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Phosphorylation of Charge Isomers (Components) of Human Myelin Basic Protein: Identification of Phosphorylated Sites


Address correspondence and reprint requests to Dr. A. Mos- carello at Research Institute, The Hospital for Sick Children, Toronto, Ontario. Canada M5G 1X8.


Abstract: Myelin basic protein isolated from normal human brain was resolved into its various components (charge isomers) by CM-52 column chromatography. Two of the components, C-1 and C-4, were phosphorylated in vitro with a soluble preparation of brain protein kinase C. For each component, the peptides phosphorylated were identified. In both components a major site of phosphorylation was found at Ser7 in the N-terminal portion of the protein. Both the specific activity and the rate of phosphorylation were greatest at this site in both components when compared with the other sites. The rate of phosphorylation of peptide 5-I3 was ∼10 times greater than that of any of the other peptides derived from C-1, while the rate of phosphorylation of peptide 5-I3 derived from C-4 was 10-20 times greater than that of any of the other peptides derived from C-4. In addition, peptide 5-13, which contained a major phosphorylation site in both C-1 and C-4, was phosphorylated at a faster rate in C-4 (460 cpm/nM/min) compared with C-1 (285 cpm/nM/min). Both the specific activity and the rate data presented in the present communication were correlated with the proportion of p-structure in a previous study. In that study, C-1, which contained about 13% & structure before phosphorylation, increased to −40% after phosphorylation. Construction of a model peptide of this N-terminal region, which included the phosphorylation site at Ser7, demonstmted that the β structure was stabilized by electrostatic interactions between the phosphate on Ser7 and the guanidy groups of Arg5 and Arg9. On the other hand, phosphorylation of C-4, which was already −34% P-structure, was twice as fast at Ser7 as for C-I, suggesting that the preexistence of 8-structure in the molecule facilitated phosphorylation at this site. In previous studies phosphorylation of C-I was shown to decrease vesicle aggregation, which we concluded to be the result of electrostatic repulsion between the phosphate on the protein and that of the lipid. The present data suggest that phosphorylation of some specific site may be playing an important role in the conformation of the protein.