G0 is a specific state for non-dividing cells. G1 and G2 are two gap phases during which various proteins are synthesized for DNA replication and mitosis respectively (Pardee, et al., 1989). The DNA replication begins once the cell enters into S-phase. In this stage, the amount of DNA in the cell doubles exactly. Mitosis is the shortest phase during the cell cycle and lasts about 1 to 2 hours. Mitosis itself is divided into 5 distinctive stages. In prophase, the DNA is condensed into chromosomes. Additionally, the nuclear envelope is broken down. This is followed by the establishment of a bipolar spindle in pro-metaphase. In metaphase, the chromosomes align along the metaphase plate. The sister chromatids can then be separated into two identical sets in anaphase by being pulled towards each spindle pole. In telophase, the chromosomes decondense and the nuclear envelope reforms around each set of DNA. After this, the contractile ring begins to assemble and the cell divides into two genetically identical daughter cells (Harper, et al., 1999).
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1.2 mechanisms of cell cycle regulation
The cell cycle is a tightly regulated process controlled by various proteins. Several kinase families have been identified to be important for that regulation. One of these kinase families is the Cdk (for cyclin-Dependent kinases). Cdks associate to their substrates cyclins, and play a critical role in the regulation of the cell cycle (Minshull, et al., 1989; Morgan, 1997), by assembling specific complexes for each stage/phase (Satyanarayana and Kaldis, 2009).
In G1:Cdk4/cyclin D or Cdk6/cyclin D complexes are required for the cell progression through the G1-phase (Sherr, et al., 1999; Malumbres and Barbacid, 2001). Cyclin D is synthesized once the cell receives mitogenic signals and coactivates Cdk4 or Cdk6, which leads to the phosphorylation of retinoblastoma (Rb) protein and other substrates, such as Smads, Cdt1. Rb protein acts as a tumor suppressor regulating the cell cycle through the interaction with E2F (transcriptional factor), histone deacetylases and chromatin remodeling complex (Cobrinik, 2005)). In late G1, Cyclin E associates to Cdk2. The active CyclinE/Cdk2 complex then inactivate Rb protein by generating an hyperphosphorylated form of Rb once the cell progresses towards S phase(Malumbres and Barbacid, 2001; Dannenberg, et al., 2000).
Cdk3 has also been reported to phosporylate Rb protein in the G0/G1transition through association to CyclinC (Malumbres, et al., 2005). Nevertheless, further studies are required to elucidate the functions of Ckd3 in the cell cycle (Ye, et al., 2001).
In S: CyclinE/Cdk2 complex is essential for the cell to complete the G1phase, and is required to trigger the DNA replication in S-phase (Malumbres, et al., 2005). This complex initiates the DNA duplication by loading MCM (minimicrotubule maintenance) on the original replication sites. The degradation of Cyclin by proteasome plays an equal role in S phase to ensure that the DNA replication occurs only once per cell cycle (Hwang, and Clurman, 2005). After the degradation of Cyclin E at the beginning of S phase, Cdk2 binds to CyclinA and phosphorylates various proteins in order to promote the progression in S phase (Malumbres, et al., 2005; Satyanarayana, et al., 2009).
In G2/M: In G2, Cyclin A is degraded by ubiquitin-mediated proteolysis, and CyclinB is synthesized. It has been shown that cyclinB/Cdk1 complex regulates the G2/M transition through association with a range of various substrates. In prophase, it interacts with Eg5 triggering the separation of chromosomes. Furthermore, CyclinB/Cdk1 is also involved in promoting the chromosomal condensation and the nuclear envelope breakdown. As for other cyclin/Cdk complexes, the interaction of Cyclin B/Cdk1 is critical for mitotic exit (Harper, 2002).
Interaction of the Cdks with their specific partners Cyclins contribute to regulate the cell cycle. Therefore, the activation and inactivation of each of those complexes plays an equal role in the cell cycle progression. This is regulated either by controlling the activities of Cdks or by the synthesis and the degradation of Cyclins. The activities of Cdks are controlled by both positive and negative regulators.
For instance, phosphorylation of the Thr160/161 residus in the T-loop region of Cdk1 by CAK (cyclinH/Cdk7), combined with dephosphorylation of Thr14/15 residus by CDC25C phosphatase activate CDK1 and CDK2 complex. Cdks are also negatively regulated by CDK inhibitors (CDKI). The CDKI has two families, namely INK4 and CIP/KIP. They inhibit the cyclin/Cdk complex in different ways. The INK4 CDKIs prevent the Cyclin from binding to the Cdk. The CIP/KIP CDKIs, on the other hand, bind to the Cdk/Cyclin complex and inhibit the activities of Cdk. Antiproliferative signals and activation of cell cycle checkpoints induce the expression of CDKIs and lead to cell cycle arrest. The positive and negative regulations allow the cell cell to go through mitosis only once per cell cycle. Compared to Cdks which are constantly expresses during the cell cycle, the synthesis of Cyclin is cell cycle-dependent and its degradation is carried on via the ubiquitin-dependent proteolysis. The coordination of these events ensures the progression from one phase to the rest. Most of human cancers carry mutations in upstream regulators of Cdk1; therefore Cdks have been considered as targets for cancer therapy since the last decade (Knockaert, et al., 2002).
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Towards the development of more effective cancer drugs
Until now, more than 100 distinct types of cancer have been identified. It has been demonstrated that cancer is a multistep process involving genetic alteration. Although their development has been attributed to various reasons, they share some common characteristics. One of the most important characteristic is a deregulation in the cell cycle (Weinberg, 2007). Studies in the past years have shown that cancer cells are not only hypersensitive to growth factors but also insensitive to anti-growth signals. Furthermore, almost all cancer cells are resistant to apoptosis (Weinberg, 2007). As cancer cells acquire the ability to disrupt activities of cell cycle regulators. The deregulation in the cell cycle is therefore considered one of the main routes leading to tumorigenesis.
Since 1975, the basic methods to treat cancer are chemotherapy, radiotherapy and surgery (Weinberg, 2007). However, most of these procedures lead to disappointing results (Elmore, 2007) as cancer treatment is still dependent on clinical experience (Debatin, 2002). Additionally, anti-cancer drug commonly used lack specificity towards cancer cells. Consequently, serious side-effects are observed after their use. It has also been observed that cancer cells can develop resistance to those drugs.
Consequently, in 2010, Degenhardt and Lampkin pointed out the following model stating that the next generation of anti-cancer drugs should target mainly dividing cells.
Mitosis is a crucial point in the cell cycle in which one cell divides into two genetically identical daughter cells. As mentioned before, the onset of mitosis is controlled by Cdk1 (O'Connell, 2003). Three serine/threonine kinases families acting downstream of Cdk1 are involved in the regulation of mitosis: Plk (for Polo-like kinases), Aurora kinases, and Nek kinases (for NIMA-Related kinases) (O'Connell, 2003).
So far, members of the Aurora and PLKs families have been well studied and appear as good targets for the development of anti-cancer drugs.
Aurora kinase family is composed of three serine/threonine kinases, named Aurora A, B and C, all essential for the progression in mitosis (Katayama, 2003). Although the three kinases show differences in localizations, activation, and functions in the regulation of mitosis (Bischoff, 1998), they play a crucial role in controlling sister chromatids segregation and ensuring two daughter cells receive a full set of chromosomes (Fu, 2007; Ducat, 2004). The Aurora kinases have been shown to be overexpressed in many types of human cancers (Katayama, 2003). Hence, the Aurora kinases are important targets for the anti-cancer drug development. Until now, three Aurora kinases inhibitors (Hesperadin, ZM447439 and VX-680 ) have been successfully developed (Keen, 2004).
Polo-like kinases, named after the Polo gene of Drosophila melanogaster, are one of the most important regulators to control mitosis entry, exit and cytokinesis by adjusting the activities of its substrates, such as Cdc25C phosphatase, cyclin B, subunits of the APC, MKLP-1 and other kinesin related motor proteins (Nigg 1998). Similarly to Aurora kinases, PLKs have been shown to be overexpressed in various human cancers leading to aneuploidy by promoting immature chromosome segregation (Degenhardt and Lampkin, 2010). Consequently, PLKs are therefore also described as good targets for cancer therapy.
3. a new discovery: the NEK family
3.1 emergence of the NEK family
Compared to the PLKs and Aurora, the third serine/threonine family, NIMA-related kinases (NEK) is less described (O'Connell, 2003).
In 1975, Ron Morris undertook a screen for cell cycle mutants in Aspergillus nidulans. He divided the mutants into two categories: bim mutants, which block cells in mitosis with condensed chromosomes and mitotic spindle. On the other hand, Nim mutants (for never in mitosis) arrest cells in interphase (O'Connell, Krien et al., 2003; O'Regan, et al. 2007). The nim, included several cell cycle regulators, for instance, B-type cyclin, Cdc25 phosphatase and DNA polymerases.
The nimA gene was cloned and shown to encode a kinase active in mitosis (O'Connell, Krien et al., 2003). NIMA is a nuclear protein responsible for localizing cyclinB/Cdc2 complex into the nucleus and on the spindle pole body. It has been shown that overexpression of NIMA in Aspergillus lead to a pre-mature entry in mitosis with aberrant spindle (O'Connell et al., 2003; Roig, et al., 2005). It is also suggested that NIMA controls the chromatin condensation by phosphorylating its substrate Histone H3 on ser-10 (O'Connell et al., 1994). Therefore, the ectopic expression of NIMA could promote premature chromatin condensation.
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Fin1 gene in fission yeast encodes a kinase whose functions are closely related to NIMA protein although they have some significant differences. Fin1 is not required for viability. Additionally, there is no evidence supporting that Fin1 is essential for loading CyclinB/Cdc2 complex into the nucleus and on the spindle pole body (O'Regan, et al. 2007). However, overexpression of Fin1 also induces premature chromatin condensation (Krien,et al.,1998; O'Regan, et al. 2007). Grallert (2002) has also demonstrated that Fin1 mutant results in the accumulation of monopolar spindles.
In mammals, the NIMA-related kinase (NEK) family consists of 11 members, NEK1 to NEK11. These protein kinases share about 40% to 45% of identity in their catalytic domain. However, significant differences were observed within their non-catalytic domains (O'Regan, et al. 2007) suggesting that each NEK kinase has separate functions.
To date, few data are available on NEK3, NEK4, NK5 and NEK10. NEK11 has been suggested to be involved in S-phase and DNA damage response and to be activated when DNA damage occurs. Nevertheless, the mechanism by which NEK11 is activated remains unknown. The mutations of NEK1 and NEK8 lead to polycystic kidney disease (PKD) in mice models (Liu, et al., 2002). This suggests that NEK8 and NEK1 contribute to regulate ciliogenesis (Liu, et al., 2002). NEK8 localizes to the proximal region of the primary cilia, NEK1 around the centrosome. NEK1 might respond to G2/M DNA damage and regulate the microtubule-dependent transport. The localization of those two kinases provides evidence to support that these two NIMA-related kinases have functions related to cilia formation.
Compared to these kinases, NEK2, NEK6, NEK7 and NEK9 are classified as mitotic kinases involved in mitotic progression.
NEK2 has been well studied as its catalytic region shares a high similarity with the catalytic regions with NIMA and Fin1 (Fry, 2002; O'Regan, et al. 2007,). However, NEK2 does not complement nimA mutation phenotype, suggesting that they have distinct functions (O'Connell, et al., 2003; O'Regan, et al. 2007). The expression level of NEK2 peaks in S and G2 phases. Nek2 localizes to the centrosome when the cell enters mitosis. Unlike NIMA, there is little evidence to prove that NEK2 regulates the mitotic entry by loading CyclinB/Cdk1 and Plk1 to the centrosome(Fry, et al., 1998; O'Regan, et al. 2007).
NEK2 supports the formation of bipolar spindles by promoting centrosomes splitting (O'Regan, et al. 2007). Overexpression of active NEK2 promotes centrosome splitting in interphase cells (Fry, 1998). In a recent study, NEK2 depletion by RNAi inhibits centrosome separation (Fletcher, et al., 2005). Furthermore, Yang (2006) described that NEK2 phosphorylates C-Nap1 and RooTletin, two components of the centriolar linkage, contributing to the centrosome dissociation. NEK2 also plays a role in chromatin condensation in murine cells by phosphorylating HMGA2 protein (Di Agostino S et al., 2004). Other studies report that NEK2 interacts with mitotic checkpoint components, such as Hec1 and Mad1 (Yao, et al., 2004). In addition, NEK2 was reported to localize at the midbody in late mitosis in Drosophila. The overexpression of NEK2 can stimulate the mislocalisation of actin and anillin, as well as promoting the formation of a cleavage furrow at the incorrect sites (Prigent, et al., 2005; O'Regan, et al. 2007). Hence, it is valuable to identify the substrates of NEK2 to elucidate its role in cytokinesis (O'Regan, et al. 2007).
NEK9 contains a RCC1-like domain and a coiled-coil motif in the non-catalytic region (Richards, et al., 2009; O'Regan, et al. 2007). NEK9 is expressed throughout the cell cycle, its activities increasing when the cell enters mitosis via phosphorylation of the T210 residu in the activation loop (Roig, et al., 2002). Mutations within the coiled-coil and RCC1-like domain were shown to generate an inactive or hyperactive kinase respectively (Roig, et al., 2002). Although it was observed that NEK9 contains Cdk1 phosphorylation sites, there is no proof that NEK9 is a substrate of CyclinB/Cdk1 (O'Regan, et al. 2007). NEK9 is mainly distributed in the cytoplasm in interphase and mitosis, however, the phosphorylated NEK9 localizes to spindle poles at the beginning of mitosis (Roig, et al., 2005; O'Regan, et al. 2007). Depletion assays and antibody microinjection in cells lead to the formation of aberrant mitotic spindles and reduce the formation of bipolar spindles (Roig, et al., 2005; O'Regan and Fry, 2009). This suggests that NEK9 plays a crucial role in mitotic progression and spindle formation. It is suggested that NEK9 controls spindle formation via both a centrosomal and an acentrosomal pathways (O'Regan, et al. 2007). Although NEK9 can bind to Ran-GTPase, which intern binds to importin-α, and triggers the release of TPX2 in the spindle. The mechanisms of how NEK9 and Ran cooperate in controlling the formation of spindle remain obscure (Roig et al., 2005; O'Regan, et al. 2007). In recent studies, γ-tubulin identified as a partner of NEK9 suggests that NEK9 might be involved in microtubule nucleation, anchoring to the centrosome and microtubule organization in mitotic spindles (Holland, et al., 2002; O'Regan and Fry, 2009).
NEK6 and NEK7, contain mainly a catalytic domain. NEK9 activates NEK6 and NEK7 via the phosphorylation of ser206 and ser195 residus in the activation loop respectively (Belham, et al., 2003). Therefore, NEK9, NEK6 and NEK7 act as a novel mitotic cascade to regulate the mitotic progression.
NEK6 and NEK7 show distinct subcellular localization patterns. NEK6 and NEK7 distributed both in cytoplasm and nucleus (O'Regan and Fry, 2009). However, in mitotic cells, NEK6 was observed concentrated around mitotic spindle in metaphase, and translocalize around central spindle and midbody during anaphase and cytokinasis that implicated NEK6 might play a role in the late of mitosis. On the contrary, NEK7 were detected associated with centrosome during interphase, however, it didn't notice localize to special mitotic structure (O'Regan and Fry, 2009) during mitosis. Although, other studies has shown completely opposite localization for NEK6 and NEK7 (O'Regan ,et al.,, 2007).
Even through, NEK6 and NEK7 demonstrate some common functions and properties as they share approximately 87% similarities with their catalytic domain (O'Regan and Fry. 2009). Overexpression kinase-inactive or depletion NEK6 and NKE7 by RNAi method have shown the same phenotype. Functional studies has exemplifies that kinase-inactive NEK6 or NEK7 induce apoptosis without affecting cell cycle progression (O'Regan and Fry. 2009). Nonetheless, the increase of apoptosis was cell cycle status dependent. Furthermore, the completely inactive of either NEK6 or NEK7 arrest cells in metaphase. In contrast, partially inactive these kinases delay the cytokinasis (O'Regan and Fry. 2009). It the recent studies, it suggested that the inactive or RNAi depletion of NEK6 or NEK7 decrease stability of microtubules, additionally, spindles appeared more fragile and lake of tension, which ,leading to activation of the SAC . this, to some extent, can explain its arrest cells in metaphase (O'Regan and Fry. 2009). Therefore, both NEK6 and NEK7 are required for mitotic spindle formation and bypass cytokinases (O'Regan and Fry. 2009).
The mechanism of the kinase cascade remains to be studied. It has been shown that both NEK6 and NEK7 interact with NEK9. However, NEK7 does not rescue the phenotype observed after NEK6 depletion, suggesting that NEK6 and NEK7 have distinct functions. In addition, depletion of NEK7 shows a decrease in the level of centrosomal γ-tubulin. Moreover, no substrates of NEK7 or other substrates of NEK9 have been identified to date.
Therefore, using the constructs GFP-NEK7 and GFP-NEK9, we will isolate potential substrates and partners of NEK7 and NEK9 by immunoprecipitation using the GFP-Trap method. The spindle and centrosome components will be tested alongside known partners such as NEK6 or γ-tubulin. We expect to isolate and copurify any protein that will specifically bind to our GFP constructs.
At the same time, we will generate RNAi resistant mCherry-tagged analogue-sensitive (as) NEK7 and asNEK9 constructs. Those constructs will be used to establish stable cell lines expressing these mutant kinases. We will determine the phenotype after expression of the kinases and depletion of the endogenous NEK7 and NEK9 using RNAi method.