Brander Cancer Institute, Department of Medicine, New York Medical College, Valhalla, New York, NY 10595, USA

Correspondence to: W Dai, E-mail: wei_dai@nymc.edu

Oncogene (2002) 21, 6195-6200. doi:10.1038/sj.onc.1205710

Polo; Polo-like kinases; centrosome; spindle pole body; microtubule organization center

Polo and Polo like kinases

Protein kinases play a pivotal role in the regulation of the centrosome cycle. Extensive research in the past decade or so has led to the identification of several families of protein kinases that are important to the regulation of centrosome functions. These kinase families include cyclin-dependent kinases (Cdks) (Winey, 1999; Okuda et al., 2000), Aurora kinases (Descamps and Prigent, 2001; Hannak et al., 2001), Mps1 (monopolar spindle) gene product (Hinchcliffe and Sluder, 2001; Fisk and Winey, 2001), NIMA family of kinases (Mayor et al., 1999; Fry et al., 2000), and the Polo family of protein kinases (Glover et al., 1996; Wianny et al., 1998; Glover et al., 1996). Protein kinases of the Polo family, conserved through evolution, have been described in major eukaryotic models such as Sacchromyces cerevisiae (Kitada et al., 1993), Schizosacchromyces pombe (Ohkura et al., 1995), Caenorhabditis elegans (Ouyang et al., 1999; Chase et al., 2000), Drosophila melanogaster (Sunkel and Glover, 1988; Llamazares et al., 1991), Xenopus laevis (Kumagai and Dunphy, 1996; Duncan et al., 2001), mouse (Donohue et al., 1995; Lake and Jelinek, 1993; Simmons et al., 1992), and human (Golsteyn et al., 1994; Hamanaka et al., 1995; Li et al., 1996). The founding member of this family, Polo, was originally identified in Drosophila and was shown to be a serine-threonine kinase that is required for mitosis (Llamazares et al., 1991). Subcellular localization of Polo protein and its kinase activity is cell cycle-dependent. It is predominantly cytoplasmic during interphase and is associated with condensed chromosome during mitosis (Llamazares et al., 1991) at which time its kinase activity is also at peak (Fenton and Glover, 1993). In addition to the conserved kinase domain, Polo family members all share a unique amino acid sequence termed Polo box in the carboxy-terminal half of these proteins (Li et al., 1996; Nigg, 1998). Mutations in the Polo box that do not affect the kinase activity are capable of abolishing its biological activity in vivo (Lee et al., 1998), indicating the importance of this conserved motif.

CDC5 and Plo1 are structural as well as the functional homologues of Polo in budding yeast and fission yeast, respectively (Nigg, 1998). Both genes are essential, and loss of their function leads to mitotic arrest (Kitada et al., 1993; Ohkura et al., 1995). Vertebrate cells contain three Polo-like kinases (Plk1, Plk2 and Plk3) that exhibit marked sequence homology to Polo (Table 1). The C. elegans genome also contains three Polo structural homologues (Ouyang et al., 1999; Chase et al., 2000). Interestingly, to date no additional gene products structurally homologous to Polo have been identified in Drosophila. Given the lower evolutionary hierarchy of C. elegans compared to Drosophila, it is reasonable to predict that the fruit fly may contain additional Polo homologues. There is circumstantial evidence supporting this notion. Fruit flies homozygous for the original polo¹ mutant allele are capable of developing to adulthood (Sunkel and Glover, 1988). Interpretations are that the mutation is weakly hypomorphic and that polo deficiency is partially rescued by the wild-type protein from an heterozygous mother (Donaldson et al., 2001b). An alternative explanation could be that there exists yet to be identified polo member(s) that may complement, to certain degree, polo¹ mutation.

In mammalian cells, Plk1 (alternatively named Plk (Hamanaka et al., 1995)) is the best studied Polo member to date (Donaldson et al., 2001a). Extensive investigations have uncovered a variety of cell cycle events that are regulated by Plk1, including the onset of mitosis (Toyoshima-Morimoto et al., 2001; Roshak et al., 2000), DNA-damage checkpoint activation (Smits et al., 2000), regulation of the anaphase promoting complex (Golan et al., 2002; Kotani et al., 1998), phosphorylation of the proteasome (Feng et al., 2001), as well as centrosome duplication and maturation (Golsteyn et al., 1995).

Mammalian Plk3 (alternatively named Prk (Li et al., 1996) and Fnk (Donohue et al., 1995)), was originally shown to be an immediate early gene product. The amount of Plk3 mRNA (but not Plk1 mRNA) is rapidly and transiently increased in response to mitogenic stimulation; in cycling cells, Plk3 kinase activity, but not its protein level, fluctuates significantly, and its kinase activity appears to peak during late S and G2 phase (Li et al., 1996). Both Plk3 and Plk1 are capable of rescuing CDC5 temperature sensitive (ts) mutants at restrictive temperature, indicating functional conservation of these kinases throughout evolution (Ouyang et al., 1997; Lee and Erikson, 1997). At present, it remains unclear which mammalian Polo-like kinase represents the true ortholog of Cdc5. In addition to its role in regulation of mitosis, Plk3 is activated during DNA damage checkpoint activation (Xie et al., 2001b) and severe oxidative stress (Xie et al., 2001a).

Plk2 (alternatively named Snk (Simmons et al., 1992)) was originally identified as the product of an immediate early gene, and it is the least well characterized of the mammalian Polo homologs. Recent studies have shown that Plk2 and Plk3 each associate with CIB, a calmodulin-related protein (Kauselmann et al., 1999). Both Plk2 and Plk3 have been implicated in long-term synaptic plasticity and thus may have important post-mitotic functions.

Cdc5, Plo1 and spindle pole bodies

In yeast, the functional counterparts to centrosomes are the spindle pole bodies (SPBs). CDC5, MOB1 and MPS1 are some of the essential genes in S cerevisiae that are involved in regulating SPBs. Mob1p is known to physically interact with Mps1p (Luca and Winey, 1998), the latter being a protein kinase essential for duplication of SPBs (Schutz et al., 1997) and centrosomes (Fisk and Winey, 2001) as well as for spindle checkpoint activation (Weiss and Winey, 1996). In fact, Mob1p is a phosphorylation target of Mps1p in vitro (Luca and Winey, 1998). Mob1p undergoes dynamic subcellular relocation during mitosis. It primarily localizes to SPBs during mid-anaphase and then moves to a ring at the budding neck prior to cytokinesis; however, in the absence of CDC5, this translocation process is blocked (Luca et al., 2001), suggesting dependence of Mob1p on upstream phosphorylation events during subcellular relocation. CDC5 also functionally interacts with several other genes such as DBF2 and CDC15 whose products are localized to SPBs (Menssen et al., 2001). Dbf2p is localized to SPBs for much of the cell cycle and relocates to the bud neck during late mitosis, suggesting that it participates in cytokinesis (Frenz et al., 2000). Cdc15p localizes to SPBs in a unique pattern. Cdc15p is associated with SPB of the mother cell much of the cell cycle until late mitosis when it is also associated with SPB of the daughter cell. It appears that the binding of Cdc15p to the daughter spindle pole is essential for triggering cytokinesis (Menssen et al., 2001). Thus, Cdc5p may regulate translocation of Cdc15p to and from SPBs through phosphorylation. The most direct evidence indicating a role for CDC5 in regulating SPBs comes from the study of meiosis in the budding yeast (Schild and Byers, 1980). A ts mutant of CDC5 fails to complete meiosis I due to arrest at a stage after SPB duplication and separation at the restrictive temperature. In these mutant cells, SPBs lack the normal spindle microtubules that are characteristic of meiosis I in wild-type cells.

In fission yeast, Plo1 is required for the assembly and function of the mitotic spindle. Both loss of Plo1 function and over-expression of this gene results in formation of cells in which condensed chromosomes are associated with monopolar spindles (Ohkura et al., 1995), indicating a failure in bipolar spindle formation. Plo1p localizes to SPBs and mitotic spindles and its subcellular localization is cell cycle-dependent (Mulvihill et al., 1999). Plo1p is not associated with SPBs during interphase. However, it becomes strongly associated with SPBs during early stages of mitosis until anaphase. Association of Plo1p with SPBs appears to require the activity of mitosis-promoting factor. In certain yeast mutants, Plo1p becomes inappropriately associated with SPBs in the cell cycle. For example, in Stf1.1 mutants Plo1p interacts with SPBs during interphase bypassing the requirement for Cdc25, an activator for the mitosis-promoting factor; conversely, when septation occurs due to deregulated Spg1 pathway, Plo1p is not associated with SPBs during mitosis (Mulvihill et al., 1999).

Although no definitive SPB target(s) of Cdc5p or Plo1p has been identified, the observed changes in their subcellular localization and existing genetic studies are consistent with a role of these kinases in SPB function. It is likely that Cdc5p and Plo1p serve to coordinate spindle pole functions with other cellular events throughout the cell cycle, especially during mitosis. The identification of physiological substrates of these Polo-like kinases in SPBs would greatly facilitate the elucidation of their mode of action in cell division in the lower eukaryotes.

Polo and centrosomes

Drosophila gene Polo is required for cytokinesis as well as for organization of spindle poles. Fruit flies homozygous for the original polo¹ allele exhibit certain abnormalities in spindle poles during development (Sunkel and Glover, 1988). These defects include multiple branched spindles in syncytial polo-derived embryos, spindles with broad poles and fewer circular mitotic figures in larval neuroblasts. In strongly hypomorphic mutants (polo^9/10), a majority of cells are arrested in a metaphase-like stage (Donaldson et al., 2001b). The arrested cells lack asters at each spindle pole although they all possess bipolar spindles with robust arrays of microtubules. This may be partly due to the involvement of Polo in recruiting centrosomal components. In polo^9/10 mutants, CP190 and -tubulin, two centrosome-specific antigens, do not concentrate on centrosomes; instead they scatter throughout the mitotic spindles (Donaldson et al., 2001b). The absence (or greatly diminished levels) of these proteins in centrosomes is not due to reduced levels of these proteins in the cells (Donaldson et al., 2001b). Thus, Polo kinase activity is required for localization of these proteins to the microtubule organization center. In contrast, centrosomin and abnormal spindle protein (Asp) are present at spindle poles in these mutant cells (Donaldson et al., 2001b), indicating an independence of their subcellular localization on Polo.

Genetic studies have identified several mutants that lie in the same pathway as Polo (Gonzalez et al., 1998; do Carmo et al., 2001). Both Polo and Asp mutants share similar phenotypes and there exists a synergism between these mutants (Gonzalez et al., 1998), suggesting a common role in spindle pole function. Asp mutant cells contain bipolar spindles but with disorganized broad poles, and the distribution of -tubulin in these cells is also abnormal. Asp is a microtubule-associated protein that appears to mediate the interaction of centrosomes with spindle microtubules. Biochemical analyses show that Polo interacts with and phosphorylates Asp in vitro, resulting in an MPM-2 epitope (do Carmo et al., 2001). Furthermore, extracts of polo¹-deprived embryos are incapable of supporting microtubule aster nucleation by salt-stripped centrosomes; however, addition of phosphorylated Asp or kinase-active Polo protein can complement this defect (do Carmo et al., 2001), indicating a common role of Polo and Asp in promoting the mitotic organizing activity of centrosomes. Given that subcellular localization of Asp to centrosomes is independent of Polo (Donaldson et al., 2001b) it is reasonable to suggest that phosphorylation of Asp by Polo mainly affects the nucleating activity of this spindle protein.

Similar to the situation in yeast, Polo is also required for spindle pole function during meiosis because chromosome nondisjunction, as well as failure to undergo cytokinesis, is observed during meiosis in polo¹ mutants (Riparbelli et al., 2000). A close examination reveals that there are several major defects that are related to the centrosome cycle or centrosomal function. Although no appreciable defects in spindle poles during meiosis I are observed, the spindles of meiosis II are abnormal. The growth of sperm aster microtubules is also significantly compromised. In addition, sperm centrosome duplication is uncoupled from female meiosis in polo¹ mutants.

The stability of Polo also contributes to centrosomal function (de Carcer et al., 2001). Heat shock protein, Hsp90, is required for proper centrosomal function in Drosophila as well as in vertebrate cells (Lange et al., 2000). Polo was identified as one of those proteins that are stabilized by this chaperone (de Carcer et al., 2001). Hsp90 and Polo are found to be part of a protein complex. Inhibition of Hsp90 leads to the disappearance of Polo protein and thus inactivation of Polo kinase activity. It also renders protein extracts prepared from these cells incapable of complementing salt-stripped centrosomal preparations to nucleate microtubules. Consistently, supplementation of the kinase active recombinant Polo to the in vitro nucleation assays restores the ability of those centrosomes to nucleate microtubules. Hence, Polo appears to affect not only the organization (assembling) of centrosomes by recruiting key components but also their nucleating ability.

Mammalian Plks and centrosomes

Plk1

Several mammalian Plks are involved in regulating centrosomal function although the molecular basis of their roles remains unclear. Plk1 was first implicated in the centrosome cycle because of its subcellular localization (Golsteyn et al., 1995). Subsequent expression of a recombinant green fluorescence protein confirmed the centrosomal localization of Plk1 (Arnaud et al., 1998). However, while Plk1 is concentrated at the centrosomal region during interphase, as the cell cycle progresses, Plk1 associates with mitotic spindle poles, and eventually, from metaphase on, relocates to the midzone from which the midbody eventually derives as the cell goes through telophase (Golsteyn et al., 1995).

Inhibition of the activity of Plk1 by antibody injection results in cells with monoastral microtubules nucleated from duplicated but unseparated centrosomes in both transformed and non-transformed cells (Lane and Nigg, 1996), suggesting that Plk1 may be involved in regulating centrosome maturation not duplication. This notion is supported by the observations that Plk1 antibody injection does not block DNA synthesis (Lane and Nigg, 1996) and that DNA replication and centrosome duplication are coordinated and regulated by Cdk2-cyclin E (Winey, 1999). Although the mechanism controlling centrosome maturation remains to be elucidated HsEg5, a plus end-directed kinesin-related motor protein, appears to be an important mediator for centrosome separation and bipolar spindle formation (Blangy et al., 1995, 1997). HsEg5 is phosphorylated by Cdk1 and the phosphorylated form specifically associates with centrosomes and mitotic spindles (Blangy et al., 1995).

Plk1 is capable of activating Cdk1 kinase activity by phosphorylation of cyclin B at the onset of mitosis resulting in translocation of this protein to the nucleus, forming an active Cdk1/cyclin B complex (Toyoshima-Morimoto et al., 2001). Thus, Plk1's role in the centrosomal cycle may be through direct regulation of centrosomal components and through activation of other protein kinases, which in turn regulate the activity of centrosomal targets.

Plk3

Using a Plk3-specific monoclonal antibody, we have observed that during interphase Plk3 primarily localizes, as a concentrated dot, to the microtubule organization center as shown by co-staining with -tubulin (Figure 1). As the cell cycle progresses through S, G2, and M phases, Plk3 appears to comigrate with duplicated centrosomes; in all stages of the cell cycle Plk3 is associated with centrosomes or mitotic spindle poles (Wang et al., 2002). In addition, the localization of Plk3 to centrosomal region is microtubule-dependent because depolymerization of microtubules by cold or nocodazole treatment disrupts its centrosomal localization (Wang et al., 2002). It remains unclear why both Plk1 and Plk3 are localized to centrosomal regions during interphase. Given that the kinase activity of Plk1, as well as its protein level, peak at mitosis it is likely that Plk3, but not Plk1, may play a major role in controlling centrosomal function during interphase. This is consistent with the observation that levels of Plk3 do not fluctuate greatly during the cell cycle and that its kinase activity peaks around late S and G2 phase (Ouyang et al., 1997). Thus, Plks may fulfill different functions in regulating microtubule dynamics during the cell cycle just as Cdks play different roles in regulating various events associated with cell cycle progression.

Plks, the Golgi complex, and microtubule organization center

Our recent studies indicate that Plk3 appears to be associated with the Golgi apparatus as well during the cell cycle in many cell types (Figure 2). This is not surprising because the Golgi complex is closely associated with centrosomes. The striking co-localization of Plk3 with giantin, a Golgi-specific membrane protein, indicates a function of this Polo-family kinase in regulating the dynamics of the Golgi complex. Given that Golgi cisternae undergo extensive reorganization during mitosis and that the contents of the Golgi apparatus also need to be equally partitioned during cell division (Seemann et al., 2002), Plks may play a key role in fragmentation, dispersal and reassembling of the Golgi apparatus during mitosis of animal cells.

Besides their close proximity, a functional relation exists between the Golgi apparatus and centrosomes. Drug-induced disruption of microtubules results in disruption of the Golgi apparatus; the microtubule-dependent motor proteins cytoplasmic dynein and kinesin bind to the Golgi membrane and are implicated in vesicular transport within the complex (Thyberg and Moskalewski, 1999). On the other hand, the Golgi complex also has a microtubule-organizing capability. The Golgi complex can directly stimulate microtubule nucleation both in vivo and in vitro; the nucleation of Golgi-based microtubules in interphase cells involves -tubulin (Chabin-Brion et al., 2001).

Plk3 and other Plks may have adapted during evolution to coordinate disassemble and reassemble of the Golgi complex during cell division through protein phosphorylation. In fact, Plk1 interacts with and phosphorylates GRASP65 (Golgi reassembly stacking protein of 65 kDa) (Lin et al., 2000) which appears to be required for mitosis-specific fragmentation of the Golgi apparatus (Sutterlin et al., 2001). Clearly, centrosomes and the Golgi apparatus share some common regulatory molecules during cell division. Plks appear to be one major group of protein kinases that control the dynamics of these cellular organelles.

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Figure 1 Colocalization of Plk3 with -tubulin. Lung carcinoma A549 cells were double-stained with antibodies to Plk3 (red) and -tubulin (green), a centrosome-specific marker. Cellular DNA was stained with DAPI. Fluorescence microscopy was performed on a Nikon microscope and images were captured using a digital camera (Optronics) using Optronics MagFire and Image-Pro Plus softwares

Figure 2 Colocalization of Plk3 with a Golgi-specific antigen. Interphase A549 cells were double-stained with antibodies to Plk3 (red) and giantin (green), a Golgi-specific marker. Cellular DNA was stained with DAPI

Table 1 Polo family of kinases