Nek6 controls mitotic progression through regulating EML3 localisation to spindle microtubules

• Main Author: Montgomery, Jessica M.
• Other Authors: Fry, Andrew
• Published: University of Leicester 2016
• Subjects: 572
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.689351

EMLs are a highly conserved family of microtubule-associated proteins that play a role in microtubule stability. In humans there are six EMLs. EML1, EML2, EML3 and EML4 consist of a largely unstructured basic N-terminal domain (NTD) that contains a short coiled-coil mediating trimerisation, and a highly structured C-terminal domain (CTD) named the TAPE domain. Microtubule binding is conferred through the NTD whilst the TAPE domain binds to soluble tubulin dimers. EML5 and EML6 lack the N-terminal region but have three continuous TAPE domains encoded within a single polypeptide. Previous proteomic studies had identified EML3 as a binding partner of Nek6, a serine/threonine kinase that promotes mitotic spindle assembly. In this study, we have explored the microtubule binding properties of EML3 and its regulation by Nek6. Using a stable cell line expressing YFP-EML3, fixed and time lapse imaging revealed that EML3 associates along the length of microtubules and exhibits rapid recovery following photobleaching. Whilst microtubule binding of the related EML1-NTD was reduced upon incubation of microtubules with the protease subtilisin in vitro. This suggests an electrostatic interaction between the basic EML-NTD and the acidic tubulin C-terminal tails. Immunoprecipitation experiments confirmed the interaction between EML3 and Nek6 and revealed that this interaction is increased in mitosis. Unexpectedly, while EML3 bound strongly to interphase microtubules, it exhibited much lower affinity for microtubules in mitosis. Depletion of Nek6 led to accumulation of EML3 on spindle microtubules in mitosis, whilst expression of a constitutively active Nek6 displaced EML3 from interphase microtubules. By mass spectrometry a number of phosphorylation sites were identified within the basic NTD of EML3 upon incubation with Nek6. Furthermore, introduction of acidic charge into the basic NTD of EML3 perturbed its association with interphase microtubules. Hence, we propose that phosphorylation of EML3 by Nek6 disturbs electrostatic interactions between the basic N-terminus and the acidic surface of MTs. This leads to reduced association of EML3 with microtubules in mitosis. We speculate that this decreases microtubule stability, facilitating mitotic spindle assembly and search and capture of condensed chromosomes.