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Journal of Neuroscience, Vol 10, 3714-3726, Copyright © 1990 by Society for Neuroscience
Transfected rat high-molecular-weight neurofilament (NF-H) coassembles with vimentin in a predominantly nonphosphorylated form
SS Chin and RK Liem
Department of Pathology, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
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.
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