995, Greenwood et al 1993, Nixon 1993, Goldstein et al 1987, Fiumelli et al 2008, Grant et al 2001) These collective phosphorylation events are mediated by an interactive network of kinases and phosphatases that regulate NF transport and incorporation into the axonal cytoskeleton including: cyclin-dependent kinase (cdk5), mitogen activated kinases (MAPKs), casein kinase 1 and 2 (CK1 and CK2), glycogen synthase kinase 3α and 3β (GSK3α and GSK3β), p38 MAPK, c-Jun N-terminal kinase (JNKs) and protein phosphatases 1, 2A and 2B (PP1, PP2A, PP2B, respectively) These collective phosphorylation events are mediated by an interactive network of kinases and phosphatases that regulate NF transport and incorporation into the axonal cytoskeleton including: cyclin-dependent kinase (cdk5), mitogen activated kinases (MAPKs), casein kinase 1 and 2 (CK1 and CK2), glycogen synthase kinase 3α and 3β (GSK3α and GSK3β), p38 MAPK, c-Jun N-terminal kinase (JNKs) and protein phosphatases 1, 2A and 2B (PP1, PP2A, PP2B, respectively) (Lee et al 2014; Pant & Veeranna 1995, and refs. therein)

Results

Discussion

Conclusion