text:
The NIMA-related kinase family
Mitotic functions for human NEKs
Do 'mitotic' NEKs also function in interphase?
Allosteric regulatory mechanisms and chemical inhibitors
NEKs as anti-cancer targets
Conclusions and future perspectives
COMMENTARY| 01 October 2012
Cell cycle regulation by the NEK family of protein kinases
Andrew M. Fry,
Andrew M. Fry *
Department of Biochemistry, University of Leicester
Lancaster Road, Leicester LE1 9HN
*Author for correspondence (amf5@le.ac.uk)
Laura O'Regan,
Laura O'Regan
Sarah R. Sabir,
Sarah R. Sabir
Richard Bayliss
Andrew M. Fry, Laura O'Regan, Sarah R. Sabir, Richard Bayliss; Cell cycle regulation by the NEK family of protein kinases. J Cell Sci 1 October 2012; 125 (19): 4423–4433. doi: https://doi.org/10.1242/jcs.111195
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.
NEK, NIMA, Mitosis, Centrosome, Microtubule, Mitotic spindle, DNA damage response, Cilia
Maintaining 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 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 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 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 targeted by an upstream kinase (Belham et al., 2003; Bertran et al., 2011; Rellos et al., 2007; Roig et al., 2002). In terms of a phosphorylation consensus sequence, early studies found that NIMA has a strong preference for phenylalanine at position −3 (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 preference, with both NEK2 and NEK6 preferring a hydrophobic residue, ideally phenylalanine or leucine, at the −3 position (F/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.
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.
In contrast with the conserved catalytic domains, the C-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 a coiled-coil, which promotes autophosphorylation and activation. Autophosphorylation can occur within the activation loop of the kinase domain. However, autophosphorylation is also likely to occur elsewhere in the protein, as supported by the finding that both NEK8 and NEK9 autophosphorylate in the non-catalytic C-terminal region to regulate their localisation and/or activation (Bertran et al., 2011; Zalli et al., 2012). Several NEKs, including Aspergillus NIMA and vertebrate NEK2, have also been shown to contain destruction motifs within the non-catalytic regions that target the protein for degradation (Hames et al., 2001; Pu and Osmani, 1995). NEK2 contains both a KEN (Lys-Glu-Asn) box and 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 with core subunits of the APC/C, thereby triggering NEK2 degradation in early mitosis in a manner that is independent of the spindle assembly checkpoint (SAC) (Hayes et al., 2006). NEK6 and NEK7 notably lack a C-terminal domain. They consist solely of a kinase domain 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 certainly contribute to substrate recognition, as well as regulation, and this is illustrated, for example, by the recognition of NEK6 and NEK7 by a sequence in the C-terminus of NEK9 (Roig et al., 2002).
Overexpression of NIMA not only causes Aspergillus cells to initiate mitotic events during any stage of the cell cycle, but also induces aspects of mitotic entry when introduced into fission yeast, 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 higher eukaryotes and effort is now being invested to explore these functions (Fig. 2). Furthermore, details about the pathways that control the activity of cell-cycle-regulated NEKs are emerging and are summarised in Fig. 3.
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.
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 γ-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 β-TrCP, which leads to the ubiquitylation and degradation of CDC25A, which, in turn, prevents CDK1–cyclin-B activation and mitotic entry. MTs, microtubules.
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 that take 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 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 β-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 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 γ-tubulin ring complex (γ-TuRC), which initiates microtubule nucleation, and phosphorylates the γ-TuRC adaptor protein, NEDD1/GCP-WD, whereas NEK7 might be required to recruit γ-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 γ-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 protein that associates with dynein and facilitates nuclear envelope breakdown at mitosis onset (Holland et al., 2002).
NEKs and cytokinesis
As mentioned above, Fin1 has a key role in regulating cytokinesis in fission yeast (Grallert et al., 2004). Meanwhile, in Drosophila, NEK2 localises to the midbody of late mitotic cells, and its overexpression 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 be involved in the final stages of cell division. First, NEK2B, a short NEK2 splice variant that lacks the APC/C-mediated degradation motifs, persists into late mitosis, and its depletion by RNAi results in delayed mitotic exit (Fletcher et al., 2005). Second, when NEK6- or NEK7-depleted cells succeed in progressing beyond metaphase, 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 expressing hypomorphic mutants of NEK6 or NEK7 with reduced kinase activity. NEK6 and NEK7 concentrate at the midbody in late mitotic cells and the kinase activity of NEK6 is maximal during cytokinesis (Kim et al., 2007; O'Regan and Fry, 2009; Rapley et al., 2008). Furthermore, Nek7-knockout mice die in late embryogenesis or early post-natal stages, and fibroblasts derived from Nek7−/− embryos show defects that are indicative of cytokinesis failure (Salem et al., 2010). Mechanistically, it is possible 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 spindle microtubules in a similar manner to their role in the regulation of microtubules 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 checkpoints that arrest the cell cycle in response to DNA damage. Whereas some NEKs, such as NEK2 and NEK6, are checkpoint targets that are inhibited by DNA damage (Fletcher et al., 2004; Lee et 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-M transitions (Chen et al., 2011; Chen et al., 2008; Pelegrini et al., 2010; Polci et al., 2004). Depleting cells of NEK1 causes failure of 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 this pathway. However, NEK1 activation is not dependent on the ataxia telangiectasia mutated (ATM) or ataxia telangiectasia and Rad3-related protein (ATR) kinases, which suggests that NEK1 might act as an independent transducer of the damage signal.
NEK10 and NEK11 have both been implicated in the G2-M DDR checkpoint. In response to UV irradiation, but not to mitogenic growth factors, NEK10 forms a trimeric complex with MEK1 and RAF1, where RAF1 is required for the interaction between NEK10 and MEK1 (Moniz and Stambolic, 2011). Although NEK10 does not alter RAF1 kinase activity, it does promote MEK1 activation, which leads to phosphorylation of extracellular-signal-regulated kinase 1/2 (ERK1/2) and G2-M arrest. Indeed, NEK10 depletion impairs activation of MEK1 and/or ERK1/2 in response to UV treatment. NEK11 exhibits a cell-cycle-dependent expression pattern, and its expression remains 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 and ATR 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 surrounding Ser82. This leads to binding of the SCF(β-TrCP) ubiquitin ligase, which targets CDC25A for proteasomal degradation and thereby causes G2-M arrest (Melixetian et al., 2009).
NEK2 might also have a role in the SAC, as it can interact with SAC 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 phosphorylated on Ser165 when present on the kinetochores of misaligned chromosomes and it has been suggested that this phosphorylation event is mediated by NEK2. Although not required for kinetochore localisation of Hec1, phosphorylation at this site might regulate kinetochore–microtubule binding (Du et al., 2008). However, to date, there is little evidence that manipulation of NEK2 expression or activity obviously interferes with SAC integrity.
As alluded to above, some members of the NEK family have roles in non-dividing cells in regulating aspects of ciliary function. This is true both for unicellular organisms, such as Chlamydomonas and Tetrahymena, and for mammals, where mutations in NEK1 and NEK8 can lead to ciliopathy-related disorders. This aspect of NEK function is discussed in more detail in Box 1 and in the excellent review by Quarmby and Mahjoub (Quarmby and Mahjoub, 2005). However, the focus here is on those NEKs implicated in cell cycle control.
Recent data has suggested that NEK7 might have a role in regulating the centrosome cycle during interphase. NEK7 can be weakly 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 not obviously concentrate in this location unless they are fused to a centrosome-targeting motif (Kim et al., 2011). Nevertheless, it has been proposed that NEK7 contributes to centrosome duplication because its depletion leads to loss of centrosomes, whereas its overexpression induces formation of additional centrosomes in a kinase-dependent manner (Kim et al., 2011). NEK7 also promotes the recruitment of pericentriolar material (PCM) to centrosomes in G1 and S phase and this could explain its contribution to centrosome duplication, because the amount of PCM directly influences duplication efficiency (Loncarek et al., 2008). This result is intriguing, because it suggests that NEK7 has interphase functions that are dependent on its kinase activity, when the current view is that it is specifically activated in mitosis. Whether this is achieved through a basal level of activity that is present during interphase that is independent of NEK9 or whether there are alternative interphase-specific NEK7 activators that are perhaps localised to specific sites, remains to be seen. Interphase roles for NEK2 have also been suggested. First, in response to G1-S arrest, autophosphorylated NEK2 can phosphorylate NEK11, which results in it opening up and thus the activation of an otherwise inhibited conformation of NEK11 (Noguchi et al., 2004). Second, NEK2 phosphorylates centrobin and limits the recruitment of this daughter centriole-specific protein, which is essential for centrosome duplication, to the centrosome (Jeong et al., 2007). However, the mechanistic details for these putative roles remain unknown.
Crystal structures of the NEK2 and NEK7 catalytic domains have been determined (Rellos et al., 2007; Richards et al., 2009; Westwood et al., 2009). These structures have revealed inactive states 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 selective inhibitors that target NEK2 (Henise and Taunton, 2011; Innocenti et al., 2012; Whelligan et al., 2010). Crystal structures were particularly important in revealing the changes in NEK2 conformation that are induced by the inhibitors. In addition, they made it possible to design compounds that have improved potency against NEK2 and selectivity over other mitotic kinases. Two of the inhibitor scaffolds bind reversibly to the kinase, whereas a third one reacts to form a covalent linkage with Cys22 within the active site, thus irreversibly inhibiting catalytic activity. Consistent with RNAi studies, the use of the irreversible inhibitor indicates that NEK2 does not have an essential role in the mitotic progression of A549 cells. Further studies with the available inhibitors will enable the precise roles of NEK2 during mitosis in different cell types to be delineated, and the experience of designing NEK2 inhibitors will facilitate the generation of chemical inhibitors targeting other members of the NEK family.
The structure of NEK7, either bound to ADP or with a vacant active site, has revealed a new inhibited conformation for the kinase, in which the side-chain of Tyr97, which is located within the 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 active kinase. Indeed, mutation of this residue to alanine activates the kinase. Even the mutation of Tyr97 to a phenylalanine residue, which retains the aromatic ring but can no longer form a stabilising hydrogen bond with the backbone of Leu180, leads to some activation. Strikingly, the addition of a C-terminal fragment of NEK9 stimulates the kinase activity of wild-type NEK7, but not the already active NEK7 Y97A mutant. Similar results were obtained with NEK6, which has a tyrosine residue at the equivalent position (Tyr108). On the basis of these data, it has been proposed that NEK9 induces an allosteric activation of NEK7 (and NEK6) that is independent of its ability to activate these 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 of ADP, the side-chain of this residue points upwards and out of the active site, that is, it is not inhibitory. Nonetheless, structures of NEK2 in the presence of ATP-competitive inhibitors have revealed a Tyr-down conformation, which indicates that this residue 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 at this 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 yet to 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 s−1). 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.
Microtubule 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 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.
It 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 cells (Salem et al., 2010). NEK3 has a role in prolactin-mediated signalling, 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 both the 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 protein NEDD9 (also known as HEF1 and Cas-L), which also happens be a regulator of NEK2 (Pugacheva and Golemis, 2005; Pugacheva et al., 2007).
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 α- or β-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.
From a cancer biology perspective, overexpression of the mitotic NEKs in various tumour types suggests that they could act as important prognostic biomarkers. Furthermore, from what we currently know about their functions, inhibition of the mitotic or DDR NEKs has the potential to selectively interfere with the proliferation of cancer cells. However, greater structural detail of the NEKs is clearly required to understand their mechanisms of activation and to design specific inhibitors. Meanwhile, the development of knockout animals for the different NEKs will be an important step in defining their key physiological roles, as well as the potential side effects of NEK inhibitors. Finally, defining therapeutic windows, synthetic lethal interactions and potential combination treatments is a huge challenge that would clearly benefit from the development of cell-permeable inhibitors as tools. In summary, there is still a very long way to go in terms of understanding the basic biology of this kinase family. Only with the development of this understanding will the mechanisms through which NEKs contribute to human disease become clear and only then will we be in a position to fully exploit them as therapeutic targets.
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.
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.
We thank Sharon Yeoh for comments on the manuscript and sincerely apologise to authors whose work we have not had space to cite.
The work of A.M.F. is supported by the Wellcome Trust; Cancer Research UK; and the Association for International Cancer Research (AICR). R.B. acknowledges support from Cancer Research UK; and the Royal Society. The authors are members of the Leicester Cancer Research UK Experimental Cancer Medicine Centre.
Joughin
Hegemann
J. R.
Ehrenberger
Hudecz
Nigg
Spatial exclusivity combined with positive and negative selection of phosphorylation motifs is the basis for context-dependent mitotic signaling.
Sci. Signal.
https://doi.org/10.1126/scisignal.2001796
C. J.
Mortensen
Proteomic characterization of the human centrosome by protein correlation profiling.
https://doi.org/10.1038/nature02166
Stierhof
Y. D.
Leiss
Rootletin forms centriole-associated filaments and functions in centrosome cohesion.
Bahmanyar
D. D.
Deluca
O'Toole
E. T.
Winey
E. D.
W. J.
A. I.
beta-Catenin is a Nek2 substrate involved in centrosome separation.
Genes Dev.
Belham
Roig
B. E.
Avruch
A mitotic cascade of NIMA family kinases. Nercc1/Nek9 activates the Nek6 and Nek7 kinases.
Bertran
Sdelci
Regué
Caelles
Nek9 is a Plk1-activated kinase that controls early centrosome separation through Nek6/7 and Eg5.
https://doi.org/10.1038/emboj.2011.179
Bettencourt–Dias
Hildebrandt
Pellman
Godinho
Centrosomes and cilia in human disease.
Trends Genet.
https://doi.org/10.1016/j.tig.2011.05.004
Blangy
H. A.
d'Hérin
Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo.
Quarmby
L. M.
A NIMA-related kinase, Cnk2p, regulates both flagellar length and cell size in Chlamydomonas.
https://doi.org/10.1242/jcs.02455
The NIMA-family kinase Nek3 regulates microtubule acetylation in neurons.
Jiang
Never-in-mitosis related kinase 1 functions in DNA damage response and checkpoint control.
https://doi.org/10.4161/cc.7.20.6815
Nek1 kinase functions in DNA damage response and checkpoint control through a pathway independent of ATM and ATR.
https://doi.org/10.4161/cc.10.4.14814
Coene
K. L.
D. A.
Boldt
Gloeckner
van Reeuwijk
Bolat
Roosing
Letteboer
T. A.
F. P.
The ciliopathy-associated protein homologs RPGRIP1 and RPGRIP1L are linked to cilium integrity through interaction with Nek4 serine/threonine kinase.
https://doi.org/10.1093/hmg/ddr280
Daviter
Smerdon
Pfuhl
An undecided coiled coil: the leucine zipper of Nek2 kinase exhibits atypical conformational exchange dynamics.
https://doi.org/10.1074/jbc.M110.196972
De Souza
L. P.
J. L.
Mitotic histone H3 phosphorylation by the NIMA kinase in Aspergillus nidulans.
Di Agostino
Chieffi
Fusco
Geremia
Phosphorylation of high-mobility group protein A2 by Nek2 kinase during the first meiotic division in mouse spermatocytes.
Mol. Biol. Cell
https://doi.org/10.1091/mbc.E03-09-0638
The mitotic checkpoint kinase NEK2A regulates kinetochore microtubule attachment stability.
Oncogene
https://doi.org/10.1038/onc.2008.34
Faragher
Nek2A kinase stimulates centrosome disjunction and is required for formation of bipolar mitotic spindles.
Cerniglia
G. J.
Yend
T. J.
Inhibition of centrosome separation after DNA damage: a role for Nek2.
Radiat. Res.
https://doi.org/10.1667/RR3211
Live cell imaging reveals distinct roles in cell cycle regulation for Nek2A and Nek2B.
Biochim. Biophys. Acta
https://doi.org/10.1016/j.bbamcr.2005.01.007
Meraldi
C-Nap1, a novel centrosomal coiled-coil protein and candidate substrate of the cell cycle-regulated protein kinase Nek2.
A centrosomal function for the human Nek2 protein kinase, a member of the NIMA family of cell cycle regulators.
https://doi.org/10.1093/emboj/17.2.470
Activity of the human centrosomal kinase, Nek2, depends on an unusual leucine zipper dimerization motif.
Gascoigne
K. E.
S. S.
Cancer cells display profound intra- and interline variation following prolonged exposure to antimitotic drugs.
https://doi.org/10.1016/j.ccr.2008.07.002
Goshima
New look inside the spindle: microtubule-dependent microtubule generation within the spindle.
Curr. Opin. Cell Biol.
https://doi.org/10.1016/j.ceb.2009.11.012
Grallert
I. M.
Schizosaccharomyces pombe NIMA-related kinase, Fin1, regulates spindle formation and an affinity of Polo for the SPB.
https://doi.org/10.1093/emboj/cdf294
Krapp
Bagley
Simanis
Recruitment of NIMA kinase shows that maturation of the S. pombe spindle-pole body occurs over consecutive cell cycles and reveals a role for NIMA in modulating SIN activity.
https://doi.org/10.1101/gad.296204
Graser
Cep68 and Cep215 (Cdk5rap2) are required for centrosome cohesion.
Wattam
Yamano
Bacchieri
APC/C-mediated destruction of the centrosomal kinase Nek2A occurs in early mitosis and depends upon a cyclin A-type D-box.
https://doi.org/10.1093/emboj/20.24.7117
Kimata
Early mitotic degradation of Nek2A depends on Cdc20-independent interaction with the APC/C.
Nat. Cell Biol.
https://doi.org/10.1038/ncb1410
R. B.
The centrosomal kinase Nek2 displays elevated levels of protein expression in human breast cancer.
Cancer Res.
https://doi.org/10.1158/0008-5472.CAN-04-0960
N. R.
H. M.
P. T.
NIMA-related kinase 2 (Nek2), a cell-cycle-regulated protein kinase localized to centrosomes, is complexed to protein phosphatase 1.
Biochem. J.
https://doi.org/10.1042/0264-6021:3490509
Henise
J. C.
Irreversible Nek2 kinase inhibitors with cellular activity.
J. Med. Chem.
https://doi.org/10.1021/jm200222m
Garka
C. T.
Virca
G. D.
Purification, cloning, and characterization of Nek8, a novel NIMA-related kinase, and its candidate substrate Bicd2.
K. M.
Solanki
Mas–Droux
Yeoh
Boxall
Design of potent and selective hybrid inhibitors of the mitotic kinase Nek2: structure-activity relationship, structural biology, and cellular activity.
https://doi.org/10.1021/jm201683b
Targeted anti-mitotic therapies: can we improve on tubulin agents?
Nat. Rev. Cancer
https://doi.org/10.1038/nrc2049
H. J.
Yun
Nek6 overexpression antagonizes p53-induced senescence in human cancer cells.
https://doi.org/10.4161/cc.9.23.14059
Jeon
Y. J.
K. Y.
Y. Y.
H. G.
Role of NEK6 in tumor promoter-induced transformation in JB6 C141 mouse skin epidermal cells.
Characterization of NIP2/centrobin, a novel substrate of Nek2, and its potential role in microtubule stabilization.
L. N.
M. E.
Active and inactive protein kinases: structural basis for regulation.
Microtubules as a target for anticancer drugs.
NEK7 is a centrosomal kinase critical for microtubule nucleation.
Biochem. Biophys. Res. Commun.
NEK7 is essential for centriole duplication and centrosomal accumulation of pericentriolar material proteins in interphase cells.
Kokuryo
Nagino
Nimura
Hamaguchi
Nek2 as an effective target for inhibition of tumorigenic growth and peritoneal dissemination of cholangiocarcinoma.
Komlodi–Pasztor
Sackett
Fojo
Mitosis is not a key target of microtubule agents in patient tumors.
Nat. Rev. Clin. Oncol.
https://doi.org/10.1038/nrclinonc.2010.228
Krien
Palatsides
Asouline
Morimyo
A NIMA homologue promotes chromatin condensation in fission yeast.
R. R.
U. P.
Koniaras
The fission yeast NIMA kinase Fin1p is required for spindle function and nuclear envelope integrity.
https://doi.org/10.1093/emboj/21.7.1713
Theerthagiri
Bodenmiller
Horvath
Aebersold
Kutay
Phosphorylation of Nup98 by multiple kinases is crucial for NPC disassembly during mitotic entry.
M. Y.
J. I.
Nek6 is involved in G2/M phase cell cycle arrest through DNA damage-induced phosphorylation.
Obara
Kuida
Lehoczky
I. A.
A defect in a novel Nek-family kinase causes cystic kidney disease in the mouse and in zebrafish.
https://doi.org/10.1242/dev.00173
Lizcano
Deak
Morrice
Kieloch
Hastie
Schutkowski
Molecular basis for the substrate specificity of NIMA-related kinase-6 (NEK6). Evidence that NEK6 does not phosphorylate the hydrophobic motif of ribosomal S6 protein kinase and serum- and glucocorticoid-induced protein kinase in vivo.
Loncarek
Hergert
Magidson
Khodjakov
Control of daughter centriole formation by the pericentriolar material.
Zereshki
NEK2A interacts with MAD1 and possibly functions as a novel integrator of the spindle checkpoint signaling.
Evidence for a NIMA-like mitotic pathway in vertebrate cells.
A. R.
Identification of substrate specificity determinants for the cell cycle-regulated NIMA protein kinase.
Mahjoub
Montpetit
Finst
The FA2 gene of Chlamydomonas encodes a NIMA family kinase with roles in cell cycle progression and microtubule severing during deflagellation.
B. R.
Scholz
S. R.
Schiebel
Components of the Hippo pathway cooperate with Nek2 kinase to regulate centrosome disjunction.
Agircan
F. G.
Plk1 controls the Nek2A-PP1γ antagonism in centrosome disjunction.
https://doi.org/10.1016/j.cub.2011.05.047
Melixetian
D. K.
Sørensen
C. S.
NEK11 regulates CDC25A degradation and the IR-induced G2/M checkpoint.
Raghunath
P. N.
Tomaszewski
Clevenger
C. V.
Nek3 kinase regulates prolactin-mediated cytoskeletal reorganization and motility of breast cancer cells.
https://doi.org/10.1038/sj.onc.1210264
Moniz
Stambolic
Nek10 mediates G2/M cell cycle arrest and MEK autoactivation in response to UV irradiation.
Mol. Cell. Biol.
https://doi.org/10.1128/MCB.00648-10
Dutt
Nek family of kinases in cell cycle, checkpoint control and cancer.
Cell Div.
https://doi.org/10.1186/1747-1028-6-18
Nassirpour
Flanagan
Jallal
Smeal
Nek6 mediates human cancer cell transformation and is a potential cancer therapeutic target.
Mol. Cancer Res.
https://doi.org/10.1158/1541-7786.MCR-09-0291
Nek11, a new member of the NIMA family of kinases, involved in DNA replication and genotoxic stress responses.
Nucleolar Nek11 is a novel target of Nek2A in G1/S-arrested cells.
Premature chromatin condensation upon accumulation of NIMA.
Never say never. The NIMA-related protein kinases in mitotic control.
Trends Cell Biol.
Mitotic regulation by NIMA-related kinases.
The Nek6 and Nek7 protein kinases are required for robust mitotic spindle formation and cytokinesis.
A mutation in Aspergillus nidulans that blocks the transition from interphase to prophase.
https://doi.org/10.1083/jcb.96.4.1155
R. T.
Mitotic induction and maintenance by overexpression of a G2-specific gene that encodes a potential protein kinase.
Parallel activation of the NIMA and p34cdc2 cell cycle-regulated protein kinases is required to initiate mitosis in A. nidulans.
Schultheiss
U. T.
Helou
NEK8 mutations affect ciliary and centrosomal localization and may cause nephronophthisis.
Mooers
Phylogenetic analysis of the Neks reveals early diversification of ciliary-cell cycle kinases.
Pelegrini
A. L.
Ledur
P. F.
Maques
G. P.
Henriques
Nek1 silencing slows down DNA repair and blocks DNA damage-induced cell cycle arrest.
https://doi.org/10.1093/mutage/geq026
Polci
NIMA-related protein kinase 1 is involved early in the ionizing radiation-induced DNA damage response.
Glover
Giet
Drosophila Nek2 protein kinase knockdown leads to centrosome maturation defects while overexpression causes centrosome fragmentation and cytokinesis failure.
Mitotic destruction of the cell cycle regulated NIMA protein kinase of Aspergillus nidulans is required for mitotic exit.
Pugacheva
Golemis
The focal adhesion scaffolding protein HEF1 regulates activation of the Aurora-A and Nek2 kinases at the centrosome.
Jablonski
T. R.
Henske
HEF1-dependent Aurora A activation induces disassembly of the primary cilium.
Caught Nek-ing: cilia and centrioles.
Nicolàs
The NIMA-family kinase Nek6 phosphorylates the kinesin Eg5 at a novel site necessary for mitotic spindle formation.
Rellos
Nott
Fedorov
Q. Y.
Structure and regulation of the human Nek2 centrosomal kinase.
M. W.
Hoelder
Bayliss
An autoinhibitory tyrosine motif in the cell-cycle-regulated Nek7 kinase is released through binding of Nek9.
Mol. Cell
https://doi.org/10.1016/j.molcel.2009.09.038
Mikhailov
Nercc1, a mammalian NIMA-family kinase, binds the Ran GTPase and regulates mitotic progression.
Active Nercc1 protein kinase concentrates at centrosomes early in mitosis and is necessary for proper spindle assembly.
Rachmin
Yissachar
Lavi
Motro
Nek7 kinase targeting leads to early mortality, cytokinesis disturbance and polyploidy.
https://doi.org/10.1038/onc.2010.162
Sawin
Mitchison
Mutations in the kinesin-like protein Eg5 disrupting localization to the mitotic spindle.
https://doi.org/10.1073/pnas.92.10.4289
Nek9, Nek6, Nek7 and the separation of centrosomes.
https://doi.org/10.4161/cc.10.22.18226
Schütz
Pinyol
Nek9 phosphorylation of NEDD1/GCP-WD contributes to Plk1 control of γ-tubulin recruitment to the mitotic centrosome.
Spalluto
Hearn
Nek2 localises to the distal portion of the mother centriole/basal body and is required for timely cilium disassembly at the G2/M transition.
Eur. J. Cell Biol.
https://doi.org/10.1016/j.ejcb.2012.03.009
Novel combination treatment for colorectal cancer using Nek2 siRNA and cisplatin.
Cancer Sci.
Giessl
Dimmler
Shalev
von der Haar
Zahnleiter
Stöss
Beinder
NEK1 mutations cause short-rib polydactyly syndrome type majewski.
Am. J. Hum. Genet.
https://doi.org/10.1016/j.ajhg.2010.12.004
Tsunoda
Nek2 as a novel molecular target for the treatment of breast carcinoma.
Upadhya
Birkenmeier
E. H.
Mutations in a NIMA-related kinase gene, Nek1, cause pleiotropic effects including a progressive polycystic kidney disease in mice.
https://doi.org/10.1073/pnas.97.1.217
Vaz Meirelles
Ferreira Lanza
D. C.
Santana Bernachi
Paes Leme
Kobarg
Characterization of hNek6 interactome reveals an important role for its short N-terminal domain and colocalization with proteins at the centrosome.
J. Proteome Res.
https://doi.org/10.1021/pr100562w
Clinical and pathologic findings in two new allelic murine models of polycystic kidney disease.
W. H.
Phosphorylation of the Ndc80 complex protein, HEC1, by Nek2 kinase modulates chromosome alignment and signaling of the spindle assembly checkpoint.
Cheary
van Montfort
R. L.
Insights into the conformational variability and regulation of human Nek2 kinase.
J. Mol. Biol.
https://doi.org/10.1016/j.jmb.2008.12.033
Whelligan
D. W.
K-M.
Barillari
Grummitt
C. G.
Aminopyrazine inhibitors binding to an unusual inactive conformation of the mitotic kinase Nek2: SAR and structural characterization.
https://doi.org/10.1021/jm1008727
Wloga
Camba
Rogowski
Jerka–Dziadosz
Gaertig
Members of the NIMA-related kinase family promote disassembly of cilia by multiple mechanisms.
X. L.
Small molecule targeting the Hec1/Nek2 mitotic pathway suppresses tumor cell growth in culture and in animal.
Mirabito
A role for NIMA in the nuclear localization of cyclin B in Aspergillus nidulans.
Fardilha
da Cruz e Silva
Alternative splicing controls nuclear translocation of the cell cycle-regulated Nek2 kinase.
Adamian
Rootletin interacts with C-Nap1 and may function as a physical linker between the pair of centrioles/basal bodies in cells.
C. K.
Nek1 and TAZ interact to maintain normal levels of polycystin 2.
Tennenbaum
Nek7 kinase is enriched at the centrosome, and is required for proper spindle assembly and mitotic progression.
FEBS Lett.
https://doi.org/10.1016/j.febslet.2006.10.069
Zalli
The Nek8 protein kinase, mutated in the human cystic kidney disease nephronophthisis, is both activated and degraded during ciliogenesis.


text:
Heklina returns: drag fave to perform a new solo show at Oasis
by Jim Provenzano
Tuesday March 1, 2022
Heklina goes solo in her new cabaret show.
In an auspicious return to her former nightlife roost, Heklina will perform a new solo show with stories and live —yes, live singing— on March 10 and 11 at Oasis.
"It's sort of the story of my life, told with songs by Stephen Sondheim, some classics made famous by Liza Minnelli," said Heklina (aka Stefan Grygelko), from her home in Palm Springs. Local pianist Tom Shaw will accompany her onstage.
"I wanted to challenge myself. I've done Trannyshack, Mother, films, podcasts, plays, so this is something new."
Since the early 1990s, Heklina has been a mainstay in Bay Area queer nightlife. From the first irreverent drag nights at The Stud, to Trannyshack's expansion at DNA Lounge that included annual contests, Heklina has often hosted the most prominent drag and nightlife events which included her own numbers.
In 2015, along with D'Arcy Drollinger and other investors, Heklina opened Oasis in South of Market; the same building that once housed the original Oasis. The new nightclub has become popular for not only drag shows and DJed dance nights, but comic plays and musicals, cabaret concerts and community fundraisers. Heklina recently sold her share of Oasis ownership and moved to Palm Springs, while still keeping a foothold on the Bay Area's nightlife scene.
And of course, Heklina's deadpan line delivery as Dorothy (Bea Arthur's character) in stage productions of episodes of the classic sitcom The Golden Girls have become an annual tradition.
Having endured shutdowns, closures, and online DJ Zoom parties —"They never really worked," admits Heklina, the time for a personal and in-person show is now.
"You know, things have changed," she said of 2020's early pandemic outbreak. "We were all trying to pretend that everything was okay and that they said it was going to be temporary. I remember the early days of the pandemic, we were online, acting like it would be over soon."
Now, with health precautions shifting yet again, while some may be understandably hesitant to attend nightlife events, many are returning to parties and drag shows with their proof of vaccination and facemasks.
"I'm really excited about my solo show," said Heklina, who admits she is not a trained singer, but can carry a tune.
"I sent vocal tracks to Tom Shaw for tempo, and he replied, 'Oh, this is fine, but what key are you singing?'
Heklina's multi-tasking —including recently being a contestant on the TV game show The Price is Right— during her upcoming visit includes returning to host the 23rd annual The Mother Star Search Competition on March 13 at Oasis, and the seasonal monthly dance party Daytime Realness at El Rio on March 20 with DJs Carnita and Stanley Frank.
"It's my favorite party, with a lot of energy, and you know, we don't see a lot of young people here in Palm Springs."
Heklina performing at Oasis
Heklina did note that aside from the age variance and hot weather, it's mostly easy living in Palm Springs.
"You just get so spoiled. You can drive anywhere, and you know if you're running late you know you can find parking. It's a lot easier than life in San Francisco."
Asked about the extremely hot summers that many residents escape, Heklina mentioned her return to San Francisco and Iceland, her birth country, with its famous volcano that inspired Grygelko's drag name Heklina.
"I love returning to Iceland. But last time everything shut down just as I arrived." Heklina's hosted Pride events in Reykjavík, the country's capital. "I'm their 'queen who made it in America.'
On her 2020 visit, with travel limited and nearly everything closed, she noted, "I got to see so much of the country without any tourists. That was a nice caveat. I was finally able to go around the country like I've not been able to do since I was a child the first time in years."
Heklina's Grand Opening, March 10 & 11, 7pm, at Oasis, 398 11th St. www.sfoasis.com
The Mother Star Search Competition, March 13, 6pm at Oasis, 398 11th St. www.sfoasis.com
Hekina hosts Daytime Realness at El Rio; Sunday March 20 at El Rio, 3158 Mission St. www.elriosf.com
Heklina's online TV show: 'Live From the Apocalypse'
Over the Coals :: Drag Icon Heklina Gets Roasted
Sherry Vine: parody song queen returns to Oasis
Oaklash serves up drag fun from SF Oasis May 28-30
Going Out, Homing's In, March 4-11, 2022

redpajama_set_name: RedPajamaCommonCrawl


How much content have you written in the past month?
Believe me, I get it. Creating a high amount of high quality content on a regular basis is hard.
But the traffic, subscribers, and eventual sales you get from it make creating content a necessity for most businesses.
The first two are often out of your control or not possible at this time. The third one, however, is almost always possible.
If you haven't tried to optimize your writing habits, consider doing it now. It's possible to double your productivity after some tweaks. Imagine being able to produce an article in half the time it currently takes you?
Hopefully, by the end of this post, you'll have picked up a few tips that you can apply right away to significantly decrease the amount of time you spend creating content.
I'm going to share with you six tips, many of which are a bit unconventional but can work wonders.
Content is a necessary part of modern marketing for most businesses.
And yet, it's always difficult to find the time to produce content on a regular basis.
That's where learning how to create content faster comes in.
I've shown you six different ways to create content faster. Just try one or two for now, and come back for the rest later. Measure your content creation times before and after, and you should see a big difference.


Nyaaa has attained level 66!
SmellyAfro has been killed by Duncan Idaho.
SmellyAfro has been killed by Atreides Officer.
Best Leveler today Nyaaa with 1 levels!
Most victorious mobile today Duncan Idaho with 1 kills!


Eaton Federal Savings Bank is proud to announce the hiring of Senior Loan Officer, Garrett Bensinger. Garrett comes to Eaton Federal with 15 years experience in the banking industry having worked in a variety of different positions with his former employer.
Currently, Garrett serves on the Board of Directors for the Charlotte Chamber of Commerce, #CharlotteRising and Good Stewards, Inc.
A graduate of Charlotte High School, Alma College, and most recently the University of Wisconsin, Graduate School of Banking, Garrett now lives in Charlotte with his wife and three kids. When he's not working he can be found coaching youth sports, fishing and spending time with family and friends.
Eaton Federal plans to use 2017 to prepare Garrett for his future position of Chief Lending Officer, currently held by Dan Kares, who plans on retiring in the spring of 2018.


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For an Intrepid Few, Doing Your Own Taxes is Just Part of Running an Eyecare Biz
Do You Or Don't You
"I'd rather spend my valuable time running my business."
INVISION Staff Invision March 2018 Issue
THIS MONTH'S QUESTION: DO YOU DO YOUR OWN TAXES?
Yes: 13%
Lengthy, depressing and expensive. Billy Isgett, Eyecare of Florence, Florence, SC
Not hard to do my own taxes. Will Taylor, Eye 2 Eye Contact, Northville, MI
My husband is a finance guy so he totally gets how to do all of the finance for the shop. He geeks out on it. We are a good team. Nancy Revis, Uber Optics, Petaluma, CA
It's a necessary evil. Rick Rickgauer, Vision Associates, Girard, PA
This is a half truth, I give them to my husband and then walk in the other room with a bottle of wine to make it a bit more bearable. Sherry Berry Eye against Eye, Philadelphia, PA
My husband is a CPA and he is the CFO of my business, so it's kind of like I do it myself. Either way, it's not outsourced and I don't pay for it. Chani Miller, OD, Park Eye Center, Highland Park, NJ
I did taxes on the side professionally for 14 years. Martha Davenport, Safe Vision, Wheatfield, IN
NO: 87%
We do all our accounting, payroll, and finances other than our annual taxes, which keeps us pretty aware of how things work overall. We pay a local CPA to handle our annual returns, because we find that getting an outside perspective on our books once a year can really help us and keep us up-to-date. We're experts in eyecare, they're experts in money and laws. It's a no-brainer. Jen Heller, Pend Oreille Vision Care, Sandpoint, ID
A CPA … Best advice I ever took: "Know what you don't know." I'd rather spend my valuable time running my business and leave the tax laws to someone trained in that field. I'm too pretty for jail. Kate Giroux, MacPherson Opticians, Arlington, VA
Attorney CPA specializing in medical practices. She is a retired CFO of Sutter Medical Group. Texas L. Smith, OD, Dr. Texas L. Smith & Assoc, Citrus Heights, CA
I pay a CPA that can do monetary math and work with charts and lets me worry about glasses and comic books. Angel Miller, Cynthiana Vision Center, Cynthiana, KY
My wife. She is the smart one and bookkeeper. Ron Catterson, Clear View Optix, The Villages, FL
I don't do my own taxes just like I don't let my accountant do his/her own eye exam. There are lots of things I do not understand about tax law, deductions, Section 179, and the like. I realize that some may think they are going to save money by doing it themselves; but you could make a lot more money in the time it takes them to figure out how to do the taxes than you would pay the accountant to actually do the taxes. Now if you enjoy that kind of thing and are really good at it, be my guest. It is a lot like me representing myself in a courtroom on trial. If I did, I'd have a fool for a lawyer. Ted McElroy, OD, Vision Source Tifton, Tifton, GA
An accountant and frankly I feel like I should be looking for someone who specializes. No way I can do it on my own. Zachary Dirks, OD, St. Peter and Belle Plaine Eyecare Centers, Saint Peter, MN
I use a CPA. She has saved me a lot of worry and stress, and helps me prioritize what I can use my money for personally and professionally to improve my life and plan for a comfortable life later on. She's worth every penny. Sarah Jerome, OD, Look+See Eye Care, Minneapolis, MN
My cousin is an accountant. Even if he wasn't I wouldn't do my own. Too stressful! Jennifer Leuzzi, Mill Creek Optical, Dansville, NY
I have a business of my own and an accountant who can deal with the numbers. Betty Aretz, The Eyecare Boutique, Wexford, PA
What's the Brain Squad?
If you're the owner or top manager of a U.S. eyecare business serving the public, you're invited to join the INVISION Brain Squad. By taking one five-minute quiz a month, you can get a free t-shirt, be featured prominently in this magazine, and make your voice heard on key issues affecting eyecare professionals. Good deal, right? Sign up here.
Related Topics:Do You Or Don't YouINVISION March '18
It's a 50/50 Split on Whether You Guys Hold Clearance Sales
Your Customers Are Constantly on Their Phones, Why Are More Than 60% of You Not Grabbing Their Attention There?
INVISION Staff
Since launching in 2014, INVISION has won 23 international journalism awards for its publication and website. Contact INVISION's editors at [email protected].
Ask INVISION10 hours ago
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Affordable and glamorous are two words that could
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Attracting about 2,000 patrons, this fund-raising tour in support of David Lubin School has become a Mother's Day weekend tradition. See seven gardens that became works of art. A tea garden and artisan boutique also are offered.


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Colourful Easter Parade in Strokestown
Auction this Saturday night in the Tulsk Inn January 1, 2010
Prime farmland in Athleague January 1, 2010
There was lots of colour in Strokestown on Sunday last, as the town hosted its biggest ever Easter Parade. 43 floats and hundreds of people turned up for the 10 th annual event, run by Strokestown Community Development Association. The vintage display was particularly popular with young and old. Speaking of youngsters, Strokestown Community Playschool had a very innovative float, which won the overall prize in the fancy dress category. Calla Floral Design was another eye-catching float and it was rewarded with a prize for originality. Other prize winners included Laragan Quarries, David Kennedy Construction, Féile Frank McGann, Percy French Hotel and Johnny Duke with his vintage tractors. Strokestown bikers, who go on their Achill bike run in July in aid of the Hospice were also prize winners. Chairman of Strokestown Community Development Association Eugene Murphy said that the committee were very happy with this year's parade, but were greatly disappointed that not one environmental type float was entered. 'It was sad that two trophies were left on stage after the event the only category that was not contested, and it proves to me that there is no real interest in the environment, it's all talk in relation to the issue. The committee will put in the category next year in the hope that someone will enter. Cllr. Murphy, on behalf of the Strokestown group, also thanked all young people from the GAA Community Games, Ballyboro Scouts, St. Barry's and other groups who participated on the day. 'The parade was led by McCartan's Pipe Band from Monaghan and also the Boyle Marching Band. He also praised the traders for their input and sponsorship, the Garda Síochána for their excellent stewardship this year, and also John Reynolds from Roscommon Co. Council and Bernard Devine who had worked from early morning to ensure traffic was under control for the duration of the parade which took one hour and ten minutes to pass through the town. Also thanks to all our own stewards for their assistance on the day.'


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Category: Turn Backaround
Turn Backaround: Revisiting the Nintendo Switch
This is a quick return to the Nintendo Switch I thought would be interesting to those of you still on the fence about picking up a Switch. It turns out sales of the console have started to decay and there has been news of Nintendo bringing out an updated version in the near future.
So far the Switch is the console I have most used in this generation since I changed my trucking job a couple years ago. I don't have a lot of time to play my XBOX One S or my Playstation 4 as I am only home for usually one day of the week. That usually leaves me time to do a podcast and play a little on my computer "ZPM".
So mobile has become very big for me and that's saying a lot as before this I had dabbled with the Nintendo Gameboy, DS, and 3DS (and Sonys PSP and Vita) but they were never more than Pokemon machines for me.
The Switch is something different. It has a ton of great console grade games that I can take anywhere with me on the road not requiring a television of any kind. That's not to say the experience on TV is bad. It's not. It's just not where the Switch excels.
The battery life leaves me a bit wanting but other than that I am incredibly happy with the Switch. Games like Octopath Traveler and Super Mario Odyssey look incredible on the small and big screen alike and the games themselves are great.
Right now, if Nintendo had better third party support, I would tell you to buy a Switch. Period. No question. But the third party support is lagging. Yeah there are some newish games out there but when a game like Skyrim is like a big thing to be released on your console when the game itself was released in 2011 that really isn't a big deal.
One does have to wonder why Nintendo still bothers with their 3DS/2DS line of handhelds. I can only surmise at this point that they are on life-support. I am guessing at this point if there is new Switch hardware in the works they will be giving us more battery life and going fully mobile with perhaps the option to still dock it if you want to play on the big screen.
What has your experience been with the Switch? Or are you still on the fence?
Author JeffPosted on October 20, 2018 October 20, 2018 Categories Editorial, Gaming, Turn BackaroundLeave a comment on Turn Backaround: Revisiting the Nintendo Switch


If it hasn't happened to you, it's happened to someone you know. Experiencing a merger or acquisition firsthand has become commonplace in today's cutthroat business climate. The environmental industry in particular has been experiencing a wave of consolidations that has left few businesses unaffected.
Inevitably, corporate restructuring impacts the employees of the companies involved. Fortunately, there are a number of steps you can take to reduce your stress and increase your job security.
Most people caught in a merger or acquisition feel helpless. To stay in charge of the situation, remember that you are still the master of your own destiny. What will happen depends a great deal on the actions you take during this crucial time.
This is a good time to take stock of your career. Ask yourself where you want it to go and what you want to be doing in five years. Think about whether your goals can be met at your restructured company or whether it's time to move on.
Karen DeWitt became an account manager with Kelly Scientific Resources, Houston, Texas, when her former position in the oil and gas industry was eliminated in a merger. DeWitt advised, "When a company merges, you have to make a conscious decision: Do I want to work for this company or not? That's the first step. The second is, if I don't want to work here, do I want to stay in this industry or do I want to do something different?"
If you have decided you want to stay with the company, you can promote your job stability by employing certain tactics. The following are good habits to practice throughout your professional career, even if you never experience a corporate reorganization.
Promote yourself. H. Stephen Jones, president of Executive Projects Consultants, an environmental project management company based in Ellicott City, Md., has been on both sides of the fence in corporate restructuring. In 1998, as senior manager for a national real estate provider, he made the difficult decision of whom to lay off during a major downsizing; later, he became a victim of management restructuring when his own job was eliminated.
During a transition period, it is vital that you make yourself visible. The two best ways to be tapped for a reduction are to been seen as a negative force — or not to be seen at all.
Jones offered some tips on surviving corporate reorganization. "You can't control your fate, but you can proactively influence it," he said. "The people who do that are the ones who achieve and become valuable resources. The ones who don't, and just let things happen to them, become the pawns in the game and become subject to dismissal based on just a head count."
The sad truth is that your continued success on the job often does not depend on how hard you work or on your competence. It depends in large part on your ability to impress the decision-makers at your company.
During a transition period, it is vital that you make yourself visible. Stay upbeat and active at work. The two best ways to be tapped for a reduction are to be seen as a negative force — or not to be seen at all.
It is your responsibility to let people know your skills, education and experience. One way to do so is to have a one-on-one meeting with your new boss. Create a personal marketing portfolio that includes a brief personal introductory statement, a list of accomplishments and an updated resume. Offering this information allows you to self-promote and gives the boss a better idea of how you will fit into the restructured company's new goals. During the meeting, ask your manager what is expected of you, and make sure those expectations are clear.
Keep a list of your professional achievements. Performance reviews often play an important role in the decision of who goes and who stays. "When I've had to make decisions about who I keep and who I don't keep, the employee's file is pulled out and their performance reviews are looked at to see what their contributions were," Jones said.
Keep your professional achievements updated quarterly. Don't wait for your annual review to write up everything you accomplished over the year. "Too often, in a performance review, the only things you focus on are what happened in the last three months," Jones said. Employees who do a good job of documenting their achievements gain a key advantage.
Participate in planning sessions. When organizations undergo significant change, fresh approaches and ideas are usually welcomed. This is your chance to shine. Being able to make your opinion heard also increases your sense of control in an uncertain situation.
Network. "Do you have strategic relationships? So many times it becomes who you know — and who those people know — that becomes a strength for you in a situation when you decide who you keep and who you don't keep," Jones said.
Many people feel squeamish about networking, misunderstanding it as exploitative. However, networking is a two-way street — it is collaborative and reciprocal. The main goal of networking is to develop meaningful relationships that benefit both parties.
Expand your network of contacts at your own company. While the acquiring company usually makes the decisions on who stays and who goes, it might seek input from the acquired company's management. It is vitally important that you have a good relationship with your boss and other co-workers who can vouch for you and the value you bring to the department.
It is equally important to build relationships with new co-workers. Act as if you are going to work for a new company and make every effort to meet your new colleagues. Your new company or department will expect you to integrate with current employees and perform as a team.
Nurture relationships and networks outside your company, too. You don't want to be someone who only shows up in hard times. Keep your network active — offer to help others and they will wind up helping you.
"A lot of people don't like to sell themselves. They're scientists, they're technicians, they are experts in their field. They're not in sales and marketing. It's different for them to have to wear a hat to market themselves," DeWitt said. "There are people and agencies out there that can help these individuals — they just have to be willing to go out there and let people know what their circumstances are."
The victims of corporate downsizing include not only those who lose their jobs but also those who are left behind to pick up the slack.
Update your skills. This is essential whether you aim to keep your old job or venture into the market for a new one. Examine yourself as critically as you would a business problem.
"Education is a process, not an achievement. Every day I learn something new," Jones said. Keep up with the latest technology by taking classes or workshops. You can also stay on top of what's going on in your industry through professional organizations and the business press.
Evaluate your financial situation. Talk to your financial advisor and calmly analyze your financial situation. The fear of financial calamity is often far worse than the situation warrants. Find out how long you could afford to hunt for a job you really want. Develop a financial reserve; you should have at least three months living expenses should your position be eliminated. This will help you avoid taking the first opportunity that comes along in order to make ends meet.
Keep a positive attitude. You are now part of a larger company and there are bound to be more activities, responsibilities and opportunities for everyone. Management will value employees who see the glass as half full instead of half empty.
"Instead of being a road block, be a catalyst," Jones advised. "If you embrace change, you are a rare individual. Any time you restructure a company, 80 percent of the employees are going to look at the negative side. But there's 20 percent, maybe even less, that see the change as an opportunity to move from where they are to a new position. They like the newness, the challenges that come with it, and those people have a real leadership quality that you're looking for."
Keeping a positive attitude will help you see the benefits that reorganization offers. For example, the transition period offers a great negotiating opportunity. Management is highly receptive to the morale needs of employees when layoffs occur, allowing you a rare chance to take care of those "me" issues.
Even if you don't stay with your present company, keeping a positive attitude is key to your continued success.
"You can't move forward if you have a vindictive, negative attitude. By having a good attitude when things happened, I was able to respond much more quickly than some people," DeWitt said.
You will be facing a significant change, regardless on which side of the downsizing equation you may find yourself. The victims of corporate downsizing include not only those who lose their jobs but also those who are left behind to pick up the slack. Studies show that surviving the ax can be as stressful as being let go. The problem, of course, stems from the extra workload created by the newly departed. The double-edged sword of making the cut may mean greater career opportunities — but at the price of longer hours, more stress and potential burnout.
Whether you go or stay, the upside is that you can use the experience for personal growth and self-renewal. How you decide to respond will determine your future rewards much more than the event itself. It is simply a matter of keeping your own sense of purpose intact even when the world is changing around you.
Try adopting a Zen Buddhist approach — keep your mind open to every possibility. Don't resist change — it's inevitable. If you stay in your position, be prepared for the fact that your job requirements will probably change; management will place a high value on employees with the flexibility to adapt to new circumstances.
All change, even positive change, is stressful. Acknowledge that the situation is not your fault. Try to keep everything in its proper perspective, and don't lose your sense of humor.
Ask for family and friends' support. Use stress control techniques that are helpful for you, such as exercise, meditation or prayer. Managing your stress will clear your mind, allowing you to make better decisions.
At work, don't try to cover all the bases by yourself. If you are in a position to delegate, do so. This is an opportunity to build teams and forge strong alliances. Support others and they will support you.
Stay in balance with the rest of your life. Despite being loyal to your company, your first loyalty must be to yourself. Your family, your health and your peace of mind must always take precedence over your employer's requirements.
If your position is eliminated, remember that although working through a layoff is difficult, the final result is often rewarding. "Those that are proactive will move forward and will find out there's always a life after the company they left. They did not realize how much happier they could be somewhere else," DeWitt said.
Laid-off workers often end up with better positions: higher pay, more opportunity for advancement and a more stimulating environment. Flexible employees can view the change as a chance to move up.
"If you've kept yourself positioned well, you literally can look at a pink slip as a new opportunity for yourself, just like the merger is a new opportunity for those still with the company. Keep that positive attitude," Jones advised.
Surviving a corporate restructuring requires preparation, communication skills and adaptability. Developing these attributes will not only help you in a time of career crisis, but throughout your entire professional life.
This article appeared in Environmental Protection magazine, August 2000, Vol. 11, No. 8, p. 48.


The answers varied wildly depending on country. 48 percent said they would trust a driverless car to ferry around their children. In general, western Europe was less accepting of vehicle autonomy t.
Here is an easy-to-understand guide to Brexit – beginning with the basics, then a look at the current negotiations, followed.
Study: World is getting fatter, needs to stop – Only 1 in 10 women there were categorized as obese, while high levels of car use in the U.S. contributed to obesity. as Swindon says, "There seems much more a willingness to spend money in drugs an.
Answer: Don't expect collectors for scam artists to help you out. Amy Nofziger, regional director for the AARP Foundation, recommends you contact your state's attorney general to file. recover comm.
The problem is that how COLA is calculated is a complete joke when it comes. going toward gas or any car-related costs. What does affect Social Security beneficiaries' cost of living is healthcare.
Judging both from comments on this blog and from some of my mail, a significant number of Americans believe that the answer to our health care problems. which is why private insurance has much high.
Now I want to purchase health insurance. Do I need to disclose this information? Will it make me ineligible for insurance? Rakesh Jain ED and CEO, Reliance General Insurance replies. Our panel of e.
In places like Houston or Atlanta, where home prices have not risen much compared with underlying rents. and the federal government believed that austerity and cutting spending was the answer to re.
The question is, by how much? Until recently, those answers were hard to come by. Credit bureaus were uncommunicative about expressing, in points, just how much impact different foreclosure types of m.
We asked how much student debt. how do you buy a car, a home, or start a family (i.e. put the "consumer" into our "consumer economy"), if you can't even afford a monthly student loan payment? You a.
"People know more about how to buy a car than they do about selecting a doctor," says Powell. It's not so much a matter of labeling a doctor. Having roughly determined the general category of physi.
While most people have health insurance to cover medical costs, celebrities — who have so much riding on their physical appearance. entire cast for a movie with a $50 million budget. In general, i.


Cosmo.ph x Blo Bar Bloggers Blowout!
Taken in Tondo yo! This was immediately after my meeting with Freego, outside their office hehe. Bilang waley na time jan nalang nagpic. Anyway, wore this preppy number because I wanted a business casual look lang. Yun lng. Simple noh? Haha. Okay, went for plaid & plaid! How awesome is my Suiteblanco bag? Bida sha palagi! It even got featured on Cosmo.ph website which I'll share later. Also love the personalized bracelet Silverworks sent. Now I'm not scared to get lost. Labo haha.
Anyway, here are more photos from that day!
I registered upon arrival and I was immediately escorted to my assigned seat and my assigned blo-er. Corny haha. I was given a mini book where there are illustrations and explanations of the different hairstyles you could avail. I picked Holly Would coz I wanted volume & curls!
They shampooed me first and then it's Blo time!
Alyssa loved her new 'do!
Blo Bar might be a new concept here in Manila but it sure is a hit among the ladies! It is not a salon ha. I repeat, it is not a salon. So don't go there asking for a haircut or a manicure. They basically just do professional hairstyling. If you have an event, shoot, date, you can run to Blo Bar and have an instant makeover for just P500! They could also do more complicated styles for a higher fee, just don't forget to bring an inspiration photo!:) Oh, did I mention they also do home service and offer party packages? Yuuuup. A fun way to bond with your girlfriends!
Danika and me! Thanks Blo Bar for our new hair hehe and Island Rose for the beautiful flowers!
I think it's perfect and fitting how during our Blo makeover, Cosmo.ph introduced to us their newly revamped site! The Cosmo team had a one on one with the bloggers and they personally showed us how to navigate the site. Sweet!
Love the new, clean, uncluttered look of Cosmo.ph! It's also super easy to navigate. They have segregated topics into sections which you can browse according to your liking! Read the hottest articles under beauty, fashion, fitness, entertainment, etc. Plus, they update daily! Woot.
Thanks again Cosmo.ph and Blo Bar for the fun afternoon!
It was nice meeting all of you! Cosmo team: Publishing Assistant Sam Uy, Associate Publishers Tiffany Santosand Risa De Sagun, Cosmo.ph Managing Editor Jillian Gatcheco, Cosmo.ph Editorial Assistants Sandra Pineda and Gianna Banzon, and Group Publisher Christine Ongteco.
Here's my "Purse Paparazzi" feature on Cosmo.ph!
A great sense of fashion styles.


These are little 3 x 3″ gift cards, quick and easy to put together, a stamped sentiment, a couple of punches, all layered on a cheerful washi background. One thing I do like about this size card as opposed to a gift tag is that there's room to write a little message and they're cute enough to be displayed with the rest of the Christmas cards.
I used Thick Whisper White cardstock (cut at 3 x 6″, scored @ 3″) for the base and another layer cut at 2-3/4 x 2-3/4″, the extra layer is optional, I liked it in case I messed up the washi layer (it happened a couple of times ), that way I hadn't ruined the whole card.
I used the Candy Cane Lane Washi Tape (reduced in the Year-End Closeouts) for these ones but they will look just as sweet using any of the others, just use the coordinating cardstock and ink and the results will be perfect (Stampin' Up! is renowned for their product coordination, it's why I joined, they make it look effortless).
The sentiments are from the Tin of Tags stamped with Real Red ink, punched out with the 1-3/4″ Circle Punch and layered on Real Red cardstock punched out with the Decorative Label Punch. I popped this up on the washi tape background with dimensionals and added a few Rhinestone Basic Jewels for some Christmas sparkle.
I didn't make envelopes for these but could easily make some using the Envelope Punch Board and some coordinating paper or even just copy paper if you're running low.
These little cards are too small to post but are just the right size to add to a gift (big or small), some flowers or could make a sweet gift themselves (who hasn't run out of gift cards on Christmas Eve?) They don't have to just be for Christmas, change the sentiments and they could cover birthdays, babies, weddings, anniversaries or pop them into a lunch box for a little hug from home.
I'll see you tomorrow with Day 8.


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AVZ Wealth Management
About AVZ
Mission Statement & Privacy Policy
AVZ Team
Economic Surveys
2020 Inflation Adjustments for Pension Plans, Retirement Accounts Released
The 2020 cost-of-living adjustments (COLAs) were released for
-pension plan dollar limitations, and
-other retirement-related provisions.
Highlights of Changes for 2020
The contribution limit increased from $19,000 to $19,500 for employees who take part in:
-401(k),
-403(b),
-most 457 plans, and
-the federal government's Thrift Savings Plan
The annual limit on contributions to an IRA remains unchanged at $6,000.
The catch-up contribution limit for individuals 50 and over remains $1,000.
The income ranges increased for determining eligibility to make deductible contributions to:
-IRAs,
-ROTH IRAs, and
-to claim the Saver's Credit.
Phase-Out Ranges
Taxpayers can deduct contributions to a traditional IRA if they meet certain conditions. The deduction phases out if the taxpayer or their spouse takes part in a retirement plan at work. The phase out depends on the taxpayer's filing status and income.
-Single taxpayers covered by a workplace retirement plan, the phase-out range is $65,000 and $75,000, increased from between $64,000 and $74,000.
-Joint filers, when the spouse making the contribution takes part in a workplace retirement plan, the phase-out range is $104,000 and $124,000.
-An IRA contributor, who is not covered by a workplace retirement plan but their spouse is, the phase out is between $196,000 and $206,000.
-A married individual covered by a workplace plan filing a separate return, the phase-out range remains $0 to $10,000. The phase-out ranges for Roth IRA contributions are
-$124,000 to $139,000 for singles and heads of household
-$196,000 to $206,000, for joint filers, and
-$0 to $10,000 for married separate filers.
Finally, the income limit for the Saver' Credit is:
-$64,000 for joint filers,
-$48,000 for heads of household, and
-$32,500 for singles and married separate filers.
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