CYTOPLASMIC O-GLCNAC MODIFICATION OF THE HEAD DOMAIN AND THE KSP REPEAT MOTIF OF THE NEUROFILAMENT PROTEIN NEUROFILAMENT-H

Neurofilaments, the major intermediate filaments in large myelinated n eurons, are essential for specifying proper axonal caliber. Mammalian neurofilaments are obligate heteropolymers assembled from three polype ptides, neurofilament (NF)-H, NF-M, and NF-L, each of which undergoes phosphorylation at multiple sites. NF-M and NF-L are known to be modif ied by O-linked N-acetylglucosamine (O-GlcNAc) (Dong, D. L.-Y., Xu, Z. -S., Chevrier, M. R., Cotter, R. J., Cleveland, D. W., and Hart, G. W. (1993) J. Biol. Chem. 268, 16679-16687), Here we further report that NF-H is extensively modified by O-GlcNAc at Thr(53), Ser(54), and Ser( 56) in the head domain and, somewhat surprisingly, at multiple sites w ithin the Lys-Ser-Pro repeat motif in the tail domain, a region in ass embled neurofilaments known to be nearly stoichiometrically phosphoryl ated on each of the similar to 50 KSP repeats. Beyond the earlier iden tified sites on NF-M and NF-L, O-GlcNAc sites on Thr(19) and Ser(34) o f NF-M and Ser(34) and Ser(48) of MF-L are also determined here, all o f which are localized in head domain sequences critical for filament a ssembly, The proximity of O-GlcNAc and phosphorylation sites in both h ead and tail domains of each subunit indicates that these modification s may influence one another and play a role in filament assembly and n etwork formation.