EML4: Cell Cycle-Dependent Regulation And Function

Abstract

EMLs are a highly conserved family of microtubule-associated proteins. However, whether and how they regulate microtubule dynamics remains far from clear. Human cells express six members of this family, EML1 to EML6. EML1 to 4 have a similar canonical structure, consisting of an N-terminal region containing a coiled-coil trimerization motif and C-terminal tandem b-propeller (TAPE) domain, whereas EML5 and EML6 lack the coiled-coil but have three repeats of the TAPE domain. Structure-function studies have revealed that the TAPE domain allows interaction with soluble tubulin dimers, whereas the N-terminal region of EML1 to 4 including the coiled-coil promotes microtubule binding. In this project, we explored the microtubule binding of endogenous EML4 and its regulation by Nek mitotic kinases. Using localization and depletion approaches, we found that EML4 decorates the interphase microtubule lattice as punctate foci, while it exhibits reduced binding affinity to spindle microtubules. We also showed that endogenous EML4 stabilises microtubules, promotes microtubule acetylation and detyrosination, and protects microtubules from nocodazoleinduced depolymerisation. Depletion of EML4 disturbs mitotic spindle organisation and results in loss of K-fibres suggesting that it also stabilises microtubules in mitosis. We discovered that phosphorylation of EML4 within its N-terminal region at S144 and S146 by the Nek6 and Nek7 mitotic kinases weakens the affinity of EML4 for microtubules. We propose that this phosphorylation of EML4 by Nek kinases disturbs electrostatic interactions between the basic EML4 N-terminal region and the acidic C-terminal tubulin tails exposed on the surface of microtubules. This leads to reduced association of EML4 with microtubules in mitosis enabling the increase in microtubule dynamics that is required for chromosome capture and congression. Consistent with this model, overexpression of an EML4 phosphonull mutant, S144/146A, led to inappropriate retention of EML4 on spindle microtubules disrupting spindle organization and chromosome congresssion