Identification of six phosphorylation sites in the COOH-terminal tail region of the rat neurofilament protein M.
The COOH-terminal tail domain of the neurofilament polypeptide M from rat nervous tissue contains approximately six molecules of phosphate. We report here that protein kinases in a crude cytoskeleton preparation of rat nervous tissue phosphorylated a set of tryptic peptides of M similar (but not identical) to those phosphorylated by living dorsal root ganglion cells in culture. Using these phosphopeptides as markers, we purified these same peptides from rat spinal cord and identified six specific phosphorylation sites in M by enzymatic and chemical criteria. These sites, serines 502, 506, 536, 606, 608, and 666, are all located in the COOH-terminal tail domain. Four are embedded in the repeated motif KSP whereas two are within variants of this motif, KSD and ESP. All of the sites that were preceded by lysine were resistant to alkaline phosphatase prior to modification of the lysine with citraconic anhydride. The identification of these sites should aid in investigations of the function of the phosphorylation of this protein and provides criteria for identifying the relevant kinases.
Xu
Z S
ZS
Department of Anatomy and Neurobiology and Biochemistry, Washington University School of Medicine, St. Louis, Missouri 63110.
Liu
W S
WS
Willard
M B
MB
D000595:Amino Acid Sequence; D000818:Animals; D003599:Cytoskeleton / Q000201:enzymology; D005727:Ganglia, Spinal / Q000378:metabolism; D008969:Molecular Sequence Data; D016900:Neurofilament Proteins / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D013116:Spinal Cord / Q000166:cytology / Q000201:enzymology; D014357:Trypsin / Q000378:metabolism
D016900:Neurofilament Proteins; C061195:neurofilament protein M; D011494:Protein Kinases; D014357:Trypsin