Discrete nuclear structures in actively growing neuroblastoma cells are revealed by antibodies raised against phosphorylated neurofilament proteins.
BACKGROUND
Nuclear objects that have in common the property of being recognized by monoclonal antibodies specific for phosphoprotein epitopes and cytoplasmic intermediate filaments (in particular, SMI-31 and RT-97) have been reported in glial and neuronal cells, in situ and in vitro. Since neurofilament and glial filaments are generally considered to be restricted to the cytoplasm, we were interested in exploring the identity of the structures labeled in the nucleus as well as the conditions under which they could be found there.


RESULTS
Using confocal microscopy and western analysis techniques, we determined 1) the immunolabeled structures are truly within the nucleus; 2) the phosphoepitope labeled by SMI-31 and RT-97 is not specific to neurofilaments (NFs) and it can be identified on other intermediate filament proteins (IFs) in other cell types; and 3) there is a close relationship between DNA synthesis and the amount of nuclear staining by these antibodies thought to be specific for cytoplasmic proteins. Searches of protein data bases for putative phosphorylation motifs revealed that lamins, NF-H, and GFAP each contain a single tyrosine phosphorylation motif with nearly identical amino acid sequence.


CONCLUSIONS
We therefore suggest that this sequence may be the epitope recognized by SMI-31 and RT-97 mABs, and that the nuclear structures previously reported and shown here are likely phosphorylated lamin intermediate filaments, while the cytoplasmic labeling revealed by the same mABs indicates phosphorylated NFs in neurons or GFAP in glia.
Weigum
Shannon E
SE
Department of Biology, Southwest Texas State University, San Marcos 78666, USA. sweigum@mail.utexas.edu
García
Dana M
DM
Raabe
Timothy D
TD
Christodoulides
Nicholas
N
Koke
Joseph R
JR
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000918:Antibody Specificity; D015153:Blotting, Western; D002455:Cell Division / Q000502:physiology; D002467:Cell Nucleus / Q000378:metabolism / Q000648:ultrastructure*; D022003:Cell Nucleus Structures / Q000276:immunology / Q000648:ultrastructure*; D004247:DNA / Q000096:biosynthesis; D000939:Epitopes / Q000276:immunology; D005904:Glial Fibrillary Acidic Protein / Q000096:biosynthesis / Q000276:immunology; D006801:Humans; D007381:Intermediate Filament Proteins / Q000096:biosynthesis / Q000276:immunology*; D034882:Lamins / Q000096:biosynthesis / Q000276:immunology; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000378:metabolism / Q000648:ultrastructure*; D016900:Neurofilament Proteins / Q000096:biosynthesis / Q000276:immunology*; D010766:Phosphorylation; D051381:Rats
D000911:Antibodies, Monoclonal; D000939:Epitopes; D005904:Glial Fibrillary Acidic Protein; D007381:Intermediate Filament Proteins; D034882:Lamins; D016900:Neurofilament Proteins; C051774:neurofilament protein H; D004247:DNA