The NIMA-family kinase Nek6 phosphorylates the kinesin Eg5 at a novel site necessary for mitotic spindle formation

Abstract

Summary Nek6 and Nercc1/Nek9 belong to the NIMA family of protein kinases. Nercc1 is activated in mitosis whereupon it binds, phosphorylates and activates Nek6. Interference with Nek6 or Nercc1 in mammalian cells causes prometaphase/metaphase arrest, and depletion of XNercc from Xenopus egg extracts prevents normal spindle assembly. Herein we show that Nek6 is constitutively associated with Eg5, a kinesin necessary for spindle bipolarity. Nek6 phosphorylates Eg5 at several sites in vitro, and one of these sites, Ser1033, is phosphorylated in vivo during mitosis. While Cdk1 phosphorylates nearly all Eg5 during mitosis at Thr926, Nek6 phosphorylates ~3% of Eg5, primarily at the spindle poles. Eg5 depletion arrests cells with a monopolar spindle; this can be rescued by Eg5 wildtype but not by Eg5(Thr926Ala). Eg5(Ser1033Ala) rescues half as well as wildtype whereas an Eg5(Ser1033Asp) mutant is nearly as effective. Thus Nek6 phosphorylates a subset of Eg5 polypeptides during mitosis at a conserved site, whose phosphorylation is critical for the mitotic function of Eg5.

Nek7 kinase targeting leads to early mortality, cytokinesis disturbance and polyploidy

“…The higher incidence of chromosomal lagging and micronuclei formation in the mutant cells could be attributed to defects in mitotic spindle formation and organization. Notably, in this regard, it has been demonstrated recently that Nek6 interacts and can phosphorylate the bipolar mitotic kinesin Eg5 at ser1033 (Rapley et al, 2008). The mutation of ser1033 resulted in monopolar spindle formation (Rapley et al, 2008).…”

“…Notably, in this regard, it has been demonstrated recently that Nek6 interacts and can phosphorylate the bipolar mitotic kinesin Eg5 at ser1033 (Rapley et al, 2008). The mutation of ser1033 resulted in monopolar spindle formation (Rapley et al, 2008). Given the high similarity between Nek6 and Nek7, it is plausible that Eg5 is also targeted by Nek7 kinase.…”

DYNLL/LC8 Protein Controls Signal Transduction through the Nek9/Nek6 Signaling Module by Regulating Nek6 Binding to Nek9

“…Plasmids and Reagents-Different Nek9 and Nek6 expression plasmids have been described elsewhere (13,15,35). cDNAs coding for the human Nek9 Kin (1-346), RCC1 (347-726), and Ct (721-979) were subcloned into pGBKT7 (Clontech).…”

“…Although it is not clear whether both kinases have specific roles in different cell types, they seem to be functionally equivalent in most instances; thus, when adequate, the two kinases will be collectively referred to as Nek6/7.) In vitro, Nek9 is capable of autoactivating through autophosphorylation; in vivo, the kinase is inactive during interphase and is activated at centrosomes and spindle poles during mitosis, when it binds Nek6 and Nek7 and is then able to directly phosphorylate these kinases in the activation loop, in turn inducing their activation (13)(14)(15). Nek6/7 binding to Nek9 has in addition been reported to directly increase the activity of Nek6 and Nek7 by disrupting an autoinhibited conformation of these kinases (16).…”

“…3 Despite the proven importance of Nek9, Nek6, and Nek7 for proper progression through mitosis, a precise description of their physiological roles is presently missing. We have suggested that Nek9 could be controlling spindle organi- zation in part through the action of the downstream Nek6 and Nek7 and their ability to phosphorylate the kinesin Eg5 at a site necessary for normal mitotic progression (15), but possibly more substrates of the signaling module await to be discovered. LC8 is a cytoplasmic, ubiquitous, essential, and extraordinarily conserved protein (ϳ90% sequence identity between homologues from mammalians, Drosophila, Caenorhabditis, and Chlamydomonas) that interacts with many different proteins and protein complexes (LC8a and LC8b are highly similar and collectively referred to LC8 herein).…”