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 FJglycogen synthase kinase-3 (F,/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,/GSK-3 were further determined by two-dimensional electrophoresis/TLC, phosphoamino acid analysis, tryptic peptide mapping, Edman degradation, and direct sequencing. Kinase F,/GSK-3 phosphorylates MBP on both threonine and serine residues. Three tryptic phosphopeptide peaks were resolved by C,8 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 Thrg7-Pro, one of the in vivo phosphorylation sites in MBP, as the major site phosphorylated by kinase FJGSK-3, implicating a physiologically relevant role of F,/GSK-3 in the regulation of brain myelin function. By using the same approach, we also identified NIVTg4(p)PR as the phosphorylation site sequence in the second major tryptic phosphopeptide derived from [32P]MBP phosphorylated by kinase FJGSK-3, further indicating that kinase FJ GSK-3 represents a Thr-Pro motif-directed MBP kinase involved in the phosphorylation of brain myelin. Key Words: Myelin basic protein-Protein kinase F,/glycogen synthase kinase-3-Phosphorylation site-Consensus sequence motif. J. Neurochern. 62, 1596-1 603 (1 994).

Regulation of c-Myc Protein Abundance by a Protein Phosphatase 2A-Glycogen Synthase Kinase 3 -Negative Feedback Pathway

“…35 As shown in Figure 1D, GSK3β derived from P493-6 cells grown in decreasing amounts of Tet displayed significantly increased kinase activity. This activity was inhibited by treatment with the specific GSK3β inhibitor BIO.…”

Classic 18.5- and 21.5-kDa Myelin Basic Protein Isoforms Associate with Cytoskeletal and SH3-Domain Proteins in the Immortalized N19-Oligodendroglial Cell Line Stimulated by Phorbol Ester and IGF-1

“…The PMA-stimulation also induced phosphorylation of MBP at Thr98, the site phosphorylated by both MAPK and GSK-3β [56,57]. (This threonyl residue in the human 18.5-kDa MBP sequence corresponds to residues Thr95/Thr97 in the murine/bovine 18.5-kDa MBP sequences, respectively [11,15,35,74]).…”

“…Using a phospho-specific antibody toward Thr98 (human 18.5-kDa sequence; bovine Thr97; murine Thr95) of MBP, an identified MAPK [56] and GSK-3β [57] phosphorylation site, we found that there was an overall increase in Thr98-phosphorylated MBP-C1 in PMA-stimulated N19-OLGs, including at ruffling sites (Fig. 2b).…”

Myelin basic protein: a multifunctional protein

“…MBP can be phosphorylated in vitro by protein kinase C, protein kinase A, glycogen synthase kinase (GSK), calmodulin-dependent kinase, and mitogen-activated protein kinase (MAPK), enzymes all present in myelin [4,[78][79][80][81][82]. The in vitro sites of phosphorylation differ in some cases from those found to be phosphorylated in myelin MBP in vivo [56,67], but one of the in vivo sites, Thr97 (bovine sequence), is phosphorylated by MAPK and GSK in vitro [78,79]. However, the sites of phosphorylation of MBP in OLs, as opposed to mature myelin, are not known and may include some of the in vitro phosphorylation sites.…”