A molecular study on phospho-regulation of microtubule depolymerase MCAK via GTSE1

Microtubules (MT) are essential components of the cellular cytoskeleton. During cell division, they form a highly dynamic structure called the mitotic spindle that is necessary for proper alignment and segregation of chromosomes. Hence it becomes crucial for the cell to ensure that the microtubule dynamics are strictly regulated in a spatio-temporal manner. This regulation is often orchestrated by the interplay of various Microtubule Associated Proteins (MAPs) and mitotic kinases in a complex and poorly understood coordination. The kinesin 13 family proteins (Kif2A, Kif2B and Kif2C/MCAK) are MAPs that execute a regulatory function on MT dynamics via their potent MT depolymerase activity. MCAK is required to establish the length and stability of astral microtubules during spindle assembly as well as to facilitate any microtubule-kinetochore attachment error correction process. We previously discovered a novel MCAK-inhibiting protein, GTSE1, whose loss in cells causes microtubule destabilization, resulting in spindle positioning defects and chromosome mis-alignment. We do not, however, understand how GTSE1 inhibits MCAK activity mechanistically, nor how it is regulated in cells. Here, we aim to elucidate this inhibitory mechanism at the molecular level, which we suspect will define a novel mechanism for the poorly understood, yet essential, cellular control of the potent Kinesin 13 MT depolymerase activities. We found that the interaction of GTSE1 with MCAK requires phosphorylation of GTSE1 on its T165 site by either of the mitotic kinases Aurora A or Aurora B in vitro. Further examination revealed a differential regulation of MCAK activity by GTSE1 that is dependent on the phosphorylation status of GTSE1. Phosphorylated GTSE1 was found to modulate MCAK activity in a more nuanced manner, suggesting a finely tuned regulatory mechanism, which could be vital for maintaining the fidelity of mitotic processes. This work will further our understanding of the spatio-temporal regulation of microtubule dynamics in mitosis.