Dissecting the molecular mechanism of microtubule depolymerising kinesinsTools Belsham, Hannah R. (2020) Dissecting the molecular mechanism of microtubule depolymerising kinesins. PhD thesis, University of Nottingham.
AbstractMitotic Centromere Associated Kinesin (MCAK) is a microtubule depolymerase from the Kinesin-13 family of molecular motors. In cells it plays an important role in regulating microtubule length and in ensuring replicated chromosomes are accurately divided between the two daughter cells during mitosis. MCAK activity is tightly regulated and here I show how phosphorylation of MCAK, at different sites by two different kinases, acts to regulate MCAK’s activity through two distinct mechanisms. Firstly I show how phosphorylation of MCAK at T537 by Cdk1 prevents MCAK from recognising the microtubule end, a key function for its depolymerisation activity. Secondly, while phosphorylation of MCAK at S621 by Plk1 has been shown to lead to a reduction in MCAK’s activity in cells, I show that this is not reproduced in vitro. In this case phosphorylation affects MCAK’s activity in cells by affecting the rate of degradation of MCAK. Finally I introduce three MCAK residues, important in Kinesin-13 for microtubule end recognition, into Kinesin-1, at the core interface between the kinesin motor domain and the microtubule. This induces this translocating kinesin to pause at the microtubule end. Together, these data provide further information on both the depolymerisation mechanism of MCAK and the structural distinctions between microtubule regulating and translocating kinesins.
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