A Cdk1 phosphomimic mutant of MCAK impairs microtubule end recognition
Hannah R Belsham1, Claire T Friel1
1School of Life Sciences, University of Nottingham, Nottingham, United Kingdom.
Abstract:
The microtubule depolymerising kinesin-13, MCAK, is phosphorylated at residue T537 by Cdk1. This is the only known phosphorylation site within MCAK's motor domain. To understand the impact of phosphorylation by Cdk1 on microtubule depolymerisation activity, we have investigated the molecular mechanism of the phosphomimic mutant T537E. This mutant significantly impairs microtubule depolymerisation activity and when transfected into cells causes metaphase arrest and misaligned chromosomes. We show that the molecular mechanism underlying the reduced depolymerisation activity of this phosphomimic mutant is an inability to recognise the microtubule end. The microtubule-end residence time is reduced relative to wild-type MCAK, whereas the lattice residence time is unchanged by the phosphomimic mutation. Further, the microtubule-end specific stimulation of ADP dissociation, characteristic of MCAK, is abolished by this mutation. Our data shows that T537E is unable to distinguish between the microtubule end and the microtubule lattice.
Insights
Phosphorylation of kinesin-13, MCAK, at T537 by Cdk1 impairs microtubule depolymerization. The T537E mutant cannot recognize microtubule ends, causing cell cycle arrest.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Microtubule dynamics are crucial for cell division.
- Kinesin-13 family motor proteins, like MCAK, depolymerize microtubules.
- Phosphorylation regulates motor protein function.
Purpose of the Study:
- Investigate the effect of Cdk1-mediated phosphorylation at T537 on MCAK activity.
- Elucidate the molecular mechanism of MCAK's reduced depolymerization.
- Understand the role of T537 phosphorylation in cell cycle regulation.
Main Methods:
- Site-directed mutagenesis to create T537E phosphomimic mutant.
- In vitro assays measuring microtubule depolymerization rates.
- Analysis of MCAK's binding and dissociation kinetics on microtubules.
- Cell transfection to observe effects on mitosis.
Main Results:
- The T537E mutant significantly reduces microtubule depolymerization activity.
- MCAK T537E shows impaired recognition of microtubule ends.
- Residence time at microtubule ends is decreased, while lattice binding is unaffected.
- The characteristic stimulation of ADP dissociation at microtubule ends is abolished in T537E.
- Transfection of T537E causes metaphase arrest and chromosome misalignment.
Conclusions:
- Phosphorylation of MCAK at T537 by Cdk1 is critical for its microtubule depolymerization function.
- The T537E mutation disrupts MCAK's ability to distinguish microtubule ends from the lattice.
- Dysregulation of MCAK activity by T537 phosphorylation impacts mitotic progression and chromosome alignment.
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