Identification of serine 473 as a major phosphorylation site in the neurofilament polypeptide NF-L

Xu, ZS; Liu, WS; Willard, Mark

doi:10.1523/jneurosci.10-06-01838.1990

Cited by 33 publications

(29 citation statements)

References 41 publications

(41 reference statements)

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“…Anti-TL IgG might abolish the effect of the negative charge and accelerate the lateral adhesion [29]. The reported major phosphorylation site, Ser473, might be concerned with regulation by the tail region [33].…”

Section: Discussionmentioning

confidence: 96%

Acceleration of bovine neurofilament L assembly by deprivation of acidic tail domain

Nakamura

Takeda

Aimoto

et al. 1993

European Journal of Biochemistry

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Functions of the tail region of neurofilament L have, to date, not been clearly elucidated. Bovine neurofilament L was cleaved into tail-less neurofilament L (50 kDa) and a tail fragment (19 kDa), by thrombin. Tail-less neurofilament L was deficit of the highly acidic domain of the tail region (=77 % of the entire tail region). Assembly of tail-less neurofilament L was observed to be accelerated by both fluorometric and centrifugal measurements, compared with intact neurofilament L. The critical concentration of tail-less neurofilament L, which constitutes the constant unassembled pool, was approximately 0.25-times lower than that of neurofilament L. Under physiological conditions, tail-less neurofilament L formed a ribbon-like structure, whereas tail-less neurofilament L could form 10-nm filaments in an extremely low ionic-strength buffer in the presence of 1 mM MgCl,. An affinity-purified antibody directed against the tail fragment also accelerated neurofilament L assembly. The tail fragment neither coassembled with neurofilament L nor affect neurofilament L assembly. The acidic domain of the tail region may regulate neurofilament assembly and may be involved in 10-nm filament formation under physiological conditions.Intermediate filament proteins (IF), which had been believed to be static, have recently been revealed to be dynamic [ 1 -51. However, the regulation of IF dynamics have, to date, not been fully elucidated. IF are composed of three regions, the amino-terminal head region, the rod region and the carboxy-terminal tail region [6]. The head region has been reported to regulate IF assembly by phosphorylation and dephosphorylation [7-131. The rod region is highly conserved and constitutes the body of a 10-nm filament [6].Expression of tail-less keratins in living cells indicate that the tail region of keratin is involved in lateral packing, elongation and the formation of a network of the filaments [14-161. Tail-less desmin formed a ribbon-like structure in vitro under conditions where intact desmin formed 10-nm filaments [17]. Experiments using a peptide of desmin residues 436-442 and an antibody directed against it, suggested that the tail region is involved in the parallel stacking of subunits [19]. These observations demonstrate that the tail regions of keratin and desmin are involved in filament assembly. acidic tail fragment could be easily obtained by thrombin digestion of neurofilament L. Assembly of IF can be closely monitored using fluorescently labeled IF [l, 2, 101. In this report, the characteristics of tail-less neurofilament L and the tail fragment were investigated in detail using fluorometric measurement of assembly, electron-microscopic observation and an antibody raised against the tail fragment. The functions of the acidic domain of the tail region were discussed. MATERIALS AND METHODS Purification of tail-less neurofilament L and the tail fragmentNeurofilament L was purified from bovine spinal cords by DE52 (Whatman) and hydroxyapatite (Bio-Rad) chromatographies [I, 101. Neurofilame...

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Section: Discussionmentioning

confidence: 96%

Acceleration of bovine neurofilament L assembly by deprivation of acidic tail domain

Nakamura

Takeda

Aimoto

et al. 1993

European Journal of Biochemistry

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“…RM: 677-845 actually contains one KSP sequence (GDK-SPQE, 717-723). However this is not evolutionarily conserved, is not preceded by neutral or hydrophobic amino acids, and is apparently not an in vivo phosphorylation site (Xu et al, 1989). It seems that this tripeptide arose fortuitously and is probably not related to the other KSP sequences.…”

Section: Fusion Protein Studiesmentioning

confidence: 92%

A molecular dissection of the carboxyterminal tails of the major neurofilament subunits NF‐M and NF‐H

Harris

Ayyub

Shaw

1991

J of Neuroscience Research

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We have initiated a multidisciplinary project that aims to dissect and ultimately define the functions of the long and unusual C-terminal "tail" sequences of the two high molecular weight neurofilament subunits, NF-M and NF-H. A series of recombinant fusion proteins containing selected NF-M and NF-H tail sequences were constructed using appropriate cDNAs. These fusion proteins were used to further define the epitopes for a variety of widely used neurofilament antibodies, including NN18 and N52, which are now available commercially from several companies. We also measured the SDS-PAGE mobility of the fusion proteins and found that, like the native neurofilament tails, the fusion proteins ran considerably slower than predicted from their molecular weight. Since all fusion proteins produced so far exhibit this characteristic we conclude that all segments of the NF-M and NF-H tail share this unusual property. Finally we were able to produce novel and potentially useful polyclonal and monoclonal antibodies to selected segments of NF-M and NF-H sequence. These antibody studies showed that the extreme C-termini of NF-M and NF-H are immunologically absolutely distinct from one another and also indicate that the extreme C-terminus of NF-M is immunologically much more conserved than the analogous region of NF-H. These findings are in complete agreement with our conclusions derived from amino acid sequence analysis, and further underline the possible functional importance of the extreme C-terminus of NF-M. We also show that the unusual immunological properties of the bovine NF-M tail we have previously observed do not extend to the extreme C-terminal region, which appears immunologically no different from the analogous region of other NF-M molecules. The peculiarities of bovine NF-M could be explained by the presence of a KSP motif that resembles the NF-H KSP prototype.

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“…Purification of NF Triplet Proteins-Total NFs comprising mainly NF triplet proteins were prepared from about 15 g of frozen rat spinal cord as described (33,44,45). The Triton X-100 insoluble pellet from repeated sucrose cushions was resuspended in 20 mM sodium phosphate, pH 7.0, 8.0 M urea, and 0.1% ␤-mercaptoethanol, filtered through a 0.20-m filter, and loaded onto a 45-ml DE52 cellulose column.…”

Section: Methodsmentioning

confidence: 99%

Cytoplasmic O-GlcNAc Modification of the Head Domain and the KSP Repeat Motif of the Neurofilament Protein Neurofilament-H

Dong

Hart

et al. 1996

Journal of Biological Chemistry

105

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Identification of serine 473 as a major phosphorylation site in the neurofilament polypeptide NF-L

Cited by 33 publications

References 41 publications

Acceleration of bovine neurofilament L assembly by deprivation of acidic tail domain

Acceleration of bovine neurofilament L assembly by deprivation of acidic tail domain

A molecular dissection of the carboxyterminal tails of the major neurofilament subunits NF‐M and NF‐H

Cytoplasmic O-GlcNAc Modification of the Head Domain and the KSP Repeat Motif of the Neurofilament Protein Neurofilament-H

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