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Identification of Ser‐Pro and Thr‐Pro Phosphorylation Sites in Chicken Neurofilament‐M Tail Domain
Author(s) -
Bennett Gudrun S.,
Quintana Ricardo
Publication year - 1997
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1997.68020534.x
Subject(s) - neurofilament , phosphorylation , identification (biology) , domain (mathematical analysis) , biology , chemistry , microbiology and biotechnology , computational biology , immunohistochemistry , immunology , mathematics , mathematical analysis , botany
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 [ 32 P]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. 32 P‐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.