Volume 272, Number 20,
Issue of May 16, 1997
pp. 12922-12927
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Phosphorylation Sites on Neurofilament Proteins
by Nanoelectrospray Mass Spectrometry
(Received for publication, November 7, 1996, and in revised form, February 27, 1997)
Joanna C.
Betts
§
,
Walter P.
Blackstock
§
,
Malcolm A.
Ward
§
and
Brian H.
Anderton
From the 
Department of Neuroscience, The Institute of
Psychiatry, De Crespigny Park, London, SE5 8AF and
§ Glaxo Wellcome Research and Development,
Stevenage SG1 2NY, United Kingdom
Neurofilament (NF) proteins are intermediate
filaments found in the neuronal cytoskeleton. Phosphorylation of these
proteins is considered an important factor in the assembly of filaments and determination of filament caliber and stability. Mammalian neurofilaments are composed of three polypeptide subunits, NF-L, NF-M,
and NF-H, all of which are phosphorylated. Here we used techniques for
the mass spectrometric sequencing of proteins from polyacrylamide gels
to analyze in vivo phosphorylation sites on NF-M and NF-L.
Neurofilaments were isolated from rat brain and enzymatically digested
in gel. The resulting peptides were analyzed and sequence data obtained
by nanoelectrospray mass spectrometry. Four phosphorylation sites have
been found in the C-terminal domain of NF-M: serines 603, 608, 666, and
766. Two of these are found in lysine-serine-proline (KSP) motifs and
two in the variant motifs, glutamic acid-serine-proline (ESP) and
valine-serine-proline (VSP). Serine 55 in NF-L was not found to be
phosphorylated, which confirms the possible role of phosphorylation and
dephosphorylation of this site in early neurofilament assembly. The
techniques used enable sequence data and characterization of
posttranslational modifications to be obtained for each individual
subunit directly from polyacrylamide gels.
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