[CANCER RESEARCH 60, 6826–6831, December 15, 2000] Advances in Brief Incomplete Cytokinesis and Induction of Apoptosis by Overexpression of the Mammalian Polo-Like Kinase, Plk31 Christopher W. Conn, Robert F. Hennigan, Wei Dai, Yolanda Sanchez, and Peter J. Stambrook2 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 of mitotic progression including centrosome maturation (11), proper assembly of the mitotic spindle (11), and activation of the anaphase- The polo-like kinases (Plks) are a family of conserved serine/threonine promoting complex (12). In contrast to Plk1, Plk3 and Plk2 are less kinases that play a critical role in the normal progression of cells through well defined, and a role for these kinases in cell cycle regulation is mitosis. The Plk3 serine/threonine kinase is a mammalian member of this presently unknown. One difference between Plk1 and the other two family. Overexpression of Plk3 in mammalian cells suppresses prolifera- tion and inhibits colony formation. Subsequent analysis demonstrated Plks is that both Plk2 and Plk3 were originally identified as immedi- that overexpression of Plk3 induces chromatin condensation and apopto- ate-early genes (8, 9), whereas Plk1 does not share this characteristic. sis. This phenotype could not be inhibited by coexpression of Bcl-2 and The Plks have been implicated in the genesis or progression of tumors. was partially dependent on the COOH-terminal domain of Plk3 but not on Plk3 has been suggested as a candidate tumor suppressor (13), and its the catalytic activity of Plk3. Analysis of EGFP-Plk3 subcellular localiza- expression is down-regulated or absent in lung carcinomas (10) and tion revealed that Plk3 localizes to the cellular cortex and to the cell squamous cell carcinomas of the head and neck (13). Similarly, Plk1 midbody during exit from mitosis and is consistent with a role in cytoki- has been suggested to have prognostic value for squamous cell car- nesis. These data suggest that overexpression or ectopic suppression of cinoma of the head and neck (14), esophageal carcinoma (15), and Plk3 interferes with cellular proliferation by impeding cytokinesis. 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 Introduction are different. The data currently available for Plk3 and Plk1 suggest Progression through the cell cycle is a tightly regulated series of that the two kinases are likely to have both overlapping and unique events that are carried out by the reversible phosphorylation and functions within the cell cycle. This is based in part on the ability of ubiquitin-mediated degradation of key substrates (1, 2). In addition, a Plk3 and Plk1 to complement a temperature-sensitive allele of the cell has mechanisms to ensure that a given event in the cell cycle is budding yeast polo-like kinase, cdc5-1 (17, 18). We have demon- not executed until the preceding event has been faithfully completed. strated previously that Plk3, similar to Plk1, is likely to have a role in These safeguards, known as checkpoints, ensure that a cell does not mitosis (17). However, unlike Plk1, the identification of Plk3 as an undergo division until its genome has been replicated completely (3). immediate-early gene (8) suggests additional roles for Plk3 in the G1 The ordered progression through the cell cycle is mediated by the phase of the cell cycle. Here we have assessed the effects of overex- periodic synthesis and degradation of cyclins in conjunction with the pression of Plk3 in an attempt to begin to understand the biological periodic activation of their cognate partners, the cyclin-dependent function of this member of the Plk family. kinases (1). An emerging family of protein serine/threonine kinases Materials and Methods 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 Cell Culture and Transfections. All cells were maintained in DMEM highly conserved serine/threonine kinases named for their founding supplemented with 10% FBS and penicillin/streptomycin (Life Technologies, member POLO, from Drosophila melanogaster (6), and are repre- Inc., Gaithersburg, MD). HeLa cells that stably express Bcl-2 were generated sented in many organisms ranging from yeast to humans (4, 5). They by transfecting cells with mpZEN-hBcl-2-neo (provided Dr. David Askew, have been implicated in various aspects of cell cycle progression University of Cincinnati, Cincinnati, OH) or vector control and selecting for a including centrosome maturation, assembly of the mitotic spindle, G418-resistant population. Transfections were performed using Fugene 6 (Roche Biochemicals, Indianapolis, IN), according to the manufacturer’s rec- regulation of the anaphase-promoting complex, and cytokinesis (4, 5). ommendation. Transfection efficiencies at 24 h were around 95, 36, 25, and To date, three Plks have been identified in mammals, Plk1, Snk, and 51% for vector control, pEGFP-Plk3, pEGFP-Plk3D164A, and pEGFP- Prk/Fnk (7–10). In accordance with the designation of Glover et al. Plk3⌬CT, respectively. For colony formation assays, cells were seeded at (5) in 1998, we have adopted the nomenclature of Plk1, Plk2, and Plk3 1 ϫ 105 cells/60-mm plate and allowed to attach overnight. Cells were then for Plk1, Snk, and Prk/Fnk, respectively. Of these, the most exten- transfected with the indicated constructs and puromycin selection of 0.5–1.5 sively studied has been Plk1, which is important for numerous aspects ␮g/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 aspar- Received 6/16/00; accepted 10/31/00. The costs of publication of this article were defrayed in part by the payment of page tic acid 164 to and alanine in the kinase domain of Plk3. Site-directed charges. This article must therefore be hereby marked advertisement in accordance with mutagenesis to produce pIRES-Plk3D164A and pEGFP-Plk3D164A was car- 18 U.S.C. Section 1734 solely to indicate this fact. ried out with a Quick Change Site Directed Mutagenesis kit according to the 1 Supported by NIH Grants P01 ES-05652 and RO1 DK38185. C. W. C. is supported, manufacturer’s recommendation (Stratagene, La Jolla, CA). The primers used in part, by NIH Training Grant ES-07250 and a fellowship from the Albert J. Ryan Ј Foundation. to produce Plk3D164A in these constructs are 5 -GGGTATCTTACACA- 2 To whom requests for reprints should be addressed, at University of Cincinnati GAGCTCTCAAGCTGGG-3Ј and 5Ј-CCCAGCTTGAGAGCTCTGTGTAA- College of Medicine, Department of Cell Biology, Neurobiology, and Anatomy, Vontz GATACCC-3Ј. To produce Plk3⌬CT lacking 142 amino acids of the COOH- Center for Molecular Studies, 3125 Eden Avenue, Cincinnati, OH 45267-0521. Phone: terminal domain of Plk3, full-length murine Plk3 cDNA was cut with EcoRI (513) 558-5685; Fax: (513) 558-4454; E-mail: [email protected]. 3 The abbreviations used are: Plk, polo-like kinase; GFP, green fluorescent protein; and BbsI, cloned into pEGFP-C1 (Clontech, Palo Alto, CA), and cut with EGFP, enhanced GFP. EcoRI and SmaI to produce an NH2-terminal fusion between EGFP and 1.5 kb 6826 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2000 American Association for Cancer Research. INCOMPLETE CYTOKINESIS AND INDUCTION OF APOPTOSIS BY Plk3 of the 5Ј-end of the murine Plk3 open reading frame. All constructs were linked to a neo resistance marker were unsuccessful, suggesting that verified by sequencing. Plk3 overexpression was lethal. Because attempts to produce cells Immunoblotting. For Western blot analysis, cells were lysed in ice-cold with inducible Plk3 were also unsuccessful, we tested this proposition TGN lysis buffer containing [50 mM Tris (pH 7.5), 50 mM ␤-glycerophosphate, by cloning the full-length murine Plk3 cDNA into the bicistronic 150 mM NaCl, 10% glycerol, 1% Tween 20, 1 mM NaF, 1 mM sodium pIRES-puro vector (Clontech, Palo Alto, CA), which allows concom- orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 2 ␮g/ml pepstatin A, 5 itant translation of Plk3 and puromycin resistance from a common ␮g/ml leupeptin, 10 ␮g/ml aprotinin, and 1 mM DTT]. Lysates were then sonicated and precleared by centrifugation. Protein concentration was deter- transcript. Selection with low concentrations of puromycin should mined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, allow survival of cells that express Plk3 at low levels. To test this, CA), and 20 ␮g of lysate were fractionated on 8% acrylamide gels. Proteins cells plated at equal densities were transfected with either pIRES- were then transferred from the gel to an Immobilon-P polyvinylidene difluo- Plk3, a control vector that contains EGFP in the place of the Plk3 ride (Millipore, Bedford, MA) membrane, followed by blotting with mono- cDNA, or a catalytically inactive mutant of Plk3, pIRES-Plk3D164A clonal antibodies directed against either GFP (Boehringer Mannheim, Inc., (Fig. 1, A and B). After 24 h, puromycin was added to cells in doses Indianapolis, IN), Fnk (Plk3; Transduction Labs, Lexington, KY), or Bcl-2 from 0.5 to 1.5 ␮g/ml. Selection was maintained until colonies (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 ϫ 105 cells/well into six-well plates, allowed to attach overnight, and then transfected with the indicated constructs.