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Updated: Aug 14, 2026
Oligopeptide Competition Assay for Phosphorylation Site Determination
Published on: May 18, 2017
Peptide mapping of phosphorylated vimentin. Evidence for a site-specific alteration in mitotic cells
Abstract:
Vimentin, the subunit protein of one type of intermediate filament, has been isolated from 32Pi-labeled nonmitotic and mitotic mouse L-929 cells. Analysis of tryptic phosphopeptides by two-dimensional maps indicates that vimentin is phosphorylated at multiple sites in mitotic cells. Comparison of nonmitotic and mitotic vimentin phosphotryptic peptides indicated that in addition to the 6-7 major phosphorylated tryptic peptides found in nonmitotic cells, vimentin isolated from mitotic cells contained an additional 2 distinct phosphorylated peptides following trypsin digestion. Partial acid hydrolysis and one-dimensional phosphoamino acid analysis indicates that phosphoserine is present in all 9 major phosphopeptides. Treatment of L-929 cells with 8-bromo-cAMP did not result in a qualitative change in the phosphopeptide map of vimentin isolated from a normal cell population. These results suggest that the reorganization of vimentin filaments during mitosis is accompanied by a site-specific change in phosphorylation.
Insights
Vimentin phosphorylation changes during cell division. Mitotic cells show additional phosphorylation sites on vimentin, suggesting a role in filament reorganization during mitosis.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Vimentin is a key intermediate filament protein.
- Intermediate filaments play crucial roles in cellular structure and function.
- Understanding vimentin's regulation is vital for cell biology.
Purpose of the Study:
- To investigate vimentin phosphorylation patterns in mitotic versus nonmitotic cells.
- To identify specific phosphorylation sites altered during mitosis.
- To explore the relationship between vimentin phosphorylation and filament reorganization.
Main Methods:
- Isolation of vimentin from 32Pi-labeled mouse L-929 cells (mitotic and nonmitotic).
- Analysis of tryptic phosphopeptides using two-dimensional mapping.
- Partial acid hydrolysis and phosphoamino acid analysis.
Main Results:
- Vimentin is phosphorylated at multiple sites in mitotic cells.
- Mitotic vimentin exhibits 2 additional distinct phosphorylated peptides compared to nonmitotic vimentin.
- Phosphoserine is present in all identified major phosphopeptides.
- 8-bromo-cAMP treatment did not alter vimentin phosphopeptide maps.
Conclusions:
- Mitosis is associated with site-specific changes in vimentin phosphorylation.
- Altered vimentin phosphorylation likely accompanies filament reorganization during cell division.
