@article{ecb4aa5980f843dd8bda27473006a65d,
title = "Functional independency between catalytic activity and subcellular targeting of polo-like kinase-1. Phenotypes of ectopic overexpression of various mutants",
abstract = "The mammalian polo-like kinase-1 (Plk1) plays key role in the progression of the M-phase. As cells enter the M-phase, Plk1 is phosphorylated by an upstream kinase on Thr-210 in N-terminal region, which is essential for Plk1 activation. In addition to catalytic activation, the subcellular localization is also an important task for Plk1, and the C-terminal polo-boxes are responsible for Plk1 targeting. Although both catalytic activation and targeting might be essential and work concurrently, it is still unclear which process precedes the other in mitotic progression, and which is more essential for the function of Plk1 through the M-phase. In order to investigate this mechanism, this study introduced several point mutations in the catalytic and polo-box domains, and overexpressed these constructs in HeLa cells. Although the cellular effects by ectopic expression were different in the cells containing various Plk1 constructs, phosphorylation itself, not the catalytic activity, was found to be important for the progression of the M-phase. Plk1 phosphorylation was also responsible for targeting by polobox, and compensated for the polo-box mutation. These results suggested that Plk1 is phosphorylated in the starting M-phase, and this phosphorylation induces polo-box targeting through a possible conformational change. In this situation, catalytic activation does not appear to be a critical factor for the function of Plk1.",
keywords = "Cell cycle, Ectopic expression, Mitosis, Polo-box, Polo-like kinase-1",
author = "Ji, {Jae Hoon} and Jang, {Young Joo}",
note = "Funding Information: the FL1 signal (GFP)] and analyzed using Cell QuestTMsoftware Ji JH, Jang YJ. Screening of domain-specific target proteins of in the negative cells. The data was collected [~30,000 events with U-2 OS cells. J Biol Chem 2002; 277:32282-93..polo-like kinase 1: construc- (Becton Dickinson) and Modfit LT 2.0TM (Verity Software House, tion and application of centrosome/kinetochore-specific targeting peptide. J Biochem Mol Topsham, ME).16,26,27 For fluorescence microscopy, the HeLa Jang YJ, Lin CY, Ma S, Erikson RL. Functional studies on the role of the C-terminal Biol 2006; 39:709-16. cells were seeded onto cover slips and were transfected 24 h later, domain of mammalian polo-like kinase. Proc Natl Acad Sci USA 2002; 99:1984-9. as previously described. Forty hours after transfection, the cells tions with multiple proteins in fission yeast polo kinase. J Cell Sci 2003; 116:1377-87.Reynolds N, Ohkura H. Polo boxes form a single functional domain that mediates interac- were fixed in a 4% paraformaldehyde-PBS solution followed by a LeeKS,EriksonRL.PlkisafunctionalhomologofSaccharomycescerevisiaeCdc5,and treatment with ice-cold methanol. After washing with PBS-0.1% elevated Plk activity induces multiple septation structures. Mol Cell Biol 1997; 17:3408-17. Triton X-100 (PBST), the cells were incubated for 2 h at room 137 in the regulation of mammalian polo-like kinase. J Biol Chem 2002; 277:44115-20.JangYJ,MaS,TeradaY,EriksonRL.Phosphorylationofthreonine210andtheroleofserine temperature in PBST supplemented with DAPI (10 μg/ml) for the Mundt KE, Golsteyn T DISTRIBUTERM, Lane HA, Nigg EA. On the regulation and function of human detection of chromosomes. Immunostaining with γ-tubulin was polo-like kinase 1 (PLK1): effects of overexpression on cell cycle progression. Biochem carried out using a mouse anti-γ-tubulin antibody (Sigma, T6557) 18. Jang YJ, Kim YS, Kim WH. Oncogenic effect of Polo-like kinase 1 expression in human Biophys Res Commun 1997; 239:377-85. and a Cy3-conjugated goat anti-mouse antibody (Jackson Immuno gastric carcinomas. Int J Oncol 2006; 29:589-94. Research). The mounted coverslips were analyzed by fluorescence 19. Knecht R, Elez R, Oechler M, Solbach C, von Ilberg C, Strebhardt K. Prognostic signifi-microscopy, and the visualized cells and images were captured on a neck. Cancer Res 1999; 59:2794-7.cance of polo-like kinase (PLK) expression in squamous cell carcinomas of the head and LSM510 imaging system (Zeiss). 20.Simizu S, Osada H. Mutations in the Plk gene lead to instability of Plk protein in human . DO NO Preparation of cell lysates. The cells were lysed in a Nonidet P-40 tumour cell lines. Nat Cell Biol 2000; 2:852-4. extraction buffer (0.5% Nonidet P-40, 50 mM Hepes, pH 7.4, 150 21. Smith MR, Wilson ML, Hamanaka R, Chase D, Kung H, Longo DL, Ferris DK. mM NaCl, 1 mM DTT, 5 mM EGTA, 1 mM EDTA) supplemented polo-like kinase. Biochem Biophys Res Commun 1997; 234:397-405.Malignant transformation of mammalian cells initiated by constitutive expression of the with protease inhibitors. The lysate was centrifuged at 12,000 rpm Tang J, Erikson RL, Liu X. Ectopic expression of Plk1 leads to activation of the spindle for 20 min, and the supernatant was recovered. The extracts were checkpoint. Cell Cycle 2006; 5:2484-8. used in western blot analysis and immunoprecipitation. polo-like kinase-1 polo box domain and its phospho-peptide complex. EMBO J 2003; Cheng KY, Lowe ED, Sinclair J, Nigg EA, Johnson LN. The crystal structure of the human Immunoprecipitation and kinase reactions. Approximately 1 22:5757-68. mg in 1 ml of clarified cell lysates was immunoprecipitated for the Chen C, Okayama H. High-efficiency kinase reactions. The following antibodies and dilution conditions Lee KS, Grenfell TZ, Yarm FR, Erikson RL. Mutation of the polo-box disrupts localization DNA. Mol Cell Biol 1987; 7:2745-52. were used: monoclonal anti-FLAG (Sigma) at 2 μg/ml; monoclonal and mitotic functions of the mammalian polo kinase Plk. Proc Natl Acad Sci USA 1998; anti-Plk (Zymed) at 2.5 μg/ml. Protein Aor G-Sepharose (Zymed) 95:9301-6. was added and incubated for an additional 2–3 h in order to absorb targeted GFP. Biotechniques 1998; 24:349-50.Jiang W, Hunter T. Analysis of cell cycle profiles in transfected cells using a membrane- the antibody. The kKinase reactions were carried out as described Pestov DG, Polonskaia M, Lau LF. Flow cytometric analysis of the cell cycle in transfected elsewhere.16 Briefly, the kinase activity of Plk was measured in a cells without cell fixation. Biotechniques 1999; 26:102-6. solution (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 5 mM DTT, Rev Mol Cell Biol 2004; 5:429-40.28.Barr FA, Sillje HH, Nigg EA. Polo-like kinases and the orchestration of cell division. Nat 2 mM EGTA, 0.5 mM Na3VO4, 20 mM p-nitrophenyl phosphate) 29. Elia AE, Rellos P, Haire LF, Chao JW, Ivins FJ, Hoepker K, Mohammad D, Cantley LC, supplemented with 4 μg of casein (Sigma) and 25 μM ATP (10 μCi Smerdon SJ, Yaffe MB. The molecular basis for phosphodependent substrate targeting and of [γ-32P]ATP; 1 Ci = 37 GBq) at 30°C for 30 min. 30. Song S, Grenfell TZ, Garfield S, Erikson RL, Lee KS. Essential function of the polo box regulation of Plks by the Polo-box domain. Cell 2003; 115:83-95. Acknowledgements{\textcopyright}2008 LANDES BIOSCIENCE of Cdc5 in subcellular localization and induction of cytokinetic structures. Mol Cell Biol This work was supported by grant R01-2007-000-20380-0 from 31. Feng Y, Hodge DR, Palmieri G, Chase DL, Longo DL, Ferris DK. Association of polo-2000; 20:286-98. Korea Science and Engineering Foundation. like kinase with alpha-, beta-and gamma-tubulins in a stable complex. Biochem J 1999;",
year = "2008",
month = jun,
day = "1",
doi = "10.4161/cc.7.11.5897",
language = "English (US)",
volume = "7",
pages = "1597--1603",
journal = "Cell Cycle",
issn = "1538-4101",
publisher = "Landes Bioscience",
number = "11",
}