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Updated: May 24, 2026
Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins
Published on: March 3, 2016
Control of MCAK degradation and removal from centromeres
Anutosh Ganguly1, Rajat Bhattacharya, Fernando Cabral
1Department of Integrative Biology and Pharmacology, University of Texas Medical School, Houston, Texas 77030, USA.
Abstract:
Mitotic centromere associated kinesin (MCAK) is a kinesin related protein with the ability to stimulate microtubule depolymerization. It is found at spindle poles, where it may be involved in poleward microtubule flux, and at kinetochores and centromeres where it plays a role in correcting chromosome alignment errors. Its microtubule depolymerase activity and recruitment to centromeres is regulated by phosphorylation, but little is known about how MCAK is maintained at appropriate levels. We previously reported that MCAK accumulates during the cell cycle and is then degraded during mitosis. Using proteomic analysis, we have now identified a new phosphorylation site on MCAK that is responsible for its degradation. Mutation of the site to prevent phosphorylation prolonged the stability of the protein beyond the metaphase to anaphase transition and into the subsequent cell cycle whereas a phosphomimetic mutation accelerated degradation. Unexpectedly, the mutation that prevented phosphorylation also inhibited the removal of MCAK from centromeres causing it to remain attached throughout the cell cycle. Even low expression of phosphorylation-resistant MCAK delayed mitosis and interfered with cell division. Mitotic defects were also observed by overexpressing a green fluorescent protein-tagged version of wild-type MCAK that similarly escaped degradation and accumulated to toxic levels, but did not remain associated with kinetochores during interphase. The results demonstrate that degradation is an important mechanism for controlling the activity of MCAK.
Insights
Degradation of mitotic centromere-associated kinesin (MCAK) controls its activity. A newly identified phosphorylation site drives MCAK degradation, and preventing it causes mitotic defects and cell division interference.
Area of Science:
- Cell Biology
- Molecular Biology
- Kinesin motor proteins
Background:
- Mitotic centromere-associated kinesin (MCAK) regulates microtubule dynamics and chromosome alignment.
- MCAK levels and activity are tightly controlled, but the mechanisms are not fully understood.
- Previous work showed MCAK accumulates during the cell cycle and degrades during mitosis.
Purpose of the Study:
- Identify the molecular mechanisms regulating MCAK degradation.
- Investigate the role of phosphorylation in MCAK stability and function.
- Determine the consequences of altered MCAK degradation on mitosis and cell division.
Main Methods:
- Proteomic analysis to identify phosphorylation sites on MCAK.
- Site-directed mutagenesis to create phosphorylation-deficient and phosphomimetic MCAK variants.
- Microscopy and cell cycle analysis to assess MCAK localization, stability, and mitotic progression.
- Expression of wild-type and mutant MCAK in cells.
Main Results:
- A novel phosphorylation site on MCAK was identified as crucial for its degradation.
- Mutating this site to prevent phosphorylation stabilized MCAK beyond mitosis and caused it to remain at centromeres.
- Phosphorylation-resistant MCAK expression delayed mitosis and disrupted cell division.
- Overexpression of wild-type MCAK also led to mitotic defects due to accumulation.
Conclusions:
- Phosphorylation-dependent degradation is a key mechanism for controlling MCAK activity and ensuring proper cell division.
- Dysregulation of MCAK degradation leads to significant mitotic errors.
- Targeting MCAK degradation pathways could offer therapeutic strategies for mitotic disorders.
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