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Phosphorylation of a 62 kd porcine alpha-internexin, a newly identified intermediate filament protein
1Department of Neurophysiology, Tokyo Metropolitan Institute of Gerontology, Japan.
Insights
Phosphorylation of alpha-internexin by protein kinase A inhibits its ability to form 10nm filaments. This suggests that phosphorylation controls the polymerization of alpha-internexin, a key brain protein.
Area of Science:
- Neuroscience
- Cell Biology
- Protein Biochemistry
Background:
- Alpha-internexin is a protein found in the brain.
- It is known to form filaments.
Purpose of the Study:
- To investigate the role of phosphorylation in alpha-internexin filament formation.
- To identify the specific sites of phosphorylation and their effect on filament assembly.
Main Methods:
- Purification of a 62 kDa protein from porcine brain white matter.
- In vitro filament formation assays.
- Phosphorylation using cAMP-dependent protein kinase.
- SDS-PAGE for mobility shift analysis.
- Peptide fragmentation and amino acid sequencing.
- Amino acid analysis of phosphopeptides.
Main Results:
- The purified 62 kDa protein formed 10nm filaments in vitro.
- Phosphorylation by cAMP-dependent protein kinase shifted the protein's molecular weight to 66 kDa and abolished filament formation.
- Peptide sequencing confirmed the protein's identity as alpha-internexin.
- Phosphorylation sites were localized to the amino-terminal head domain.
Conclusions:
- Porcine alpha-internexin polymerizes into 10nm filaments in vitro.
- Phosphorylation of the amino-terminal domain by cAMP-dependent protein kinase regulates alpha-internexin polymerizability.
Abstract:
A 62 kd protein was purified from the Triton-insoluble fraction of porcine brain white matter. This protein formed 10nm filaments, in vitro. The phosphorylation of the 62 kd protein by cAMP-dependent protein kinase caused electrophoretic mobility to shift to 66 kd on SDS-PAGE and a complete loss of the filament forming ability ensued. Amino acid sequences of four peptide fragments obtained from the 62 kd protein by lysylendopeptidase were identical with that of a 66 kd rat brain alpha-internexin. Amino acid analyses of the phosphopeptide fragment derived from phosphorylated porcine alpha-internexin revealed that the phosphorylation sites by cAMP-dependent protein kinase located in the amino-terminal head domain of this protein. These results strongly suggest that alpha-internexin polymerizes into 10nm filaments in vitro and that phosphorylation of the amino-terminal domain of alpha-internexin controls its polymerizability.