Autophosphorylation-dependent protein kinase predominantly phosphorylates Ser115, the in vivo site in brain myelin basic protein.
In a previous report [Yang et 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 high-performance liquid chromatography. Sequential manual Edman degradation together with direct sequence analysis revealed that FS(p)WGAEGQKPGFGYGGR is the phosphorylation site sequence (molar ratio approximately 1.0) for the first major phosphopeptide peak. When mapping with bovine brain MBP sequence, we finally demonstrate Ser115, one of the in 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 approximately 0.9) and TT(p)HYGSLPQK (molar ratio approximately 0.8) as the major phosphorylation site sequences in 32P-MBP phosphorylated by autokinase, further indicating that -Arg-X-Ser/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.
Yang
S D
SD
Institute of Biomedical Sciences, National Tsing Hua University, Hsinchu, Taiwan.
Huang
T J
TJ
Huang
J J
JJ
D000595:Amino Acid Sequence; D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D012694:Serine / Q000378:metabolism*; D013552:Swine
D004676:Myelin Basic Protein; D012694:Serine; D017346:Protein Serine-Threonine Kinases