Journ

alof

Cell

Scie

nce

Cell cycle regulation by the NEK family of proteinkinases

Andrew M. Fry*, Laura O’Regan, Sarah R. Sabir and Richard BaylissDepartment of Biochemistry, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK

*Author for correspondence (amf5@le.ac.uk)

Journal of Cell Science 125, 4423–4433� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.111195

SummaryGenetic 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 beenidentified 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 otheraspects of mitotic progression, such as chromatin condensation, nuclear envelope breakdown, spindle assembly checkpoint signallingand 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-dependentprocesses in both dividing and non-dividing cells. Here, we review the functions of the human NEKs, with particular emphasis on thosefamily 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

IntroductionMaintaining genome stability is crucial to the health of an organism.

The vast majority of human cancer cells are aneuploid, meaning

they have too few or, more commonly, too many chromosomes.

Furthermore, many cancers exhibit chromosome instability,

whereby there is frequent loss or gain of chromosomes at each

cell division. Together, these defects provide tumours with a rapid

evolutionary potential that can lead to the acquisition of malignant

properties and drive selection of drug resistance. Typically,

aneuploidy and chromosome instability arise as a result of errors

in the process of chromosome segregation that occurs on the

microtubule-based spindle that assembles during mitosis. Not

surprisingly, cells have developed complex, and often redundant,

mechanisms that enable them to undergo mitosis without errors.

Reversible phosphorylation is among the best understood of

the mechanisms of mitotic control. Cell-cycle-dependent protein

kinases, along with their counteracting phosphatases, regulate the

phosphorylation status of many hundreds of substrate proteins that,

in turn, dictate the events that orchestrate mitosis. To date, a

relatively small number of protein kinase families that regulate

mitosis have been identified. These include the cyclin-dependent

kinases (CDKs), Aurora kinases and Polo-like kinases (PLKs).

However, there is another, less-well-characterised protein kinase

family, whose members have key roles in mitosis: the NIMA-related

kinases or NEKs (Moniz et al., 2011; O’Connell et al., 2003;

O’Regan et al., 2007; Quarmby and Mahjoub, 2005). Here, we

review our current knowledge of how NEKs contribute to the

process of cell division. Moreover, because targeting mitosis is a

proven approach to cancer treatment, we also debate the potential

use of NEKs as therapeutic targets in human cancer.

The NIMA-related kinase familyThe founding member of the NEK family is the never-in-mitosis

A (NIMA) protein of Aspergillus nidulans, which was identified

by Ron Morris and colleagues in a genetic screen for cell division

cycle mutants (Oakley and Morris, 1983). Loss-of-function

mutations in nimA cause G2 arrest, whereas overexpression

leads to cells attempting to enter mitosis prematurely (Osmani

et al., 1991; Osmani et al., 1988). It has been subsequently

discovered that degradation of NIMA is essential for mitotic exit,

which puts it on a par with the Cdc2–cyclin-B complex as a

master regulator of mitotic progression in Aspergillus (Pu and

Osmani, 1995). Aspergillus express a single NIMA-related gene,

as do the yeasts Saccharomyces cerevisiae (called kin3) and

Schizosaccharomyces pombe (called fin1; note that this is

unrelated to the budding yeast protein Fin1 that localises to the

mitotic spindle). However, although these yeast NEKs contribute

to various aspects of cell cycle progression, including chromatin

condensation, spindle assembly and cytokinesis, they are not

required for viability (De Souza et al., 2000; Grallert and Hagan,

2002; Grallert et al., 2004; Krien et al., 1998; Wu et al., 1998).

NEKs have now been identified in a wide range of organisms

from protists, such as Chlamydomonas, Plasmodium and

Tetrahymena, to multicellular eukaryotes, including Drosophila,

Xenopus, mice and humans. Human cells express eleven genes

that encode NEK1 to NEK11 (Fig. 1). The defining feature of

this protein family is an N-terminal catalytic domain that contains

all the motifs that are typical of a serine/threonine kinase and

shares closer sequence similarity with Aspergillus NIMA than

any other class of protein kinase. NEK10 breaks this rule by

having a centrally located kinase domain, but with regards to its

Commentary 4423

Journ

alof

Cell

Scie

nce

amino acid sequence it clearly belongs to the NEK family.

Generally, the NEK kinase domains are only moderately

conserved, with ,40–50% identity on the amino acid level both

to the kinase domain of NIMA and, on the whole, to each other.

NEK6 and NEK7 are unusual in this respect, because their kinase

domains share more than 85% sequence identity. All eleven human

NEKs contain a His-Arg-Asp (HRD) motif within the catalytic

domain, which is usually found in kinases that are positively

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.

Journal of Cell Science 125 (19)4424

Journ

alof

Cell

Scie

nce

regulated through phosphorylation (Johnson et al., 1996), and theyall possess a serine or threonine residue within the activation loop,

which is a probable site for an activating modification. In someNEKs, this residue is autophosphorylated, whereas in others it istargeted by an upstream kinase (Belham et al., 2003; Bertran et al.,

2011; Rellos et al., 2007; Roig et al., 2002). In terms of aphosphorylation consensus sequence, early studies found thatNIMA has a strong preference for phenylalanine at position 23(i.e. FxxS/T, where ‘x’ is any amino acid) (Lu et al., 1994). More

recent studies have indicated that human NEKs have a similarpreference, with both NEK2 and NEK6 preferring a hydrophobicresidue, ideally phenylalanine or leucine, at the 23 position (F/

LxxS/T) (Alexander et al., 2011; Lizcano et al., 2002). However,these are not strict requirements, as phosphorylation sites that donot fall into this motif have been mapped on NEK substrates.

In contrast with the conserved catalytic domains, theC-terminal regions of the NEKs are highly divergent in length,sequence and domain organisation (Fig. 1). The one relatively

common feature is an oligomerisation motif, usually acoiled-coil, which promotes autophosphorylation and activation.Autophosphorylation can occur within the activation loop of the

kinase domain. However, autophosphorylation is also likely tooccur elsewhere in the protein, as supported by the finding thatboth NEK8 and NEK9 autophosphorylate in the non-catalyticC-terminal region to regulate their localisation and/or activation

(Bertran et al., 2011; Zalli et al., 2012). Several NEKs, includingAspergillus NIMA and vertebrate NEK2, have also been shown tocontain destruction motifs within the non-catalytic regions that

target the protein for degradation (Hames et al., 2001; Pu andOsmani, 1995). NEK2 contains both a KEN (Lys-Glu-Asn) boxand a C-terminal methionine-arginine dipeptide (MR)-tail, which

allow recognition by the anaphase-promoting complex/cyclosome(APC/C). Unusually, the MR-tail mediates a direct interaction withcore subunits of the APC/C, thereby triggering NEK2 degradation

in early mitosis in a manner that is independent of the spindleassembly checkpoint (SAC) (Hayes et al., 2006). NEK6 and NEK7notably lack a C-terminal domain. They consist solely of a kinasedomain with a short N-terminal extension that might be important

for substrate recognition (Vaz Meirelles et al., 2010). Indeed,the non-catalytic regions of the other NEKs almost certainlycontribute to substrate recognition, as well as regulation, and this is

illustrated, for example, by the recognition of NEK6 and NEK7 bya sequence in the C-terminus of NEK9 (Roig et al., 2002).

Mitotic functions for human NEKsOverexpression of NIMA not only causes Aspergillus cells to

initiate mitotic events during any stage of the cell cycle, but alsoinduces aspects of mitotic entry when introduced into fissionyeast, Xenopus oocytes or human cells (Lu and Hunter, 1995;O’Connell et al., 1994). This observation provided the first clue

that NEKs might have roles in cell cycle control in highereukaryotes and effort is now being invested to explore thesefunctions (Fig. 2). Furthermore, details about the pathways that

control the activity of cell-cycle-regulated NEKs are emergingand are summarised in Fig. 3.

The role of NEKs in mitotic entry

No single human NEK is absolutely required for mitotic entry.

Nonetheless, it has now been well established that NEK2, NEK6,NEK7 and NEK9 participate in the architectural changes thattake place as cells move from interphase into mitosis. This

includes functions in centrosome separation and mitotic spindle

assembly and, potentially, chromatin condensation, nuclear pore

complex (NPC) disassembly and nuclear envelope breakdown.

Of the human NEKs, NEK2 is the most closely related to

Aspergillus NIMA and is the first NEK that has been studied in

any depth. Like NIMA, NEK2 is cell cycle regulated, and its

expression and activity peak in S and G2 phase. NEK2 is a core

component of the human centrosome (Andersen et al., 2003; Fry

et al., 1998b), where it regulates a key step in the centrosome

cycle, namely centrosome disjunction (O’Regan et al., 2007).

Importantly, many NEKs, from fungi to humans, are found

at the respective microtubule-organising centres (MTOCs) (for

examples, see De Souza et al., 2000; Krien et al., 2002; Mahjoub

et al., 2002; Prigent et al., 2005; Wloga et al., 2006). This crucial

piece of evidence has led to the hypothesis that NEKs exert their

functions through regulation of centrosomes and the microtubule

structures that they organise, and that this might not be restricted

to dividing cells (Box 1).

In human cells, centrosomes are composed of two

microtubule-based barrels, called centrioles, to which proteins

involved in microtubule nucleation are recruited. During

interphase, the two centrioles are held in close proximity

through a loose tether of filamentous proteins. This tether, or

inter-centriolar linker, extends between the proximal ends of the

mother and daughter centrioles and remains in place during S and

G2 while the two centrioles duplicate. The role of NEK2 is to

disassemble the tether at the onset of mitosis to facilitate

centrosome separation and the establishment of the mitotic

spindle. The organisation of the tether and how it is regulated by

G1

S

G2

M

Cilliary functionNEK1NEK8NEK4

G0

Proliferation andsignallingNEK3

G2-M checkpointNEK1NEK10NEK11

Mitotic progressionNEK2NEK6NEK7NEK9

Fig. 2. Roles for NEKs in cell cycle progression. Human NEKs contribute

to different events during cell cycle progression and differentiation. Our

current understanding is that during mitosis, NEK2, NEK6, NEK7 and NEK9

cooperate to ensure formation of a robust bipolar spindle that is capable of

satisfying the SAC. They might also contribute to other aspects of mitotic

progression, including chromatin condensation, nuclear envelope breakdown

and cytokinesis. Beyond mitosis, NEK3 contributes to prolactin-dependent

signalling and proliferation, whereas NEK1, NEK10 and NEK11 have all

been implicated in DDR signalling, particularly at the G2-M transition.

Finally, there is evidence that NEK1, NEK8 and possibly NEK4, have key

roles in cilia in post-mitotic cells.

NEKs in cell cycle regulation 4425

Journ

alof

Cell

Scie

nce

NEK2 have not yet been fully elucidated. However, two major

components of the linker, which are related in sequence, are the

large coiled-coil proteins, C-Nap1 (also known as CEP250) and

rootletin (Bahe et al., 2005; Faragher and Fry, 2003; Fry et al.,

1998a; Yang et al., 2006). These can be phosphorylated by

NEK2, and the current hypothesis is that their phosphorylation

triggers the dissolution of the tether. However, it is likely that

there are other components of this linker structure that might also

be regulated by NEK2, including both CEP68 and b-catenin

(Bahmanyar et al., 2008; Graser et al., 2007).

Whereas disjunction of centrosomes by NEK2 facilitates

spindle pole separation at the onset of mitosis, NEK6, NEK7

and NEK9 have other important roles in generating the mitotic

spindle (O’Regan et al., 2007; Sdelci et al., 2011). These three

proteins act in concert, whereby NEK9 is an upstream activator

of NEK6 and NEK7 (Belham et al., 2003). NEK9 was originally

PLK1

NEK6

-TuRC Eg5 MTs

NEK7

Mitotic entry

Spindle formation

Mitotic exit

NEK9

CDK1

PLK1 CDK1

MST2 T2NEK2

C-Nap1 ap1Rootletin

PP1

HEC1 HEECC1

NEK2

NEK11

-TrCP

ATM/ATR

Proteasomal degradation

Cyclin B Cyclin B

CDK1

CHK1

CDC25A

RAF1

MEK1

ERK1/2

G2 M

RA

ME

NEK10

MAD2

NEK2

Ser165-P

CDDC25AACD

EK11

UV IR

A Centrosome disjunction B SAC signaling

C Spindle assembly D DNA damage response

NEK

MAD1

Sister chromatids Inter-centriolar

linker

Centriole pair Kinetochore

Ser273-P

Ser82-P Ser76-P

Fig. 3. Pathways regulating NEKs in cell cycle control. (A) During interphase, NEK2 exists in a complex with protein phosphatase 1 (PP1) and mammalian

STE20-like protein kinase 2 (MST2), and is maintained in an inactive state through dephosphorylation by PP1 (Helps et al., 2000; Mardin et al., 2010). Following

the onset of mitosis, PLK1 phosphorylates MST2, thereby preventing PP1 from binding to the MST2–NEK2 complex (Mardin et al., 2011). This allows NEK2 to

autophosphorylate and become active. NEK2 then phosphorylates the inter-centriolar linker proteins C-Nap1 and rootletin, causing their displacement from the

centrosome. (B) NEK2 might also localise to the kinetochore of unaligned sister chromatids during mitosis and carry out a role in the SAC through

phosphorylating HEC1 on Ser165 and interacting with MAD1 (Du et al., 2008; Lou et al., 2004; Wei et al., 2011). (C) At the onset of mitosis, CDK1

phosphorylates a number of sites in NEK9, which primes NEK9 for subsequent phosphorylation and activation by PLK1 (Bertran et al., 2011). Activated NEK9

can then phosphorylate NEK6 and NEK7, which subsequently phosphorylate components (Eg5, microtubules and the c-TuRC) that are necessary for proper

mitotic spindle formation (Sdelci et al., 2011). (D) In response to UV-induced DNA damage, NEK10 forms a trimeric complex with MEK1 and RAF1. This

causes activation of MEK1, which then phosphorylates and activates ERK1/2. This leads to cell cycle arrest; however, the pathway by which this occurs has not

yet been clarified. DNA breaks caused by IR activate the kinases ATM and ATR, which phosphorylate and activate CHK1. CHK1 phosphorylates NEK11 on

Ser273, thereby activating it, and CDC25A on Ser76, thereby priming DSG motifs (e.g. Ser82) in CDC25A for phosphorylation by NEK11. This promotes binding

of b-TrCP, which leads to the ubiquitylation and degradation of CDC25A, which, in turn, prevents CDK1–cyclin-B activation and mitotic entry. MTs,

microtubules.

Journal of Cell Science 125 (19)4426

Journ

alof

Cell

Scie

nce

identified through its physical association with NEK6. This

interaction is more prominent during mitosis and requires NEK6

to be active (Belham et al., 2003; Rapley et al., 2008; Roig et al.,

2002). Furthermore, NEK9 can phosphorylate NEK6 in vitro on a

site in the activation loop that is important for NEK6 activity.

Thus, on a first level, NEK9 acts as an upstream kinase for

NEK6. On the basis of the high sequence similarity between

NEK6 and NEK7, it has been assumed that NEK9 activates

NEK7 in the same manner (Belham et al., 2003). However, on a

second level, direct interaction with the non-catalytic domain of

NEK9 appears to allosterically activate both NEK6 and NEK7 by

disrupting an auto-inhibitory conformation of these kinases

(Richards et al., 2009). The fact that NEK6 activity promotes

interaction with NEK9 also raises the possibility of a feedback

mechanism, whereby NEK6 (and NEK7) phosphorylates NEK9,

although this remains to be investigated.

The kinase activity of all three of these NEKs is elevated in

mitosis and early functional studies using overexpressed proteins

and antibody microinjection provided evidence that these

proteins had a role in spindle formation (Roig et al., 2002;

Yissachar et al., 2006). Such a role has been confirmed through

RNA interference (RNAi) depletion studies, which have revealed

that the loss of NEK6, NEK7 or NEK9 leads to failure of

centrosome separation in prophase and/or formation of weak

mitotic spindles with reduced microtubule density and interpolar

distances (Bertran et al., 2011; Kim et al., 2007; O’Regan and

Fry, 2009). Importantly, these changes activate the SAC and

thereby lead to mitotic arrest with cells frequently initiating

apoptosis as a result. The simplest explanation for these spindle

defects would be a reduction in microtubule nucleation from the

centrosomes or spindle poles in mitosis. Indeed, NEK9 associates

with multiple components of the c-tubulin ring complex (c-

TuRC), which initiates microtubule nucleation, and

phosphorylates the c-TuRC adaptor protein, NEDD1/GCP-WD,

whereas NEK7 might be required to recruit c-tubulin to the poles

(Kim et al., 2007; Roig et al., 2005; Sdelci et al., 2012). However,

NEK6 does not obviously concentrate at spindle poles, but does

weakly associate with the microtubules of the mitotic spindle

itself, and both NEK6 and NEK7 co-sediment with microtubules

(O’Regan and Fry, 2009). Moreover, depletion of NEK9 from

Xenopus egg extracts prevents microtubule aster formation

through either the centrosome- or the chromatin-mediated

pathway, which suggests that NEK9 activity is not restricted to

centrosomes (Roig et al., 2005). Thus, we speculate that these

kinases might control microtubule nucleation not only at spindle

poles but also within the spindle itself, possibly by regulating the

augmin complexes that specifically recruit c-TuRCs to spindle

fibres (Goshima and Kimura, 2010).

Another route through which these kinases might control

spindle formation is phosphorylation of microtubule-associated

proteins, such as Eg5 (also known as kinesin-like protein KIF11),

a member of the BimC kinesin family. Eg5 is a plus-end-directed

motor that crosslinks and slides microtubules across one another

in an anti-parallel manner, thereby driving spindle poles apart.

Recruitment of Eg5 to spindle microtubules depends on its

phosphorylation by CDK1 (Blangy et al., 1995; Sawin and

Mitchison, 1995). However, Eg5 is also phosphorylated by

NEK6, and this appears to be required for its function in

centrosome separation. This could explain why depletion of

either NEK6 or NEK9 can lead to monopolar spindle formation

(Bertran et al., 2011; Rapley et al., 2008). It is also worth bearing

in mind that NEK6 and NEK7 are capable of phosphorylating

tubulin in vitro, which raises the prospect of direct regulation of

microtubule dynamics through tubulin phosphorylation (O’Regan

and Fry, 2009).

Beyond spindle formation, NEK2, NEK6, NEK7 and NEK9

might also have additional functions in mitotic progression. Data

supporting a role for NEK2 in chromatin condensation have come

from studies on mouse meiotic spermatocytes (Di Agostino et al.,

2004). In these cells, chromatin condensation, as well as the

activation of NEK2 and phosphorylation of the chromatin

architecture protein HMGA2, is under the control of the

mitogen-activated protein kinase (MAPK) pathway. NEK2 can

interact with HMGA2 and phosphorylate it in vitro, which

decreases the affinity of HMG2A for DNA. Thus, NEK2 might

contribute to the release of HMGA2 from chromatin as cells

progress from the pachytene stage of G2 into M phase.

Interestingly, the NEK2C splice variant has a functional nuclear

localisation signal that is absent from other splice variants, which

supports the idea of a specific nuclear function for this isoform

(Wu et al., 2007). However, the way in which NEK2 is activated

by the MAPK pathway is not known. NEKs might also contribute

to disassembly of nuclear pore complexes (NPCs) and nuclear

envelope breakdown, as phosphorylation and release of the NPC

component, Nup98, is driven by both CDK1 and the mitotic NEKs

(Laurell et al., 2011). Moreover, NEK9 interacts with BICD2, a

Box 1. NEKs in ciliary function and ciliopathies

Roles for NEKs in cilia were first revealed through studies in ciliated

unicellular eukaryotes, such as Chlamydomonas and Tetrahymena

(Bradley and Quarmby, 2005; Mahjoub et al., 2002; Wloga et al.,

2006). Compared with non-ciliated fungi, the complement of genes

encoding NEKs in these organisms has expanded considerably,

and functional studies have suggested that these NEKs might be

crucial in regulating ciliary length (Parker et al., 2007). In

vertebrates, primary cilia have key roles in regulating signalling

pathways that are initiated at the cell surface, and it is now

understood that defective cilia underlie a wide range of inherited

human genetic disorders, which are collectively referred to as

ciliopathies (Bettencourt-Dias et al., 2011). A typical characteristic

of these disorders is polycystic kidney disease (PKD), and studies

on two mouse PKD models have led to the identification of

mutations in the Nek1 and Nek8 genes (Liu et al., 2002; Upadhya et

al., 2000; Vogler et al., 1999). Since then, mutations in NEK1 and

NEK8 have also been identified in human ciliopathy patients; these

NEKs are thought to have roles in the post-mitotic process of cilia

assembly and/or function (Otto et al., 2008; Quarmby and Mahjoub,

2005; Thiel et al., 2011). Interestingly, NEK4 has also recently been

implicated in cilium stability (Coene et al., 2011). Centrosomes and

microtubules are intimately involved both in the assembly of the

mitotic spindle and in ciliogenesis, which provides a plausible

explanation for why NEKs have evolved to contribute to both

processes. It has even been suggested that NEKs might somehow

coordinate these processes to either induce cilia formation following

cell cycle exit or to stimulate cilia resorption on cell cycle re-entry

(Quarmby and Mahjoub, 2005; Spalluto et al., 2012). Moreover,

besides defective ciliary signalling, aberrant cell cycle regulation or

spindle orientation could directly contribute to PKD. Hence, some of

the disease symptoms that result from the mutation or loss of NEK1

and NEK8 could result from the loss of a cell cycle function of these

two kinases.

NEKs in cell cycle regulation 4427

Journ

alof

Cell

Scie

nce

protein that associates with dynein and facilitates nuclear envelopebreakdown at mitosis onset (Holland et al., 2002).

NEKs and cytokinesis

As mentioned above, Fin1 has a key role in regulating cytokinesisin fission yeast (Grallert et al., 2004). Meanwhile, in Drosophila,NEK2 localises to the midbody of late mitotic cells, and itsoverexpression results in the mislocalisation of actin and anillin at

ectopic sites of cleavage furrow formation (Prigent et al., 2005).There is some evidence that vertebrate NEKs might also beinvolved in the final stages of cell division. First, NEK2B, a short

NEK2 splice variant that lacks the APC/C-mediated degradationmotifs, persists into late mitosis, and its depletion by RNAi resultsin delayed mitotic exit (Fletcher et al., 2005). Second, when

NEK6- or NEK7-depleted cells succeed in progressing beyondmetaphase, they frequently fail to complete abscission (Kim et al.,2007; O’Regan and Fry, 2009). Consistent with this, abscission

defects are the most common phenotype in cells expressinghypomorphic mutants of NEK6 or NEK7 with reduced kinaseactivity. NEK6 and NEK7 concentrate at the midbody in latemitotic cells and the kinase activity of NEK6 is maximal during

cytokinesis (Kim et al., 2007; O’Regan and Fry, 2009; Rapleyet al., 2008). Furthermore, Nek7-knockout mice die in lateembryogenesis or early post-natal stages, and fibroblasts derived

from Nek72/2 embryos show defects that are indicative ofcytokinesis failure (Salem et al., 2010). Mechanistically, it ispossible that NEK6 and NEK7 regulate the localisation or activity

of factors that are directly required for cytokinesis. Alternatively,these kinases could regulate the dynamics of central spindlemicrotubules in a similar manner to their role in the regulation ofmicrotubules during spindle assembly (O’Regan and Fry, 2009).

The role of NEKs in cell cycle checkpoints

Cell cycle progression is monitored by a series of checkpointsthat arrest the cell cycle in response to DNA damage. Whereassome NEKs, such as NEK2 and NEK6, are checkpoint targets

that are inhibited by DNA damage (Fletcher et al., 2004; Leeet al., 2008), others have more integral roles in DNA-damage-response (DDR) signalling. NEK1 is involved in both the sensing

and the repair of DNA strand breaks at the G1-S and G2-Mtransitions (Chen et al., 2011; Chen et al., 2008; Pelegrini et al.,2010; Polci et al., 2004). Depleting cells of NEK1 causes failureof checkpoint kinase 1 and 2 (CHK1 and CHK2; also known as

CHEK1 and CHEK2) activation in response to ultraviolet (UV)light and ionising radiation (IR), which places NEK1 early in thispathway. However, NEK1 activation is not dependent on the

ataxia telangiectasia mutated (ATM) or ataxia telangiectasia andRad3-related protein (ATR) kinases, which suggests that NEK1might act as an independent transducer of the damage signal.

NEK10 and NEK11 have both been implicated in the G2-MDDR checkpoint. In response to UV irradiation, but not tomitogenic growth factors, NEK10 forms a trimeric complex with

MEK1 and RAF1, where RAF1 is required for the interactionbetween NEK10 and MEK1 (Moniz and Stambolic, 2011).Although NEK10 does not alter RAF1 kinase activity, it does

promote MEK1 activation, which leads to phosphorylation ofextracellular-signal-regulated kinase 1/2 (ERK1/2) and G2-Marrest. Indeed, NEK10 depletion impairs activation of MEK1

and/or ERK1/2 in response to UV treatment. NEK11 exhibits acell-cycle-dependent expression pattern, and its expressionremains high from S phase to the G2-M transition (Noguchi

et al., 2002). However, NEK11 activity specifically increases in

response to DNA replication inhibitors and genotoxic stresses,and this activation is lost following the inhibition of the ATM andATR kinases (Melixetian et al., 2009; Noguchi et al., 2002).

Following exposure to IR, ATM and ATR phosphorylate CHK1,which then activates NEK11 by phosphorylating it on Ser273,while also phosphorylating CDC25A on Ser76 (Melixetian et al.,2009). Phosphorylation of CDC25A on Ser76 primes it for

further phosphorylation by active NEK11 within Asp-Ser-Gly(DSG) motifs of CDC25A, notably in the motif surroundingSer82. This leads to binding of the SCF(b-TrCP) ubiquitin ligase,

which targets CDC25A for proteasomal degradation and therebycauses G2-M arrest (Melixetian et al., 2009).

NEK2 might also have a role in the SAC, as it can interact withSAC components and phosphorylate the kinetochore complex

protein HEC1 (also known as NDC80) (Du et al., 2008; Lou et al.,2004; Wei et al., 2011). Specifically, HEC1 is phosphorylatedon Ser165 when present on the kinetochores of misaligned

chromosomes and it has been suggested that this phosphorylationevent is mediated by NEK2. Although not required for kinetochorelocalisation of Hec1, phosphorylation at this site might regulate

kinetochore–microtubule binding (Du et al., 2008). However, todate, there is little evidence that manipulation of NEK2 expressionor activity obviously interferes with SAC integrity.

Do ‘mitotic’ NEKs also function in interphase?As alluded to above, some members of the NEK family haveroles in non-dividing cells in regulating aspects of ciliary

function. This is true both for unicellular organisms, such asChlamydomonas and Tetrahymena, and for mammals, wheremutations in NEK1 and NEK8 can lead to ciliopathy-relateddisorders. This aspect of NEK function is discussed in more

detail in Box 1 and in the excellent review by Quarmby andMahjoub (Quarmby and Mahjoub, 2005). However, the focushere is on those NEKs implicated in cell cycle control.

Recent data has suggested that NEK7 might have a role inregulating the centrosome cycle during interphase. NEK7 can beweakly detected at interphase centrosomes (Kim et al., 2007;Yissachar et al., 2006), although it was not detected in a proteomic

analysis of centrosomes, and recombinant proteins do notobviously concentrate in this location unless they are fused to acentrosome-targeting motif (Kim et al., 2011). Nevertheless, it has

been proposed that NEK7 contributes to centrosome duplicationbecause its depletion leads to loss of centrosomes, whereas itsoverexpression induces formation of additional centrosomes in a

kinase-dependent manner (Kim et al., 2011). NEK7 also promotesthe recruitment of pericentriolar material (PCM) to centrosomes inG1 and S phase and this could explain its contribution tocentrosome duplication, because the amount of PCM directly

influences duplication efficiency (Loncarek et al., 2008). Thisresult is intriguing, because it suggests that NEK7 has interphasefunctions that are dependent on its kinase activity, when the current

view is that it is specifically activated in mitosis. Whether this isachieved through a basal level of activity that is present duringinterphase that is independent of NEK9 or whether there are

alternative interphase-specific NEK7 activators that are perhapslocalised to specific sites, remains to be seen. Interphase roles forNEK2 have also been suggested. First, in response to G1-S arrest,

autophosphorylated NEK2 can phosphorylate NEK11, whichresults in it opening up and thus the activation of an otherwiseinhibited conformation of NEK11 (Noguchi et al., 2004). Second,

Journal of Cell Science 125 (19)4428

Journ

alof

Cell

Scie

nce

NEK2 phosphorylates centrobin and limits the recruitment of this

daughter centriole-specific protein, which is essential forcentrosome duplication, to the centrosome (Jeong et al., 2007).However, the mechanistic details for these putative roles remain

unknown.

Allosteric regulatory mechanisms andchemical inhibitorsCrystal structures of the NEK2 and NEK7 catalytic domains havebeen determined (Rellos et al., 2007; Richards et al., 2009;Westwood et al., 2009). These structures have revealed inactivestates of these proteins that have provided insights into their

conformational flexibility and regulatory mechanisms (Fig. 1).Furthermore, they have enabled the generation of the first selectiveinhibitors that target NEK2 (Henise and Taunton, 2011; Innocenti

et al., 2012; Whelligan et al., 2010). Crystal structures wereparticularly important in revealing the changes in NEK2conformation that are induced by the inhibitors. In addition, they

made it possible to design compounds that have improved potencyagainst NEK2 and selectivity over other mitotic kinases. Two ofthe inhibitor scaffolds bind reversibly to the kinase, whereas a thirdone reacts to form a covalent linkage with Cys22 within the active

site, thus irreversibly inhibiting catalytic activity. Consistent withRNAi studies, the use of the irreversible inhibitor indicates thatNEK2 does not have an essential role in the mitotic progression of

A549 cells. Further studies with the available inhibitors will enablethe precise roles of NEK2 during mitosis in different cell types tobe delineated, and the experience of designing NEK2 inhibitors

will facilitate the generation of chemical inhibitors targeting othermembers of the NEK family.

The structure of NEK7, either bound to ADP or with a vacantactive site, has revealed a new inhibited conformation for the

kinase, in which the side-chain of Tyr97, which is located withinthe N-terminal lobe, points downwards into the active site(Fig. 1B) (Richards et al., 2009). This ‘Tyr-down’ conformation

blocks a number of key interactions that are required for an activekinase. Indeed, mutation of this residue to alanine activates thekinase. Even the mutation of Tyr97 to a phenylalanine residue,

which retains the aromatic ring but can no longer form astabilising hydrogen bond with the backbone of Leu180, leads tosome activation. Strikingly, the addition of a C-terminal fragmentof NEK9 stimulates the kinase activity of wild-type NEK7, but

not the already active NEK7 Y97A mutant. Similar results wereobtained with NEK6, which has a tyrosine residue at theequivalent position (Tyr108). On the basis of these data, it has

been proposed that NEK9 induces an allosteric activation ofNEK7 (and NEK6) that is independent of its ability to activatethese kinases through activation loop phosphorylation. Analysis

of the kinase domain of NEK2 structure has revealed that it, too,has a tyrosine at this position (Fig. 1C, Tyr70). In the presence ofADP, the side-chain of this residue points upwards and out of the

active site, that is, it is not inhibitory. Nonetheless, structures ofNEK2 in the presence of ATP-competitive inhibitors haverevealed a Tyr-down conformation, which indicates that thisresidue is flexible and can be switched up or down (Richards

et al., 2009; Whelligan et al., 2010). Eight out of eleven NEKs,and ,10% of human kinases in total, have a tyrosine residue atthis position in their catalytic domains, thus it is possible that this

mode of regulation applies to a number of these enzymes as well.

Structural studies of the non-catalytic domains of NEKs are yetto really begin but have the potential to reveal many more

important modes of regulation. Biophysical and nuclear magnetic

resonance (NMR)-based studies have been performed on the

coiled-coil motif of NEK2, which lies just downstream of the

catalytic domain (Croasdale et al., 2011). This motif promotes

homodimerisation and activation of NEK2, probably by trans-

autophosphorylation (Fry et al., 1999). According to sequence

analysis, this motif adopts a rather unusual leucine zipper that

could dimerise through one of two registers, and the NMR data

strongly support a model whereby this motif interchanges

between these two registers on a relatively slow timescale

(17 s21). On one hand, this argues that it will be challenging to

obtain a crystal structure of the full-length protein; on the other

hand, it opens up the possibility that interactions or post-

translational modifications could regulate NEK2 by favouring

adoption of one register over the other.

NEKs as anti-cancer targetsMicrotubule poisons such as taxanes and vinca alkaloids are

effective therapies in the treatment of many cancers, including

breast, ovarian and lung tumours (Jordan and Wilson, 2004).

However, their clinical use is limited by side effects, such as

neuropathy, poor responses in a subset of patients and drug

resistance. Microtubule poisons induce mitotic arrest by interfering

with microtubule dynamics, which eventually leads to cell death.

Patients would clearly benefit from compounds that trigger a similar

cellular response but do not affect the functions of microtubules in

nerve cells, and such a treatment could be used either to

complement, or as an alternative to, microtubule poisons (Jackson

et al., 2007). Members of the NEK family are attractive drug targets

because they are involved in specific aspects of microtubule

Box 2. Mitotic kinases as anti-cancer targets

There is an ever-present need for new chemotherapeutics to provide

effective therapies for cancer patients. As a consequence, there is

currently considerable interest in mitotic kinases as potential cancer

drug targets. Intensive medicinal chemistry research has resulted in

the development of a number of selective inhibitors for CDK1, PLK1,

Aurora A and Aurora B. Results from clinical trials on these

compounds have shown modest anti-tumour activity. To allow

further progress to be made in the development of anti-mitotic

cancer drugs, it is crucial to explain why these compounds have not

lived up to the promise from studies in cell and animal models. The fact

that mitotic kinase inhibitors cause haematological toxicity shows that

they are active against their targets in patient bone marrow cells,

and, indeed, haematological malignancies have shown the most

encouraging responses to mitotic kinase inhibitors. It has therefore

been suggested that targeting mitosis alone might not form a sufficient

basis for the effective therapy of solid tumours, which have a very low

rate of cell duplication (Komlodi-Pasztor et al., 2011). Furthermore,

mitotic kinases have varied and complex roles in cell cycle checkpoint

and cell death pathways, and cells treated with mitotic kinase inhibitors

have diverse fates (Gascoigne and Taylor, 2008). Because the

functions of many NEKs are poorly characterised, it is not yet clear how

we could define the desired selectivity profile of a successful and safe

NEK inhibitor, although we might predict that it would be undesirable to

inhibit NEKs that have been implicated in ciliary function. Nevertheless,

the development of selective compounds for mechanistic studies is

clearly a high priority if we are to understand how NEK inhibitors might

be developed as effective clinical treatments.

NEKs in cell cycle regulation 4429

Journ

alof

Cell

Scie

nce

function and/or the DNA damage checkpoint, which is another

precedented target pathway for cancer drug discovery (Box 2).

To date, the potential of NEKs as anti-cancer targets is relatively

unexplored and only limited target validation studies have been

carried out on a few members of the family. NEK2 is a potential

drug target in breast cancer (Hayward et al., 2004; Tsunoda et al.,

2009; Wu et al., 2008), as well as cholangiocarcinoma and

colorectal cancer (Kokuryo et al., 2007; Suzuki et al., 2010). The

availability of NEK2-selective inhibitors should enable further

target validation studies in these and other cancers. There is also

evidence in support of NEK6 as a tumour-promoting protein and

potential cancer drug target. NEK6 activity promotes anchorage-

independent growth; depletion of NEK6 leads to death in cancer cell

lines but is tolerated by normal fibroblasts (Nassirpour et al., 2010).

Overexpression of NEK6 also inhibits p53-dependent cellular

senescence (Jee et al., 2010). The molecular mechanisms that

underlie these observations are unknown, and could reflect a non-

mitotic function of NEK6. For example, NEK6 might affect the

transcriptional programme of a cancer cell through phosphorylation

of STAT3 on Ser727, an event that is required for maximal

transcriptional activity (Jeon et al., 2010). Although kinases are

generally regarded as druggable, developing NEK6-selective

compounds will be a challenge, because the active site is identical

to that of NEK7. Furthermore, these two kinases are particularly

insensitive to ATP-competitive inhibitors, which suggests that an

alternative approach to drug design might be required.

Conclusions and future perspectivesIt has now been well established that some human NEKs have

roles in mitotic progression, whereas others contribute to cilia

function (Quarmby and Mahjoub, 2005). The common theme that

underlies these two processes is the regulation of microtubule-

dependent processes, MTOCs or microtubules themselves

(Fig. 4). Phylogenetic analysis has indicated that the last

common ancestor of eukaryotes, which was a ciliated cell,

probably expressed as many as five NEKs, of which some

regulated cell division and others regulated cilia (Parker et al.,

2007). This analysis has also indicated that the NEKs can be

divided into sub-families that might predict functional similarity.

For example, NEK1, NEK3 and NEK5 fall into the same sub-

family, whereas NEK2 is indeed probably the orthologue of

NIMA and the yeast NEKs. The hypothesis is that those

organisms without cilia, for example, Aspergillus, have lost the

cilia-related NEKs, whereas organisms with complex cilia

arrangements, for example, Tetrahymena, have expanded the

repertoire of cilia-related NEKs. Human cells, of course, possess

NEKs that are devoted to each process. However, it is interesting

to consider that some NEKs might directly coordinate the two

events, as seems to be the case for some of the Chlamydomonas

NEKs (Bradley and Quarmby, 2005; Mahjoub et al., 2002). For

example, NEK7 is intimately linked with mitosis, but the

percentage of MEFs derived from Nek7-knockout mice that

bear primary cilia is reduced compared with that in wild-type

+TIP

A B

C

PP

NEK6 6

NEK7

P

P

NEK2

NEK9

NEK6 andNEK7

NEK1

NEK8

+TIP

+TIP

P

P

P

Eg5Eg5

NEK6

NEK6

NEK7

Tubulin C-terminal tails

NEK6

P

Glu

NEK7

Glu Glu

Primarycilium

Microtubule

Minus end

Plus end

βα

Inter-centriolarlinker

Centriolepair

Tubulin

Fig. 4. Potential roles for NEKs in

microtubule organisation. This diagram

illustrates how NEKs might have

evolved to contribute to different aspects

of microtubule organisation in cells.

(A) NEKs can regulate assembly of the

microtubule-based mitotic spindle.

NEK2 promotes loss of cohesion

between the interphase centrosomes,

whereas NEK6 and NEK7, under the

control of NEK9, contribute to robust

bipolar spindle assembly. (B) Some

NEKs, such as NEK1 and NEK8, have

roles in cilia, possibly through regulation

of axonemal microtubules and/or basal

bodies to control ciliary length.

However, they might impact on ciliary

function through alternative routes such

as regulating the stability of proteins

such as polycystin 2 (Yim et al., 2011).

(C) We speculate that NEKs could have

direct roles in regulating microtubules

themselves. For example, NEK6 and

NEK7 could phosphorylate a- or

b-tubulin or microtubule-associated

proteins, such as +TIP tracking proteins,

to regulate microtubule dynamics, or

they could modulate the activity of

microtubule-based motor proteins, such

as Eg5, or tubulin-modifying enzymes,

such as those that polyglutamylate

(indicated by ‘Glu’) the C-terminal tails

of tubulin.

Journal of Cell Science 125 (19)4430

Journ

alof

Cell

Scie

nce

cells (Salem et al., 2010). NEK3 has a role in prolactin-mediatedsignalling, but also potentially regulates the acetylation status of

microtubules in neurons (Chang et al., 2009; Miller et al., 2007).Furthermore, there is good evidence that NEK1 has roles in boththe DDR and cilia function, and NEK2 has recently been

proposed to promote cilium disassembly at the onset of mitosis(Spalluto et al., 2012). Another mitotic kinase, Aurora A,coordinates cilia resorption with mitotic entry, and this requires

activation of Aurora A by the focal adhesion scaffolding proteinNEDD9 (also known as HEF1 and Cas-L), which also happens bea regulator of NEK2 (Pugacheva and Golemis, 2005; Pugachevaet al., 2007).

From a cancer biology perspective, overexpression of themitotic NEKs in various tumour types suggests that they could

act as important prognostic biomarkers. Furthermore, from whatwe currently know about their functions, inhibition of the mitoticor DDR NEKs has the potential to selectively interfere with the

proliferation of cancer cells. However, greater structural detail ofthe NEKs is clearly required to understand their mechanisms ofactivation and to design specific inhibitors. Meanwhile, thedevelopment of knockout animals for the different NEKs will be

an important step in defining their key physiological roles, aswell as the potential side effects of NEK inhibitors. Finally,defining therapeutic windows, synthetic lethal interactions and

potential combination treatments is a huge challenge that wouldclearly benefit from the development of cell-permeable inhibitorsas tools. In summary, there is still a very long way to go in terms

of understanding the basic biology of this kinase family. Onlywith the development of this understanding will the mechanismsthrough which NEKs contribute to human disease become clear

and only then will we be in a position to fully exploit them astherapeutic targets.

AcknowledgementsWe thank Sharon Yeoh for comments on the manuscript andsincerely apologise to authors whose work we have not had space tocite.

FundingThe work of A.M.F. is supported by the Wellcome Trust; CancerResearch UK; and the Association for International Cancer Research(AICR). R.B. acknowledges support from Cancer Research UK; andthe Royal Society. The authors are members of the Leicester CancerResearch UK Experimental Cancer Medicine Centre.

ReferencesAlexander, J., Lim, D., Joughin, B. A., Hegemann, B., Hutchins, J. R., Ehrenberger,

T., Ivins, F., Sessa, F., Hudecz, O., Nigg, E. A. et al. (2011). Spatial exclusivitycombined with positive and negative selection of phosphorylation motifs is the basisfor context-dependent mitotic signaling. Sci. Signal. 4, ra42.

Andersen, J. S., Wilkinson, C. J., Mayor, T., Mortensen, P., Nigg, E. A. and Mann,M. (2003). Proteomic characterization of the human centrosome by proteincorrelation profiling. Nature 426, 570-574.

Bahe, S., Stierhof, Y. D., Wilkinson, C. J., Leiss, F. and Nigg, E. A. (2005). Rootletinforms centriole-associated filaments and functions in centrosome cohesion. J. Cell

Biol. 171, 27-33.Bahmanyar, S., Kaplan, D. D., Deluca, J. G., Giddings, T. H., Jr, O’Toole, E. T.,

Winey, M., Salmon, E. D., Casey, P. J., Nelson, W. J. and Barth, A. I. (2008). beta-Catenin is a Nek2 substrate involved in centrosome separation. Genes Dev. 22, 91-105.

Belham, C., Roig, J., Caldwell, J. A., Aoyama, Y., Kemp, B. E., Comb, M. and

Avruch, J. (2003). A mitotic cascade of NIMA family kinases. Nercc1/Nek9activates the Nek6 and Nek7 kinases. J. Biol. Chem. 278, 34897-34909.

Bertran, M. T., Sdelci, S., Regue, L., Avruch, J., Caelles, C. and Roig, J. (2011).Nek9 is a Plk1-activated kinase that controls early centrosome separation throughNek6/7 and Eg5. EMBO J. 30, 2634-2647.

Bettencourt-Dias, M., Hildebrandt, F., Pellman, D., Woods, G. and Godinho, S. A.(2011). Centrosomes and cilia in human disease. Trends Genet. 27, 307-315.

Blangy, A., Lane, H. A., d’Herin, P., Harper, M., Kress, M. and Nigg, E. A. (1995).

Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo. Cell 83, 1159-1169.

Bradley, B. A. and Quarmby, L. M. (2005). A NIMA-related kinase, Cnk2p, regulates

both flagellar length and cell size in Chlamydomonas. J. Cell Sci. 118, 3317-3326.

Chang, J., Baloh, R. H. and Milbrandt, J. (2009). The NIMA-family kinase Nek3

regulates microtubule acetylation in neurons. J. Cell Sci. 122, 2274-2282.

Chen, Y., Chen, P. L., Chen, C. F., Jiang, X. and Riley, D. J. (2008). Never-in-mitosis

related kinase 1 functions in DNA damage response and checkpoint control. Cell

Cycle 7, 3194-3201.

Chen, Y., Chen, C. F., Riley, D. J. and Chen, P. L. (2011). Nek1 kinase functions inDNA damage response and checkpoint control through a pathway independent ofATM and ATR. Cell Cycle 10, 655-663.

Coene, K. L., Mans, D. A., Boldt, K., Gloeckner, C. J., van Reeuwijk, J., Bolat, E.,

Roosing, S., Letteboer, S. J., Peters, T. A., Cremers, F. P. et al. (2011). Theciliopathy-associated protein homologs RPGRIP1 and RPGRIP1L are linked to cilium

integrity through interaction with Nek4 serine/threonine kinase. Hum. Mol. Genet. 20,3592-3605.

Croasdale, R., Ivins, F. J., Muskett, F., Daviter, T., Scott, D. J., Hardy, T., Smerdon,

S. J., Fry, A. M. and Pfuhl, M. (2011). An undecided coiled coil: the leucine zipperof Nek2 kinase exhibits atypical conformational exchange dynamics. J. Biol. Chem.

286, 27537-27547.

De Souza, C. P., Osmani, A. H., Wu, L. P., Spotts, J. L. and Osmani, S. A. (2000).Mitotic histone H3 phosphorylation by the NIMA kinase in Aspergillus nidulans. Cell

102, 293-302.

Di Agostino, S., Fedele, M., Chieffi, P., Fusco, A., Rossi, P., Geremia, R. and Sette,

C. (2004). Phosphorylation of high-mobility group protein A2 by Nek2 kinase duringthe first meiotic division in mouse spermatocytes. Mol. Biol. Cell 15, 1224-1232.

Du, J., Cai, X., Yao, J., Ding, X., Wu, Q., Pei, S., Jiang, K., Zhang, Y., Wang, W.,

Shi, Y. et al. (2008). The mitotic checkpoint kinase NEK2A regulates kinetochoremicrotubule attachment stability. Oncogene 27, 4107-4114.

Faragher, A. J. and Fry, A. M. (2003). Nek2A kinase stimulates centrosomedisjunction and is required for formation of bipolar mitotic spindles. Mol. Biol. Cell

14, 2876-2889.

Fletcher, L., Cerniglia, G. J., Nigg, E. A., Yend, T. J. and Muschel, R. J. (2004).Inhibition of centrosome separation after DNA damage: a role for Nek2. Radiat. Res.

162, 128-135.

Fletcher, L., Cerniglia, G. J., Yen, T. J. and Muschel, R. J. (2005). Live cell imaging

reveals distinct roles in cell cycle regulation for Nek2A and Nek2B. Biochim.

Biophys. Acta 1744, 89-92.

Fry, A. M., Mayor, T., Meraldi, P., Stierhof, Y. D., Tanaka, K. and Nigg, E. A.

(1998a). C-Nap1, a novel centrosomal coiled-coil protein and candidate substrate ofthe cell cycle-regulated protein kinase Nek2. J. Cell Biol. 141, 1563-1574.

Fry, A. M., Meraldi, P. and Nigg, E. A. (1998b). A centrosomal function for the humanNek2 protein kinase, a member of the NIMA family of cell cycle regulators. EMBO J.

17, 470-481.

Fry, A. M., Arnaud, L. and Nigg, E. A. (1999). Activity of the human centrosomalkinase, Nek2, depends on an unusual leucine zipper dimerization motif. J. Biol. Chem.

274, 16304-16310.

Gascoigne, K. E. and Taylor, S. S. (2008). Cancer cells display profound intra- andinterline variation following prolonged exposure to antimitotic drugs. Cancer Cell 14,

111-122.

Goshima, G. and Kimura, A. (2010). New look inside the spindle: microtubule-

dependent microtubule generation within the spindle. Curr. Opin. Cell Biol. 22, 44-49.

Grallert, A. and Hagan, I. M. (2002). Schizosaccharomyces pombe NIMA-relatedkinase, Fin1, regulates spindle formation and an affinity of Polo for the SPB. EMBO

J. 21, 3096-3107.

Grallert, A., Krapp, A., Bagley, S., Simanis, V. and Hagan, I. M. (2004). Recruitmentof NIMA kinase shows that maturation of the S. pombe spindle-pole body occurs overconsecutive cell cycles and reveals a role for NIMA in modulating SIN activity.

Genes Dev. 18, 1007-1021.

Graser, S., Stierhof, Y. D. and Nigg, E. A. (2007). Cep68 and Cep215 (Cdk5rap2) are

required for centrosome cohesion. J. Cell Sci. 120, 4321-4331.

Hames, R. S., Wattam, S. L., Yamano, H., Bacchieri, R. and Fry, A. M. (2001). APC/

C-mediated destruction of the centrosomal kinase Nek2A occurs in early mitosis anddepends upon a cyclin A-type D-box. EMBO J. 20, 7117-7127.

Hayes, M. J., Kimata, Y., Wattam, S. L., Lindon, C., Mao, G., Yamano, H. and Fry,

A. M. (2006). Early mitotic degradation of Nek2A depends on Cdc20-independentinteraction with the APC/C. Nat. Cell Biol. 8, 607-614.

Hayward, D. G., Clarke, R. B., Faragher, A. J., Pillai, M. R., Hagan, I. M. and Fry,

A. M. (2004). The centrosomal kinase Nek2 displays elevated levels of proteinexpression in human breast cancer. Cancer Res. 64, 7370-7376.

Helps, N. R., Luo, X., Barker, H. M. and Cohen, P. T. (2000). NIMA-related kinase 2(Nek2), a cell-cycle-regulated protein kinase localized to centrosomes, is complexed

to protein phosphatase 1. Biochem. J. 349, 509-518.

Henise, J. C. and Taunton, J. (2011). Irreversible Nek2 kinase inhibitors with cellularactivity. J. Med. Chem. 54, 4133-4146.

Holland, P. M., Milne, A., Garka, K., Johnson, R. S., Willis, C., Sims, J. E., Rauch,

C. T., Bird, T. A. and Virca, G. D. (2002). Purification, cloning, and

characterization of Nek8, a novel NIMA-related kinase, and its candidate substrateBicd2. J. Biol. Chem. 277, 16229-16240.

NEKs in cell cycle regulation 4431

Journ

alof

Cell

Scie

nce

Innocenti, P., Cheung, K. M., Solanki, S., Mas-Droux, C., Rowan, F., Yeoh, S.,

Boxall, K., Westlake, M., Pickard, L., Hardy, T. et al. (2012). Design of potent andselective hybrid inhibitors of the mitotic kinase Nek2: structure-activity relationship,structural biology, and cellular activity. J. Med. Chem. 55, 3228-3241.

Jackson, J. R., Patrick, D. R., Dar, M. M. and Huang, P. S. (2007). Targeted anti-mitotic therapies: can we improve on tubulin agents? Nat. Rev. Cancer 7, 107-117.

Jee, H. J., Kim, A. J., Song, N., Kim, H. J., Kim, M., Koh, H. and Yun, J. (2010).Nek6 overexpression antagonizes p53-induced senescence in human cancer cells. Cell

Cycle 9, 4703-4710.

Jeon, Y. J., Lee, K. Y., Cho, Y. Y., Pugliese, A., Kim, H. G., Jeong, C. H., Bode,

A. M. and Dong, Z. (2010). Role of NEK6 in tumor promoter-induced transformationin JB6 C141 mouse skin epidermal cells. J. Biol. Chem. 285, 28126-28133.

Jeong, Y., Lee, J., Kim, K., Yoo, J. C. and Rhee, K. (2007). Characterization of NIP2/centrobin, a novel substrate of Nek2, and its potential role in microtubulestabilization. J. Cell Sci. 120, 2106-2116.

Johnson, L. N., Noble, M. E. and Owen, D. J. (1996). Active and inactive proteinkinases: structural basis for regulation. Cell 85, 149-158.

Jordan, M. A. and Wilson, L. (2004). Microtubules as a target for anticancer drugs.Nat. Rev. Cancer 4, 253-265.

Kim, S., Lee, K. and Rhee, K. (2007). NEK7 is a centrosomal kinase critical formicrotubule nucleation. Biochem. Biophys. Res. Commun. 360, 56-62.

Kim, S., Kim, S. and Rhee, K. (2011). NEK7 is essential for centriole duplication andcentrosomal accumulation of pericentriolar material proteins in interphase cells.J. Cell Sci. 124, 3760-3770.

Kokuryo, T., Senga, T., Yokoyama, Y., Nagino, M., Nimura, Y. and Hamaguchi,

M. (2007). Nek2 as an effective target for inhibition of tumorigenic growth andperitoneal dissemination of cholangiocarcinoma. Cancer Res. 67, 9637-9642.

Komlodi-Pasztor, E., Sackett, D., Wilkerson, J. and Fojo, T. (2011). Mitosis is not akey target of microtubule agents in patient tumors. Nat. Rev. Clin. Oncol. 8, 244-250.

Krien, M. J., Bugg, S. J., Palatsides, M., Asouline, G., Morimyo, M. and O’Connell,

M. J. (1998). A NIMA homologue promotes chromatin condensation in fission yeast.J. Cell Sci. 111, 967-976.

Krien, M. J., West, R. R., John, U. P., Koniaras, K., McIntosh, J. R. and O’Connell,

M. J. (2002). The fission yeast NIMA kinase Fin1p is required for spindle functionand nuclear envelope integrity. EMBO J. 21, 1713-1722.

Laurell, E., Beck, K., Krupina, K., Theerthagiri, G., Bodenmiller, B., Horvath, P.,Aebersold, R., Antonin, W. and Kutay, U. (2011). Phosphorylation of Nup98 bymultiple kinases is crucial for NPC disassembly during mitotic entry. Cell 144, 539-550.

Lee, M. Y., Kim, H. J., Kim, M. A., Jee, H. J., Kim, A. J., Bae, Y. S., Park, J. I.,Chung, J. H. and Yun, J. (2008). Nek6 is involved in G2/M phase cell cycle arrestthrough DNA damage-induced phosphorylation. Cell Cycle 7, 2705-2709.

Liu, S., Lu, W., Obara, T., Kuida, S., Lehoczky, J., Dewar, K., Drummond, I. A. andBeier, D. R. (2002). A defect in a novel Nek-family kinase causes cystic kidneydisease in the mouse and in zebrafish. Development 129, 5839-5846.

Lizcano, J. M., Deak, M., Morrice, N., Kieloch, A., Hastie, C. J., Dong, L.,

Schutkowski, M., Reimer, U. and Alessi, D. R. (2002). Molecular basis for thesubstrate specificity of NIMA-related kinase-6 (NEK6). Evidence that NEK6 does notphosphorylate the hydrophobic motif of ribosomal S6 protein kinase and serum- andglucocorticoid-induced protein kinase in vivo. J. Biol. Chem. 277, 27839-27849.

Loncarek, J., Hergert, P., Magidson, V. and Khodjakov, A. (2008). Control ofdaughter centriole formation by the pericentriolar material. Nat. Cell Biol. 10, 322-328.

Lou, Y., Yao, J., Zereshki, A., Dou, Z., Ahmed, K., Wang, H., Hu, J., Wang, Y. andYao, X. (2004). NEK2A interacts with MAD1 and possibly functions as a novelintegrator of the spindle checkpoint signaling. J. Biol. Chem. 279, 20049-20057.

Lu, K. P. and Hunter, T. (1995). Evidence for a NIMA-like mitotic pathway invertebrate cells. Cell 81, 413-424.

Lu, K. P., Kemp, B. E. and Means, A. R. (1994). Identification of substrate specificitydeterminants for the cell cycle-regulated NIMA protein kinase. J. Biol. Chem. 269,6603-6607.

Mahjoub, M. R., Montpetit, B., Zhao, L., Finst, R. J., Goh, B., Kim, A. C. and

Quarmby, L. M. (2002). The FA2 gene of Chlamydomonas encodes a NIMA familykinase with roles in cell cycle progression and microtubule severing duringdeflagellation. J. Cell Sci. 115, 1759-1768.

Mardin, B. R., Lange, C., Baxter, J. E., Hardy, T., Scholz, S. R., Fry, A. M. and

Schiebel, E. (2010). Components of the Hippo pathway cooperate with Nek2 kinaseto regulate centrosome disjunction. Nat. Cell Biol. 12, 1166-1176.

Mardin, B. R., Agircan, F. G., Lange, C. and Schiebel, E. (2011). Plk1 controls theNek2A-PP1c antagonism in centrosome disjunction. Curr. Biol. 21, 1145-1151.

Melixetian, M., Klein, D. K., Sørensen, C. S. and Helin, K. (2009). NEK11 regulatesCDC25A degradation and the IR-induced G2/M checkpoint. Nat. Cell Biol. 11, 1247-1253.

Miller, S. L., Antico, G., Raghunath, P. N., Tomaszewski, J. E. and Clevenger, C. V.(2007). Nek3 kinase regulates prolactin-mediated cytoskeletal reorganization andmotility of breast cancer cells. Oncogene 26, 4668-4678.

Moniz, L. S. and Stambolic, V. (2011). Nek10 mediates G2/M cell cycle arrest andMEK autoactivation in response to UV irradiation. Mol. Cell. Biol. 31, 30-42.

Moniz, L., Dutt, P., Haider, N. and Stambolic, V. (2011). Nek family of kinases in cellcycle, checkpoint control and cancer. Cell Div. 6, 18.

Nassirpour, R., Shao, L., Flanagan, P., Abrams, T., Jallal, B., Smeal, T. and Yin,M. J. (2010). Nek6 mediates human cancer cell transformation and is a potentialcancer therapeutic target. Mol. Cancer Res. 8, 717-728.

Noguchi, K., Fukazawa, H., Murakami, Y. and Uehara, Y. (2002). Nek11, a newmember of the NIMA family of kinases, involved in DNA replication and genotoxicstress responses. J. Biol. Chem. 277, 39655-39665.

Noguchi, K., Fukazawa, H., Murakami, Y. and Uehara, Y. (2004). Nucleolar Nek11is a novel target of Nek2A in G1/S-arrested cells. J. Biol. Chem. 279, 32716-32727.

O’Connell, M. J., Norbury, C. and Nurse, P. (1994). Premature chromatincondensation upon accumulation of NIMA. EMBO J. 13, 4926-4937.

O’Connell, M. J., Krien, M. J. and Hunter, T. (2003). Never say never. The NIMA-related protein kinases in mitotic control. Trends Cell Biol. 13, 221-228.

O’Regan, L., Blot, J. and Fry, A. M. (2007). Mitotic regulation by NIMA-relatedkinases. Cell Div. 2, 25.

O’Regan, L. and Fry, A. M. (2009). The Nek6 and Nek7 protein kinases are requiredfor robust mitotic spindle formation and cytokinesis. Mol. Cell. Biol. 29, 3975-3990.

Oakley, B. R. and Morris, N. R. (1983). A mutation in Aspergillus nidulans that blocksthe transition from interphase to prophase. J. Cell Biol. 96, 1155-1158.

Osmani, S. A., Pu, R. T. and Morris, N. R. (1988). Mitotic induction and maintenanceby overexpression of a G2-specific gene that encodes a potential protein kinase. Cell

53, 237-244.

Osmani, A. H., McGuire, S. L. and Osmani, S. A. (1991). Parallel activation of theNIMA and p34cdc2 cell cycle-regulated protein kinases is required to initiate mitosisin A. nidulans. Cell 67, 283-291.

Otto, E. A., Trapp, M. L., Schultheiss, U. T., Helou, J., Quarmby, L. M. and

Hildebrandt, F. (2008). NEK8 mutations affect ciliary and centrosomal localizationand may cause nephronophthisis. J. Am. Soc. Nephrol. 19, 587-592.

Parker, J. D., Bradley, B. A., Mooers, A. O. and Quarmby, L. M. (2007).Phylogenetic analysis of the Neks reveals early diversification of ciliary-cell cyclekinases. PLoS ONE 2, e1076.

Pelegrini, A. L., Moura, D. J., Brenner, B. L., Ledur, P. F., Maques, G. P.,

Henriques, J. A., Saffi, J. and Lenz, G. (2010). Nek1 silencing slows down DNArepair and blocks DNA damage-induced cell cycle arrest. Mutagenesis 25, 447-454.

Polci, R., Peng, A., Chen, P. L., Riley, D. J. and Chen, Y. (2004). NIMA-relatedprotein kinase 1 is involved early in the ionizing radiation-induced DNA damageresponse. Cancer Res. 64, 8800-8803.

Prigent, C., Glover, D. M. and Giet, R. (2005). Drosophila Nek2 protein kinaseknockdown leads to centrosome maturation defects while overexpression causescentrosome fragmentation and cytokinesis failure. Exp. Cell Res. 303, 1-13.

Pu, R. T. and Osmani, S. A. (1995). Mitotic destruction of the cell cycle regulatedNIMA protein kinase of Aspergillus nidulans is required for mitotic exit. EMBO J. 14,995-1003.

Pugacheva, E. N. and Golemis, E. A. (2005). The focal adhesion scaffolding proteinHEF1 regulates activation of the Aurora-A and Nek2 kinases at the centrosome. Nat.

Cell Biol. 7, 937-946.

Pugacheva, E. N., Jablonski, S. A., Hartman, T. R., Henske, E. P. and Golemis, E. A.

(2007). HEF1-dependent Aurora A activation induces disassembly of the primarycilium. Cell 129, 1351-1363.

Quarmby, L. M. and Mahjoub, M. R. (2005). Caught Nek-ing: cilia and centrioles.J. Cell Sci. 118, 5161-5169.

Rapley, J., Nicolas, M., Groen, A., Regue, L., Bertran, M. T., Caelles, C., Avruch,

J. and Roig, J. (2008). The NIMA-family kinase Nek6 phosphorylates the kinesinEg5 at a novel site necessary for mitotic spindle formation. J. Cell Sci. 121, 3912-3921.

Rellos, P., Ivins, F. J., Baxter, J. E., Pike, A., Nott, T. J., Parkinson, D. M., Das,

S., Howell, S., Fedorov, O., Shen, Q. Y. et al. (2007). Structure and regulation of thehuman Nek2 centrosomal kinase. J. Biol. Chem. 282, 6833-6842.

Richards, M. W., O’Regan, L., Mas-Droux, C., Blot, J. M., Cheung, J., Hoelder, S.,

Fry, A. M. and Bayliss, R. (2009). An autoinhibitory tyrosine motif in the cell-cycle-regulated Nek7 kinase is released through binding of Nek9. Mol. Cell 36, 560-570.

Roig, J., Mikhailov, A., Belham, C. and Avruch, J. (2002). Nercc1, a mammalianNIMA-family kinase, binds the Ran GTPase and regulates mitotic progression. Genes

Dev. 16, 1640-1658.

Roig, J., Groen, A., Caldwell, J. and Avruch, J. (2005). Active Nercc1 protein kinaseconcentrates at centrosomes early in mitosis and is necessary for proper spindleassembly. Mol. Biol. Cell 16, 4827-4840.

Salem, H., Rachmin, I., Yissachar, N., Cohen, S., Amiel, A., Haffner, R., Lavi,

L. and Motro, B. (2010). Nek7 kinase targeting leads to early mortality, cytokinesisdisturbance and polyploidy. Oncogene 29, 4046-4057.

Sawin, K. E. and Mitchison, T. J. (1995). Mutations in the kinesin-like protein Eg5disrupting localization to the mitotic spindle. Proc. Natl. Acad. Sci. USA 92, 4289-4293.

Sdelci, S., Bertran, M. T. and Roig, J. (2011). Nek9, Nek6, Nek7 and the separation ofcentrosomes. Cell Cycle 10, 3816-3817.

Sdelci, S., Schutz, M., Pinyol, R., Bertran, M. T., Regue, L., Caelles, C., Vernos, I.

and Roig, J. (2012). Nek9 phosphorylation of NEDD1/GCP-WD contributes to Plk1control of c-tubulin recruitment to the mitotic centrosome. Curr. Biol. 22, 1516-1523.

Spalluto, C., Wilson, D. I. and Hearn, T. (2012). Nek2 localises to the distal portion ofthe mother centriole/basal body and is required for timely cilium disassembly at theG2/M transition. Eur. J. Cell Biol. 91, 675-686.

Suzuki, K., Kokuryo, T., Senga, T., Yokoyama, Y., Nagino, M. and Hamaguchi,

M. (2010). Novel combination treatment for colorectal cancer using Nek2 siRNA andcisplatin. Cancer Sci. 101, 1163-1169.

Thiel, C., Kessler, K., Giessl, A., Dimmler, A., Shalev, S. A., von der Haar,

S., Zenker, M., Zahnleiter, D., Stoss, H., Beinder, E. et al. (2011). NEK1 mutationscause short-rib polydactyly syndrome type majewski. Am. J. Hum. Genet. 88, 106-114.

Journal of Cell Science 125 (19)4432

Journ

alof

Cell

Scie

nce

Tsunoda, N., Kokuryo, T., Oda, K., Senga, T., Yokoyama, Y., Nagino, M., Nimura,Y. and Hamaguchi, M. (2009). Nek2 as a novel molecular target for the treatment ofbreast carcinoma. Cancer Sci. 100, 111-116.

Upadhya, P., Birkenmeier, E. H., Birkenmeier, C. S. and Barker, J. E. (2000).Mutations in a NIMA-related kinase gene, Nek1, cause pleiotropic effects including aprogressive polycystic kidney disease in mice. Proc. Natl. Acad. Sci. USA 97, 217-221.

Vaz Meirelles, G., Ferreira Lanza, D. C., da Silva, J. C., Santana Bernachi, J., PaesLeme, A. F. and Kobarg, J. (2010). Characterization of hNek6 interactome revealsan important role for its short N-terminal domain and colocalization with proteins atthe centrosome. J. Proteome Res. 9, 6298-6316.

Vogler, C., Homan, S., Pung, A., Thorpe, C., Barker, J., Birkenmeier, E. H. and

Upadhya, P. (1999). Clinical and pathologic findings in two new allelic murinemodels of polycystic kidney disease. J. Am. Soc. Nephrol. 10, 2534-2539.

Wei, R., Ngo, B., Wu, G. and Lee, W. H. (2011). Phosphorylation of the Ndc80complex protein, HEC1, by Nek2 kinase modulates chromosome alignment andsignaling of the spindle assembly checkpoint. Mol. Biol. Cell 22, 3584-3594.

Westwood, I., Cheary, D. M., Baxter, J. E., Richards, M. W., van Montfort, R. L.,Fry, A. M. and Bayliss, R. (2009). Insights into the conformational variability andregulation of human Nek2 kinase. J. Mol. Biol. 386, 476-485.

Whelligan, D. K., Solanki, S., Taylor, D., Thomson, D. W., Cheung, K.-M., Boxall,

K., Mas-Droux, C., Barillari, C., Burns, S., Grummitt, C. G. et al. (2010).Aminopyrazine inhibitors binding to an unusual inactive conformation of the mitotickinase Nek2: SAR and structural characterization. J. Med. Chem. 53, 7682-7698.

Wloga, D., Camba, A., Rogowski, K., Manning, G., Jerka-Dziadosz, M. andGaertig, J. (2006). Members of the NIMA-related kinase family promotedisassembly of cilia by multiple mechanisms. Mol. Biol. Cell 17, 2799-2810.

Wu, G., Qiu, X. L., Zhou, L., Zhu, J., Chamberlin, R., Lau, J., Chen, P. L. and Lee,

W. H. (2008). Small molecule targeting the Hec1/Nek2 mitotic pathway suppressestumor cell growth in culture and in animal. Cancer Res. 68, 8393-8399.

Wu, L., Osmani, S. A. and Mirabito, P. M. (1998). A role for NIMA in the nuclearlocalization of cyclin B in Aspergillus nidulans. J. Cell Biol. 141, 1575-1587.

Wu, W., Baxter, J. E., Wattam, S. L., Hayward, D. G., Fardilha, M., Knebel, A.,

Ford, E. M., da Cruz e Silva, E. F. and Fry, A. M. (2007). Alternative splicingcontrols nuclear translocation of the cell cycle-regulated Nek2 kinase. J. Biol. Chem.

282, 26431-26440.Yang, J., Adamian, M. and Li, T. (2006). Rootletin interacts with C-Nap1 and may

function as a physical linker between the pair of centrioles/basal bodies in cells.Mol. Biol. Cell 17, 1033-1040.

Yim, H., Sung, C. K., You, J., Tian, Y. and Benjamin, T. (2011). Nek1 and TAZinteract to maintain normal levels of polycystin 2. J. Am. Soc. Nephrol. 22, 832-837.

Yissachar, N., Salem, H., Tennenbaum, T. and Motro, B. (2006). Nek7 kinase isenriched at the centrosome, and is required for proper spindle assembly and mitoticprogression. FEBS Lett. 580, 6489-6495.

Zalli, D., Bayliss, R. and Fry, A. M. (2012). The Nek8 protein kinase, mutated in thehuman cystic kidney disease nephronophthisis, is both activated and degraded duringciliogenesis. Hum. Mol. Genet. 21, 1155-1171.

NEKs in cell cycle regulation 4433