Polo-box domain

An amino-terminal Serine/Threonine kinase domain antecedes a carboxy-terminal Polo-box domain (PBD) can be seen in all Plks. Most of PBD acquired two Polo boxes that can create a binding pocket for phosphorylated motifs in targeted proteins (Lu and Yu 2009).This is an exception for PLK4 as only one Polo-box is conserved and the interaction between PLK4 and its targeted protein is still in investigation. Polo boxes in PLK1 can interact with each other but this does not happen between cryptic polo box and the polo box in PLK4 although these two take part in intermolecular dimerization. The mitotic entry and exit and cytokinesis in Saccharomyces cerevisiae or budding yeast is regulated by a Plk called cell division cycle 5 (Cdc5) (Schleker et al. 2010) and in Schizosaccharomyces pombe or fission yeast, this process is regulated by Plo1. In metazoans, at least two Plks can be found. For instance in Drosophila melanogaster, Polo (Figure 1) is responsible for many mitotic functions while PLK4 or known as SAK hold designated roles in centrioles duplication. Polo is said to be closely related to mammalian PLK1 and yeast Plks. Plk1 is well-characterised compared to other Plks. Correct Plk1 cellular activity and concentration is important in order to ensure precise cell division coordination. Over expression of Plk1 can be found in many types of cancer and it is related to tumorogenesis. Plk1 exhibits a promising candidate to develop an avant-garde drug therapy for cancer as it provides two functionally essential target sites in one molecule that display distinct properties. These target sites are the kinase domain, common in a few members of protein kinases superfamily and the unique polo-box domain or known as PBD (van de Weerdt et al. 2008). The perplexity of serum-inducible kinase (SAK) or PLK2 and fibroblast growth factor- inducible kinase (Fnk) or PLK3 functions in vertebrates are aligned with the complexness of the cell-cycle although the symmetry of PLK1 shows similarity with PLK2 and PLK3 symmetry. Their functions are less understood. It is thought that PLK2 may carry out its function in S phase. Meanwhile, PLK3 culminates during G1-S phase transition and is localised in interphase. Concurrently with cyclin-dependent kinase 2 (CDK2), accumulation of cyclin E is promoted by PLK3 in activation of DNA replication.PLK3 also able to promote DNA replication via activation of cell division cycle 25 A (Cdc25A) in the vicinity of CDK2. Damage in DNA and mitotic stress can trigger activation of PLK3 which leads to cell-cycle to be arrested and causing apoptosis to occur via a few mechanisms.

Below is the summarized table of Plks and their main functions in organisms that are most commonly studied in this field:

The localization of Polo kinases is different throughout cell cycle event in organisms. During anaphase in mammals in (a), PLK1 is localized to centromeres, centrosomes (Kishi et al. 2009), kinetochores and spindle midzone in mitosis. For PLK2 and PLK4, they are localized near or at the centrioles from the beginning to end of the cell-cycle while PLK3 can be found in nucleolus. In (b), Polo resembles the same localization as PLK1 in Drosophila melanogaster but at the microtubules outside of mitosis. Before the breakdown of nuclear envelope, Polo cannot be seen on the centromeres. Meanwhile, PLK4 or SAK is localized on centrioles. In Saccharomyces cerevisiae or budding yeast (c), cell division cycle (Cdc5) localizes on the spindle pole bodies (SPBs) all the time and it is localized on mother bud neck during mitosis and cytokinesis. Cdc5 is also discovered to be a scattered nuclear protein but in G1 phase, this does not occur when Cdc5 is targeted by Cdc20 homologue 1 (Cdh1)-activated anaphase promoting complex for degradation. For Plo1 in Schizosaccharomyces pombe or fission yeast (d), it is localized on SPBs during mitosis except interphase and cytokinesis. Plo1 also detected more delicately on the spindle during mitosis but in anaphase, more to the future site of cellular fission (Archambault and Glover 2009). Plks constantly found on spindle poles and near cytokinesis site, parallel to its important and broadly conserved functions to these sites. Adversely, Plks are not localized at kinetochores or centromeres in yeasts, indicating that the roles of Plks on these sites are less important for appropriate cell division.

The comparison of activity of Plks in distinct organisms shows that they take part in the important roles in cell cycle of each individual in which the mechanisms can be different in variant organisations but the occurrence may be similar. As mentioned before, Plks constantly found on several sites during cell cycle, thus their functions on these sites may be the key to drug development for cancer.

Plks are believed to hold a fundamental part of cell cycle engine as they helps to regulate several enzymes at the essence of cell cycle. It involves in maintaining the periodic changes of the activities in phosphatase, kinase and ubiquitin ligase for cell division to happen. Cyclin-dependent kinase (CDK1) controls the entry into M phase when it is activated and determines the nuclear breakdown and promotion of chromosome condensation. The activity of CDK1 (Jones 2010) can be control using the inhibitory action of WEE1 and MYT1 kinases by phosphorylation (Figure 3) whereas cell division cycle 25 (CDC25) (Rudolph 2010) does the opposite as it helps to dephosphorylate CDK1 on the similar sites which assist the mitotic entry. PLK1 has positive feedback part in activation of CDK1; hence both PLK1 and CDK1 can affect the activity of CDC25. In fission yeast, recruitment of Plo1 to spindle pole bodies (SPBs) by Cut12 protein is required for activation of CDK1 pivot pathway in entry into M phase. Furthermore, Cdc5 in budding yeast can counteract SWE1 activity (Jiang and Kang 2003) which has the same role with WEE1 in fission yeast, controlling the activity of Cdk1 and this control system can be seen in almost vertebrates. Using these criteria, these Plks are targeted to control the regulation of cell-cycle. Cut 12- Plo1 in fission yeast for instance, the network is triggered by rapamycin signalling (Nakashima et al. 2008) that operates via mitogen-activated protein kinase stress pathway to induce precise mitotic entry based on nutrient accessibility and stress. In human, PLK1 is inactivated when DNA is damaged and there is a process called recovery where another kinase called Aurora A kinase together with its activator BORA(27) (Madeira et al. 2003) help to activate PLK1 to resume the cell cycle. Based on this knowledge, Polo kinases may be the crucial switches in coordination of mitotic entry.

Microtubule-organising centre (MTOC) can be found at the spindle poles in yeast and animal cells but with distinct structure. In yeast, spindle pole bodies (SPBs) are made of molecular plaques inserted in the nuclear envelope. Meanwhile, centrosomes in animal cells at the spindle poles consist of two centrioles enclosed with pericentriolar material (PCM) that involves in cytoplasmic microtubules (MT) nucleation. Centrioles are linked to basal bodies of flagella and cilia where they become compatible to each other. In the presence of centrioles, PCM cannot be coordinated by spindle MT minus ends in some mutant cells. PCM and centrioles are both regulated by Plks at yeast SPBs. When anti-Plks antibodies are injected into Drosophila melanogaster and human cells which lead to defective Polo and PLK1, monopolar spindles were detected. The similar outcome is expected from fission yeast as Plo1 will fail to function in the presence of anti-Plks antibodies but in budding yeast, bipolar spindles were detected in Cdc5 mutants. The assembly of bipolar spindles and SPB separation in yeast that do not involve the breakdown of nuclear envelope is less understood and still under investigation. The functions of Plks in animal cells are even more complex than in yeast. So far, it is known that the two proteins involve at the spindle poles sites are motor proteins and MT-associated proteins (MAPs) and the regulation of these two proteins are coordinated by Polo and Aurora kinases by phosphorylation. During mitosis in Xenopus laevis, the assembly of spindle is promoted via inhibition of MT-destabilising protein Op18, also called stathmin 1 (Andersen et al. 1997)by Plx1. In contrast, at the mitotic entry, MT-stabilizing protein TCTP or TPT1 is negatively regulated by PLK1 that amplifies MT dynamics. It is conceptualized that the labyrinthine of connection in phosphodependent event of MAPs and motor proteins throughout mitosis explains the precise spindle MT dynamics regulation by Plks.

Centrosome maturation occurs in animal cells where MTs recruited to enter the M phase and this process is mainly involved Plks regardless that their molecular pathways are not fully understood. At the centrosomal minus ends of Drosophila melanogaster, the recruitment of γ-tubulin ring complex (γ-TuRC) and Abnormal spindle (ASP) by Polo (Figure 4) is important for MTs nucleation. γ-TuRC is also recruited in metazoan and yeast cells for cytoplasmic MTs nucleation that is needed for spindle positioning and orientation throughout the mitosis event.

In human cells, PLK1 phosphorylates a centrosomal protein during interphase and also a microtubule nucleation regulator called NLP. This will inhibit dynein-dynactic-dependent transport by NLP into centrosome.PLK1 helps in the preservation of PCM cohesion by phosphorylating centrosomal protein kizuna and also in Aurora A localization (Carmena, Ruchaud and Earnshaw 2009)that is crucial for maturation and function of centrosome. By interacting with its target proteins, PLK1 is localized on centrosome which it can intervene the disassemblies of pericentriolar Golgi apparatus that may be participate in the daughter cells division event.

On the other hand, PLK4 involves in the duplication of centrioles in each cell cycle. When PLK4 is inhibited or mutated, this will decrease the number of centrioles in human and Drosophila melanogaster cells but when it is over expressed, the centrioles number would be massive. It is said that PLK2 may be involved in this process in human, but its function is less understood. The cohesion of centrioles is mediated by PLK1 by promoting the sSGO1 association (Archambault and Glover 2009). This shows that duplication of centrioles in human involve many Plks with different roles. Conversely, this event in Drosophila melanogaster only involves PLK4 or sometimes with Polo.

PLK1 in vertebrates promote the sister chromatid cohesion protein 1 (SCC1) cleavage and also involved in the phosphorylation of SA2 cohesin subunit in prophase for its dissociation from chromosome arms. The same event occurs in Saccharomyces cerevisiae where Cdc5 plays similar role but it happens in metaphase-anaphase transition.

Segregation of chromosome in yeast and Drosophila melanogaster is much more complex than in vertebrates. Cohesions removal from chromosome arms, cross-over resolution and homologous chromosomes co-orientation during meiosis 1 in budding yeast requires Cdc5 to make these happen. Shugoshin (SGO1 and SGO2) in vertebrates is triggered by protein phosphatase 2A (PP2a) and MEI-S332 by Polo in Drosophila melanogaster help to protect sister chromatid cohesion around centromeres from being split by cohesin until it reaches anaphase II (Figure 5). Plks have different functions at kinetochores in distinct organisms. PLK1 and Plx1 help to create phosphoepitope called 3F3/2 that is associated with tension deficient during sister kinetochores

attachment to spindle. PLK1 is required for a stable kinetochores and MTs attachment to phosphorylate BUBR1 (Izumi et al. 2009) that functions in spindle attachment checkpoint (SAC) and also interact with a dynein-associated protein called NUDC. PLK1 is not necessarily needed for SAC as if it is inhibited, this may induce cell-cycle arrest. Plx1 recruits in the kinetochores as it is required to regulate BUBR1 in SAC and indirectly can Aurora B, thus promote precise attachment of bipolar MT. In contrast, Cdc5 in budding yeast and Plo1 in fission yeast are not detected at the kinetochores as their functions may not be needed at this site.

CDK1-cyclin B kinase needs to be inactivated for mitotic exit to happen and this can be achieved by mitotic cyclins degradation. This process involves a ligase called anaphase promoting complex (APC) ubiquitin ligase that is activated by polo kinases in vertebrates. In human cells, PLK1 phosphorylates APC inhibitor named mitotic inhibitor 1 (EMI1), creating degradation signal for EMI1 so it will be degraded. The same process happens in Xenopus laevis to regulate mitotic inhibitor 2 (EMI2) by Plx1 where it will bind to EMI2, aid by CAMKII. Yeasts use a mechanism akin to those in vertebrates and Xenopus laevis mechanism without the presence of the APC inhibitors and EMI. Cdc5 localized in spindle poles activates the mitotic entry network (MEN) that is crucial to promote mitotic exit and cytokinesis in budding yeast. Plo1 in fission yeast plays its role in septation initiation network (SIN) similar to MEN with extended role in cytokinesis. It also promotes export of Dmf1 in nucleus that induces actin-based medial ring assembly around nucleus. The overexpression of Plo1 in fission yeast leads to septation where it can occur at any stage throughout the mitosis while mutants carrying defect plo1 gene cannot septate at all. Animal polo kinases interact with mitotic kinesin-like protein2 (MKLP2) and protein regulator of cytokinesis 1 (PRC1) that explain their localization in into central spindle. The functions of Plks in cytokinesis may be their most conserved role compared to in other processes.

The frequency of PLK1 expression is used as the predictive value of tumour aggressiveness in cancer patients. It has been shown that downregulation of PLK1 by microinjection with anti-PLK1 antibodies can inhibit the activity of cancer cells (Spaenkuch-Schmitt et al. 2002). When cellular PLK1 level is downregulated, there is some progression in the cell cycle to a degree of about 30% in G2/M phase. With suitable doses, the activity of anti-PLK1 antibodies can be applied for cancer therapeutic and this knowledge has been applied in the medicine field nowadays. The effects of PLK1 activity when it is depleted in cancer cells and also PLK1 activity leads to its potential treatment for cancer will be discussed further below.

The depletion of PLK1 can lead to apoptosis in cancerous cells. This process can be shown using pBS/U6-Plk1 and pBabe- puro at the ratio of 10:1 where it will deplete Plk1 in human cells (Liu and Erikson 2003). After transfection with pBS/U6-Plk1, cell proliferation of HeLa cells was inhibited actively (Figure 7a) and only approximately 10% attached cells present. Transfection with control vector did not affect HeLa cells growth activity and it has very small effect on the cells viability.

Depletion of PLK1 can affect the cell cycle progression especially in G2/M phase (Degenhardt and Lampkin 2010)and this can be analysed using Fluorescence activated cell sorting (FACS). The percentage of cells in 4N DNA content showed an increase after the transfection and after 3 days, the 4N DNA content increased to about 40% and later together with sub-G1 increment (Figure 8a). Using confocal microscopy, there were three populations of cells observed after the transfection; normal, dumb-bell shape or fragmented (Figure 8b). Dumb-bell shape and fragmented nuclei show that the cells are undergoing apoptosis. Using these populations of cells, five independent experiments were carried out to show the percentage of cells with different chromatin structures. About 31% of the cells carried dumb-bell shape chromatin structure and about 36% with fragmented nuclei. Additionally, after 6 days, sister chromatids were unable to detach in anaphase.

A protein called p-53 tumour-suppressor protein (Dias et al. 2009) with different stress stimuli has a crucial role in apoptosis and also the arrest of cell cycle was believed to be dependent on PLK1. When PLK1 depleted in cancer cells, the p53 pathways were very stable but undetectable in control cells (Chopra et al. 2010).The pathways involved were death-receptor pathway and mitochondrial pathway and both induce apoptosis event in cancer cells. On the other hand, ataxia telangiectasia (ATM) protein kinase holds an important part in cellular events and when it is inhibited, this will promote the fatality of PLK1 depletion. Caffeine and wortannin are ATM inhibitors and when PLK1-depleted cells were treated with pBS/U6-Plk1 using GenePORTER reagent together with these inhibitors, apoptosis occurred in almost 60% of the cells after one day of treatment. This shows that ATM cells are extremely sensitive with depletion of PLK1. Nowadays most of the cancer treatments are based on genotoxic agents, for examples UV-mimetic agents, DNA-cross-linking and alkylating agents, DNA intercalators, and topoisomerase inhibitors as these agents can cause

damage in DNA and induce apoptosis in cancerous cells. Meanwhile, caffeine and wortannin act as radiosensitizers to alter the checkpoints for DNA damage for stimulation of cancer cells killing.

PLK1 has crucial parts in various events in cell cycle, a very important regulator during mitosis but its physiological characteristics are not fully understood. On the other hand, correlation between PLK1 and prognosis and the aggressive level of cancer cells can be marked as a new diagnostic for certain, but not all, types of cancer such as non-small-cell lung cancer (NSCLC), head and neck cancer, endometrial cancer, colorectal cancer and thyroid cancer. Using newly improved techniques and suitable compounds, inhibition of PLK1 may be the promising remarks to cancer treatment to kill cancer cells in vitro and also in vivo as activity of anti-PLK1 antibodies were well-tolerated during clinical trials of advanced cancer patients. Not only the activity of PLK1, but the functions and other polo kinases roles should be taken into account in considering the precise therapeutic for cancer patients.

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