Protein Kinase F_A/Glycogen Synthase Kinase‐3 Predominantly Phosphorylates the In Vivo Site Thr⁹⁷‐Pro in Brain Myelin Basic Protein: Evidence for Thr‐Pro and Ser‐Arg‐X‐X‐Ser as Consensus Sequence Motifs

Abstract: 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 C₁₈ 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 Thr⁹⁷‐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 NIVT⁹⁴(p)PR as the phosphorylation site sequence in the second major tryptic phosphopeptide derived from [³²P]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.