The protein kinase NIMA is an indispensable pleiotropic regulator of mitotic progression in Aspergillus. Although several mammalian NIMA-like kinases (Neks) are known, none appears to have the broad importance for mitotic regulation attributed to NIMA. Nercc1 is a new NIMA-like kinase that regulates chromosome alignment and segregation in mitosis. Its NIMA-like catalytic domain is followed by a noncatalytic tail containing seven repeats homologous to those of the Ran GEF, RCC1, a Ser/Thr/Pro-rich segment, and a coiled-coil domain. Nercc1 binds to another NIMA-like kinase, Nek6, and also binds specifically to the Ran GTPase through both its catalytic and its RCC1-like domains, preferring RanGDP in vivo. Nercc1 exists as a homooligomer and can autoactivate in vitro by autophosphorylation. Nercc1 is a cytoplasmic protein that is activated during mitosis and is avidly phosphorylated by active p34 Cdc2 . Microinjection of anti-Nercc1 antibodies in prophase results in spindle abnormalities and/or chromosomal misalignment. In Ptk2 cells the outcome is prometaphase arrest or aberrant chromosome segregation and aneuploidy, whereas in CFPAC-1 cells prolonged arrest in prometaphase is the usual response. Nercc1 and its partner Nek6 represent a new signaling pathway that regulates mitotic progression.

The NIMA-family kinases Nek9/Nercc1, Nek6 and Nek7 form a signalling module required for mitotic spindle assembly. Nek9, the upstream kinase, is activated during prophase at centrosomes although the details of this have remained elusive. We now identify Plk1 as Nek9 direct activator and propose a two-step activation mechanism that involves Nek9 sequential phosphorylation by CDK1 and Plk1. Furthermore, we show that Plk1 controls prophase centrosome separation through the activation of Nek9 and ultimately the phosphorylation of the mitotic kinesin Eg5 at Ser1033, a Nek6/7 site that together with the CDK1 site Thr926 we establish contributes to the accumulation of Eg5 at centrosomes and is necessary for subsequent centrosome separation and timely mitosis. Our results provide a basis to understand signalling downstream of Plk1 and shed light on the role of Eg5, Plk1 and the NIMA-family kinases in the control of centrosome separation and normal mitotic progression.

During interphase in Drosophila neuroblasts, the Centrobin (CNB)-positive daughter centriole retains pericentriolar material (PCM) and organizes an aster that is a key determinant of the orientation of cell division. Here we show that daughter centrioles depleted of CNB cannot fulfil this function whereas mother centrioles that carry ectopic CNB can. CNB co-precipitates with a set of centrosomal proteins that include γ-TUB, ANA2, CNN, SAS-4, ASL, DGRIP71, POLO and SAS-6. Following chemical inhibition of POLO or removal of three POLO phosphorylation sites present in CNB, the interphase microtubule aster is lost. These results demonstrate that centriolar CNB localization is both necessary and sufficient to enable centrioles to retain PCM and organize the interphase aster in Drosophila neuroblasts. They also reveal an interphase function for POLO in this process that seems to have co-opted part of the protein network involved in mitotic centrosome maturation.

SummaryThe function of microtubules depends on their arrangement into highly ordered arrays. Spatio-temporal control over the formation of new microtubules and regulation of their properties are central to the organization of these arrays. The nucleation of new microtubules requires c-tubulin, an essential protein that assembles into multi-subunit complexes and is found in all eukaryotic organisms. However, the way in which c-tubulin complexes are regulated and how this affects nucleation and, potentially, microtubule behavior, is poorly understood. c-tubulin has been found in complexes of various sizes but several lines of evidence suggest that only large, ring-shaped complexes function as efficient microtubule nucleators. Human c-tubulin ring complexes (cTuRCs) are composed of c-tubulin and the c-tubulin complex components (GCPs) 2, 3, 4, 5 and 6, which are members of a conserved protein family. Recent work has identified additional unrelated cTuRC subunits, as well as a large number of more transient cTuRC interactors. In this Commentary, we discuss the regulation of cTuRC-dependent microtubule nucleation as a key mechanism of microtubule organization. Specifically, we focus on the regulatory roles of the cTuRC subunits and interactors and present an overview of other mechanisms that regulate cTuRC-dependent microtubule nucleation and organization.

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.