Identification of Ser-Pro and Thr-Pro phosphorylation sites in chicken neurofilament-M tail domain.
The tail domain of the midsize chicken neurofilament polypeptide (NF-M) contains several different types of Ser-Pro and Thr-Pro putative phosphorylation sites. We determined which of these sites are actually phosphorylated in vivo. Chick sensory neuron cultures were incubated in [32P]phosphate, and the cytoskeletal fraction was mixed with a neurofilament fraction prepared from adult chicken brain. NF-M was purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and digested with chymotrypsin, and two large fragments were isolated. These were individually cleaved with trypsin, endoprotease Lys-C, or endoprotease Glu-C, and peptides separated by two-dimensional high-voltage electrophoresis and thin-layer chromatography. 32P-labeled phosphopeptides were eluted from the cellulose plates and subjected to microsequencing and mass spectometry. We found that of 21 potential Ser-Pro and Thr-Pro phosphoacceptor sites, at least 20 are phosphorylated in vivo: all four Lys-Ser-Pro sites and at least 16 of the 17 Lys-Xaa-Xaa-Ser/Thr-Pro repeats. In addition, a novel Ser-Pro site in the extreme carboxy terminus is phosphorylated. This site, which has no proximal Lys residue, is also found in mammalian NF-M, but has not been reported to be phosphorylated. Together with three casein kinase I sites we have found recently in the acidic amino-terminal segment of the tail, a total of 24 or 25 Ser and Thr phosphoacceptor sites have now been located in the chicken NF-M tail.
Bennett
G S
GS
Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, USA.
Quintana
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001923:Brain Chemistry; D002642:Chick Embryo; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D016900:Neurofilament Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000032:analysis; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011392:Proline / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism
D016900:Neurofilament Proteins; D010446:Peptide Fragments; D010748:Phosphopeptides; D010761:Phosphorus Radioisotopes; C061195:neurofilament protein M; D013912:Threonine; D012694:Serine; D011392:Proline; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase