Site-specific phosphorylation of the middle molecular weight human neurofilament protein in transfected non-neuronal cells.
We expressed the human midsized neurofilament subunit (NF-M) using genomic DNA in mouse L cells and showed that it is transcribed and translated into a protein capable of assembly into the cytoskeleton and of forming a filamentous network that colocalizes with the endogenous vimentin filaments. Moreover, human NF-M expressed in L cells is phosphorylated at sites within the multiphosphorylation repeat (MPR), i.e., the major sites of phosphorylation of NF-M in vivo. We also expressed a genomic construct lacking the MPR domain in the native molecule and showed that this MPR(-) protein also was expressed and formed a filamentous network despite diminished incorporation of radiolabeled phosphate. Two major conclusions emerged from the work described in this paper: human NF-M is translated, assembled, and phosphorylated at physiological sites without the need of any other specific neuronal proteins; phosphorylation sites other than the MPR are present within NF-M which may play a role in synthesis, assembly, and degradation of NF protein in humans.
Department of Pathology, University of Pennsylvania School of Medicine, Philadelphia 19104-4283.
S J
SJ
Pleasure
V M
VM
Lee
D L
DL
Nelson
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D015180:Electrophoresis, Gel, Two-Dimensional; D005455:Fluorescent Antibody Technique; D005796:Genes; D006801:Humans; D007381:Intermediate Filament Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D007739:L Cells / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D016900:Neurofilament Proteins; D010766:Phosphorylation; D010957:Plasmids; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger; D015183:Restriction Mapping; D014162:Transfection
D000911:Antibodies, Monoclonal; D007381:Intermediate Filament Proteins; D009419:Nerve Tissue Proteins; D016900:Neurofilament Proteins; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA