Mitotic Roles of Polo Like Kinases
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Mitotic Roles of Polo Like Kinases
Cell division is characterized by orchestrated events of chromosome segregation, distribution of cellular organelles, and the eventual partitioning and separation of the two daughter cells. Mitosis is a highly regulated process that assures the proper allotment of genetic material between each pair of daughter cells. It proceeds through successive stages of well-defined and coordinated sub-processes. Entry into mitosis is regulated by the CDC2 (Cell Division Cycle-2)/Cyclin-B heterodimer. CDC2/Cyclin-B activity drives the events of early mitosis, such as nuclear breakdown, chromosome condensation and spindle formation by phosphorylating cellular substrates. While CDC2 (the catalytic subunit of the heterodimer) is required to drive the events of early mitosis, protein kinases structurally distinct from CDKs (Cyclin-Dependent Kinases) also make important contributions to cell cycle progression. The PLKs (Polo-Like Kinases) make up an evolutionarily conserved, newly emerging family of essential cell-cycle regulators (Ref.1).

PLKs are present in fungi and higher organisms and are characterized by the presence of a distinct region of homology in the C-Terminal noncatalytic domain, termed the Polo-box (amino acids 410-439 in PLK), which is critical for the subcellular localization of Polo Kinases. Members of this subfamily include mammalian PLK (human Polo-Like Kinase-1 [PLK1], PLK2, and PLK3), Snk (Serum-inducible Kinase), and Fnk (Fibroblast Growth Factor-Inducible Kinase)/PRK (Proliferation-Related Kinase), Xenopus laevis Plx1, Drosophila melanogaster Polo, Schizosaccharomyces pombe Plo1, and Saccharomyces cerevisiae CDC5. Polo Kinases regulate diverse cellular and biochemical events at various stages of M-Phase (Ref.2). These are required at several key points through mitosis, starting from control of the G2/M transition through phosphorylation of CDC25C and mitotic cyclins and a role in the DNA damage checkpoint adaptation to prevent entry into mitosis. At the beginning of mitosis, various proteins are recruited to the centrosomes, a maturation process which requires Polo Kinases. Polo Kinases are also required for the establishment of a bipolar spindle. Equally, Polo Kinases are important for exit from mitosis. It has a role in the Metaphase to Anaphase transition via its interaction with the APC (Anaphase Promoting Complex)/Cyclosome. In addition to these roles, Polo Kinases also play important role(s) in the regulation of cytokinesis (Ref.3).

At the onset of mitosis, the eukaryotic cells undergo profound structural rearrangements that are regulated by protein phosphorylation. Prominent among the kinases responsible for regulating entry into mitosis is the CDC2 (Cell Division Cycle-2) kinase, the first member of the evolutionarily conserved CDKs family (Ref.4). CDC2 has a basal phosphatase activity that partially activates CDC2 by removing the inhibitory phosphates added by Wee1 and Myt1. The amplification of CDC2 activity needed for mitotic entry does not occur until PLK becomes activated by SLK (STE20-Like Kinase). CDC25 is then further activated, which results in the burst of CDC2 activation needed for mitotic entry. PLK directly activate CDC25 and plays a role in the positive feedback loop that operates during CDC2 activation at the G2-M transition (Ref.5). Several protein kinases such as Chk2/Rad53, PP2A (Protein Phosphatase-2A) inhibit PLK activation of CDC25. Growth factors like TGF-Beta (Transforming Growth Factor-Beta) also inhibit the phosphorylation of CDC25 through a different signaling mechanism. PLKs have a role in centrosome maturation and separation. In human cells PLK stimulate the centrosome’s microtubule-nucleating activity upon mitotic entry. Furthermore, it facilitates recruitment of Gamma-Tubulin and activates Asp (a microtubule-associated protein) at the centrosome. The chaperone HSP90 (Heat Shock Protein-90) is pivotal for stability and function of PLK (Ref.13). Another microtubule-associated protein, the plus-end-directed motor HsEg5, is involved in separation of the centrosomes to opposite poles of the cell and the organization of mitotic asters. HsEg5 is phosphorylated and activated by CDC2-Cyclin-B, which in turn is activated by PLK (Ref.5). During M-Phase the PLKs activate certain functions of the APC/Cyclosome, an E3 Ubiquitin-protein Ligase that directs the proteasomal degradation of Anaphase inhibitors (Ref.4). Both Securin and CDC2s activating subunit Cyclin-B are ubiquitinated at the onset of Anaphase by the APC/Cyclosome, leading to their proteasome-dependent degradation and to Separase activation (Ref.6). The substrate specificity of the APC is regulated by its interactions with FZR1 (Fizzy/Cell Division Cycle-20 Related-1(Drosophila)). When the APC is in a complex with FZR1, it directs the breakdown of components that inhibit sister chromatid separation, such as the Securin Pds1. This leads to activation of the Separin Esp1, which cleaves the Cohesin Scc1 (Ref.5). Cohesin is a protein complex that is highly conserved in evolution and consists of at least four subunits: two SMC (Structural Maintenance of Chromosomes) proteins, SMC1 and SMC3, the so-called “kleisin” subunit Scc1 (also called Rad21 or Mcd1), and Scc3. Cells of humans, Xenopus, and other higher eukaryotes contain two mitotic orthologs of Scc3, called SA1 and SA2. Cohesin complexes in these cells contain either SA1 or SA2, but not both (Ref.9, 10 & 11). Proper cohesion of sister chromatids is a prerequisite for the correct segregation of chromosomes during cell division. Most of the cohesin complexes dissociate from the chromosomes before mitosis, although those complexes at the kinetochore remain. PTTG1 (Pituitary Tumor-Transforming-1) which is a substrate of APC is associated with Esp1 until activation of the APC (Ref.7 & 8). Another microtubule-associated protein, PRC1 (Protein Regulator of Cytokinesis-1) is also required to maintain the spindle midzone and for the cleavage process, but their specific roles in cell cleavage have not as yet been defined (Ref.14).

In addition to the described roles for PLKs during entry into and exit from mitosis, PLKs also promote the onset of cytokinesis. PLK1 interacts with MKLP1 (Mitotic Kinesin-Like Motor Protein-1) during Anaphase and Telophase. These two proteins localize to the interzone during Anaphase and the mid-body during Telophase and Cytokinesis (Ref.12), which is required for the organization of the central spindle, formation of a contractile ring. Over-expression of yeast or human PLKs leads to the formation of multiple septa at any stage of the cell cycle. The septum-inducing network has a two-part GAP (GTPase-activating protein) and a GTP-binding protein, which signals septum formation through the kinase CDC7. In S. pombe, the GAP comprises CDC16 and Byr4 and the GTP-binding protein is Spg1. PLK regulates this pathway and, once activated, the pathway feeds back and inhibits PLK. In Drosophila, Polo reorganise the central region of the spindle in late M-phase, co-localizing with Pav-KLP, a microtubule motor protein of the kinesin family, at the spindle midzone and the midbody during cytokinesis (Ref.5).

In contrast to PLK1, both PLK2 and PLK3 are immediate early genes, implying a function in interphase cells (Ref.4). PLK3 remains relatively constant during the cell cycle, and its kinase activity peaks during late S- and G2-phases. Furthermore, PLK3 phosphorylates CDC25C resulting in inhibition of the activity of this protein, whereas phosphorylation of Cyclin-B by PLK1 results in its translocation from the cytosol to the nucleus, thus activating CDC2 kinase. PLK2 and PLK3 also function in the dendrites and somata of post-mitotic neurons. Deregulated expression of PLK3 induces a change in cell morphology due to the disruption of the cellular F-Actin network (Ref.4). Snk and Fnk associate with CIB, a Calmodulin-related protein and have been implicated in long-term synaptic plasticity and thus may perform post mitotic functions.

Polo Kinases are key regulators of the cell cycle that are conserved from lower eukaryotes through mammalian species. Mutations in Drosophila Polo cause abnormalities in mitosis. In human cells, maximal PLK activity is reached in the M-phase of the cell cycle, and microinjection of anti-PLK antibody into living cells induce a mitotic abnormality that contributes to the generation of aneuploidy. PLK1 is a proliferation marker with prognostic value in human lung and head and neck cancers. Over expression of a dominant-negative PLK1 mutant containing a functional Polo-box caused apoptosis specific to tumor cells. Since the Polo-Box is a unique and essential domain for Polo Kinase function, these inhibitors provide tools to selectively target proliferating cells and are good candidates for antitumor agents. The involvement of the Polo Kinase family with the regulation of several different aspects of mitosis is important in gaining insight into the process of oncogenesis and evaluating their potential as future targets for anticancer therapies (Ref.4).