Disease relevance of Nef3

• Two-dimensional gel analysis and immunoblotting of total proteins from adult rat dorsal root ganglion verified mAb specificity in situ, and showed that differentially phosphorylated isoforms of NF-M and NF-H occur in vivo [1].
• The transient induction of NF-M mRNA is also apparent in dedifferentiating Schwann cells during Wallerian degeneration [2].
• Using antibodies that were directed at all three neurofilament (NF) epitopes, NF-L, NF-M, and NF-H, we attempted to determine whether these neurons would have an altered cytoskeletal profile following the stress of transplantation, because previous studies have shown such changes following ischemia or direct brain injury [3].
• We examined the short-term regulation of the phosphorylation of the mid-sized neurofilament subunit (NF-M) by kinases which were activated in rat pheochromocytoma (PC12) cells by nerve growth factor (NGF) and/or 12-O-tetradecanoylphorbol 13-acetate (TPA) [4].
• Phosphorylation of the neuronal cytoskeletal proteins NF-H, NF-M and tau is important for normal axonal development, and is involved in axonal injury and neurodegenerative diseases [5].

High impact information on Nef3

• To examine this and to further investigate the function of the carboxyl-terminal tail domain of NF-M, we made various deletion mutants that lacked part of their tail domains, and we expressed these with NF-L [6].
• These data demonstrate that Schwann cells remodel their IFs by expressing NF-M before acquiring the myelin-forming phenotype and that IF proteins of the neurofilament-type are not restricted to neurons in the vertebrate nervous system [7].
• Schwann cells of the myelin-forming phenotype express neurofilament protein NF-M [7].
• Immunological comparisons with other IF proteins revealed a close similarity between p145 and the neurofilament protein NF-M; the identification of p145 as NF-M was confirmed by isolating and sequencing a full-length clone from a Schwann cell cDNA library [7].
• The cultured cells expressed two neuronal traits, tyrosine hydroxylase [L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2] and the neuron-specific 160-kDa neurofilament subunit protein, but were devoid of glial fibrillary acidic protein, a marker for non-myelin-forming Schwann cells in ganglia [8].

Biological context of Nef3

• The amino acid sequence of rat NF-M shows the conserved rod segment present in all intermediate filament proteins [9].
• To test this hypothesis, we studied the effect of PKA phosphorylation of the NF-M head domain on phosphorylation of tail domain KSP sites [10].
• Within 24 hr of axotomy, there was a down-regulation of the 68 kDa (NF-L) and 145 kDa (NF-M) NF subunits [11].
• Detection of NF-L and NF-M mRNA in injured nerve, however, indicated that there was an up-regulation in response to nerve injury [12].
• Restoration of axonal contact in the growing nerve stimulates the recapitulation of Schwann cell differentiation including the elevation of NF-M and NF-L mRNA expression [2].

Anatomical context of Nef3

• Partial phosphorylation, denoted P[+], of NF-M was already present in perikarya and neurites of E12 neurons [13].
• The results indicated that postnatal malnutrition imposed during the brain growth spurt period did not alter the expression of IF proteins of the cerebral cortex in 21-d-old rats, but increased the expression of NF-L and NF-M proteins in adult rats [14].
• Left sciatic nerves were transected at postnatal day 3 (P3), 6 (P6), 8 (P8), or 10 (P10). mRNA and protein levels of the light (NF-L), mid-sized (NF-M), and heavy (NF-H) NF proteins were compared in L4 and L5 DRGs from the transected (left) vs. control (right) sides of the same animals at varying intervals after transection [15].
• These findings suggest that the expression of the NF-M and NF-L polypeptides is an important characteristic of those Schwann cells that will contribute to the repair of damaged peripheral nerves [2].
• The pellet contained the bulk of the cytoskeleton proteins from tissue, identified as the 150- and 68-kDa subunits of neurofilaments (NF-M and NF-L, respectively), the 66-kDa associated protein, the 57-kDa intermediate filament-like protein, and the 50-kDa glial fibrillary acidic protein [14].

Associations of Nef3 with chemical compounds

• The molecule also contains an unusual C-terminal extension with stretches of glutamic acid, which could contribute to the anomalous migration of this protein on SDS-PAGE and the fact that NF-M does not readily assemble into filaments [9].
• To demonstrate the site specificity of PKA phosphorylation and its effect on tail domain phosphorylation, we transfected NIH3T3 cells with NF-M mutated at PKA-specific head domain serine residues [10].
• Chronic, but not acute, administration of either morphine or cocaine was found to decrease levels of the three NF proteins, NF-200 (NF-H), NF-160 (NF-M), and NF-68 (NF-L), by between 15% and 50% in the VTA by back phosphorylation, immunolabeling, and Coomassie blue staining [16].
• Here we used techniques for the mass spectrometric sequencing of proteins from polyacrylamide gels to analyze in vivo phosphorylation sites on NF-M and NF-L [17].
• NF-M and NF-L are known to be modified 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) [18].

Other interactions of Nef3

• Epidermal growth factor stimulation of cells with mutant NF-M in the presence of forskolin exhibited no inhibition of NF-tail domain phosphorylation compared with the wild type NF-M-transfected cells [10].
• On the other hand, chronic MMA administration induced a decreased 32P in vitro incorporation into NF-M, while chronic treatment with propionate decreased the in vitro phosphorylation of NF-M and alpha-tubulin [19].
• Immunoelectron microscopic analysis revealed NF-M and vimentin proteins to be colocalized on the same intermediate filaments [20].
• Labeling for the 160 kDa neurofilament subunit, calbindin, and parvalbumin varied [21].

Analytical, diagnostic and therapeutic context of Nef3

• Using matrix-assisted laser desorption-ionization (MALDI) and nano-electrospray mass spectrometry, we report the identification of several novel in vitro cAMP-dependent protein kinase (PKA) phosphorylation sites in the low (NF-L) and middle (NF-M) molecular mass neurofilament subunits [22].
• Northern blot analysis of severed nerve detected NF-L and NF-M, but not NF-H [12].
• The present study examined degenerating retinal terminals by means of immunohistochemistry and antibodies specific for the triplet of neurofilament proteins of low (NF-L), medium (NF-M), and high (NF-H) molecular weight class [23].
• Acrylamide given acutely (100 mg/kg, single intraperitoneal injection) causes a selective increase in NF-M mRNA (approximately 50%) compared to controls [24].
• Results of Northern blotting of total RNA obtained from the DRG indicated that NF-L and NF-M mRNA levels had largely returned to control levels at 12 weeks following crush axotomy but were still substantially depressed following cut/ligation injury of the sciatic nerve at that time [25].

References