Characterisation of the Nek6 and Nek7 mitotic protein kinases

Entry into mitosis results in a dramatic reorganization of the cellular architecture to allow for segregation of duplicated DNA to nascent daughter cells. This complex biomechanical feat is orchestrated by members of the Cyclin-dependent, Aurora, Polo-like and NIMA-related kinase families. Four NIMA-related kinase proteins are implicated in regulation of mitotic progression, Nek2, Nek9, Nek6 and Nek7. Nek6 and Nek7 are closely related in sequence, encode little more than a catalytic domain and have been implicated in a mitotic NIMA-related kinase cascade downstream of Nek9. Functional data on Nek9 has implicated it in regulation of mitotic spindle architecture and thus Nek6 and Nek7 are also thought likely to function in spindle organization. However, functional data validating such a role is sparse and the roles of Nek6 and Nek7 remain poorly defined. In this thesis I set out to carry out a detailed functional analysis of Nek6 and Nek7, focusing on their proposed roles in mitotic progression. We show that expression of Nek6 and Nek7 mutants whose kinase activity is compromised results in mitotic arrest leading to apoptosis. However, whilst mutants with no activity cause an arrest at metaphase with fragile mitotic spindles, hypomorphic mutants, which retain intermediate levels of activity, result in an arrest in late mitosis. Nek6 and Nek7 interact with γ-tubulin, and interference with Nek6 or Nek7 disturbs the centrosomal localization of γ-tubulin. Nek6 localizes to the microtubules of the mitotic spindle and RNAi depletion of Nek6 leads to destabilization of the spindle microtubules. Thus, Nek6 and Nek7 may function during metaphase to regulate microtubule nucleation both from the spindle poles and from within the spindle itself. Furthermore, we identified the spindle components, Hsp70 and β-tubulin as Nek6 substrates, providing a possible mechanism by which Nek6 may achieve such a role. Finally, we also identified Cortactin A, a regulator of actin dynamics, as a Nek6 substrate. Cortactin A and Nek6 localize to the cleavage furrow of late mitotic cells raising the possibility that Nek6 may be involved in the regulation of membrane dynamics during cytokinesis. Together, the functional data suggests that Nek6 and Nek7 may regulate multiple events during mitosis and the identification of Nek6 substrates provides possible mechanisms by which they might achieve these functions

Hsp70 proteins in mitosis and disease.

Hsp70 proteins in mitosis and disease

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

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

EML Proteins in Microtubule Regulation and Human Disease

The EMLs are a conserved family of microtubule-associated proteins (MAPs). The founding member was discovered in sea urchins as a 77-kDa polypeptide that co-purified with microtubules. This protein, termed EMAP for echinoderm MAP, was the major non-tubulin component present in purified microtubule preparations made from unfertilized sea urchin eggs [J. Cell Sci. (1993) 104, 445–450; J. Cell Sci. (1987) 87(Pt 1), 71–84]. Orthologues of EMAP were subsequently identified in other echinoderms, such as starfish and sand dollar, and then in more distant eukaryotes, including flies, worms and vertebrates, where the name of ELP or EML (both for EMAP-like protein) has been adopted [BMC Dev. Biol. (2008) 8, 110; Dev. Genes Evol. (2000) 210, 2–10]. The common property of these proteins is their ability to decorate microtubules. However, whether they are associated with particular microtubule populations or exercise specific functions in different microtubule-dependent processes remains unknown. Furthermore, although there is limited evidence that they regulate microtubule dynamics, the biochemical mechanisms of their molecular activity have yet to be explored. Nevertheless, interest in these proteins has grown substantially because of the identification of EML mutations in neuronal disorders and oncogenic fusions in human cancers. Here, we summarize our current knowledge of the expression, localization and structure of what is proving to be an interesting and important class of MAPs. We also speculate about their function in microtubule regulation and highlight how the studies of EMLs in human diseases may open up novel avenues for patient therapy

Alternative Treatment Options to ALK Inhibitor Monotherapy for EML4-ALK-Driven Lung Cancer

EML4-ALK is an oncogenic fusion protein that accounts for approximately 5% of NSCLC cases. Targeted inhibitors of ALK are the standard of care treatment, often leading to a good initial response. Sadly, some patients do not respond well, and most will develop resistance over time, emphasizing the need for alternative treatments. This review discusses recent advances in our understanding of the mechanisms behind EML4-ALK-driven NSCLC progression and the opportunities they present for alternative treatment options to ALK inhibitor monotherapy. Targeting ALK-dependent signalling pathways can overcome resistance that has developed due to mutations in the ALK catalytic domain, as well as through activation of bypass mechanisms that utilise the same pathways. We also consider evidence for polytherapy approaches that combine targeted inhibition of these pathways with ALK inhibitors. Lastly, we review combination approaches that use targeted inhibitors of ALK together with chemotherapy, radiotherapy or immunotherapy. Throughout this article, we highlight the importance of alternative breakpoints in the EML4 gene that result in the generation of distinct EML4-ALK variants with different biological and pathological properties and consider monotherapy and polytherapy approaches that may be selective to particular variants

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

Proteins of the echinoderm microtubule associated protein-like (EML) family contribute to formation of the mitotic spindle and interphase microtubule (MT) network. EML1-4 consist of 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 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 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 Nek7(Y97F) bound to Nek9(810-828) reveals a binding site on the C-lobe of the Nek7 kinase domain. Nek7(Y97F) 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

EML4-ALK V3 oncogenic fusion proteins promote microtubule stabilisation and accelerated migration through NEK9 and NEK7

EML4-ALK is an oncogenic fusion present in ∼5% non-small cell lung cancers. However, alternative breakpoints in the EML4 gene lead to distinct variants with different patient outcomes. Here, we show in cell models that EML4-ALK variant 3 (V3), which is linked to accelerated metastatic spread, causes microtubule stabilization, formation of extended cytoplasmic protrusions and increased cell migration. It also recruits the NEK9 and NEK7 kinase to microtubules via the N-terminal EML4 microtubule-binding region. Overexpression of wild-type EML4 as well as constitutive activation of NEK9 also perturb cell morphology and accelerate migration in a microtubule-dependent manner that requires the downstream kinase NEK7 but not ALK activity. Strikingly, elevated NEK9 expression is associated with reduced progression-free survival in EML4-ALK patients. Hence, we propose that EML4-ALK V3 promotes microtubule stabilization through NEK9 and NEK7 leading to increased cell migration. This represents a novel actionable pathway that could drive metastatic disease progression in EML4-ALK lung cancer.</p