Novel axonal distribution of neurofilament-H phosphorylated at the glycogen synthase kinase 3beta-phosphorylation...

The Ser493 residue in the E-segment of the rat neurofilament heavy chain (NF-H) is phosphorylated by glycogen synthase kinase 3beta (GSK3 beta) in vitro and in spinal cord. We examined Ser493 phosphorylation by analyzing developmental changes and cellular distribution of phospho-Ser493 using phosphorylation-site-specific antibodies. This residue was phosphorylated in NF-H prepared from human, rat, and mouse spinal cord, all species in which the amino acid sequence of NF-H is known. Phosphorylated Ser493 appeared on postnatal day 2 in rat brain, at the same time when NF-H is first detected. It gradually increased together with the increase in total NF-H during brain development. Phospho-Ser493 was detected on the phosphorylated form of NF-H at multiple Lys-Ser-Pro (KSP) repeats, which are distributed mainly in axons. In rat ventral horn, phosphorylated Ser493 was localized in axons but not in cell bodies or dendrites. However, the distributions of phosphorylated Ser493 and KSP phosphorylation in axons were not identical. Ser493 was continuously phosphorylated at nodes of Ranvier, whereas the KSP sites were dephosphorylated. Ser493 was also phosphorylated in unmyelinated regions of optic nerve axons. A biochemical difference in phosphorylation between Ser493 and KSP repeats was also found; the subtle phosphorylation at Ser493 was detected in NF-H unphosphorylated at the KSP repeats by immunoblotting cerebral cortex extracts. These results indicate that Ser493 in the NF-H E-segment is a novel site that is phosphorylated in both the myelinated and the unmyelinated regions of axons.