Transfected rat high-molecular-weight neurofilament (NF-H) coassembles with vimentin in a predominantly nonphosphorylated form.
A fully encoding cDNA for the high-molecular-weight rat neurofilament protein (NF-H) has been isolated from a lambda gt11 library, sequenced and subcloned into eukaryotic expression vectors. Sequence analysis shows that rat NF-H has an overall homology of 72 and 88% with human and mouse NF-H, respectively. The head and rod domains are almost entirely identical, and the divergences are due to differences in the long C-terminal extensions of the molecule. The consensus phosphorylation sequence for neurofilaments Lys-Ser-Pro (KSP) is present 52 times. The predicted molecular mass of the protein is 115 kDa, 42% lower than that observed by SDS-PAGE. Upon transfection into vimentin-containing fibroblasts, such as L tk-, L929, and 3T6 cells, NF-H is seen distributed with vimentin by light and electron microscopic examinations indicating that copolymers of NF-H and vimentin are formed in these cells. Only a negligible proportion of the cells is positive when stained with a number of antibodies directed against phosphorylated NF-H epitopes. This is in contrast with the middle molecular weight NF protein (NF-M) transfected into L tk- and L929 cells, which can readily be detected by antibodies against phosphorylated neurofilament epitopes. The mobilities of the transfected protein on 1- and 2-dimensional gels confirm that NF-H is predominantly in a nonphosphorylated form. These results indicate that phosphorylation of NF-H, but not NF-M, on the KSP sequence is due to protein kinases, which are not present in fibroblasts and are presumably NF-H specific. The stable NF-H-expressing cell lines can therefore be used to study these putative neurofilament kinases in vitro and in vivo.
Chin
S S
SS
Department of Pathology, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
Liem
R K
RK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism*; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D005455:Fluorescent Antibody Technique; D015723:Gene Library; D006801:Humans; D007381:Intermediate Filament Proteins / Q000235:genetics / Q000378:metabolism*; D007382:Intermediate Filaments / Q000378:metabolism; D007739:L Cells / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016900:Neurofilament Proteins; D010766:Phosphorylation; D014176:Protein Biosynthesis; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D014162:Transfection; D014746:Vimentin / Q000378:metabolism*
D007381:Intermediate Filament Proteins; D016900:Neurofilament Proteins; D014746:Vimentin; C051774:neurofilament protein H; D004247:DNA