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.
Highlights
Mitotic Centromere Associated Kinesin (MCAK) is a member of the kinesin-13 family of microtubule depolymerising kinesins
These data indicate that the phosphomimic mutant folds correctly as it turns over ATP at a similar rate to wild-type both in solution and in the presence of unpolymerized tubulin
The difference between the ATPase rates in the presence of unpolymerized tubulin compared to the presence of microtubules for wild-type MCAK has previously been shown to be due to the acceleration of ADP dissociation caused by microtubule ends (Friel & Howard, 2011)
Summary
Mitotic Centromere Associated Kinesin (MCAK) is a member of the kinesin-13 family of microtubule depolymerising kinesins. MCAK plays crucial roles in the cell cycle both in building the mitotic spindle and in correcting erroneous microtubule-kinetochore attachments Both the localisation and depolymerisation activity of MCAK must be tightly regulated throughout the cell cycle. MCAK is regulated through the action of various mitotic kinases, including the aurora kinases, polo like kinase 1 and p21 activated kinase 1 (Andrews et al, 2004; Lan et al, 2004; Pakala et al, 2012; Zhang et al, 2011; Zhang, Ems-McClung & Walczak, 2008) These kinases phosphorylate MCAK at various sites within the N and C-terminal domains and in the neck region. A phosphomimic mutant at this position, T537E, has reduced depolymerisation activity and overexpression of this mutant in cells leads to misaligned chromosomes, metaphase arrest and reduced intercentromeric distances (Sanhaji et al, 2010)
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