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Endogenous phosphorylation of basic protein in myelin of varying degrees of compaction
P Schulz1, T F Cruz, M A Moscarello
1Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
Abstract:
Fractions containing myelin of varying degrees of compaction were prepared from human white matter. Protein kinase activity in these fractions was measured by using both endogenous and exogenous myelin basic protein (MBP) as substrates. In both cases, less compact myelin fractions possessed higher levels of protein kinase activity than the compact myelin fraction. In addition, the specific activity of phosphorylated basic protein was greater in the loosely compacted fractions than in compact multilamellar myelin. When basic protein in compact myelin or the myelin fractions was phosphorylated by the endogenous kinase, approximately 70% of the [32P]phosphate was incorporated at a single site, identified as Ser-102. The remaining 30% was found in three other minor sites. Electron microscopy of less compact myelin showed it was composed of fewer lamellae which correlated with a relative decrease in the proportion of cationic charge isomers (microheteromers) when MBP was subjected to gel electrophoresis at alkaline pH. The shift in charge microheterogeneity of basic protein to the less cationic isomers in the less compact myelin fractions correlated with an increase in protein kinase activity and a greater specific activity of phosphorylated basic protein.
Insights
Less compact myelin fractions exhibit higher protein kinase activity and increased phosphorylation of myelin basic protein (MBP) compared to compact myelin. This suggests a link between myelin structure, protein kinase activity, and MBP phosphorylation.
Area of Science:
- Neuroscience
- Biochemistry
- Cell Biology
Background:
- Myelin, a lipid-rich sheath, insulates nerve fibers in the central nervous system.
- Myelin basic protein (MBP) is a major component of myelin, crucial for its structure and stability.
- Protein kinases play vital roles in cellular signaling and protein modification, including phosphorylation.
Purpose of the Study:
- To investigate the relationship between myelin compaction and protein kinase activity.
- To determine the impact of myelin structure on the phosphorylation of myelin basic protein (MBP).
- To identify phosphorylation sites on MBP within different myelin fractions.
Main Methods:
- Preparation of human white matter myelin fractions with varying degrees of compaction.
- Assay of endogenous and exogenous protein kinase activity using MBP as a substrate.
- Phosphorylation site analysis of MBP using radiolabeled phosphate ([32P]).
- Electron microscopy to assess myelin lamellar structure.
- Gel electrophoresis to analyze MBP charge microheterogeneity.
Main Results:
- Less compact myelin fractions showed significantly higher protein kinase activity than compact myelin.
- The specific activity of phosphorylated MBP was greater in loosely compacted fractions.
- Phosphorylation predominantly occurred at Ser-102 on MBP, with minor phosphorylation at other sites.
- Reduced lamellarity in less compact myelin correlated with altered MBP charge microheterogeneity and increased kinase activity.
Conclusions:
- Myelin compaction influences protein kinase activity and MBP phosphorylation patterns.
- Alterations in MBP charge microheterogeneity in less compact myelin are associated with increased protein kinase activity.
- These findings suggest a dynamic interplay between myelin structure and enzymatic modifications of its components.