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Regulation of a major microtubule-associated protein by MPF and MAP kinase
N Shiina1, T Moriguchi, K Ohta
1Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Japan.
Abstract:
The interphase-M phase transition of microtubule dynamics is thought to be induced by phosphorylation reactions mediated by MPF and by MAP kinase functioning downstream of MPF. We have now identified and purified from Xenopus eggs a major microtubule-associated protein, p220, that may be a target protein for these two M phase-activated kinases. p220, when purified from interphase cells, potently bound to microtubules and stimulated tubulin polymerization, whereas p220 purified from M phase cells showed little or no such activities. Cell staining with a monoclonal anti-p220 antibody revealed that p220 is localized on cytoplasmic microtubule networks during interphase, while it is distributed rather diffusely throughout the cell during M phase. We have further found that p220 is phosphorylated specifically in M phase. Moreover, p220 purified from interphase cells served as a good substrate for MAP kinase and MPF in vitro, and two-dimensional phosphopeptide mapping pattern of the p220 phosphorylated in vitro was very similar to that of p220 phosphorylated at M phase in vivo. These results suggest that the drastic change in p220 activity during the transition from interphase to M phase may be induced by its phosphorylation in M phase probably catalyzed by MAP kinase and MPF.
Insights
A key microtubule-associated protein, p220, changes activity between cell cycle phases. Phosphorylation by MPF and MAP kinase in M phase inactivates p220, altering microtubule dynamics.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Microtubule dynamics are crucial for cell division, regulated by protein phosphorylation.
- Maturation-promoting factor (MPF) and MAP kinase are key regulators of M phase entry.
Purpose of the Study:
- To identify and characterize proteins involved in regulating microtubule dynamics during the cell cycle.
- To investigate the role of a novel microtubule-associated protein, p220, in the interphase-M phase transition.
Main Methods:
- Purification of p220 from Xenopus eggs at different cell cycle stages.
- In vitro kinase assays using MPF and MAP kinase.
- Immunofluorescence microscopy to determine p220 localization.
- Two-dimensional phosphopeptide mapping.
Main Results:
- p220 binds microtubules and promotes polymerization in interphase but not M phase.
- p220 is phosphorylated specifically during M phase.
- In vitro phosphorylation of p220 by MPF and MAP kinase mimics in vivo M phase phosphorylation.
- p220 localization shifts from cytoplasmic networks in interphase to diffuse in M phase.
Conclusions:
- p220 is a direct target of MPF and MAP kinase.
- Phosphorylation of p220 by MPF and MAP kinase during M phase leads to its inactivation.
- This phosphorylation-dependent inactivation of p220 contributes to the regulation of microtubule dynamics during the cell cycle.