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Cell Cycle Regulation by the NEK Family of Protein Kinases

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Cell Cycle Regulation by the NEK Family of Protein Kinases Commentary 4423 Cell cycle regulation by the NEK family of protein kinases Andrew M. Fry*, Laura O’Regan, Sarah R. Sabir and Richard Bayliss Department of Biochemistry, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK *Author for correspondence ([email protected]) Journal of Cell Science 125, 4423–4433 ß 2012. Published by The Company of Biologists Ltd doi: 10.1242/jcs.111195 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. Key words: NEK, NIMA, Mitosis, Centrosome, Microtubule, Mitotic spindle, DNA damage response, Cilia Introduction The NIMA-related kinase family Maintaining genome stability is crucial to the health of an organism. The founding member of the NEK family is the never-in-mitosis The vast majority of human cancer cells are aneuploid, meaning A (NIMA) protein of Aspergillus nidulans, which was identified they have too few or, more commonly, too many chromosomes. by Ron Morris and colleagues in a genetic screen for cell division Journal of Cell Science Furthermore, many cancers exhibit chromosome instability, cycle mutants (Oakley and Morris, 1983). Loss-of-function whereby there is frequent loss or gain of chromosomes at each mutations in nimA cause G2 arrest, whereas overexpression cell division. Together, these defects provide tumours with a rapid leads to cells attempting to enter mitosis prematurely (Osmani evolutionary potential that can lead to the acquisition of malignant et al., 1991; Osmani et al., 1988). It has been subsequently properties and drive selection of drug resistance. Typically, discovered that degradation of NIMA is essential for mitotic exit, aneuploidy and chromosome instability arise as a result of errors which puts it on a par with the Cdc2–cyclin-B complex as a in the process of chromosome segregation that occurs on the master regulator of mitotic progression in Aspergillus (Pu and microtubule-based spindle that assembles during mitosis. Not Osmani, 1995). Aspergillus express a single NIMA-related gene, surprisingly, cells have developed complex, and often redundant, as do the yeasts Saccharomyces cerevisiae (called kin3) and mechanisms that enable them to undergo mitosis without errors. Schizosaccharomyces pombe (called fin1; note that this is Reversible phosphorylation is among the best understood of unrelated to the budding yeast protein Fin1 that localises to the the mechanisms of mitotic control. Cell-cycle-dependent protein mitotic spindle). However, although these yeast NEKs contribute kinases, along with their counteracting phosphatases, regulate the to various aspects of cell cycle progression, including chromatin phosphorylation status of many hundreds of substrate proteins that, condensation, spindle assembly and cytokinesis, they are not in turn, dictate the events that orchestrate mitosis. To date, a required for viability (De Souza et al., 2000; Grallert and Hagan, relatively small number of protein kinase families that regulate 2002; Grallert et al., 2004; Krien et al., 1998; Wu et al., 1998). mitosis have been identified. These include the cyclin-dependent NEKs have now been identified in a wide range of organisms kinases (CDKs), Aurora kinases and Polo-like kinases (PLKs). from protists, such as Chlamydomonas, Plasmodium and However, there is another, less-well-characterised protein kinase Tetrahymena, to multicellular eukaryotes, including Drosophila, family, whose members have key roles in mitosis: the NIMA-related Xenopus, mice and humans. Human cells express eleven genes kinases or NEKs (Moniz et al., 2011; O’Connell et al., 2003; that encode NEK1 to NEK11 (Fig. 1). The defining feature of O’Regan et al., 2007; Quarmby and Mahjoub, 2005). Here, we this protein family is an N-terminal catalytic domain that contains review our current knowledge of how NEKs contribute to the all the motifs that are typical of a serine/threonine kinase and process of cell division. Moreover, because targeting mitosis is a shares closer sequence similarity with Aspergillus NIMA than proven approach to cancer treatment, we also debate the potential any other class of protein kinase. NEK10 breaks this rule by use of NEKs as therapeutic targets in human cancer. having a centrally located kinase domain, but with regards to its 4424 Journal of Cell Science 125 (19) Journal of Cell Science Fig. 1. The human NIMA-related protein kinase (NEK) family. (A) A schematic view of the eleven human NEKs, highlighting their domain organisation. Shown are the kinase domains (purple), coiled-coils (green), degradation motifs (red), RCC1 (regulator of chromatin condensation 1) domains (light blue) and armadillo repeats (yellow). A summary of what is known about the activation, localisation and function of the kinases is included. aa, amino acids. (B) Crystal structure of human NEK7 (PDB code 2WQN). Tyr97, which points down into the active site, is coloured orange and ADP is coloured red. (C) Crystal structure of human NEK2 (PDB code 2W5A). Tyr70 in the ‘upward conformation’ is coloured orange and ADP is coloured red. (D) Magnified view of NEK2 bound to a potent and selective ‘hybrid’ inhibitor that induces an inactive conformation of the activation loop (PDB code 4A4X). Atoms in the inhibitor are coloured as follows: carbon, grey; nitrogen, blue; sulphur, yellow; oxygen, red; fluorine, cyan. The ATP-binding pocket of NEK2 has a bulky gatekeeper residue (Met86) and a phenylanine residue at the base (Phe148). This is a rare combination, which severely constrains the design of ATP-competitive inhibitors. However, it is present in several NEKs. amino acid sequence it clearly belongs to the NEK family. NEK6 and NEK7 are unusual in this respect, because their kinase Generally, the NEK kinase domains are only moderately domains share more than 85% sequence identity. All eleven human conserved, with ,40–50% identity on the amino acid level both NEKs contain a His-Arg-Asp (HRD) motif within the catalytic to the kinase domain of NIMA and, on the whole, to each other. domain, which is usually found in kinases that are positively NEKs in cell cycle regulation 4425 regulated through phosphorylation (Johnson et al., 1996), and they all possess a serine or threonine residue within the activation loop, which is a probable site for an activating modification. In some NEKs, this residue is autophosphorylated, whereas in others it is G2-M checkpoint Mitotic progression targeted by an upstream kinase (Belham et al., 2003; Bertran et al., NEK1 NEK2 2011; Rellos et al., 2007; Roig et al., 2002). In terms of a NEK10 NEK6 NEK11 NEK7 phosphorylation consensus sequence, early studies found that NEK9 NIMA has a strong preference for phenylalanine at position 23 M (i.e. FxxS/T, where ‘x’ is any amino acid) (Lu et al., 1994). More recent studies have indicated that human NEKs have a similar G2 G1 preference, with both NEK2 and NEK6 preferring a hydrophobic residue, ideally phenylalanine or leucine, at the 23 position (F/ S G0 LxxS/T) (Alexander et al., 2011; Lizcano et al., 2002). However, these are not strict requirements, as phosphorylation sites that do not fall into this motif have been mapped on NEK substrates. Cilliary function NEK1 In contrast with the conserved catalytic domains, the NEK8 Proliferation and NEK4 C-terminal regions of the NEKs are highly divergent in length, signalling NEK3 sequence and domain organisation (Fig. 1). The one relatively common feature is an oligomerisation motif, usually a coiled-coil, which promotes autophosphorylation and activation. Fig. 2. Roles for NEKs in cell cycle progression. Human NEKs contribute Autophosphorylation can occur within the activation loop of the to different events during cell cycle progression and differentiation. Our kinase domain. However, autophosphorylation is also likely to current understanding is that during mitosis, NEK2, NEK6, NEK7 and NEK9 occur elsewhere in the protein, as supported by the finding that cooperate to ensure formation of a robust bipolar spindle that is capable of both NEK8 and NEK9 autophosphorylate in the non-catalytic satisfying the SAC. They might also contribute to other aspects of mitotic C-terminal region to regulate their localisation and/or activation progression,
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