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Phosphorylation-related accumulation of the 125K nuclear matrix protein mitotin in human mitotic cells
N Z Zhelev1, I T Todorov, R N Philipova
1Department of Molecular Genetics, Bulgarian Academy of Sciences, Sofia.
Abstract:
The preparation of mammalian cells for entry into mitosis is related to a cascade of G2 phase phosphorylations of several nuclear proteins driven by mitosis-specific protein kinases. Using a monoclonal antibody we have identified previously in mammalian cells a 125K/pI6.5 protein, associated with the nuclear matrix, and markedly increased in mitotic cells, which was named 'mitotin'. Here, we show by short-term [35S]methionine labeling of cell cycle synchronized cells that this protein is synthesized at comparable rates throughout interphase. However, upon cycloheximide block of protein synthesis mitotin labeled during S phase is rapidly degraded, while the degradation of mitotin labeled during late G2 phase is abolished, resulting in its net and marked increase. The accumulation of mitotin in premitotic and mitotic cells is related to its phosphorylation and the metabolic stability of its two phosphorylated forms. The metabolic stabilization and accumulation of a nuclear matrix protein upon phosphorylation suggests the operation of a novel mechanism among the complex events preparing the cell for mitosis.
Insights
Mitotin, a nuclear matrix protein, accumulates in mitotic cells due to selective degradation during the G2 phase. Phosphorylation stabilizes mitotin, suggesting a novel mechanism for cell division preparation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Cell preparation for mitosis involves G2 phase protein phosphorylation.
- A 125K/pI6.5 nuclear matrix protein, 'mitotin', increases in mitotic cells.
Purpose of the Study:
- Investigate the synthesis and degradation of mitotin during the cell cycle.
- Elucidate the role of mitotin phosphorylation in its accumulation during mitosis.
Main Methods:
- Synchronized mammalian cell cultures.
- [35S]methionine labeling to track protein synthesis.
- Cycloheximide treatment to block protein synthesis and assess degradation.
- Monoclonal antibody for mitotin detection.
Main Results:
- Mitotin is synthesized at constant rates throughout interphase.
- Mitotin labeled during S phase is rapidly degraded.
- Degradation of mitotin labeled during late G2 is inhibited, leading to accumulation.
- Phosphorylation of mitotin correlates with its metabolic stabilization and accumulation.
Conclusions:
- Mitotin accumulation in mitosis is regulated by cell cycle-dependent degradation and stabilization.
- Phosphorylation of mitotin on the nuclear matrix is linked to its stability.
- This suggests a novel mechanism for regulating nuclear matrix proteins during mitotic entry.