Laura O'Regan

Mitotic regulation by NIMA-related kinases

The NIMA-related kinases represent a family of serine/threonine kinases implicated in cell cycle control. The founding member of this family, the NIMA kinase of Aspergillus nidulans, as well as the fission yeast homologue Fin1, contribute to multiple aspects of mitotic progression including the timing of mitotic entry, chromatin condensation, spindle organization and cytokinesis. Mammals contain a large family of eleven NIMA-related kinases, named Nek1 to Nek11. Of these, there is now substantial evidence that Nek2, Nek6, Nek7 and Nek9 also regulate mitotic events. At least three of these kinases, as well as NIMA and Fin1, have been localized to the microtubule organizing centre of their respective species, namely the centrosome or spindle pole body. Here, they have important functions in microtubule organization and mitotic spindle assembly. Other Nek kinases have been proposed to play microtubule-dependent roles in non-dividing cells, most notably in regulating the axonemal microtubules of cilia and flagella. In this review, we discuss the evidence that NIMA-related kinases make a significant contribution to the orchestration of mitotic progression and thereby protect cells from chromosome instability. Furthermore, we highlight their potential as novel chemotherapeutic targets.

Cell cycle regulation by the NEK family of protein kinases

Summary Genetic screens for cell division cycle mutants in the filamentous fungus Aspergillus nidulans led to the discovery of never-in-mitosis A (NIMA), a serine/threonine kinase that is required for mitotic entry. Since that discovery, NIMA-related kinases, or NEKs, have been identified in most eukaryotes, including humans where eleven genetically distinct proteins named NEK1 to NEK11 are expressed. Although there is no evidence that human NEKs are essential for mitotic entry, it is clear that several NEK family members have important roles in cell cycle control. In particular, NEK2, NEK6, NEK7 and NEK9 contribute to the establishment of the microtubule-based mitotic spindle, whereas NEK1, NEK10 and NEK11 have been implicated in the DNA damage response. Roles for NEKs in other aspects of mitotic progression, such as chromatin condensation, nuclear envelope breakdown, spindle assembly checkpoint signalling and cytokinesis have also been proposed. Interestingly, NEK1 and NEK8 also function within cilia, the microtubule-based structures that are nucleated from basal bodies. This has led to the current hypothesis that NEKs have evolved to coordinate microtubule-dependent processes in both dividing and non-dividing cells. Here, we review the functions of the human NEKs, with particular emphasis on those family members that are involved in cell cycle control, and consider their potential as therapeutic targets in cancer.

The Nek6 and Nek7 Protein Kinases Are Required for Robust Mitotic Spindle Formation and Cytokinesis

ABSTRACT Nek6 and Nek7 are members of the NIMA-related serine/threonine kinase family. Previous work showed that they contribute to mitotic progression downstream of another NIMA-related kinase, Nek9, although the roles of these different kinases remain to be defined. Here, we carried out a comprehensive analysis of the regulation and function of Nek6 and Nek7 in human cells. By generating specific antibodies, we show that both Nek6 and Nek7 are activated in mitosis and that interfering with their activity by either depletion or expression of reduced-activity mutants leads to mitotic arrest and apoptosis. Interestingly, while completely inactive mutants and small interfering RNA-mediated depletion delay cells at metaphase with fragile mitotic spindles, hypomorphic mutants or RNA interference treatment combined with a spindle assembly checkpoint inhibitor delays cells at cytokinesis. Importantly, depletion of either Nek6 or Nek7 leads to defective mitotic progression, indicating that although highly similar, they are not redundant. Indeed, while both kinases localize to spindle poles, only Nek6 obviously localizes to spindle microtubules in metaphase and anaphase and to the midbody during cytokinesis. Together, these data lead us to propose that Nek6 and Nek7 play independent roles not only in robust mitotic spindle formation but also potentially in cytokinesis.

An Autoinhibitory Tyrosine Motif in the Cell-Cycle-Regulated Nek7 Kinase Is Released through Binding of Nek9

Summary Mitosis is controlled by multiple protein kinases, many of which are abnormally expressed in human cancers. Nek2, Nek6, Nek7, and Nek9 are NIMA-related kinases essential for proper mitotic progression. We determined the atomic structure of Nek7 and discovered an autoinhibited conformation that suggests a regulatory mechanism not previously described in kinases. Additionally, Nek2 adopts the same conformation when bound to a drug-like molecule. In both structures, a tyrosine side chain points into the active site, interacts with the activation loop, and blocks the αC helix. Tyrosine mutants of Nek7 and the related kinase Nek6 are constitutively active. The activity of Nek6 and Nek7, but not the tyrosine mutant, is increased by interaction with the Nek9 noncatalytic C-terminal domain, suggesting a mechanism in which the tyrosine is released from its autoinhibitory position. The autoinhibitory conformation is common to three Neks and provides a potential target for selective kinase inhibitors.

Crystal structure of EML1 reveals the basis for Hsp90 dependence of oncogenic EML4-ALK by disruption of an atypical β-propeller domain

Significance Echinoderm microtubule-associated protein (EMAP)-like (EML) proteins normally function in the cytoskeleton. In some lung cancers, genetic abnormalities generate the oncogenic fusion protein EML4-anaplastic lymphoma kinase (ALK) on which the cancer cells depend for survival. We have determined the molecular structure of a conserved, tubulin-binding region of EML1 that reveals an unexpected protein fold. This region is disrupted in ∼70% of EML4-ALK fusions found in patients, causing them to be sensitive to drugs that target Hsp90, a cellular factor that stabilizes misfolded protein. Our findings will potentially enable more effective, stratified therapy of EML4-ALK nonsmall cell lung cancer and suggest that the truncation of a globular domain at the translocation breakpoint may prove generally predictive of Hsp90 inhibitor sensitivity in cancers driven by fusion oncogenes. Proteins of the echinoderm microtubule-associated protein (EMAP)-like (EML) family contribute to formation of the mitotic spindle and interphase microtubule network. They contain a unique hydrophobic EML protein (HELP) motif and a variable number of WD40 repeats. Recurrent gene rearrangements in nonsmall cell lung cancer fuse EML4 to anaplastic lymphoma kinase (ALK), causing expression of several fusion oncoprotein variants. We have determined a 2.6-Å crystal structure of the representative ∼70-kDa core of EML1, revealing an intimately associated pair of β-propellers, which we term a TAPE (tandem atypical propeller in EMLs) domain. One propeller is highly atypical, having a discontinuous subdomain unrelated to a WD40 motif in place of one of its blades. This unexpected feature shows how a propeller structure can be assembled from subdomains with distinct folds. The HELP motif is not an independent domain but forms part of the hydrophobic core that joins the two β-propellers. The TAPE domain binds α/β-tubulin via its conserved, concave surface, including part of the atypical blade. Mapping the characteristic breakpoints of each EML4-ALK variant onto our structure indicates that the EML4 TAPE domain is truncated in many variants in a manner likely to make the fusion protein structurally unstable. We found that the heat shock protein 90 (Hsp90) inhibitor ganetespib induced degradation of these variants whereas others lacking a partial TAPE domain were resistant in both overexpression models and patient-derived cell lines. The Hsp90-sensitive EML4-ALK variants are exceptions to the rule that oncogenic fusion proteins involve breakpoints in disordered regions of both partners.

Microtubule association of EML proteins and the EML4-ALK variant 3 oncoprotein require an N-terminal trimerization domain

Proteins of the echinoderm microtubule (MT)-associated protein (EMAP)-like (EML) family contribute to formation of the mitotic spindle and interphase MT network. EML1-4 consist of Trp-Asp 40 (WD40) repeats and an N-terminal region containing a putative coiled-coil. Recurrent gene rearrangements in non-small cell lung cancer (NSCLC) fuse EML4 to anaplastic lymphoma kinase (ALK) causing expression of several oncogenic fusion variants. The fusions have constitutive ALK activity due to self-association through the EML4 coiled-coil. We have determined crystal structures of the coiled-coils from EML2 and EML4, which describe the structural basis of both EML self-association and oncogenic EML4-ALK activation. The structures reveal a trimeric oligomerization state directed by a conserved pattern of hydrophobic residues and salt bridges. We show that the trimerization domain (TD) of EML1 is necessary and sufficient for self-association. The TD is also essential for MT binding; however, this property requires an adjacent basic region. These observations prompted us to investigate MT association of EML4-ALK and EML1-ABL1 (Abelson 1) fusions in which variable portions of the EML component are present. Uniquely, EML4-ALK variant 3, which includes the TD and basic region of EML4 but none of the WD40 repeats, was localized to MTs, both when expressed recombinantly and when expressed in a patient-derived NSCLC cell line (H2228). This raises the question of whether the mislocalization of ALK activity to MTs might influence downstream signalling and malignant properties of cells. Furthermore, the structure of EML4 TD may enable the development of protein-protein interaction inhibitors targeting the trimerization interface, providing a possible avenue towards therapeutic intervention in EML4-ALK NSCLC.

Mechanistic basis of Nek7 activation through Nek9 binding and induced dimerization.

Mitotic spindle assembly requires the regulated activities of protein kinases such as Nek7 and Nek9. Nek7 is autoinhibited by the protrusion of Tyr97 into the active site and activated by the Nek9 non-catalytic C-terminal domain (CTD). CTD binding apparently releases autoinhibition because mutation of Tyr97 to phenylalanine increases Nek7 activity independently of Nek9. Here we find that self-association of the Nek9-CTD is needed for Nek7 activation. We map the minimal Nek7 binding region of Nek9 to residues 810–828. A crystal structure of Nek7Y97F bound to Nek9810–828 reveals a binding site on the C-lobe of the Nek7 kinase domain. Nek7Y97F crystallizes as a back-to-back dimer between kinase domain N-lobes, in which the specific contacts within the interface are coupled to the conformation of residue 97. Hence, we propose that the Nek9-CTD activates Nek7 through promoting back-to-back dimerization that releases the autoinhibitory tyrosine residue, a mechanism conserved in unrelated kinase families.

Hsp70 proteins in mitosis and disease.

Heat shock proteins (HSPs) are ATP-dependent molecular chaperones which aid folding of nascent polypeptides, maintain proteins in unstable conformations and prevent protein denaturation. These functions are essential in many biological contexts, including assembly and disassembly of macromolecular complexes, trafficking of proteins and regulation of enzyme activity [1]. Mitotic cell division is particularly complex involving rapid changes in cytoskeletal and organelle architecture. One would therefore expect it to be highly dependent on HSPs; however, much remains to be learnt about the roles of HSPs in mitosis. In a recent study, we discovered that Hsp72, an inducible cytoplasmic isoform of the Hsp70 family, is essential to build a mitotic spindle capable of efficient chromosome congression and segregation [2]. Firstly, Hsp72 contributes to generation of stable kinetochore (K)-fibres. K-fibres are bundles of microtubules that connect the spindle poles with the kinetochores and are essential for chromosome movement. Upon depletion of Hsp72 or addition of an Hsp70 inhibitor, cells exhibited reduced K-fibres. This was coincident with misaligned chromosomes, metaphase delay and a strongly active spindle assembly checkpoint. The loss of K-fibres was not caused by reduction in microtubule nucleation, but rather failure to recruit the K-fibre-stabilising proteins, ch-TOG and TACC3. Hsp72 localises to spindle poles and spindle fibres in a similar manner to ch-TOG and TACC3. Furthermore, when TACC3 was immunoprecipitated from cells treated with the Hsp70 inhibitor, association with its partner, ch-TOG, was reduced. Together, this suggests that Hsp72 stabilises K-fibres by facilitating assembly of ch-TOG and TACC3 into a complex that can then serve to bundle K-fibre microtubules [3]. Secondly, abrogation of Hsp72 function led to reduced interpolar distances, reduced astral microtubules and misoriented spindles. Astral microtubules attach the spindle to the cell cortex to maintain its shape and position. These data indicate that Hsp72 has additional functions in astral microtubule organization or cortical attachment. This is likely to be independent of the ch-TOG-TACC3 complex as this is not thought to be required for astral microtubule function. Importantly, whilst Hsp72 depletion and chemical inhibition of Hsp70 give similar phenotypes, there are some clear differences. This can be explained by that fact that chemical inhibition blocks the activity of all Hsp70 isoforms and it is possible that other Hsp70 isoforms have roles in mitosis. On the other hand, chemical inhibition does not remove the protein and so the different phenotypes may result from Hsp72 having mitotic functions as a scaffold that are independent of its catalytic activity. Hsp72 is phosphorylated in mitosis by Nek6, a protein kinase that is also required for robust spindle assembly [4, 5]. Nek6 phosphorylates Hsp72 on T66, a residue that sits within the nucleotide-binding domain just upstream of the catalytic lysine (K71). Mislocalization of a T66A mutant and failure of Hsp72 to associate with the spindle in the absence of Nek6 indicate that phosphorylation mediates localisation of Hsp72 to the spindle. Furthermore, expression of the T66A mutant resulted in reduced K-fibres, whilst the T66E mutant could rescue the K-fibre defects and loss of ch-TOG/TACC3 recruitment to K-fibres seen upon either Hsp72 or Nek6 depletion. However, understanding the mechanism through which phosphorylation regulates Hsp72 will require molecular insights on Hsp72 interactions with its mitotic partners. Interestingly, a phosphospecific antibody revealed that Hsp72 phosphorylated on T66 was not only localised to the spindle apparatus, but also enriched on spindle poles and kinetochores. This begs the question of whether phosphorylated Hsp72 has additional, perhaps distinct, substrates at the kinetochore. The absence of total Hsp72 may lead to loss of function of these proteins as well, exacerbating the K-fibre assembly and chromosome congression defects. Similarly, problems in cortical attachment of microtubules could contribute to loss of astral microtubules and spindle orientation defects. It will thus be important to ascertain whether (phosphorylated) Hsp72 may stabilize the plus ends of microtubules that contact kinetochores and the cell cortex. Besides their homeostatic function, HSPs have an important role in protecting cells from the proteotoxic stress that can arise in different pathological states [6]. These include protein-folding disorders, autoimmune diseases and cancer. In cancer, the induced expression of Hsp70 due to proteotoxic stress promotes cell survival and tumor progression. As cancer cells undergo frequent mitotic division, Hsp70 inhibitors could have therapeutic value in targeting the proliferating tumour tissue. Whilst it has been frustratingly difficult to develop potent and selective inhibitors of Hsp70 itself [7], drugging upstream regulators, such as Nek6, offers an alternative approach. In contrast to cancer, Hsp70 proteins are beneficial to the patient in slowing the onset of neurodegenerative disorders, such as Alzheimers, Huntingtons or Parkinsons disease. Here, they promote removal of misfolded proteins through autophagy or proteasomal degradation. However, as neuronal cells are post-mitotic and do not undergo division, anti-cancer therapies specifically targeting the mitotic form of Hsp70 would have the advantage of not precipitating the onset or progression of neurodegenerative diseases.

Hsp72 and Nek6 cooperate to cluster amplified centrosomes in cancer cells

Cancer cells frequently possess extra amplified centrosomes clustered into two poles whose pseudo-bipolar spindles exhibit reduced fidelity of chromosome segregation and promote genetic instability. Inhibition of centrosome clustering triggers multipolar spindle formation and mitotic catastrophe, offering an attractive therapeutic approach to selectively kill cells with amplified centrosomes. However, mechanisms of centrosome clustering remain poorly understood. Here, we identify a new pathway that acts through NIMA-related kinase 6 (Nek6) and Hsp72 to promote centrosome clustering. Nek6, as well as its upstream activators polo-like kinase 1 and Aurora-A, targeted Hsp72 to the poles of cells with amplified centrosomes. Unlike some centrosome declustering agents, blocking Hsp72 or Nek6 function did not induce formation of acentrosomal poles, meaning that multipolar spindles were observable only in cells with amplified centrosomes. Inhibition of Hsp72 in acute lymphoblastic leukemia cells resulted in increased multipolar spindle frequency that correlated with centrosome amplification, while loss of Hsp72 or Nek6 function in noncancer-derived cells disturbs neither spindle formation nor mitotic progression. Hence, the Nek6-Hsp72 module represents a novel actionable pathway for selective targeting of cancer cells with amplified centrosomes. Cancer Res; 77(18); 4785-96. ©2017 AACR.

Novel insights into the mechanisms of mitotic spindle assembly by NEK kinases

ABSTRACT The mitotic spindle is the apparatus upon which chromosomes are segregated during cell division. We have discovered new roles for two members of the NIMA-related kinase (NEK) family in different molecular processes of spindle assembly. Moreover, loss of these proteins leads to segregation errors that drive cancer progression.