Mitotic phosphorylation by NEK6 and NEK7 reduces the microtubule affinity of EML4 to promote chromosome congression

Abstract

EML4 is a microtubule-associated protein that promotes microtubule stability. We investigated its regulation across the cell cycle and found that EML4 was distributed as punctate foci along the microtubule lattice in interphase but exhibited reduced association with spindle microtubules in mitosis. Microtubule sedimentation and cryo-electron microscopy with 3D reconstruction revealed that the basic N-terminal domain of EML4 mediated its binding to the acidic C-terminal tails of α-and β-tubulin on the microtubule surface. The mitotic kinases NEK6 and NEK7 phosphorylated the EML4 N-terminal domain at Ser 144 and Ser 146 in vitro, and depletion of these kinases in cells led to increased EML4 binding to microtubules in mitosis. An S144A-S146A double mutant not only bound inappropriately to mitotic microtubules but also increased their stability and interfered with chromosome congression.Meanwhile, constitutive activation of NEK6 or NEK7 reduced EML4 association with interphase microtubules. Together, these data support a model in which NEK6-and NEK7dependent phosphorylation promotes dissociation of EML4 from microtubules in mitosis in a manner that is required for efficient chromosome congression.! Adib et al.

EML4–ALK V3 oncogenic fusion proteins promote microtubule stabilization and accelerated migration through NEK9 and NEK7

“…S4A-C). Previous large-scale proteomic mapping had identified EML2, EML3 and EML4 as potential binding partners of NEK6 (Ewing et al, 2007), while we have recently found that EML4 can be phosphorylated by NEK6 and NEK7 in mitosis (Adib et al, 2019). Western blotting of immunoprecipitates prepared using antibodies against either NEK9 or EML4 confirmed an interaction between endogenous EML4 and NEK9 in extracts prepared from asynchronous U2OS cells (Fig.…”

The tubulin code and its role in controlling microtubule properties and functions

“…In the past ten years many novel structures of MAPs bound to the microtubule lattice have been solved (for example ref. 73,[117][118][119][120][121][122][123][124] ). As these structures reveal the precise interaction sites, i.e.…”

Physiological and Pathological Roles of Mammalian NEK7

“…NEK7 binds to and phosphorylates RGS2 and leads to the localization of RGS2 to the mitotic spindle, which is required for proper mitotic spindle organization and spindle orientation (de Souza et al, 2015). In addition, NEK6 and NEK7 promote the dissociation of EML4 from microtubules in mitosis by phosphorylating the EML4 N-terminal domain at Ser144 and Ser146, which is required for efficient chromosome congression in interphase (Adib et al, 2019). As a potential function of NEK7 in microtube regulation (Cohen et al, 2013), NEK7 was identified to regulate dendrite growth and branching, as well as spine formation and morphology, in part through phosphorylation of the kinesin Eg5/KIF11 (Freixo et al, 2018).…”