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By El Mustapha Bahassi, PhD and Robert F. Hennigan, PhD
Polo-like kinases (plks) are a family of conserved serine-threonine kinases that are emerging as important regulators of cell cycle progression.1,2 The founding member of this family is the polo gene of Drosophila melanogaster.3 Mutation of the polo gene confers a pleiotropic phenotype, initially observed to include defects in spindle formation and chromosome segregation,3,4 and more recently cytokinesis.5 Homologues of Drosophila polo have been found in a wide range of eukaryotes. These proteins, termed polo-like kinases, are characterized by a conserved N-terminal catalytic domain and a C-terminal region, the polo-box, that is involved in subcellular localization.6 Homologues of Drosophila polo include Plo1+ in Schizosaccharamyces pombe and cdc5p in Saccharomyces cerevisiae. An amphibian homologue (Plx1) has been described in Xenopus, and three Plk genes (designated Plk1, Plk2, and Plk3) have been found in mammalian cells.1,7 Of these, Plk1 is most similar in structure and function to the yeast and Drosophila polo kinases. Plks have been found to have critical roles in three distinct mitotic functions: M-phase entry, M-phase exit, and cytokinesis.1,2
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 p34cdc2 kinase, the first member of the evolutionarily conserved cyclin-dependent kinase (cdk) family. p34cdc2 activation at the G2/M transition requires dephosphorylation of threonine 14 and thyrosine 15 residues,8,9 which is accomplished by a dual specificity phosphatase cdc25C.10,11 While the key role of cdc2 kinase in triggering entry into M phase is well-established, evidence also points to the involvement of the Plks in regulating the activation of this system. The Xenopus polo-like kinase (Plx1) associates with and phosphorylates cdc25C,12 and its immunodepletion or immunoinhibition blocks the conversion of p34cdc2 into its mitotic form. Conversely, microinjection of Plx1 into oocytes accelerates activation of both cdc25C and mitosis-promoting factor (MPF). However, overexpression of Plk1 in HeLa cells does not cause premature mitosis, arguing against a role for Plk1 in triggering p34cdc2 activation.13 Instead, because Plks also must be activated by upstream kinases, it is thought that Plk1 participates in a regulatory feedback loop that amplifies the activity of p34cdc2 and does not function as the initiating event in p34cdc2 activation.1
In addition to participating in the G2/M transition described above, the Plks appear to play an essential role during M phase. Recent studies identify Plks as important upstream regulators of the ubiquitin-dependent proteolytic degradation machinery that controls passage through mitosis.14-16 This machinery controls both anaphase onset and M-phase exit by catalyzing degradation of inhibitors of sister chromatid separation (termed Pds1p in S. cerevisiae17 and cut2p in S. pombe18) and mitotic cyclins, respectively. How the proteolytic degradation of these and other proteins is regulated in time and space is not fully understood. Current studies focus primarily on the multiprotein anaphase-promoting complex/cyclosome (APC/C), and on proteins related to Drosophila fizzy and its budding yeast homologue cdc20p.19 APC is an E3 ubiquitin ligase which catalyzes the polyubiquitination of specific substrates, thereby targeting them for destruction by the proteasome. Fizzy/cdc20p and related proteins are thought to serve as substrate and/or cell cycle stage-specific, positive regulators of APC/C.
In S. cerevisiae, the polo-kinase cdc5p is both a regulator and a target of APC/C.19 Cdc5 mutants fail to complete mitosis because they fail to ubiquitinate mitotic cyclins. This indicates that cdc5p acts as a positive regulator of cyclin-specific APC/C activity. Conversely, cdc5p levels drop as wild-type yeast cells exit M phase, and this clearly results from APC/C-dependent degradation of cdc5p.19 Substrate recognition by APC/C depends on specific protein motifs, termed destruction boxes.20 Two such motifs are present in the extreme amino terminus of cdc5p, suggesting that it is, itself, a target for APC. However, no obvious destruction boxes can be discerned in Plks from other species, suggesting that organisms undergoing cytokinesis (or fission) may utilize alternative mechanisms to reduce Plk levels upon exit from mitosis. The exit from M-phase arrest in Xenopus egg extracts requires active Plx, which in turn is required for activation of APC.15 Kotani and colleagues showed that PKA and MPF-activated Plk1 plays a critical role in late mitosis progression by controlling APC activity.16 MPF-activated Plk1 phosphorylates at least three subunits of APC (cdc16, cdc27, and tsg24), which activates APC to ubiquitinate cyclin B. Conversely, PKA phosphorylates cdc27 and tsg24 and suppresses APC activity. In fission yeast, APC activation appears to be inhibited by the PKA pathway,21 whereas protein phosphatase 1 (PP1) appears to regulate APC and is essential for initiating anaphase.22 Therefore, at least two events are required for APC activation at metaphase-anaphase transition: APC phosphorylation by MPF-activated Plks and dephosphorylation of PKA phosphorylation sites in APC subunits by a specific serine/threonine phosphatase.
In addition to the described roles for Plks during entry into and exit from mitosis, Plks also may be important regulators of cytokinesis. In Drosophila, polo mutants cause cytokinesis defects at different stages of spermatogenesis.5 In addition, polo co-localizes with a kinesin-related motor protein, called Pavarotti, that is required for the organization of the central spindle, formation of a contractile ring, and cytokinesis.23 Plk1 has been localized to the spindle poles and centromeres in early mitosis and to the central spindle and mid-body during telophase and cytokinesis in animal cells.24,25 In S. cerevisiae, overexpression of the non-catalytic C-terminal domain of cdc5 results in a severe cytokinesis defect that is dependent upon an intact polo box.6 Overexpression of either wild type or catalytically-inactive Plk1 in mammalian cells increases the frequency of multinucleated cells, suggesting that Plk1 interacts with proteins that mediate cytokinesis.13 The molecular mechanism by which Plks regulate cytokinesis is unclear because critical targets for the polo kinases in cytokinesis are unknown. However, Drosophila polo is able to phosphorylate purified tubulin in vitro and the mammalian Plk1 can make a stable complex with a, b, and g tubulin in both interphase and mitotic cells.26,27
Other Polos, Other Functions
Much of the data regarding the function of polo kinases in the cell cycle was derived from lower eukaryotes and is most applicable to mammalian Plk1. Indeed, expression of human Plk1 compensates for the cdc5p mutation in S. cerevisiae, suggesting that Plk1 and cdc5p are functionally homologous. The function of the other mammalian Plk family members (i.e., Plk2 and Plk3) is less certain. In contrast to Plk1, both Plk2 and Plk3 are immediate early genes, implying a function in interphase cells. Overexpression of murine Plk1 results in oncogenic transformation,28 whereas Plk3 overexpression inhibits cell growth by inducing apoptosis.7 However, some functional overlap between Plk1 and Plk3 must exist because Plk3 also can compensate for the S. cerevisiae cdc5p mutation.29,30 Plk2 and Plk3 also have been shown to function in the dendrites and somata of post-mitotic neurons.31 Deregulated expression of Plk3 in-duces a change in cell morphology due to the disruption of the cellular F-actin network, and Plk3 co-immunoprecipitates and co-localizes with Ca2+/integrin-binding protein Cib.32 These data suggest a role for Plks in the signaling network that controls cellular adhesion and a function for polo kinases outside the cell cycle.
It is clear that the polo kinases are key regulators of the cell cycle that are conserved from lower eukaryotes through mammalian species. However, the existence of multiple homologues of the Plks in mammalian species suggests a diversification of function that is poorly understood. The finding that Plk1 levels generally are increased in rapidly proliferating cells has suggested that Plk1 is a proliferation marker with prognostic value in human lung and head and neck cancers.33-35 Overexpression of a dominant-negative Plk1 mutant containing a functional polo-box caused apoptosis specific to tumor cells.36 These data raise the intriguing possibility that conditional expression of the polo-box domain may selectively inhibit endogenous polo kinases. Since the polo-box is a unique and essential domain for polo kinase function, these inhibitors may provide tools to selectively target proliferating cells and are good candidates for antitumor agents. The possible involvement of the polo kinase family with the regulation of several different aspects of mitosis makes understanding the function of all polo kinases important in gaining insight into the process of oncogenesis and evaluating their potential as future targets for anticancer therapies. (Dr. Bahassi is Postdoctoral Assistant and Dr. Hennigan is Assistant Professor, Department of Cell Biology, University of Cincinnati College of Medicine, The Vontz Center for Molecular Studies, Cincinnati, OH.)
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