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Protein Kinase F A /Glycogen Synthase Kinase‐3 Predominantly Phosphorylates the In Vivo Site Thr 97 ‐Pro in Brain Myelin Basic Protein: Evidence for Thr‐Pro and Ser‐Arg‐X‐X‐Ser as Consensus Sequence Motifs
Author(s) -
Yu JauSong,
Yang ShiawDer
Publication year - 1994
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.1471-4159.1994.62041596.x
Subject(s) - phosphopeptide , edman degradation , gsk 3 , map2k7 , biochemistry , myelin basic protein , protein kinase a , mitogen activated protein kinase kinase , phosphorylation , biology , cyclin dependent kinase 2 , protein phosphorylation , microbiology and biotechnology , cyclin dependent kinase 9 , chemistry , peptide sequence , myelin , neuroscience , gene , central nervous system
In a previous study, protein kinase F A /glycogen synthase kinase‐3 ( F A /GSK‐3 ) was identified as a myelin basic protein (MBP) kinase associated with intact brain myelin. In this report, the phosphorylation sites of MBP by kinase F A /GSk‐3 were further determined by two‐dimensional electrophoresis/TLC, phosphoamino acid analysis, tryptic peptide mapping, Edman degradation, and direct sequencing. Kinase F A /GSK‐3 phosphorylates MBP on both threonine and serine residues. Three tryptic phosphopeptide peaks were resolved by C 18 reverse‐phase HPLC. Sequential manual Edman degradation together with direct sequence analysis revealed that T(p)PPPSQGK is the phosphorylation site sequence for the first major phosphopeptide peak. When mapping with the bovine brain MBP sequence, we finally demonstrate Thr 97 ‐Pro, one of the in vivo phosphorylation sites in MBP, as the major site phosphorylated by kinase F A /GSK‐3, implicating a physiologically relevant role of F A /GSK‐3 in the regulation of brain myelin function. By using the same approach, we also identified NIVT 94 (p)PR as the phosphorylation site sequence in the second major tryptic phosphopeptide derived from [ 32 P]MBP phosphorylated by kinase F A /GSK‐3, further indicating that kinase F A /GSK‐3 represents a Thr‐Pro motif‐directed MBP kinase involved in the phosphorylation of brain myelin.