Autophosphorylation-dependent protein kinase predominantly phosphorylates Ser115, thein vivo site in brain myelin basic protein

In a previous report [Yanget al., (1987a),J. Biol Chem.262, 7034–7040], a cyclic-AMP- and calcium-independent brain kinase which requires autophosphorylation for activity was identified as a very potent myelin basic protein (MBP) kinase. In this report, the phosphorylation sites of MBP by this autophosphorylation-dependent protein kinase (autokinase) are further determined by two-dimensional electrophoresis/thin-layer chromatography, phosphoamino acid analysis, high-performance liquid chromatography, tryptic peptide mapping, sequential manual Edman degradation, and direct peptide sequencing. Autokinase phosphorylates MBP on both threonine and serine residues. Three major tryptic phosphopeptide peaks were resolved by C₁₈-reversed phase highper-formance liquid chromatography. Sequential manual Edman degradation together with direct sequence analysis revealed that FS(p)WGAEGQKPGFGYGGR is the phosphorylation site sequence (molar ratio 1.0) for the first major phosphopeptide peak. When mapping with bovine brain MBP sequence, we finally demonstrate Ser¹¹⁵, one of thein vivo phosphorylation sites in MBP, as the major site phosphorylated by autokinase, implicating a physiologically relevant role of autokinase in the regulation of brain myelin function. By using the same approach, we also identified HRDT(p)GILDSLGR (molar ratio 0.9) and TT(p)HYGSLPQK (molar ratio 0.8) as the major phosphorylation site sequences in³²P-MBP phosphorylated by autokinase, further indicating that -Arg-XSer/Thr-(neutral amino acid)₃-(amino acid-containing hydroxyl group such as Ser/Glu/Asp)-(neutral amino acid)₂-may represent a unique consensus sequence motif specifically recognized by this autophosphorylation-dependent multisubstrate/ multifunctional protein kinase in the brain.