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Assembly Properties of Neurofilament Light Chain Ser 55 Mutants in Transfected Mammalian Cells
Author(s) -
Gibb Barry J. M.,
Robertson Janice,
Miller Christopher C. J.
Publication year - 1996
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1996.66031306.x
Subject(s) - transfection , mutant , immunoglobulin light chain , microbiology and biotechnology , neurofilament , chemistry , biology , biophysics , genetics , cell culture , gene , immunology , immunohistochemistry , antibody
Ser 55 within the head domain of neurofilament light chain (NF‐L) is transiently phosphorylated by protein kinase A, and phosphorylation of this residue is thought to regulate assembly of neurofilaments. To understand how Ser 55 phosphorylation influences NF‐L assembly, wild‐type and mutant NF‐L genes in which Ser 55 was mutated to alanine, so as to prevent phosphorylation, or to aspartate, so as to mimic permanent phosphorylation, were transfected into mammalian cells that contain or do not contain an endogenous intermediate filament network. Wild‐type and mutant NF‐Ls localised to the Triton X‐100‐insoluble fraction, which suggests that phosphorylation of Ser 55 does not inhibit assembly of NF‐L and NF‐L/vimentin polymers at or below the tetrameric stage. Immunofluorescence microscopy of transfected cells demonstrated that the wild‐type and mutant NF‐Ls all colocalised with vimentin to produce similar filamentous arrays. However, in cells lacking an endogenous intermediate filament network, the aspartate mutant produced a pattern of staining different from that of the wild‐type or alanine mutant. These results suggest that phosphorylation of NF‐L Ser 55 is not a mechanism that precludes assembly of neurofilaments from monomers into intermediate filament structures but that phosphorylation/dephosphorylation of this residue might confer more subtle characteristics on neurofilament assembly properties and architecture.