A second Polo kinase in Drosophila, SAK, is required for centriole duplication

SAK/PLK4 is a distinct member of the polo-like kinase family. SAK−/− mice die during embryogenesis, whereas SAK+/− mice develop liver and lung tumors and SAK+/− MEFs show mitotic abnormalities. However, the mechanism underlying these phenotypes is still not known. This study shows that downregulation of SAK in Drosophila cells, by mutation or RNAi, leads to loss of centrioles, the core structures of centrosomes. Such cells are able to undergo repeated rounds of cell division, but display broad disorganized mitotic spindle poles. SAK mutants lose their centrioles during the mitotic divisions preceding male meiosis but still produce cysts of 16 primary spermatocytes as in the wild-type. Mathematical modeling of the stereotyped cell divisions of spermatogenesis can account for such loss by defective centriole duplication. The majority of spermatids in SAK mutants lack centrioles and so are unable to make sperm axonemes. Depletion of SAK in human cells also prevents centriole duplication and gives rise to mitotic abnormalities. It is concluded that SAK/PLK4 is necessary for centriole duplication both in Drosophila and human cells. Drosophila cells tolerate the lack of centrioles and undertake mitosis but cannot form basal bodies and hence flagella. Human cells depleted of SAK show error-prone mitosis, likely to underlie SAK's tumor-suppressor role (Bettencourt-Dias, 2005).

Polo-like kinases (Plks) belong to a conserved family of mitotic serine-threonine protein kinases that play key roles in centrosome function and are misregulated in many human tumors. Two branches of the family have emerged in metazoans, and they are represented in Drosophila by Polo and SAK (also called Plk4). Whereas much is now known about the mitotic functions of the founder member of the family, Drosophila Polo, and its related mammalian counterparts (Plk1-3), the precise mitotic roles of SAK remain obscure. Conservation of the structures of the two types of Plks throughout evolution suggests that different roles may have been preserved. Both branches of the family have an amino-terminus kinase domain and a regulatory C-terminal domain that contains conserved polo boxes (PBs). However, whereas the two PB domains of mammalian Plk1 interact with each other to create a positively charged cleft able to bind phosphopeptides, the SAK PB forms an intermolecular homodimer in which different sequences are exposed. Moreover, SAK has a second very divergent PB (cryptic PB) that does not bind to its conserved PB. The Plk1-3 group are more akin to the single Plks found in the yeasts, such as Cdc5 in S. cerevisiae, and expression of either Plk1 or Plk3, but not SAK, rescues the mitotic defects of temperature-sensitive cdc5-1 mutant cells (Bettencourt-Dias, 2005).

Drosophila Polo and its closest mammalian counterpart, Plk1, are associated with centrosomes, kinetochores, and the late-mitotic central spindle, reflecting their functions in centrosome maturation, in the metaphase-anaphase transition, and in cytokinesis. SAK also localizes to the centrosome, and SAK−/− mice die shortly after gastrulation, showing a 20-fold increase in cell death. Elderly SAK+/− mice display a 15-times-higher incidence of spontaneous liver and lung cancers than their wild-type littermates. Similarly, the human SAK gene maps to a chromosome region, 4q28, that is frequently rearranged in hepatocellular carcinomas. Multipolar mitotic spindles have been reported in livers and MEFS of SAK+/− mice, suggesting that haploinsufficiency for tumor suppression may result from chromosome instability in the oncogenic pathway. This study addresses the underlying cause of these mitotic defects, showing that in both Drosophila and human cells, SAK is required for centriole duplication. It is therefore essential for centrosome integrity and thereby fidelity of the mitotic apparatus. Moreover, SAK is also required for development of axonemal structures, reflecting the dual nature of centrioles and basal bodies (Bettencourt-Dias, 2005).

To examine SAK function, more than 70% of SAK mRNA was depleted in cultured Drosophila cells. A 12-fold increase was observed in the percentage of mitotic cells that had no γ-tubulin at the poles (1.6%, controls versus 24.7%, SAK RNAi. The absence was observed of other proteins typically recruited to Drosophila centrosomes in mitosis: CP190 and Cnn. In a recent survey of the cell-cycle function of all the protein kinases in Drosophila, loss of function of only one other kinase, Polo, led to loss of γ-tubulin from the centrosome, consistent with the known function of Polo/Plk1 in centrosome maturation. However, in contrast to the striking metaphase arrest following polo RNAi, there was no change in mitotic index or in the flow-cytometry profile of DNA content after SAK RNAi. Thus, in S2 cells, SAK is required for centrosome integrity but not for progression through the cell cycle or cell survival (Bettencourt-Dias, 2005).

Dividing cells were examined in the central nervous system of SAK mutant larvae. Such cells showed an extremely similar phenotype to SAK RNAi cultured cells: that is, a notable absence of γ-tubulin from the poles of spindles that were often disorganized and splayed. Meiotic spindles in testes of such mutants also often lacked γ-tubulin and Cnn at the poles. It was found that Drosophila SAK localizes to centrosomes in both interphase and mitosis of wild-type cells, consistent with its having a function in ensuring centrosome integrity (Bettencourt-Dias, 2005).

In contrast to Polo-depleted cells, the poles of SAK-depleted cells were broad, similar to the mitotic figures of a Drosophila acentriolar cell line. This led to an inquiry of whether the innermost centrosomal structures, the centrioles, might be disrupted by depletion of SAK. It was found that the majority of mitotic cells that showed no γ-tubulin at the poles following SAK RNAi had no detectable pericentrin-like protein (D-PLP), normally present in both centrioles and the pericentriolar material (PCM). This was in contrast to polo or cnn RNAi cells, where D-PLP was still present in more than 75% of cells lacking γ-tubulin at both poles, reflecting the roles of Polo and Cnn in centrosome maturation but not centriolar integrity. In interphase, the majority of SAK RNAi cells had either zero or a single centrosome, whereas interphase polo or cnn RNAi cells did not show a significant change in centrosome number. The absence of D-PLP in a large proportion of interphase SAK RNAi cells suggests that they might be missing both centrioles and PCM. To confirm loss of centrioles after SAK RNAi, such cells were stained for the centriole marker centrin and then embedded and serially sectioned for transmission electron microscopy (TEM). In SAK-depleted cells in which centrin staining was absent at the spindle poles, no centrioles could be detected by TEM. Thus, the downregulation of SAK leads to loss of centrioles from affected cells, with no effect on the ability of those cells to proliferate (Bettencourt-Dias, 2005).

To determine whether loss of centrosomes also occurs in SAK mutants and still permit proliferation of diploid cells, the central nervous system of mutant larvae was examined. This revealed cells with two, one, or zero centrosomes, a phenotype identical to SAK RNAi. Moreover, the brains of mutant larvae were of normal size and the proportion of brain cells in mitosis was comparable to the wild-type, indicating no obvious defects in cell-cycle progression. SAK mutants are able to pupate, and adults eclose from the pupae. However, the majority of adults are uncoordinated and die after getting stuck in the food. This phenotype is similar to D-PLP mutations that cause defects in basal bodies, the centriolar-derived structures required for formation of cilia in neurons of type-I sensory organs that function in transduction of sensory stimuli. The lack of centriole markers in cells of the larval central nervous system of SAK mutants suggests that the uncoordinated adult phenotype is likely to reflect absence of basal bodies in sensory neurons (Bettencourt-Dias, 2005).

It was of interest to inquire whether the above defects could reflect a failure to assemble new centrosomes. This could account for the increased frequency of cells with only one centrosome in SAK mutants, if new centrosomes are not formed but cells continue to cycle. The prediction was that if cultured cells were allowed to continue dividing while being exposed to SAK dsRNA, the average number of centrosomes per cell would decrease with time. This was tested by repeatedly transfecting cells with SAK dsRNA at 4-day intervals. Successive transfections led to a progressive increase in the proportion of cells with no centrosomes, rising to greater than 93% of cells after 16 days. This dilution is consistent with SAK's having a role in centrosome assembly. Such a role was further substantiated by the finding that transfection of S2 cells with the active, but not inactive, kinase led to an increase in cells with more than two D-PLP foci. These foci are unlikely to result from aborted cell division because the majority of those cells had a single nuclei. These foci behaved as microtubule organizing centers clustering at the poles of mitotic spindles. Although additional experiments will be needed to verify the origin of the multiple foci, these results suggest that overexpression of SAK may result in multiple centrosomes (Bettencourt-Dias, 2005).

To determine whether the phenotypes observed in SAK mutant flies are associated with defective centrosome separation, abnormal centrosome inheritance, or problems in centriole duplication, spermatogenesis was examined in SAK mutant males. The germline has a stereotyped pattern of mitotic and meiotic divisions, a pattern that allows the cellular history of centrosomes and centrioles to be deduced. Additionally, their centrioles are approximately 10-fold longer than those found in other Drosophila cells and can be easily visualized by fluorescence of a GFP-PACT fusion protein harboring the centriole-targeting domain of D-PLP protein (Bettencourt-Dias, 2005).

A very high proportion of primary spermatocytes from SAK mutants had no centrioles at one or both spindle poles in meiosis I. When centrioles were absent, the spindle poles were broad and there were no astral microtubules, or very disorganized spindles were formed. Transverse TEM sections of wild-type sperm tails revealed the classic "9 + 2"'; axonemal microtubules that were missing from the majority of elongating SAK spermatids. Accordingly, the majority of sperm from SAK mutant testes are nonmotile, and males are sterile. Frequently, cells that had no axonemes had irregular size and numbers of mitochondrial derivatives per cell. This is usually associated with defective chromosome segregation and cytokinesis during meiosis. The lack of a strong spindle-assembly checkpoint in meiosis makes cells more likely to progress all the way through both meiotic divisions even in the presence of abnormalities. When axonemes were present, they appeared to be normal in structure (Bettencourt-Dias, 2005).

Wild-type primary spermatocytes enter meiosis I (MI) with a pair of centrioles at each spindle pole. Daughter cells inherit a pair of centrioles that are not duplicated in Drosophila. These centrioles separate, each generating a centrosome at the pole of the second meiotic spindle. Thus, each daughter spermatid inherits a single centriole. Failure to observe more than a single centriole in the products of meiosis II in the SAK mutant allowed the possibility of defects in centriole separation to be discarded (Bettencourt-Dias, 2005).

The cysts of 16 primary spermatocytes encapsulate the history of centriole duplication in the four preceding cell divisions. It was therefore asked whether the number of centrioles per cell in SAK mutant cysts could reflect abnormal centriole duplication. Mature cysts of primary spermatocytes in both wild-type and SAK mutant testes always contained 16 spermatocytes of comparable size, indicating success in the four rounds of premeiotic mitosis. However, whereas wild-type primary spermatocytes contained four centrioles (64 per cyst), the majority of spermatocytes from SAK mutants (68%, n = 239) had no centrioles, and a smaller proportion of cells had intermediate numbers between one and four. The absence of cells having more than two centrosomes/four centrioles (0%, n = 352) provided a second indication of the lack of defects in centrosome segregation to daughter cells. To test whether the distribution of centrioles observed resulted from a defect in centriole duplication, a mathematical model was generated to describe the centriole-duplication cycle and it was adjusted ifor the four mitotic divisions of germ cells in a cyst. Multitype branching-process theory was used to evaluate the distribution of cells with a given number of centrioles. The model has some analogies with previous analytical work on plasmid copy number in bacteria. It assumes that centrosome separation and segregation is perfect, and that the variable number of centrioles in G2 after the four germline divisions is due to partially defective centriole duplication having a probability θ. Generating functions were built to follow the dynamics of the mean number of cells with a given number of centrioles. The proportion of cells with a given number of centrioles in G2 was evaluated after four cell divisions. Finally, a value of θ was found that best fit the empirical data. The function is very peaked around 0.55, fitting the empirical data very well and giving high confidence in the estimated duplication rate. Thus, it was possible to model the reduction in centriole number during the premeiotic divisions of SAK hypomorphic mutants solely by assuming reduced success of centriole duplication (Bettencourt-Dias, 2005).

Finally, it was asked whether the role of SAK in centriole duplication was conserved in human cells and SAK siRNA conditions were developed that reduced levels of SAK transcripts by more than 70%. Such depletion resulted in a 10-fold increase of HeLa cells with just one centriole. This was associated with an increased mitotic index and a higher-than-2-fold increase in apoptosis, leading to a decrease in cell number. A similar reduction was observed in centriole number after SAK RNAi in U2OS cells. It is known that centrioles continue to replicate when U2OS cells are blocked in S phase by treatment with the DNA polymerase α-inhibitor aphidicolin (AF) or the ribonucleotide reductase inhibitor hydoxyurea (HU). Following SAK RNAi, the number of cells accumulating supernumerary centrosomes in an S phase block was found to be less than half that of control cells, as shown for other molecules required for centriole duplication. Thus downregulation of SAK reduces centriole duplication in cells blocked in S phase. There was no increase in the proportion of cells with a single centriole after SAK RNAi when cells were inhibited from dividing by treatment with AF or HU, a result consistent with inhibition of centriole duplication. It is therefore concluded that the human SAK kinase is required for centriole duplication in both HeLa and U2OS cells and for centriole reduplication in AF- or HU-treated U2OS cells (Bettencourt-Dias, 2005).

Reduced centriolar number was associated with a 6-fold increase in abnormal mitotic spindles 72 hr after SAK SiRNA in HeLa cells. These included monopolar and multipolar spindles. Similar defects were observed in U2OS cells. Curiously, in a few cases, recruitment of γ-tubulin to the acentriolar poles of multipolar spindles was seen, suggesting that acentriolar poles may organize some PCM in mammalian cells. Such cases were not observed in Drosophila. In summary, spindle organization is affected in the absence of two canonical centrosomes having two centrioles, and this is likely to contribute to the reduced chromosome-transmission fidelity that has been suggested from observations of SAK-deficient mouse cells (Bettencourt-Dias, 2005).

Human SAK/PLK4 has been described as a tumor suppressor in humans and mice, and it has been suggested that correct levels of SAK are essential for mitotic fidelity. This study shows that SAK is essential for centriole duplication in both Drosophila and human cells. The reduced centriole number arising in the absence of SAK in human cells leads to the formation of abnormal mitotic spindles, providing the first mechanistic insight for the tumorigenic role of this molecule (Bettencourt-Dias, 2005).

Drosophila has just two PLK family members: Polo, involved in centrosome maturation and mitotic progression, and SAK. Depletion of SAK, but not Polo, leads to cells with a reduced number of centrioles. Centriole loss in a cycling population of cells can arise through defective centrosome duplication, abnormal separation of centrosomes at entry to mitosis, or abnormal centrosome segregation to the daughter cells in cytokinesis. In both human and Drosophila cells there is no abnormal centrosome segregation in the absence of SAK because there is always loss but never gain of centrosomes/centrioles after cell division. Moreover, centriole reduplication in S phase is reduced after SAK knockdown in the absence of cell division in human cells. This analysis of centriole distribution in Drosophila SAK spermatids revealed no defects in centrosome separation. Moreover, the distribution of centrioles in cysts of 16 primary spermatocytes was consistent with a mathematical model assuming defects in centriole duplication. Together, these observations point to a conserved role of this member of the PLK family in centriole duplication. However, an additional function for SAK in mitosis cannot be ruled out until a complete loss-of-function mutant is isolated (Bettencourt-Dias, 2005).

Experiments in C. elegans have suggested five proteins to be important for centrosome duplication in embryogenesis, and these include one protein kinase, ZYG1. Although ZYG-1 has only low sequence similarity to Drosophila SAK, it is the closest homolog in a BLAST search, and SAK may thus represent the ortholog of ZYG-1 in flies and vertebrates (Bettencourt-Dias, 2005).

What could be the role of SAK in centriole duplication? Structurally compromised centrioles were never observed either in SAK mutants or after SAK depletion in either Drosophila or human tissue-culture cells. Moreover, overexpression of SAK, but not of the inactive kinase, leads to the formation of multiple D-PLP foci, suggestive of overduplication of centrioles. Together, these data suggest that SAK has a regulatory role in centriole duplication. ZYG-1 has been found to be high in the hierarchy of molecules necessary for centriole assembly and essential for the recruitment of SAS-6/SAS-5 to the centriole, but it remains to be discovered whether this role has been conserved in Drosophila and vertebrates (Bettencourt-Dias, 2005).

The perdurance of centrosomal structures in SAK mutants studied here may result from some residual SAK function due to the hypomorphic nature of this allele and/or from remaining wild-type maternal SAK protein provided by the heterozygous mother. Such perdurance of maternal protein to the third larval-instar stage is a common feature of mitotic mutants in Drosophila. Nonetheless, SAK mutants provide the first opportunity to assess the consequences of the absence of centrioles and centrosomes upon the development of an organism. Examination of the larval central nervous system suggests that as many as 72% of dividing cells have fewer centrosomes than expected, with 28% possessing no centrosomes at all. In cysts of primary spermatocytes, only 8% of the cells have the correct number of centrioles, whereas 68% have none. It has been proposed that centrosomes are not required for the formation of mitotic spindles, but do provide speed and fidelity to this process and so may be necessary for proper cell-cycle progression. It is concluded that in cultured Drosophila cells and in the whole organism, centrosomes are not essential for mitotic progression or cell survival. However, the absence of centrioles, and hence basal bodies, compromised both meiotic divisions and the formation of sperm axonemes. The only cilia and flagella known in the fly are found in the peripheral nervous system and in the male germline. Accordingly, SAK mutants were found to be both uncoordinated and sterile (Bettencourt-Dias, 2005).

What could be the consequences of the lack of SAK in vertebrates? Mammalian cells appear to be more sensitive to depletion of SAK and the lack of centrosomes than Drosophila cells. There was a significant increase in abnormal mitoses, in mitotic index, and in apoptosis after depletion of SAK in human cells. Mitotic abnormalities and an increase in cell death were also observed after depletion of SAS-6, a protein involved in centriole replication, in U2OS cells and may thus be a general consequence of centriole loss in vertebrates. Mice homozygotes for SAK die very early during embryogenesis, hindering study of the effects on cilia formation and development. However, these embryos showed increased cell death and a higher mitotic index. SAK+/− MEFs also show abnormal spindles and chromosome segregation, and, significantly, SAK+/− mice are more prone to develop cancer. This link between centrosome duplication, embryonic survival, and haploinsufficiency for tumor suppression is also seen with nucleophosmin, a previously suggested regulator of centrosome duplication. Together, these studies reinforce the link between centrosome defects and oncogenesis. The sensitivity with which two tumor cell lines undergo aberrant mitosis and cell death after downregulation of the SAK kinase suggests that it may be a valuable target for cancer therapy (Bettencourt-Dias, 2005).

Yeast polo kinases

S. pombe has a gene (plo1+) that shows homology to the budding yeast gene CDC5, the Drosophila gene polo, and the mammalian family of genes encoding polo-like kinases. Disruption of plo1+ has profound effects on the organism, an indication of the essential nature of this gene. Loss of plo1+ function leads to a mitotic arrest in which condensed chromosomes are associated with a monopolar spindle or to the failure of septation following the completion of nuclear division. In the latter case, cells show a failure both in the formation of an F-actin ring and in the deposition of septal material, suggesting that plo1+ function is required high in the regulatory cascade that controls septation. The overexpression of plo1+ in wild-type cells not only results in the formation of monopolar spindles, but also induces the formation of multiple septa without nuclear division. Septation can be induced in the absence of mitotic commitment and concomitant spindle formation in any of several ways by the overexpression of plo1+ in cells that have been arrested in G2 through genetic means (Ohkura, 1995).

Following chromosome segregation in anaphase, ubiquitin-dependent degradation of mitotic cyclins contributes to the exit from mitosis. A key step in this process is catalyzed by a ubiquitin-protein ligase known as the anaphase-promoting complex (APC), the regulation of which is poorly understood. The Polo-related protein kinase Cdc5 in Saccharomyces cerevisiae might encode a regulator of the APC, because cdc5 mutant cells arrest with a late mitotic phenotype similar to that observed in cells with defective cyclin destruction. The role of Cdc5 in the regulation of mitotic cyclin degradation has been investigated. In cdc5-1 mutant cells, a defect in the destruction of cyclins and a reduction in the cyclin-ubiquitin ligase activity of the APC are observed. Overexpression of CDC5 results in increased APC activity and mitotic cyclin destruction in asynchronous cells or in cells arrested in metaphase. CDC5 mutation or overexpression does not affect the degradation of the APC substrate Pds 1, which is normally degraded at the metaphase-to-anaphase transition. Cyclin-specific APC activity in cells overexpressing CDC5 is reduced in the absence of the APC regulatory proteins Hct 1 and Cdc20. In G1, Cdc5 itself is degraded by an APC-dependent and Hct1-dependent mechanism. It is concluded that Cdc5 is a positive regulator of cyclin-specific APC activity in late mitosis. Degradation of Cdc5 in G1 might provide a feedback mechanism by which the APC destroys its activator at the onset of the next cell cycle (Charles, 1998).

Plk is a mammalian serine/threonine protein kinase whose activity peaks at the onset of M phase. It is closely related to other mammalian kinases (Snk, Fnk, and Prk), as well as to Xenopus laevis Plx1, Drosophila Polo, Schizosaccharomyces pombe Plo1, and Saccharomyces cerevisiae Cdc5. The M phase of the cell cycle is a highly coordinated process that ensures the equipartition of genetic and cellular materials during cell division. To enable understanding of the function of Plk during M phase progression, various Plk mutants were generated and expressed in Sf9 cells and budding yeast. In vitro kinase assays with Plk immunoprecipitates prepared from Sf9 cells indicate that Glu206 and Thr210 play equally important roles for Plk activity and that replacement of Thr210 with a negatively charged residue elevates Plk specific activity. Ectopic expression of wild-type Plk (Plk WT) complements the cell division defect associated with the cdc5-1 mutation in S. cerevisiae. The degree of complementation correlates closely with the Plk activity measured in vitro, since it is enhanced by a mutationally activated Plk, T210D, but is not observed with the inactive forms K82M, D194N, and D194R. In a CDC5 wild-type background, expression of Plk WT or T210D, but not of inactive forms, induces a sharp accumulation of cells in G1. Consistent with elevated Plk activity, this phenomenon was enhanced by the C-terminally deleted forms WT deltaC and T210D deltaC. Expression of T210D also induces a class of cells with unusually elongated buds that develop multiple septal structures. However, this is not observed with the C-terminally deleted form T210D deltaC. It appears that the C terminus of Plk is not required for the observed cell cycle influence but may be important for polarized cell growth and septal structure formation (Lee, 1997).

Members of the polo subfamily of protein kinases play pivotal roles in cell proliferation. In addition to the kinase domain, polo kinases have a strikingly conserved sequence in the noncatalytic domain, termed the polo-box. The function of the polo-box is currently undefined. The mammalian polo-like kinase Plk is a functional homolog of Saccharomyces cerevisiae Cdc5. Plk localizes at the spindle poles and cytokinetic neck filaments. Without impairing kinase activity, a conservative mutation in the polo-box disrupts the capacity of Plk to complement the defect associated with a cdc5-1 temperature-sensitive mutation and to localize to these subcellular structures. These data provide evidence that the polo-box plays a critical role in Plk function, likely by directing its subcellular localization (Lee, 1998).

Progression through and completion of mitosis require the actions of the evolutionarily conserved Polo kinase. The levels of Cdc5p, a Saccharomyces cerevisiae member of the Polo family of mitotic kinases, are cell cycle regulated. Cdc5p accumulates in the nuclei of G2/M-phase cells, and its levels decline dramatically as cells progress through anaphase and begin telophase. Cdc5p levels are sensitive to mutations in key components of the anaphase-promoting complex (APC). Cdc5p-associated kinase activity is restricted to G2/M and this activity is posttranslationally regulated. These results further link the actions of the APC to the completion of mitosis and suggest possible roles for Cdc5p during progression through and completion of mitosis (Cheng, 1998).

The fission yeast Schizosaccharomyces pombe divides symmetrically using a medial F-actin- based contractile ring to produce equal-sized daughter cells. Mutants defective in two previously described genes, mid1 and pom1, frequently divide asymmetrically. Identification has been undertaken of three new temperature-sensitive mutants defective in localization of the division plane. All three mutants have mutations in the polo kinase gene, plo1, and show defects very similar to those of mid1 mutants in both the placement and organization of the medial ring. In both cases, ring formation is frequently initiated near the cell poles, indicating that Mid1p and Plo1p function in recruiting medial ring components to the cell center. Previously, it has been reported that during mitosis Mid1p becomes hyperphosphorylated and relocates from the nucleus to a medial ring. Mid1p first forms a diffuse cortical band during spindle formation and then coalesces into a ring before anaphase. Plo1p is required for Mid1p to exit the nucleus and form a ring, and Pom1p is required for proper placement of the Mid1p ring. Upon overexpression of Plo1p, Mid1p exits the nucleus prematurely and displays a reduced mobility on gels similar to the mobility of the hyperphosphorylated form observed previously in mitotic cells. Genetic and two-hybrid analyses suggest that Plo1p and Mid1p act in a common pathway distinct from that involving Pom1p. Plo1p localizes to the spindle pole bodies and spindles of mitotic cells and also to the medial ring at the time of its formation. Taken together, the data indicate that Plo1p plays a role in the positioning of division sites by regulating Mid1p. Given its previously known functions in mitosis and the timing of cytokinesis, Plo1p is thus implicated as a key molecule in the spatial and temporal coordination of cytokinesis with mitosis (Bahler, 1998).

At the onset of anaphase, a caspase-related protease (separase) destroys the link between sister chromatids by cleaving the cohesin subunit Scc1. During most of the cell cycle, separase is kept inactive by binding to an inhibitory protein called securin. Separase activation requires proteolysis of securin, which is mediated by an ubiquitin protein ligase called the anaphase-promoting complex. Cells regulate anaphase entry by delaying securin ubiquitination until all chromosomes have attached to the mitotic spindle. Though no longer regulated by this mitotic surveillance mechanism, sister separation remains tightly cell cycle regulated in yeast mutants lacking securin. The Polo/Cdc5 kinase phosphorylates serine residues adjacent to Scc1 cleavage sites and strongly enhances their cleavage. Phosphorylation of separase recognition sites may be highly conserved and regulates sister chromatid separation independently of securin (Alexandru, 2001).

A crucial question is whether substrate phosphorylation also regulates cleavage by separase in other eukaryotic organisms. Both cleavage sites within Rad21 (the Scc1 homolog in S. pombe: See Drosophila Rad21) contain serines in the P6 position as do potential cleavage sites within Scc1 homologs from the pathogenic yeast C. albicans and the trypanosome Leishmania. Phosphorylation of these residues might also enhance Rad21 cleavage because hyperphosphorylated forms of the protein are preferentially cleaved at the metaphase to anaphase transition. The equivalent residue is replaced by aspartic acid at a human Scc1 cleavage site and is substituted by threonine or glutamic acid within candidate cleavage sites of the Scc1 homologs in D. melanogaster and C. elegans, respectively. Thus, separase-induced cleavage might be enhanced by negatively charged residues in the P6 position in many, if not most, eukaryotic cells (Alexandru, 2001).

Phosphorylation of Scc1 in human cells during mitosis could regulate its cleavage at the onset of anaphase, but this cannot be attributed to phosphorylation of P6 serines. Cleavage of separase itself at the metaphase to anaphase transition in humans might be due to self-cleavage and have a role in activating the protease that is additional to removal of securin. Self-cleavage is thought to be important for the activation of most caspases. It is therefore remarkable that the anaphase-specific human separase cleavage occurs at a sequence that resembles yeast Scc1 cleavage sites and contains serine at the postulated P6 position. This raises the possibility that mitotic kinases like PLK1 could regulate Scc1 cleavage not only by phosphorylating Scc1 but also by phosphorylating self-cleavage sites within separase (Alexandru, 2001).

The facilitation of proteolysis through substrate phosphorylation is widespread in reactions mediated by ubiquitination but has few if any precedents in cases where proteolysis is initiated by an endopeptidase. Phosphorylation of certain caspase substrates has been previously implicated in their proteolysis, but in an inhibitory manner (Alexandru, 2001).

Polo associates with centromeres from prophase until late anaphase and with the spindle midzone in anaphase. Sister chromatid separation starts with the poleward segregation of centromeres and ends with the parting of sister telomeres at the spindle midzone. Polo is, therefore, present at precisely those locations where sister separation actually takes place. Most vertebrate cohesin leaves chromosomes during prophase in an APC/C and separase-independent manner. The mechanism underlying cohesin dissociation from chromosomes during prophase is not understood. This process correlates with cohesin phosphorylation and does not require Cdk1 kinase. It is therefore conceivable that in animal cells, Polo promotes both cohesin dissociation during prophase and its cleavage during anaphase (Alexandru, 2001).

Why do cells need a double control of anaphase? The transition from metaphase to anaphase is regulated by securin proteolysis from yeast to humans. It may be appropriate, therefore, to ask why eukaryotic cells possess more than one mechanism for regulating sister separation. Real time measurement of proteolysis in mammalian cells reveals that securin degradation starts about 30 min prior to the metaphase to anaphase transition, suggesting that securin destruction might not be sufficient to initiate sister chromatid separation. Multiple mechanisms might help to ensure that the sister separation process either is fully inhibited or goes to completion. They also provide greater opportunities for the surveillance mechanisms to regulate the anaphase onset. DNA damage in yeast not only blocks securin proteolysis, but also causes Polo modification. The former depends on the Chk1 protein kinase whereas the latter depends on the Rad53/Chk2 kinase (Drosophila homolog: loki). The delay in anaphase onset induced by DNA damage in chk1 pds1 mutants depends on Rad53. Moreover, PLK1 activity is inhibited by DNA damage in human cells. This suggests Rad53/Chk2 might delay sister separation in response to DNA damage by inhibiting Polo (Alexandru, 2001).

Polo kinases have been implicated in many aspects of mitosis, in promoting activation of Cdk1 via its Cdc25 phosphatase, in regulating centrosome function, and in promoting cytokinesis. Polo also has a crucial role in promoting sister chromatid separation. It phosphorylates Scc1 cleavage sites and enhances their affinity for separase, thereby promoting Scc1 cleavage and sister chromatid separation. It is possible that PLK overexpression in certain human tumors could contribute to their unstable karyotype by deregulating the sister separation process (Alexandru, 2001).

The fission yeast plo1+ gene encodes a polo-like kinase, a member of a conserved family of kinases that play multiple roles during the cell cycle. It has been shown that Plo1 kinase physically interacts with the anaphase-promoting complex (APC)/cyclosome through the noncatalytic domain of Plo1 and the tetratricopeptide repeat domain of the subunit, Cut23. A cut23 mutation, which specifically disrupts the interaction with Plo1, results in a metaphase arrest. This arrest can be rescued by high expression of Plo1 kinase. It is suggested that this physical interaction is crucial for mitotic progression by targeting polo kinase activity toward the APC (May, 2002).

In eukaryotes, entry into mitosis is induced by cyclin B-bound Cdk1, which is held in check by the protein kinase, Wee1. In budding yeast, Swe1 (Wee1 ortholog) is targeted to the bud neck through Hsl1 (Nim1-related kinase) and its adaptor Hsl7, and is hyperphosphorylated prior to ubiquitin-mediated degradation. Hsl1 and Hsl7 are required for proper localization of Cdc5 (Polo-like kinase homolog) to the bud neck and Cdc5-dependent Swe1 phosphorylation. Mitotic cyclin (Clb2)-bound Cdc28 (Cdk1 homolog) directly phosphorylates Swe1 and this modification serves as a priming step to promote subsequent Cdc5-dependent Swe1 hyperphosphorylation and degradation. Clb2-Cdc28 also facilitated Cdc5 localization to the bud neck through the enhanced interaction between the Clb2-Cdc28-phosphorylated Swe1 and the polo-box domain of Cdc5. It is proposed that the concerted action of Cdc28/Cdk1 and Cdc5/Polo on their common substrates is an evolutionarily conserved mechanism that is crucial for effectively triggering mitotic entry and other critical mitotic events (Asano, 2005).

This study delineates how Swe1 regulation is orchestrated by multiple components as cells progress through the cell cycle. Cla4-dependent septin filament formation early in the cell cycle permits assembly of a platform consisting of Hsl1 (Nim1-related kinase) and its adaptor Hsl7, a critical step that is required for the recruitment of Clb2-Cdc28-phosphorylated Swe1 and Cdc5 later in the cell cycle. Phosphorylated Swe1 further promotes Cdc5 localization to the platform by providing a docking site for the polo-box domain of Cdc5. The data show that both the Hsl1-Hsl7 platform and the primed Swe1 are two crucial elements for Cdc5-dependent Swe1 hyperphosphorylation and subsequent degradation at the bud neck. This coordinated, multistep, Swe1 regulation clearly provides a means to monitor the completion of earlier cell cycle events and to effectively bring about Swe1 destruction at the time of mitotic entry. Once unleashed from the Swe1-imposed G2 delay, Clb-Cdc28 can induce mitotic entry unimpeded (Asano, 2005).

In budding yeast, exit from the pachytene stage of meiosis requires the mid-meiosis transcription factor Ndt80, which promotes expression of ~200 genes. Ndt80 is required for meiotic function of polo-like kinase (PLK, Cdc5) and cyclin-dependent kinase (CDK), two cell cycle kinases implicated in pachytene exit. This study shows that ongoing CDK activity is dispensable for two events that accompany exit from pachytene: crossover formation and synaptonemal complex breakdown. In contrast, CDC5 expression in ndt80δ mutants efficiently promotes both events. Thus, Cdc5 is the only member of the Ndt80 transcriptome required for this critical step in meiotic progression (Sourirajan, 2008).

In eukaryotes, cytokinesis generally involves an actomyosin ring, the contraction of which promotes daughter cell segregation. Assembly of the contractile ring is tightly controlled in space and time. In the fission yeast, contractile ring components are first organized by the anillin-like protein Mid1 into medial cortical nodes. These nodes then coalesce laterally into a functional contractile ring. Although Mid1 is present at the medial cortex throughout G2, recruitment of contractile ring components to nodes starts only at mitotic onset, indicating that this event is cell-cycle regulated. Polo kinases are key temporal coordinators of mitosis and cytokinesis, and the Polo-like kinase Plo1 is known to activate Mid1 nuclear export at mitotic onset, coupling division plane specification to nuclear positio. This study provide evidence that yeasts Plo1 also triggers the recruitment of contractile ring components into medial cortical nodes. Plo1 binds at least two independent sites on Mid1, including a consensus site phosphorylated by Cdc2. Plo1 phosphorylates several residues within the first 100 amino acids of Mid1, which directly interact with the IQGAP Rng2, and influences the timing of myosin II recruitment. Plo1 thereby facilitates contractile ring assembly at mitotic onset (Almonacid, 2011).

APC/C-Cdh1-mediated degradation of the Polo kinase Cdc5 promotes the return of Cdc14 into the nucleolus

In the budding yeast Saccharomyces cerevisiae, the protein phosphatase Cdc14 triggers exit from mitosis by promoting the inactivation of cyclin-dependent kinases (CDKs). Cdc14's activity is controlled by Cfi1/Net1, which holds and inhibits the phosphatase in the nucleolus from G1 until metaphase. During anaphase, two regulatory networks, the Cdc14 Early Anaphase Release (FEAR) network and the Mitotic Exit Network (MEN), promote the dissociation of Cdc14 from its inhibitor, allowing the phosphatase to reach its targets throughout the cell. The molecular circuits that trigger the return of Cdc14 into the nucleolus after the completion of exit from mitosis are not known. This study shows that activation of a ubiquitin ligase known as the Anaphase-Promoting Complex or Cyclosome (APC/C) bound to the specificity factor Cdh1 triggers the degradation of the Polo kinase Cdc5, a key factor in releasing Cdc14 from its inhibitor in the nucleolus (Visintin, 2008).

Progression through mitosis is governed by ubiquitin-dependent protein degradation. Chromosome segregation at the metaphase-anaphase transition is initiated by the targeting of Securin (Pds1 in budding yeast) for degradation by a ubiquitin ligase termed the Anaphase-Promoting Complex or Cyclosome (APC/C) associated with its specificity factor Cdc20. This process liberates Separase (Esp1 in budding yeast), a protease that severs the linkages that hold sister chromatids together. The anaphase-G1 transition, also known as exit from mitosis, requires APC/C activity as well. In this cell cycle stage, APC/C-Cdc20 and APC/C-Cdh1 trigger the degradation of mitotic cyclins (Clb cyclins in yeast), thereby promoting cyclin-dependent kinase (CDK) inactivation, which in turn triggers exit from mitosis (Visintin, 2008 and references therein).

Clb-CDK inactivation is not only brought about by cyclin degradation but also by binding of the CDK inhibitor Sic1 to the Clb-CDK complex. Both events, cyclin degradation and Sic1 accumulation, are triggered by the protein phosphatase Cdc14 dephosphorylating CDK substrates. Cdc14's activity is controlled by Cfi1/Net1, which sequesters and inhibits the protein phosphatase in the nucleolus from G1 until metaphase. At the onset of anaphase, Cdc14 dissociates from its inhibitor through the action of the Cdc14 Early Anaphase Release (FEAR) network (for review, see Stegmeier and Amon 2004). During later stages of anaphase, another signaling network, the Mitotic Exit Network (MEN), further promotes Cdc14 dissociation from its inhibitor (for review, see Stegmeier, 2004; Visintin, 2008 and references therein).

The FEAR network initiates the release of Cdc14 from the nucleolus at the metaphase-anaphase transition. The network includes the protease Esp1, the Esp1 substrate and binding protein Slk19, and the nucleolar proteins Spo12 and Fob1, as well as the Polo kinase Cdc5. The FEAR network promotes the phosphorylation of Cfi1/Net1 by Clb-CDKs and appears to bring about this anaphase-specific phosphorylation by down-regulating the protein phosphatase 2A (Visintin, 2008).

The MEN is essential for exit from mitosis. In the absence of MEN function, Cdc14 is transiently released from the nucleolus during early anaphase by the FEAR network but returns into the nucleolus during late stages of anaphase, and cells fail to exit from mitosis (for review, see Stegmeier, 2004). The signaling pathway resembles a Ras-like signaling cascade and is composed of a GTPase Tem1, a GTPase-activating protein complex Bub2-Bfa1, a putative Guanine Nucleotide Exchange Factor (GEF) Lte1, and two protein kinases Cdc15 and Dbf2, as well as the Dbf2-associated factor Mob1. The scaffold protein Nud1 anchors the GTPase and its downstream protein kinases to the outer plaque of the spindle pole body destined to move into the daughter cell. There, the pathway is thought to be activated when the SPB moves into the bud during anaphase (Visintin, 2008).

The Polo kinase Cdc5 is a component of the FEAR network and a key regulator of the MEN. In cells lacking the protein kinase, Cdc14 is not released from the nucleolus at all. Because Cdc5 is required for the release of Cdc14 from the nucleolus during early anaphase, the protein kinase has been classified as a FEAR network component. Cdc5 also contributes to Cdc14 release from the nucleolus by phosphorylating the Bub2-Bfa1 complex, thereby inhibiting the GAP. Owing to Cdc5’s role in both the FEAR network and the MEN, it is not surprising that overproduction of the protein kinase can promote Cdc14 release from the nucleolus in cell cycle stages other than anaphase (Visintin, 2008).

The mechanisms that bring about the release of Cdc14 from the nucleolus have been largely elucidated. Cdc14 released by the FEAR network stimulates MEN activity, and Cdc14 released by the MEN further activates the MEN. This feed-forward mechanism coupled to a positive feedback loop results in the rapid release of Cdc14 from the nucleolus. How this activation loop is disrupted once exit from mitosis has been completed to cause Cdc14 to return into the nucleolus is not understood. This study shows that Cdc14 itself is responsible for its inactivation. By activating the APC/C-Cdh1, Cdc14 induces the degradation of Cdc5, thereby silencing both the FEAR and the MEN (Visintin, 2008).

Cdc15 integrates Tem1 GTPase-mediated spatial signals with Polo kinase-mediated temporal cues to activate mitotic exit

In budding yeast, a Ras-like GTPase signaling cascade known as the mitotic exit network (MEN) promotes exit from mitosis. To ensure the accurate execution of mitosis, MEN activity is coordinated with other cellular events and restricted to anaphase. The MEN GTPase Tem1 has been assumed to be the central switch in MEN regulation. This study shows that during an unperturbed cell cycle, restricting MEN activity to anaphase can occur in a Tem1 GTPase-independent manner. It was found that the anaphase-specific activation of the MEN in the absence of Tem1 is controlled by the Polo kinase Cdc5. It was further shown that both Tem1 and Cdc5 are required to recruit the MEN kinase Cdc15 to spindle pole bodies, which is both necessary and sufficient to induce MEN signaling. Thus, Cdc15 functions as a coincidence detector of two essential cell cycle oscillators: the Polo kinase Cdc5 synthesis/degradation cycle and the Tem1 G-protein cycle. The Cdc15-dependent integration of these temporal (Cdc5 and Tem1 activity) and spatial (Tem1 activity) signals ensures that exit from mitosis occurs only after proper genome partitioning (Rock, 2011).

Polo kinase in Xenopus

Polo-like kinases (Plks), named after the Drosophila gene product polo, have been implicated in the regulation of multiple aspects of mitotic progression, including the activation of the Cdc25 phosphatase, bipolar spindle formation and cytokinesis. Genetic analyses performed in yeast and Drosophila suggest a function for Plks at late stages of mitosis, but biochemical data to support such a function in vertebrate organisms have been lacking. Xenopus egg extracts were used for exploring the function of Plx1, a Xenopus Plk, during the cell cycle transition from M phase to interphase (I phase). The addition of a catalytically inactive Plx1 mutant to M phase-arrested egg extracts blocks their Ca2+-induced release into interphase. Concomitantly, the proteolytic destruction of several targets of the anaphase-promoting complex and the inactivation of the Cdc2 protein kinase (Cdk1) are prevented. The M to I phase transition can be abolished by immunodepletion of Plx1, but is restored upon the addition of recombinant Plx1. According to the model presented in this paper, the cMos/MAP kinase pathway promotes the activation of an inhibitor of APC/cyclosome function, The cyclosome functions in the degradation of mitotic targets. In response to Ca2+, APC is relieved from the action of this inhibitor, allowing it to degrade its substrates. Subsequently, c-Mos is degraded and the MAP kinase pathway is turned off. Plx1 is proposed to (1) mediate the Ca2+ dependent inactivation of the hypothetical APC inhibitor, (2) counteract the effect of this inhibitor, (3) maintain the activity of APC, or (4) contribute to the functional coupling between APC and co-factors of the Fzy family. These mechanisms are not mutually exclusive, and other modes of actions cannot be excluded. In particular, it would be premature to exclude regulation of the degradation machinery at the level of substrate presentation to the proteosome. These results demonstrate that the exit of egg extracts from M phase arrest requires active Plx1, and they strongly suggest an important role for Plx1 in the activation of the proteolytic machinery that controls the exit from mitosis (Descombes, 1998).

Entry into mitosis depends on activation of the dual-specificity phosphatase Cdc25C (Drosophila homolog: String), which dephosphorylates and activates the cyclin B-Cdc2 complex. Previous work has shown that the Xenopus polo-like kinase Plx1 can phosphorylate and activate Cdc25C in vitro. Plx1 is activated in vivo during oocyte maturation with the same kinetics as Cdc25C. Microinjection of wild-type Plx1 into Xenopus oocytes accelerates the rate of activation of Cdc25C and cyclin B-Cdc2. Conversely, microinjection of either an antibody against Plx1 or kinase-dead Plx1 significantly inhibits the activation of Cdc25C and cyclin B-Cdc2. This effect can be reversed by injection of active Cdc25C, indicating that Plx1 is upstream of Cdc25C. However, injection of Cdc25C, which directly activates cyclin B-Cdc2, also causes activation of Plx1, suggesting that a positive feedback loop exists in the Plx1 activation pathway. Other experiments show that injection of Plx1 antibody into early embryos, which do not require Cdc25C for the activation of cyclin B-Cdc2, results in an arrest of cleavage that is associated with monopolar spindles. These results demonstrate that in Xenopus laevis, Plx1 plays important roles both in the activation of Cdc25C at the initiation of mitosis and in spindle assembly at late stages of mitosis (Qian, 1998a).

The results reported here establish that the activity of Plx1 is cell cycle regulated and involved in the G2/M transition during Xenopus oocyte maturation. Thus Plx1 plays two roles in mitosis, regulating entry into mitosis and exit from mitosis. The inhibitory effects of Plx1 antibody injection reported here, as well as the effects of wild-type and kinase-dead Plx1 on progesterone-induced Cdc25C activation, demonstrate that Plx1 functions in the Cdc25C activation pathway. Several lines of evidence support the hypothesis that these effects occur due to direct phosphorylation of Cdc25C by Plx1. (1) Cdc25C is a substrate for Plx1 in vitro. (2) Activation of Plx1 correlates exactly with activation of Cdc25C during both meiosis I and meiosis II and Plx1 antibody or kinase-dead Plx1 significantly delays the phosphorylation and activation of Cdc25C. (3) Cdc25C itself can overcome the inhibitory effect of Plx1 antibody. These results support the hypothesis that Plx1 could be a trigger kinase, also termed kinase X, that initiates the positive feedback loop between Cdc25C and cyclin B-Cdc2. The existence of a trigger kinase had been predicted from studies in which initial activation of Cdc25C occurred prior to or in the absence of cyclin B-Cdc2. The results shown here indicate that Plx1 may be a trigger kinase, but they do not exclude the possibility that other kinases also function as trigger kinases. Since injection of Plx1 antibody almost completely inhibits Plx1 activity yet does not completely block Cdc25C activation, it is likely that, in addition to Plx1 and cyclin B-Cdc2, other protein kinases also contribute to Cdc25C activation (Qian, 1998b).

The Xenopus polo-like kinase 1 (Plx1) is essential during mitosis for the activation of Cdc25C, for spindle assembly, and for cyclin B degradation. Polo-like kinases from various organisms are activated by phosphorylation by an unidentified protein kinase. A protein kinase, polo-like kinase kinase 1 or xPlkk1, that phosphorylates and activates Plx1 in vitro was purified to near homogeneity and cloned. Phosphopeptide mapping of Plx1 phosphorylated in vitro by recombinant xPlkk1 or in progesterone-treated oocytes indicates that xPlkk1 may activate Plx1 in vivo. The xPlkk1 protein itself was also activated by phosphorylation on serine and threonine residues, and the kinetics of activation of xPlkk1 in vivo closely parallel the activation of Plx1. Moreover, microinjection of xPlkk1 into Xenopus oocytes accelerates the timing of activation of Plx1 and the transition from G2 to M phase of the cell cycle. These results define a protein kinase cascade that regulates several events of mitosis (Qian, 1998b).

During mitosis the Xenopus polo-like kinase 1 (Plx1) plays key roles in the activation of Cdc25C, in spindle assembly, and in cyclin B degradation. Previous work has shown that the activation of Plx1 requires phosphorylation on serine and threonine residues. In the present work, it is demonstrated that replacement of Ser-128 or Thr-201 with a negatively charged aspartic acid residue (S128D or T201D) elevates Plx1 activity severalfold and that replacement of both Ser-128 and Thr-201 with Asp residues (S128D/T201D) increases Plx1 activity approximately 40-fold. Microinjection of mRNA encoding S128D/T201D Plx1 into Xenopus oocytes directly induces the activation of both Cdc25C and cyclin B-Cdc2. In egg extracts T201D Plx1 delays the timing of deactivation of Cdc25C during exit from M phase and accelerates Cdc25C activation during entry into M phase. This supports the concept that Plx1 is a 'trigger' kinase for the activation of Cdc25C during the G(2)/M transition. In addition, during anaphase T201D Plx1 reduces preferentially the degradation of cyclin B2 and delayed the reduction in Cdc2 histone H1 kinase activity. In early embryos S128D/T201D Plx1 resulted in arrest of cleavage and formation of multiple interphase nuclei. Consistent with these results, Plx1 has been found to be localized on centrosomes at prophase, on spindles at metaphase, and at the midbody during cytokinesis. These results demonstrate that in Xenopus laevis activation of Plx1 is sufficient for the activation of Cdc25C at the initiation of mitosis and that inactivation of Plx1 is required for complete degradation of cyclin B2 after anaphase and completion of cytokinesis (Qian, 1999).

In the Xenopus oocyte system mitogen treatment triggers the G2/M transition by transiently inhibiting the cAMP-dependent protein kinase (PKA); subsequently, other signal transduction pathways are activated, including the mitogen-activated protein kinase (MAPK) and polo-like kinase pathways. To study the interactions between these pathways, a cell-free oocyte extract was used that carries out the signaling events of oocyte maturation after addition of PKI, the heat-stable inhibitor of PKA. PKI stimulates the synthesis of Mos and activation of both the MAPK pathway and the Plx1/Cdc25C/cyclin B-Cdc2 pathway. Activation of the MAPK pathway alone by glutathione S-transferase (GST)-Mos does not lead to activation of Plx1 or cyclin B-Cdc2. Inhibition of the MAPK pathway in the extract by the MEK1 inhibitor U0126 delays, but does not prevent, activation of the Plx1 pathway, and inhibition of Mos synthesis by cycloheximide has a similar effect, suggesting that MAPK activation is the only relevant function of Mos. Immunodepletion of Plx1 completely inhibits activation of Cdc25C and cyclin B-Cdc2 by PKI, indicating that Plx1 is necessary for Cdc25C activation. In extracts containing fully activated Plx1 and Cdc25C, inhibition of cyclin B-Cdc2 by p21Cip1 has no significant effect on either the phosphorylation of Cdc25C or the activity of Plx1. These results demonstrate that maintenance of Plx1 and Cdc25C activity during mitosis does not require cyclin B-Cdc2 activity. It is evident that Plx1 is an essential trigger kinase for Cdc25C activation at the G2/M transition. No other kinase appears to be able to substitute for this function of Plx1 in G2, although, once activated, cyclin B-Cdc2 is capable of activating Cdc25C in a positive feedback loop (Qian, 2001).

The activation of Cdc2 kinase induces the entry into M-phase of all eukaryotic cells. A cell-free system prepared from prophase-arrested Xenopus oocytes has been developed to analyze the mechanism initiating the all-or-none activation of Cdc2 kinase. Inhibition of phosphatase 2A, the major okadaic acid-sensitive Ser/Thr phosphatase in these extracts, provokes Cdc2 kinase amplification and concomitant hyperphosphorylation of Cdc25 phosphatase, with a lag of about 1 h. Polo-like kinase (Plx1 kinase) is activated slightly after Cdc2. All these events are totally inhibited by the cdk inhibitor p21(Cip1), demonstrating that Plx1 kinase activation depends on Cdc2 kinase activity. Addition of a threshold level of recombinant Cdc25 induces a linear activation of Cdc2 and Plx1 kinases and a partial phosphorylation of Cdc25. It is proposed that the Cdc2 positive feedback loop involves two successive phosphorylation steps of Cdc25 phosphatase: the first one is catalyzed by Cdc2 kinase and/or Plx1 kinase but it does not modify Cdc25 enzymatic activity; the second one requires a new kinase counteracted by phosphatase 2A. Under the conditions of this assay, Cdc2 amplification and MAP kinase activation are two independent events (Kara, 1998).

A investigation was carried out to see whether Plx1, a kinase recently shown to phosphorylate cdc25c in vitro, is required for activation of cdc25c at the G2/M-phase transition of the cell cycle in Xenopus. Using immunodepletion or the mere addition of an antibody against the C terminus of Plx1, which suppresses its activation (not its activity) at G2/M, it has been shown that Plx1 activity is required for activation of cyclin B-cdc2 kinase in both interphase egg extracts receiving recombinant cyclin B, and cycling extracts that spontaneously oscillate between interphase and mitosis. Furthermore, a positive feedback loop allows cyclin B-cdc2 kinase to activate Plx1 at the G2/M-phase transition. In contrast, activation of cyclin A-cdc2 kinase does not require Plx1 activity, and cyclin A-cdc2 kinase fails to activate Plx1 and its consequence, cdc25c activation in cycling extracts (Abrieu, 1998).

Polo-like kinases (Plks) control multiple important events during M phase progression, but little is known about their activation during the cell cycle. The activities of both mammalian Plk1 and Xenopus Plx1 peak during M phase, and this activation has been attributed to phosphorylation. However, no phosphorylation sites have previously been identified in any member of the Plk family. Tryptic phosphopeptide mapping has been combined with mass spectrometry to identify four major phosphorylation sites in Xenopus Plx1. All four sites appear to be phosphorylated in a cell cycle-dependent manner. Phosphorylations at two sites (Ser-260 and Ser-326) most likely represent autophosphorylation events, whereas two other sites (Thr-201 and Ser-340) are targeted by upstream kinases. Several recombinant kinases were tested for their ability to phosphorylate Plx1 in vitro. Whereas xPlkk1 phosphorylates primarily Thr-10, Thr-201 is readily phosphorylated by protein kinase A, and Cdk1/cyclin B was identified as a likely kinase acting on Ser-340. Phosphorylation of Ser-340 was shown to be responsible for the retarded electrophoretic mobility of Plx1 during M phase, and phosphorylation of Thr-201 was identified as a major activating event (Kelm, 2002).

During oogenesis, the Xenopus oocyte is blocked in prophase of meiosis I. It becomes competent to resume meiosis in response to progesterone at the end of its growing period (stage VI of oogenesis). Stage IV oocytes contain a store of inactive pre-MPF (Tyr15-phosphorylated Cdc2 bound to cyclin B2); the Cdc25 phosphatase that catalyzes Tyr15 dephosphorylation of Cdc2 is also present. However, the positive feedback loop that allows MPF autoamplification is not functional at this stage of oocyte growth. When cyclin B is overexpressed in stage IV oocytes, MPF autoamplification does not occur and the newly formed cyclin B-Cdc2 complexes are inactivated by Tyr15 phosphorylation, indicating that Myt1 kinase remains active and that Cdc25 is prevented from being activated. Plx1 kinase (or polo-like kinase), which is required for Cdc25 activation and MPF autoamplification in full grown oocytes is not expressed at the protein level in small stage IV oocytes. In order to determine if Plx1 could be the missing regulator that prevents MPF autoamplification, polo kinase was overexpressed in stage IV oocytes. Under these conditions, the MPF-positive feedback loop was restored. Moreover, acquisition of autoamplification competence does not require the Mos/MAPK pathway (Karaiskou, 2004).

Thus, Plx1 protein, crucial for the function of the auto-amplification feedback loop in full-grown oocytes is not expressed in small oocytes. Both Cdc25 and Myt1 are direct substrates of Plk1 during M phase. The results indicate that overexpression of Plk1 in stage IV oocytes authorizes cyclin B1 to form active complexes with Cdc2. This observation shows that in oocytes, Plk1 participates in the formation of an active MPF trigger by downregulating Myt1. Moreover, it indicates that progesterone unresponsiveness of small oocytes depends on a stable activity of Myt1 kinase, because of Plx1 absence. Although Plk1 expression prevents Tyr15 phosphorylation of Cdc2 after cyclin B overexpression, Cdc25 is not fully activated. This shows that full activation of Cdc25 requires a further regulatory mechanism. Indeed, Xenopus Cdc25 can be negatively regulated through Ser287 phosphorylation by several kinases, including Chk1 and PKA. Cdc25C, which is phosphorylated on Ser287 in Xenopus prophase oocytes, is dephosphorylated by type 1 phosphatase (PP1) at GVBD. Since the PP1 inhibitor I prevents meiotic maturation, PP1 could participate in the regulation of the MPF autoamplification loop by catalyzing the removal of the inhibitory Ser287 phosphate, and could therefore be involved in the regulation of Cdc25 during oogenesis (Karaiskou, 2004).

In competent oocytes, Plx1 action on Cdc25 is antagonized by an okadaic acid-sensitive phosphatase, involving PP2A activity. This explains why the auto-amplification mechanism can be artificially activated by okadaic acid. However, okadaic acid is unable to promote Cdc2 activation in small incompetent oocytes, showing that the loop implying Cdc2, Cdc25, Plx1 and PP2A is not functional in growing oocytes. The most probable explanation for this defect is the absence of Plx1 in stage IV oocytes. Indeed, it has been shown, both in vivo and in vitro, that expression of Plk1 is sufficient to restore the activation of MPF in response to okadaic acid in incompetent oocytes. Plx1 is therefore the missing factor explaining why the auto-amplification of MPF is defective in small oocytes (Karaiskou, 2004).

Altogether, these experiments show that the incompetence of small oocytes to resume meiosis is ensured by the absence of Plx1 resulting in a double negative control on MPF activation. (1) The formation of active complexes between Cdc2 and newly synthesized cyclins is prevented by a sustained activity of Myt1 that escapes downregulation by Plx1. (2) Cdc25 activation that is normally achieved through a feedback loop involving Plx1 is also prevented. Further investigation will be necessary to discover (1) how Plx1 expression is controlled by cell size at the end of oogenesis; (2) how PP2A controls Cdc25 activity in small oocytes, and (3) how the initial steps of the progesterone transduction pathway connect to MPF regulators, allowing the female germ cell to resume meiosis when oocyte growth is completed (Karaiskou, 2004).

The checkpoint mediator protein Claspin is essential for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing aphidicolin-induced DNA replication blocks. During this checkpoint response, Claspin becomes phosphorylated on threonine 906 (T906), which creates a docking site for Plx1, the Xenopus Polo-like kinase. This interaction promotes the phosphorylation of Claspin on a nearby serine (S934) by Plx1. After a prolonged interphase arrest, aphidicolin-treated egg extracts typically undergo adaptation and enter into mitosis despite the presence of incompletely replicated DNA. In this process, Claspin dissociates from chromatin, and Chk1 undergoes inactivation. By contrast, aphidicolin-treated extracts containing mutants of Claspin with alanine substitutions at positions 906 or 934 (T906A or S934A) are unable to undergo adaptation. Under such adaptation-defective conditions, Claspin accumulates on chromatin at high levels, and Chk1 does not decrease in activity. These results indicate that the Plx1-dependent inactivation of Claspin results in termination of a DNA replication checkpoint response (Yoo, 2004).

During the meiotic cell cycle in Xenopus oocytes, p90rsk, the downstream kinase of the Mos-MAPK pathway, interacts with and inhibits the Cdc2 inhibitory kinase Myt1/Wee1. However, p90rsk is inactivated after fertilization due to the degradation of Mos. The Polo-like kinase Plx1, instead of p90rsk, interacts with and inhibits Myt1 after fertilization of Xenopus eggs. At the M phase of the embryonic cell cycle, Cdc2 phosphorylates Myt1 on Thr478 and thereby creates a docking site for Plx1. Plx1 can phosphorylate Myt1 and inhibit its kinase activity both in vitro and in vivo. The interaction between Myt1 and Plx1 is required, at least in part, for normal embryonic cell divisions. Finally, and interestingly, Myt1 is phosphorylated on Thr478 even during the meiotic cell cycle, but its interaction with Plx1 is largely inhibited by p90rsk-mediated phosphorylation. These results indicate a switchover in the Myt1 inhibition mechanism at fertilization of Xenopus eggs, and strongly suggest that Plx1 acts as a direct inhibitory kinase of Myt1 in the mitotic cell cycles in Xenopus (Inoue, 2005).

Although the interruption of the Plx1-Myt1 interaction by T478A mutation largely impaired the Plx1 phosphorylation and inhibition of Myt1, it seemed to have a relatively small (although significant) effect on early embryonic cell divisions. This is presumably due, however, to the dramatic increase in levels of the Myt1/Wee1-antagonizing Cdc25A phosphatase during this period. In somatic cells, however, the level of Cdc25A is considerably lower; therefore, the interruption of the Plx1-Myt1 interaction in somatic cells might have a significantly larger effect on cell divisions. In human somatic cells, Myt1 localizes to the endoplasmic reticulum and Golgi complex. Therefore, the binding of Plk1/Plx1 or the Myt1 substrate Cdc2-cyclin B to Myt1 could also be responsible, at least in part, for their known localization and function at the Golgi complex. Indeed, in Drosophila embryos, Myt1 has been implicated in Golgi fragmentation (Cornwell, 2002), a mitotic event involving both Cdc2 and Plk1 (Lin, 2000; Sutterlin, 2001; Inoue, 2005 and references therein).

Mammalian Polo kinase

polo: Evolutionary Homologs part 2/2

polo: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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