The mesenchymal morphology of cells expressing the EML4-ALK V3 oncogene is dependent on phosphorylation of Eg5 by NEK7.

Echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) oncogenic fusion proteins are found in approximately 5% of non-small cell lung cancers. Different EML4-ALK fusion variants exist with variant 3 (V3) being associated with a significantly higher risk than other common variants, such as variant 1 (V1). Patients with V3 respond less well to targeted ALK inhibitors, have accelerated rates of metastasis, and have poorer overall survival. A pathway has been described downstream of EML4-ALK V3 that is independent of ALK catalytic activity but dependent on the NEK9 and NEK7 kinases. It has been proposed that assembly of an EML4-ALK V3-NEK9-NEK7 complex on microtubules leads to cells developing a mesenchymal-like morphology and exhibiting enhanced migration. However, downstream targets of this complex remain unknown. Here, we show that the microtubule-based kinesin, Eg5, is recruited to interphase microtubules in cells expressing EML4-ALK V3, whereas chemical inhibition of Eg5 reverses the mesenchymal morphology of cells. Furthermore, we show that depletion of NEK7 interferes with Eg5 recruitment to microtubules in cells expressing EML4-ALK V3 and cell length is reduced, but this is reversed by coexpression of a phosphomimetic mutant of Eg5, in a site, S1033, phosphorylated by NEK7. Intriguingly, we also found that expression of Eg5-S1033D led to cells expressing EML4-ALK V1 adopting a more mesenchymal-like morphology. Together, we propose that Eg5 acts as a substrate of NEK7 in cells expressing EML4-ALK V3 and Eg5 phosphorylation promotes the mesenchymal morphology typical of these cells.

BICD2 phosphorylation regulates dynein function and centrosome separation in G2 and M.

The activity of dynein is regulated by a number of adaptors that mediate its interaction with dynactin, effectively activating the motor complex while also connecting it to different cargos. The regulation of adaptors is consequently central to dynein physiology but remains largely unexplored. We now describe that one of the best-known dynein adaptors, BICD2, is effectively activated through phosphorylation. In G2, phosphorylation of BICD2 by CDK1 promotes its interaction with PLK1. In turn, PLK1 phosphorylation of a single residue in the N-terminus of BICD2 results in a structural change that facilitates the interaction with dynein and dynactin, allowing the formation of active motor complexes. Moreover, modified BICD2 preferentially interacts with the nucleoporin RanBP2 once RanBP2 has been phosphorylated by CDK1. BICD2 phosphorylation is central for dynein recruitment to the nuclear envelope, centrosome tethering to the nucleus and centrosome separation in the G2 and M phases of the cell cycle. This work reveals adaptor activation through phosphorylation as crucial for the spatiotemporal regulation of dynein activity.

Integrin-Mediated Adhesion Promotes Centrosome Separation in Early Mitosis.

Integrin-mediated adhesion to the extracellular matrix is a key regulator of the cell cycle, as demonstrated for the passage of the G1/S checkpoint and the completion of cytokinetic abscission. Here, integrin-dependent regulation of the cell cycle in G2 and early M phases was investigated. The progression through the G2 and M phases was monitored by live-cell imaging and immunofluorescence staining in adherent and non-adherent fibroblast cells. Non-adherent cells, as well as adherent cells lacking FAK activity due to suppressed expression or pharmacological inhibition, exhibited a prolonged G2 phase and severely defect centrosome separation, resulting in delayed progress through the early mitotic stages. The activation of the critical mitotic regulator PLK1 and its indirect target Eg5, a kinesin-family motor protein driving the centrosome separation, were reduced in the cells lacking FAK activity. Furthermore, the absence of integrin adhesion or FAK activity destabilized the structural integrity of centrosomes and often caused detachment of pericentriolar material from the centrioles. These data identify a novel adhesion-dependent mechanism by which integrins via FAK and PLK1 contribute to the regulation of the cell cycle in the G2 and early M phases, and to the maintenance of genome integrity.

The co-chaperone UNC45A is essential for the expression of mitotic kinase NEK7 and tumorigenesis.

Cumulative evidence suggests that the heat shock protein 90 (Hsp90) co-chaperone UNC-45 myosin chaperone A (UNC45A) contributes to tumorigenesis and that its expression in cancer cells correlates with proliferation and metastasis of solid tumors. However, the molecular mechanism by which UNC45A regulates cancer cell proliferation remains largely unknown. Here, using siRNA-mediated gene silencing and various human cells, we report that UNC45A is essential for breast cancer cell growth, but is dispensable for normal cell proliferation. Immunofluorescence microscopy, along with gene microarray and RT-quantitative PCR analyses, revealed that UNC45A localizes to the cancer cell nucleus, where it up-regulates the transcriptional activity of the glucocorticoid receptor and thereby promotes expression of the mitotic kinase NIMA-related kinase 7 (NEK7). We observed that UNC45A-deficient cancer cells exhibit extensive pericentrosomal material disorganization, as well as defects in centrosomal separation and mitotic chromosome alignment. Consequently, these cells stalled in metaphase and cytokinesis and ultimately underwent mitotic catastrophe, phenotypes that were rescued by heterologous NEK7 expression. Our results identify a key role for the co-chaperone UNC45A in cell proliferation and provide insight into the regulatory mechanism. We propose that UNC45A represents a promising new therapeutic target to inhibit cancer cell growth in solid tumor types.

Nek9 phosphorylation of NEDD1/GCP-WD contributes to Plk1 control of γ-tubulin recruitment to the mitotic centrosome.

The accumulation of γ-tubulin at the centrosomes during maturation is a key mechanism that ensures the formation of two dense microtubule (MT) asters in cells entering mitosis, defining spindle pole positioning and ensuring the faithful outcome of cell division. Centrosomal γ-tubulin recruitment depends on the adaptor protein NEDD1/GCP-WD and is controlled by the kinase Plk1. Surprisingly, and although Plk1 binds and phosphorylates NEDD1 at multiple sites, the mechanism by which this kinase promotes the centrosomal recruitment of γ-tubulin has remained elusive. Using Xenopus egg extracts and mammalian cells, we now show that it involves Nek9, a NIMA-family kinase required for normal mitotic progression and spindle organization. Nek9 phosphorylates NEDD1 on Ser377 driving its recruitment and thereby that of γ-tubulin to the centrosome in mitotic cells. This role of Nek9 requires its activation by Plk1-dependent phosphorylation but is independent from the downstream related kinases Nek6 and Nek7. Our data contribute to understand the mechanism by which Plk1 promotes the recruitment of γ-tubulin to the centrosome in dividing cells and position Nek9 as a key regulator of centrosome maturation.

Nek9 is a Plk1-activated kinase that controls early centrosome separation through Nek6/7 and Eg5.

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.

The gammaTuRC revisited: a comparative analysis of interphase and mitotic human gammaTuRC redefines the set of core components and identifies the novel subunit GCP8.

The γ-tubulin complex is a multi-subunit protein complex that nucleates microtubule polymerization. γ-Tubulin complexes are present in all eukaryotes, but size and subunit composition vary. In Drosophila, Xenopus, and humans large γ-tubulin ring complexes (γTuRCs) have been described, which have a characteristic open ring-shaped structure and are composed of a similar set of subunits, named γ-tubulin, GCPs 2-6, and GCP-WD in humans. Despite the identification of these proteins, γTuRC function and regulation remain poorly understood. Here we establish a new method for the purification of native human γTuRC. Using mass spectrometry of whole protein mixtures we compared the composition of γTuRCs from nonsynchronized and mitotic human cells. Based on our analysis we can define core subunits as well as more transient interactors such as the augmin complex, which associates specifically with mitotic γTuRCs. We also identified GCP8/MOZART2 as a novel core subunit that is present in both interphase and mitotic γTuRCs. GCP8 depletion does not affect γTuRC assembly but interferes with γTuRC recruitment and microtubule nucleation at interphase centrosomes without disrupting general centrosome structure. GCP8-depleted cells do not display any obvious mitotic defects, suggesting that GCP8 specifically affects the organization of the interphase microtubule network.

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

Nek6 and Nercc1 (also known as 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 Nercc1 from Xenopus egg extracts prevents normal spindle assembly. Herein we show that Nek6 is constitutively associated with Eg5 (also known as Kinesin-5 and Kif11), a kinesin that is necessary for spindle bipolarity. Nek6 phosphorylated Eg5 at several sites in vitro and one of these sites, Ser1033, is phosphorylated in vivo during mitosis. Whereas CDK1 phosphorylates nearly all Eg5 at Thr926 during mitosis, Nek6 phosphorylates approximately 3% of Eg5, primarily at the spindle poles. Eg5 depletion caused mitotic arrest, resulting in cells with a monopolar spindle. This arrest could be rescued by wild-type Eg5 but not by Eg5[Thr926Ala]. Despite substantial overexpression, Eg5[Ser1033Ala] rescued 50% of cells compared with wild-type Eg5, whereas an Eg5[Ser1033Asp] mutant was nearly as effective as wild type. Thus, during mitosis Nek6 phosphorylates a subset of Eg5 polypeptides at a conserved site, the phosphorylation of which is crucial for the mitotic function of Eg5.

Active Nercc1 protein kinase concentrates at centrosomes early in mitosis and is necessary for proper spindle assembly.

The Nercc1 protein kinase autoactivates in vitro and is activated in vivo during mitosis. Autoactivation in vitro requires phosphorylation of the activation loop at threonine 210. Mitotic activation of Nercc1 in mammalian cells is accompanied by Thr210 phosphorylation and involves a small fraction of total Nercc1. Mammalian Nercc1 coimmunoprecipitates gamma-tubulin and the activated Nercc1 polypeptides localize to the centrosomes and spindle poles during early mitosis, suggesting that active Nercc has important functions at the microtubular organizing center during cell division. To test this hypothesis, we characterized the Xenopus Nercc1 orthologue (XNercc). XNercc endogenous to meiotic egg extracts coprecipitates a multiprotein complex that contains gamma-tubulin and several components of the gamma-tubulin ring complex and localizes to the poles of spindles formed in vitro. Reciprocally, immunoprecipitates of the gamma-tubulin ring complex polypeptide Xgrip109 contain XNercc. Immunodepletion of XNercc from egg extracts results in delayed spindle assembly, fewer bipolar spindles, and the appearance of aberrant microtubule structures, aberrations corrected by addition of purified recombinant XNercc. XNercc immunodepletion also slows aster assembly induced by Ran-GTP, producing Ran-asters of abnormal size and morphology. Thus, Nercc1 contributes to both the centrosomal and the chromatin/Ran pathways that collaborate in the organization of a bipolar spindle.