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Incomplete Cytokinesis and Induction of Apoptosis by Overexpression of the Mammalian Polo-Like Kinase, Plk3¹

Departments of Cell Biology [C. W. C., R. F. H., P. J. S.] and Molecular Genetics [Y. S.], University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, and The American Health Foundation, Valhalla, New York 10595 [W. D.]

	ABSTRACT

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The polo-like kinases (Plks) are a family of conserved serine/threonine kinases that play a critical role in the normal progression of cells through mitosis. The Plk3 serine/threonine kinase is a mammalian member of this family. Overexpression of Plk3 in mammalian cells suppresses proliferation and inhibits colony formation. Subsequent analysis demonstrated that overexpression of Plk3 induces chromatin condensation and apoptosis. This phenotype could not be inhibited by coexpression of Bcl-2 and was partially dependent on the COOH-terminal domain of Plk3 but not on the catalytic activity of Plk3. Analysis of EGFP-Plk3 subcellular localization revealed that Plk3 localizes to the cellular cortex and to the cell midbody during exit from mitosis and is consistent with a role in cytokinesis. These data suggest that overexpression or ectopic suppression of Plk3 interferes with cellular proliferation by impeding cytokinesis.

	Introduction

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Progression through the cell cycle is a tightly regulated series of events that are carried out by the reversible phosphorylation and ubiquitin-mediated degradation of key substrates (1 , 2) . In addition, a cell has mechanisms to ensure that a given event in the cell cycle is not executed until the preceding event has been faithfully completed. These safeguards, known as checkpoints, ensure that a cell does not undergo division until its genome has been replicated completely (3) . The ordered progression through the cell cycle is mediated by the periodic synthesis and degradation of cyclins in conjunction with the periodic activation of their cognate partners, the cyclin-dependent kinases (1) . An emerging family of protein serine/threonine kinases that plays an important role in the cell division process is the Plk³ family (reviewed in Refs. 4 and 5 ). The Plks comprise a family of highly conserved serine/threonine kinases named for their founding member POLO, from Drosophila melanogaster (6) , and are represented in many organisms ranging from yeast to humans (4 , 5) . They have been implicated in various aspects of cell cycle progression including centrosome maturation, assembly of the mitotic spindle, regulation of the anaphase-promoting complex, and cytokinesis (4 , 5) . To date, three Plks have been identified in mammals, Plk1, Snk, and Prk/Fnk (7, 8, 9, 10) . In accordance with the designation of Glover et al. (5) in 1998, we have adopted the nomenclature of Plk1, Plk2, and Plk3 for Plk1, Snk, and Prk/Fnk, respectively. Of these, the most extensively studied has been Plk1, which is important for numerous aspects of mitotic progression including centrosome maturation (11) , proper assembly of the mitotic spindle (11) , and activation of the anaphase-promoting complex (12) . In contrast to Plk1, Plk3 and Plk2 are less well defined, and a role for these kinases in cell cycle regulation is presently unknown. One difference between Plk1 and the other two Plks is that both Plk2 and Plk3 were originally identified as immediate-early genes (8 , 9) , whereas Plk1 does not share this characteristic. The Plks have been implicated in the genesis or progression of tumors. Plk3 has been suggested as a candidate tumor suppressor (13) , and its expression is down-regulated or absent in lung carcinomas (10) and squamous cell carcinomas of the head and neck (13) . Similarly, Plk1 has been suggested to have prognostic value for squamous cell carcinoma of the head and neck (14) , esophageal carcinoma (15) , and melanoma (16) . Each of the Plks plays a role in the progression of cells through the cell cycle, but it is likely that their respective roles are different. The data currently available for Plk3 and Plk1 suggest that the two kinases are likely to have both overlapping and unique functions within the cell cycle. This is based in part on the ability of Plk3 and Plk1 to complement a temperature-sensitive allele of the budding yeast polo-like kinase, cdc5-1 (17 , 18) . We have demonstrated previously that Plk3, similar to Plk1, is likely to have a role in mitosis (17) . However, unlike Plk1, the identification of Plk3 as an immediate-early gene (8) suggests additional roles for Plk3 in the G₁ phase of the cell cycle. Here we have assessed the effects of overexpression of Plk3 in an attempt to begin to understand the biological function of this member of the Plk family.

	Materials and Methods

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Cell Culture and Transfections.
All cells were maintained in DMEM supplemented with 10% FBS and penicillin/streptomycin (Life Technologies, Inc., Gaithersburg, MD). HeLa cells that stably express Bcl-2 were generated by transfecting cells with mpZEN-hBcl-2-neo (provided Dr. David Askew, University of Cincinnati, Cincinnati, OH) or vector control and selecting for a G418-resistant population. Transfections were performed using Fugene 6 (Roche Biochemicals, Indianapolis, IN), according to the manufacturer’s recommendation. Transfection efficiencies at 24 h were around 95, 36, 25, and 51% for vector control, pEGFP-Plk3, pEGFP-Plk3D164A, and pEGFP-Plk3CT, respectively. For colony formation assays, cells were seeded at 1 x 10⁵ cells/60-mm plate and allowed to attach overnight. Cells were then transfected with the indicated constructs and puromycin selection of 0.5–1.5 µg/ml was initiated 48 h later. Colonies were allowed to form, after which they were fixed with ice-cold methanol and stained with crystal violet.

Site-directed Mutagenesis.
Plk3D164A was produced by mutating aspartic acid 164 to and alanine in the kinase domain of Plk3. Site-directed mutagenesis to produce pIRES-Plk3D164A and pEGFP-Plk3D164A was carried out with a Quick Change Site Directed Mutagenesis kit according to the manufacturer’s recommendation (Stratagene, La Jolla, CA). The primers used to produce Plk3D164A in these constructs are 5'-GGGTATCTTACACAGAGCTCTCAAGCTGGG-3' and 5'-CCCAGCTTGAGAGCTCTGTGTAAGATACCC-3'. To produce Plk3CT lacking 142 amino acids of the COOH-terminal domain of Plk3, full-length murine Plk3 cDNA was cut with EcoRI and BbsI, cloned into pEGFP-C1 (Clontech, Palo Alto, CA), and cut with EcoRI and SmaI to produce an NH₂-terminal fusion between EGFP and 1.5 kb of the 5'-end of the murine Plk3 open reading frame. All constructs were verified by sequencing.

Immunoblotting.
For Western blot analysis, cells were lysed in ice-cold TGN lysis buffer containing [50 mM Tris (pH 7.5), 50 mM ß-glycerophosphate, 150 mM NaCl, 10% glycerol, 1% Tween 20, 1 mM NaF, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml pepstatin A, 5 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM DTT]. Lysates were then sonicated and precleared by centrifugation. Protein concentration was determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA), and 20 µg of lysate were fractionated on 8% acrylamide gels. Proteins were then transferred from the gel to an Immobilon-P polyvinylidene difluoride (Millipore, Bedford, MA) membrane, followed by blotting with monoclonal antibodies directed against either GFP (Boehringer Mannheim, Inc., Indianapolis, IN), Fnk (Plk3; Transduction Labs, Lexington, KY), or Bcl-2 (Santa Cruz Biotechnology, Santa Cruz, CA). Membranes were probed with anti-tubulin (Lab Vision, Fremont, CA) to control for loading. Western blots were visualized by enhanced chemiluminescence (Amersham).

Annexin V Apoptosis Assay.
HeLa cells were seeded at 1 x 10⁵ cells/well into six-well plates, allowed to attach overnight, and then transfected with the indicated constructs. The cells were trypsinized 36 h after transfection and incubated with Annexin V biotin according to the manufacturer’s recommendation (Oncogene Research Products, Boston, MA). The cells were then incubated with streptavidin Texas Red (Oncogene Research Products, Boston, MA), placed on poly-L-lysine-coated slides, and visualized by indirect immunofluorescence. At least 100 GFP-positive cells were scored from three independent experiments.

Immuoprecipitations and Kinase Assays.
For all kinase assays, cells were lysed as described above. Lysate (250 µg) was precleared with Protein A/G-Plus agarose (Santa Cruz Biotechnology) for 1 h, followed by incubation with 2 µg of GFP monoclonal antibody (Boehringer Mannheim) and Protein A/G-agarose beads (Santa Cruz Biotechnology) for 3 h. Immunocomplexes were then washed three times in ice-cold lysis buffer, followed by one wash in kinase buffer [30 mM HEPES (pH 7.4), 10 mM MgCl₂, 7.0 MnCl₂, and 1.0 mM DTT]. Immunocomplexes were then resuspended in 20 µl of kinase buffer with 1 µg of GST-p53 (as a nonspecific substrate), 10 µM ATP, and 10 µCi of [-³²P]ATP. Reactions were then allowed to proceed for 30 min at 30°C, at which time they were extinguished by the addition of 6x SDS loading buffer. Kinase reactions were then boiled for 10 min and fractionated by SDS-PAGE on 8% acrylamide gels. Proteins were then transferred to a polyvinylidene difluoride membrane and blotted with anti-GFP monoclonal antibodies. Western blots were visualized by enhanced chemiluminescence (Amersham).

Cell Staining and Fluorescence Microscopy.
Cells growing on glass coverslips were transfected with the indicated constructs as described above. Twenty-four h after transfection, the cells were fixed in 3.7% formaldehyde for 15 min. The cells were then permeabilized in 0.2% Triton X-100 for 5 min and then stained with Alexafluor568-conjugated phalloidin (Molecular Probes, Eugene, OR) for 15 min. Coverslips were then washed three times with PBS and mounted on slides using Gel Mount (Fisher Scientific). Dual color images from 0.5-µm-thick optical sections were acquired using a Zeiss LSM 510 laser scanning confocal microscope and a x63 objective. The percentage of cells displaying a postmitotic bridge was determined by analyzing at least 100 GFP-positive cells from three independent experiments.

Time Lapse Confocal Microscopy.
HeLa cells were plated onto sterile 24-mm glass coverslips and transfected with pEGFP-C1, pEGFP-Plk3, pEGFP-Plk3D164A, or pEGFP-Plk3CT described above. After 24 h, the coverslips were transferred to a Sikes-Moore chamber and placed in an incubator stage fitted onto a Zeiss Axiovert microscope and equilibrated to 37°C. Confocal images of the EGFP fluorescence and transmitted DIC images were simultaneously acquired with the Zeiss LSM 510 and a x63 oil immersion objective fitted with an objective heater (Bioptics) that was equilibrated to 37°C.

	Results

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Plk3 Inhibits Colony Formation.
The consequences of overexpression of a putative cell cycle regulator may provide insight as to where its function is exerted during normal cell division. With this in mind, we assessed the effects of Plk3 overexpression on cell cycle progression. Initial attempts to establish stable clones of HeLa cells that constitutively overexpress the murine or human Plk3 cDNA linked to a neo resistance marker were unsuccessful, suggesting that Plk3 overexpression was lethal. Because attempts to produce cells with inducible Plk3 were also unsuccessful, we tested this proposition by cloning the full-length murine Plk3 cDNA into the bicistronic pIRES-puro vector (Clontech, Palo Alto, CA), which allows concomitant translation of Plk3 and puromycin resistance from a common transcript. Selection with low concentrations of puromycin should allow survival of cells that express Plk3 at low levels. To test this, cells plated at equal densities were transfected with either pIRES-Plk3, a control vector that contains EGFP in the place of the Plk3 cDNA, or a catalytically inactive mutant of Plk3, pIRES-Plk3D164A (Fig. 1, A and B) . After 24 h, puromycin was added to cells in doses from 0.5 to 1.5 µg/ml. Selection was maintained until colonies formed, after which, cells were fixed and stained with crystal violet (Fig. 1C) . As expected, the number of colonies formed by cells transfected with the pIRES-EGFP construct was inversely proportional to the concentration of puromycin used for selection. In contrast, no colonies were formed after transfection with the pIRES-Plk3 cDNA at any concentration of puromycin tested. Interestingly, no colonies were formed with the catalytically inactive mutant of Plk3 either, indicating that the kinase activity of Plk3 was not necessary for the suppression of colony formation. Identical results were obtained with the human fibrosarcoma cell line HT1080 as well NIH 3T3 cells (data not shown), indicating that the inhibition of cellular proliferation by Plk3 overexpression is a general phenomenon and not specific for HeLa cells.

View larger version (27K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Overexpression of Plk3 inhibits colony formation. A, HeLa cells were transiently transfected with pIRES-EGFP, pIRES-Plk3, or pIRES-Plk3D164A. Twenty-four h after transfection, cells were lysed, and immunoblotting was performed on lysates with anti-Plk3 antibody. B, HeLa cells were transiently transfected with pEGFP, pEGFP-Plk3, or pEGFP-Plk3D164A. Twenty-four h after transfection, kinase activity was measured in anti-GFP immunoprecipitates (lower panel), followed by immunoblotting with anti-GFP antibody as a loading control (upper panel). C, equal numbers of HeLa cells were transfected with pIRES-EGFP (1 µg), pIRES-Plk3 (1 µg), or pIRES-Plk3D164A (1 µg). Twenty-four h after transfection, puromycin selection was applied at doses ranging from 0.5 to 1.5 µg/ml. Selection was maintained until colonies formed, after which cells were fixed in methanol and stained with crystal violet.

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Overexpression of Plk3 promotes chromatin condensation. A, HeLa cells were transiently transfected with pEGFP, pEGFP-Plk3, pEGFP-Plk3D164A, or pEGFP-Plk3CT. Twenty-four h after transfection, cells were lysed and subjected to Western blot analysis with anti-GFP antibody. B, 36 h after the transfection, the cells were fixed with ethanol or paraformaldehyde, stained with 4',6-diamidino-2-phenylindole (DAPI) and examined by fluorescence microscopy. C, the percentage of GFP-positive cells with condensed chromatin from the above experiment was determined by counting at least 100 GFP-positive cells from three independent experiments; bars, SD.

View larger version (15K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Overexpression of Plk3 induces apoptosis. A, HeLa cells were transiently transfected with pEGFP, pEGFP-Plk3, pEGFP-Plk3D164A, or pEGFP-Plk3CT. Thirty-six h after transfection, the cells were collected and stained for Annexin V. The percentage of GFP-positive cells that stained positive for Annexin V was determined by counting at least 100 GFP-positive cells from three independent experiments; bars, SD. B, HeLa cells that stably express Bcl-2 were lysed and subjected to immunoblotting with Bcl-2 antibody to confirm expression. Expression of tubulin was monitored as a loading control. C, HeLa cells that stably express Bcl-2 cells were transfected and analyzed for Annexin V staining as described above. Bars, SD.

View larger version (49K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Subcellular localization of EGFP-Plk3. A, HeLa cells growing on glass coverslips were transiently transfected with the indicated constructs. Twenty-four h after transfection, cells were fixed and stained with phalloidin, and the subcellular localization of EGFP-Plk3 was analyzed by laser scanning confocal microscopy. B, HeLa cells were transfected with pEGFP, pEGFP-Plk3, pEGFP-Plk3D164A, or pEGFP-Plk3CT. Twenty-four h after transfection, the cells were fixed and stained as above. The percentage of cells with a postmitotic bridge was determined by counting at least 100 GFP-positive cells from three independent experiments; bars, SD. C, HeLa cells growing on glass coverslips were transfected as above. Twenty-four h after transfection (0 h), the cells were analyzed by video time lapse confocal microscopy. Arrow, postmitotic bridge with EGFP-Plk3 localization.

	Discussion

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 
The Plks comprise an evolutionarily conserved family of kinases, the involvement of which in cell cycle regulation is becoming increasingly apparent. Current data from several model systems indicate that Plks are important for multiple functions during cell cycle progression. Loss of function mutants in Drosophila, Saccharomyces cerevisiae, and Schizosaccharomyces pombe indicate roles for Plks in orchestrating the orderly progression through the mitotic phase of the cell cycle (4 , 5) . Although much has been learned regarding the function of Plks within the last few years, their function is still poorly understood, particularly in higher eukaryotic systems. The Xenopus Plk, Plx1, functions at least in part by indirectly regulating cyclin-dependent kinase 1 activity through modulation of the Cdc25C phosphatase (19) , and both Xenopus Plx1 and human Plk1 play a role in regulation of the anaphase-promoting complex (12 , 20) . However, the mechanism(s) through which Plk3 functions during the cell cycle remain largely ill defined. It is likely that the Plks will prove to be as complex and functionally diverse as the cyclin-dependent kinases. We have sought to further elucidate the role of the mammalian Plk family by investigating the function of one of its members, Plk3.

Overexpression of Plk1 induces mitotic abnormalities (21) , supporting a role for this kinase in mitosis. Additionally, its constitutive overexpression causes NIH3T3 cells to become transformed (22) . To begin elucidating the role of Plk3 in cellular proliferation and cell cycle progression, we wanted to assess the effects of overexpression of Plk3 on cell cycle progression. In contrast to Plk1, we discovered that constitutive overexpression of Plk3 inhibited proliferation in every cell line tested, and additional studies determined that this inhibition of proliferation was mediated by the induction of apoptosis. Even more intriguing is the observation that the catalytic activity of Plk3 is not necessary for this phenotype. However, it cannot be excluded that overexpression of Plk3 may be acting in a dominant-negative fashion to induce apoptosis. Further analysis into the mechanism of Plk3 function will be required before the latter interpretation can be formally tested.

Both Plk1 and Plk3 can complement the yeast cdc5-1 mutants (17 , 18) , indicating that these mammalian Plks have some common functions. However, their roles in the regulation of cell cycle progression in mammalian cells appear to have diverged. It is provocative, although not unprecedented, that aberrant expression of a putative positive regulator of the cell cycle can induce apoptosis. We and others have found that, unlike Plk1, the expression of which is restricted to the G₂-M phase of the cell cycle (23) , Plk3 expression is relatively constant during the cell cycle (24) .⁴ This suggests that to avoid deregulated Plk3 activity, Plk3 function must be tightly controlled at the posttranslational level. Indeed, Plk3 has been reported to be phosphorylated in a cell cycle-dependent manner (24) .

Data obtained in many different organisms indicate that Plks play an essential role in cytokinesis (4 , 5) . In this study, the EGFP-Plk3 protein concentrated in the midbody between the two newly divided daughter cells and was associated with the cellular cortex. Additionally, analysis by confocal microscopy revealed that EGFP-Plk3 localized to the cleavage furrow early in the cytokinetic process, during late anaphase (data not shown). Fluorescence recovery after photobleaching experiments revealed that EGFP-Plk3 is dynamically targeted to these structures and that this localization is not a result of simple diffusion (data not shown). These data are consistent with a role for Plk3 in cytokinesis.

A full understanding of the mechanism(s) by which Plks participate in the regulation of cell division will help elucidate the role of Plks in tumorigenesis and may identify new therapeutic targets. It will, therefore, be important to further dissect the roles that Plk3 plays in normal cell division and in neoplasia.

	ACKNOWLEDGMENTS

 
We are grateful to Dr. David Askew for providing mpZEN-hBcl-2neo and to Dr. Kenji Fukasawa for providing MPM-2 antibody.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by NIH Grants P01 ES-05652 and RO1 DK38185. C. W. C. is supported, in part, by NIH Training Grant ES-07250 and a fellowship from the Albert J. Ryan Foundation.

² To whom requests for reprints should be addressed, at University of Cincinnati College of Medicine, Department of Cell Biology, Neurobiology, and Anatomy, Vontz Center for Molecular Studies, 3125 Eden Avenue, Cincinnati, OH 45267-0521. Phone: (513) 558-5685; Fax: (513) 558-4454; E-mail: Peter.Stambrook{at}uc.edu

³ The abbreviations used are: Plk, polo-like kinase; GFP, green fluorescent protein; EGFP, enhanced GFP.

⁴ C. W. Conn and P. J. Stambrook, unpublished observation.

Received 6/16/00. Accepted 10/31/00.

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