Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1995 Jun 2;129(6):1617–1628. doi: 10.1083/jcb.129.6.1617

Cell cycle regulation of the activity and subcellular localization of Plk1, a human protein kinase implicated in mitotic spindle function

PMCID: PMC2291169  PMID: 7790358

Abstract

Correct assembly and function of the mitotic spindle during cell division is essential for the accurate partitioning of the duplicated genome to daughter cells. Protein phosphorylation has long been implicated in controlling spindle function and chromosome segregation, and genetic studies have identified several protein kinases and phosphatases that are likely to regulate these processes. In particular, mutations in the serine/threonine-specific Drosophila kinase polo, and the structurally related kinase Cdc5p of Saccharomyces cerevisae, result in abnormal mitotic and meiotic divisions. Here, we describe a detailed analysis of the cell cycle-dependent activity and subcellular localization of Plk1, a recently identified human protein kinase with extensive sequence similarity to both Drosophila polo and S. cerevisiae Cdc5p. With the aid of recombinant baculoviruses, we have established a reliable in vitro assay for Plk1 kinase activity. We show that the activity of human Plk1 is cell cycle regulated, Plk1 activity being low during interphase but high during mitosis. We further show, by immunofluorescent confocal laser scanning microscopy, that human Plk1 binds to components of the mitotic spindle at all stages of mitosis, but undergoes a striking redistribution as cells progress from metaphase to anaphase. Specifically, Plk1 associates with spindle poles up to metaphase, but relocalizes to the equatorial plane, where spindle microtubules overlap (the midzone), as cells go through anaphase. These results indicate that the association of Plk1 with the spindle is highly dynamic and that Plk1 may function at multiple stages of mitotic progression. Taken together, our data strengthen the notion that human Plk1 may represent a functional homolog of polo and Cdc5p, and they suggest that this kinase plays an important role in the dynamic function of the mitotic spindle during chromosome segregation.

Full Text

The Full Text of this article is available as a PDF (2.2 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Axton J. M., Dombrádi V., Cohen P. T., Glover D. M. One of the protein phosphatase 1 isoenzymes in Drosophila is essential for mitosis. Cell. 1990 Oct 5;63(1):33–46. doi: 10.1016/0092-8674(90)90286-n. [DOI] [PubMed] [Google Scholar]
  2. Bailly E., Dorée M., Nurse P., Bornens M. p34cdc2 is located in both nucleus and cytoplasm; part is centrosomally associated at G2/M and enters vesicles at anaphase. EMBO J. 1989 Dec 20;8(13):3985–3995. doi: 10.1002/j.1460-2075.1989.tb08581.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brizuela L., Draetta G., Beach D. Activation of human CDC2 protein as a histone H1 kinase is associated with complex formation with the p62 subunit. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4362–4366. doi: 10.1073/pnas.86.12.4362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brown K. D., Coulson R. M., Yen T. J., Cleveland D. W. Cyclin-like accumulation and loss of the putative kinetochore motor CENP-E results from coupling continuous synthesis with specific degradation at the end of mitosis. J Cell Biol. 1994 Jun;125(6):1303–1312. doi: 10.1083/jcb.125.6.1303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Buendia B., Draetta G., Karsenti E. Regulation of the microtubule nucleating activity of centrosomes in Xenopus egg extracts: role of cyclin A-associated protein kinase. J Cell Biol. 1992 Mar;116(6):1431–1442. doi: 10.1083/jcb.116.6.1431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Byers B., Goetsch L. Duplication of spindle plaques and integration of the yeast cell cycle. Cold Spring Harb Symp Quant Biol. 1974;38:123–131. doi: 10.1101/sqb.1974.038.01.016. [DOI] [PubMed] [Google Scholar]
  7. Clay F. J., McEwen S. J., Bertoncello I., Wilks A. F., Dunn A. R. Identification and cloning of a protein kinase-encoding mouse gene, Plk, related to the polo gene of Drosophila. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):4882–4886. doi: 10.1073/pnas.90.11.4882. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cooke C. A., Heck M. M., Earnshaw W. C. The inner centromere protein (INCENP) antigens: movement from inner centromere to midbody during mitosis. J Cell Biol. 1987 Nov;105(5):2053–2067. doi: 10.1083/jcb.105.5.2053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cyert M. S., Scherson T., Kirschner M. W. Monoclonal antibodies specific for thiophosphorylated proteins recognize Xenopus MPF. Dev Biol. 1988 Sep;129(1):209–216. doi: 10.1016/0012-1606(88)90175-3. [DOI] [PubMed] [Google Scholar]
  10. Davis F. M., Tsao T. Y., Fowler S. K., Rao P. N. Monoclonal antibodies to mitotic cells. Proc Natl Acad Sci U S A. 1983 May;80(10):2926–2930. doi: 10.1073/pnas.80.10.2926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Doonan J. H., Morris N. R. The bimG gene of Aspergillus nidulans, required for completion of anaphase, encodes a homolog of mammalian phosphoprotein phosphatase 1. Cell. 1989 Jun 16;57(6):987–996. doi: 10.1016/0092-8674(89)90337-1. [DOI] [PubMed] [Google Scholar]
  12. Draetta G., Beach D. Activation of cdc2 protein kinase during mitosis in human cells: cell cycle-dependent phosphorylation and subunit rearrangement. Cell. 1988 Jul 1;54(1):17–26. doi: 10.1016/0092-8674(88)90175-4. [DOI] [PubMed] [Google Scholar]
  13. Earnshaw W. C., Cooke C. A. Analysis of the distribution of the INCENPs throughout mitosis reveals the existence of a pathway of structural changes in the chromosomes during metaphase and early events in cleavage furrow formation. J Cell Sci. 1991 Apr;98(Pt 4):443–461. doi: 10.1242/jcs.98.4.443. [DOI] [PubMed] [Google Scholar]
  14. Earnshaw W. C., Pluta A. F. Mitosis. Bioessays. 1994 Sep;16(9):639–643. doi: 10.1002/bies.950160908. [DOI] [PubMed] [Google Scholar]
  15. Fenton B., Glover D. M. A conserved mitotic kinase active at late anaphase-telophase in syncytial Drosophila embryos. Nature. 1993 Jun 17;363(6430):637–640. doi: 10.1038/363637a0. [DOI] [PubMed] [Google Scholar]
  16. Fode C., Motro B., Yousefi S., Heffernan M., Dennis J. W. Sak, a murine protein-serine/threonine kinase that is related to the Drosophila polo kinase and involved in cell proliferation. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6388–6392. doi: 10.1073/pnas.91.14.6388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Forsburg S. L., Nurse P. Cell cycle regulation in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Annu Rev Cell Biol. 1991;7:227–256. doi: 10.1146/annurev.cb.07.110191.001303. [DOI] [PubMed] [Google Scholar]
  18. Gabrielli B. G., Roy L. M., Gautier J., Philippe M., Maller J. L. A cdc2-related kinase oscillates in the cell cycle independently of cyclins G2/M and cdc2. J Biol Chem. 1992 Jan 25;267(3):1969–1975. [PubMed] [Google Scholar]
  19. Gallant P., Nigg E. A. Cyclin B2 undergoes cell cycle-dependent nuclear translocation and, when expressed as a non-destructible mutant, causes mitotic arrest in HeLa cells. J Cell Biol. 1992 Apr;117(1):213–224. doi: 10.1083/jcb.117.1.213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Glover D. M. Mitosis in the Drosophila embryo--in and out of control. Trends Genet. 1991 Apr;7(4):125–132. doi: 10.1016/0168-9525(91)90457-2. [DOI] [PubMed] [Google Scholar]
  21. Golsteyn R. M., Schultz S. J., Bartek J., Ziemiecki A., Ried T., Nigg E. A. Cell cycle analysis and chromosomal localization of human Plk1, a putative homologue of the mitotic kinases Drosophila polo and Saccharomyces cerevisiae Cdc5. J Cell Sci. 1994 Jun;107(Pt 6):1509–1517. doi: 10.1242/jcs.107.6.1509. [DOI] [PubMed] [Google Scholar]
  22. Gorbsky G. J. Chromosome motion in mitosis. Bioessays. 1992 Feb;14(2):73–80. doi: 10.1002/bies.950140202. [DOI] [PubMed] [Google Scholar]
  23. Gorbsky G. J., Ricketts W. A. Differential expression of a phosphoepitope at the kinetochores of moving chromosomes. J Cell Biol. 1993 Sep;122(6):1311–1321. doi: 10.1083/jcb.122.6.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Gotoh Y., Nishida E., Matsuda S., Shiina N., Kosako H., Shiokawa K., Akiyama T., Ohta K., Sakai H. In vitro effects on microtubule dynamics of purified Xenopus M phase-activated MAP kinase. Nature. 1991 Jan 17;349(6306):251–254. doi: 10.1038/349251a0. [DOI] [PubMed] [Google Scholar]
  25. Hamanaka R., Maloid S., Smith M. R., O'Connell C. D., Longo D. L., Ferris D. K. Cloning and characterization of human and murine homologues of the Drosophila polo serine-threonine kinase. Cell Growth Differ. 1994 Mar;5(3):249–257. [PubMed] [Google Scholar]
  26. Heintz N., Sive H. L., Roeder R. G. Regulation of human histone gene expression: kinetics of accumulation and changes in the rate of synthesis and in the half-lives of individual histone mRNAs during the HeLa cell cycle. Mol Cell Biol. 1983 Apr;3(4):539–550. doi: 10.1128/mcb.3.4.539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Holtrich U., Wolf G., Bräuninger A., Karn T., Böhme B., Rübsamen-Waigmann H., Strebhardt K. Induction and down-regulation of PLK, a human serine/threonine kinase expressed in proliferating cells and tumors. Proc Natl Acad Sci U S A. 1994 Mar 1;91(5):1736–1740. doi: 10.1073/pnas.91.5.1736. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Hyman A. A., Mitchison T. J. Two different microtubule-based motor activities with opposite polarities in kinetochores. Nature. 1991 May 16;351(6323):206–211. doi: 10.1038/351206a0. [DOI] [PubMed] [Google Scholar]
  29. Karsenti E., Bravo R., Kirschner M. Phosphorylation changes associated with the early cell cycle in Xenopus eggs. Dev Biol. 1987 Feb;119(2):442–453. doi: 10.1016/0012-1606(87)90048-0. [DOI] [PubMed] [Google Scholar]
  30. Karsenti E. Mitotic spindle morphogenesis in animal cells. Semin Cell Biol. 1991 Aug;2(4):251–260. [PubMed] [Google Scholar]
  31. Kinoshita N., Yamano H., Le Bouffant-Sladeczek F., Kurooka H., Ohkura H., Stone E. M., Takeuchi M., Toda T., Yoshida T., Yanagida M. Sister-chromatid separation and protein dephosphorylation in mitosis. Cold Spring Harb Symp Quant Biol. 1991;56:621–628. doi: 10.1101/sqb.1991.056.01.071. [DOI] [PubMed] [Google Scholar]
  32. Kitada K., Johnson A. L., Johnston L. H., Sugino A. A multicopy suppressor gene of the Saccharomyces cerevisiae G1 cell cycle mutant gene dbf4 encodes a protein kinase and is identified as CDC5. Mol Cell Biol. 1993 Jul;13(7):4445–4457. doi: 10.1128/mcb.13.7.4445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Koshland D. Mitosis: back to the basics. Cell. 1994 Jul 1;77(7):951–954. doi: 10.1016/0092-8674(94)90432-4. [DOI] [PubMed] [Google Scholar]
  34. Krek W., Nigg E. A. Mutations of p34cdc2 phosphorylation sites induce premature mitotic events in HeLa cells: evidence for a double block to p34cdc2 kinase activation in vertebrates. EMBO J. 1991 Nov;10(11):3331–3341. doi: 10.1002/j.1460-2075.1991.tb04897.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Kuenzel E. A., Mulligan J. A., Sommercorn J., Krebs E. G. Substrate specificity determinants for casein kinase II as deduced from studies with synthetic peptides. J Biol Chem. 1987 Jul 5;262(19):9136–9140. [PubMed] [Google Scholar]
  36. Lake R. J., Jelinek W. R. Cell cycle- and terminal differentiation-associated regulation of the mouse mRNA encoding a conserved mitotic protein kinase. Mol Cell Biol. 1993 Dec;13(12):7793–7801. doi: 10.1128/mcb.13.12.7793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Liao H., Li G., Yen T. J. Mitotic regulation of microtubule cross-linking activity of CENP-E kinetochore protein. Science. 1994 Jul 15;265(5170):394–398. doi: 10.1126/science.8023161. [DOI] [PubMed] [Google Scholar]
  38. Litchfield D. W., Lüscher B., Lozeman F. J., Eisenman R. N., Krebs E. G. Phosphorylation of casein kinase II by p34cdc2 in vitro and at mitosis. J Biol Chem. 1992 Jul 15;267(20):13943–13951. [PubMed] [Google Scholar]
  39. Llamazares S., Moreira A., Tavares A., Girdham C., Spruce B. A., Gonzalez C., Karess R. E., Glover D. M., Sunkel C. E. polo encodes a protein kinase homolog required for mitosis in Drosophila. Genes Dev. 1991 Dec;5(12A):2153–2165. doi: 10.1101/gad.5.12a.2153. [DOI] [PubMed] [Google Scholar]
  40. Maridor G., Gallant P., Golsteyn R., Nigg E. A. Nuclear localization of vertebrate cyclin A correlates with its ability to form complexes with cdk catalytic subunits. J Cell Sci. 1993 Oct;106(Pt 2):535–544. doi: 10.1242/jcs.106.2.535. [DOI] [PubMed] [Google Scholar]
  41. Mayer-Jaekel R. E., Ohkura H., Gomes R., Sunkel C. E., Baumgartner S., Hemmings B. A., Glover D. M. The 55 kd regulatory subunit of Drosophila protein phosphatase 2A is required for anaphase. Cell. 1993 Feb 26;72(4):621–633. doi: 10.1016/0092-8674(93)90080-a. [DOI] [PubMed] [Google Scholar]
  42. McIntosh J. R., Hering G. E. Spindle fiber action and chromosome movement. Annu Rev Cell Biol. 1991;7:403–426. doi: 10.1146/annurev.cb.07.110191.002155. [DOI] [PubMed] [Google Scholar]
  43. McIntosh J. R., Koonce M. P. Mitosis. Science. 1989 Nov 3;246(4930):622–628. doi: 10.1126/science.2683078. [DOI] [PubMed] [Google Scholar]
  44. Meijer L., Arion D., Golsteyn R., Pines J., Brizuela L., Hunt T., Beach D. Cyclin is a component of the sea urchin egg M-phase specific histone H1 kinase. EMBO J. 1989 Aug;8(8):2275–2282. doi: 10.1002/j.1460-2075.1989.tb08353.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Nicklas R. B., Krawitz L. E., Ward S. C. Odd chromosome movement and inaccurate chromosome distribution in mitosis and meiosis after treatment with protein kinase inhibitors. J Cell Sci. 1993 Apr;104(Pt 4):961–973. doi: 10.1242/jcs.104.4.961. [DOI] [PubMed] [Google Scholar]
  46. Nigg E. A. Targets of cyclin-dependent protein kinases. Curr Opin Cell Biol. 1993 Apr;5(2):187–193. doi: 10.1016/0955-0674(93)90101-u. [DOI] [PubMed] [Google Scholar]
  47. Nislow C., Lombillo V. A., Kuriyama R., McIntosh J. R. A plus-end-directed motor enzyme that moves antiparallel microtubules in vitro localizes to the interzone of mitotic spindles. Nature. 1992 Oct 8;359(6395):543–547. doi: 10.1038/359543a0. [DOI] [PubMed] [Google Scholar]
  48. Nurse P. Universal control mechanism regulating onset of M-phase. Nature. 1990 Apr 5;344(6266):503–508. doi: 10.1038/344503a0. [DOI] [PubMed] [Google Scholar]
  49. Ohkura H., Kinoshita N., Miyatani S., Toda T., Yanagida M. The fission yeast dis2+ gene required for chromosome disjoining encodes one of two putative type 1 protein phosphatases. Cell. 1989 Jun 16;57(6):997–1007. doi: 10.1016/0092-8674(89)90338-3. [DOI] [PubMed] [Google Scholar]
  50. Palladino F., Laroche T., Gilson E., Axelrod A., Pillus L., Gasser S. M. SIR3 and SIR4 proteins are required for the positioning and integrity of yeast telomeres. Cell. 1993 Nov 5;75(3):543–555. doi: 10.1016/0092-8674(93)90388-7. [DOI] [PubMed] [Google Scholar]
  51. Pines J., Hunter T. Human cyclins A and B1 are differentially located in the cell and undergo cell cycle-dependent nuclear transport. J Cell Biol. 1991 Oct;115(1):1–17. doi: 10.1083/jcb.115.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Pinna L. A. Casein kinase 2: an 'eminence grise' in cellular regulation? Biochim Biophys Acta. 1990 Sep 24;1054(3):267–284. doi: 10.1016/0167-4889(90)90098-x. [DOI] [PubMed] [Google Scholar]
  53. Rattner J. B. Mapping the mammalian intercellular bridge. Cell Motil Cytoskeleton. 1992;23(4):231–235. doi: 10.1002/cm.970230402. [DOI] [PubMed] [Google Scholar]
  54. Rattner J. B., Rao A., Fritzler M. J., Valencia D. W., Yen T. J. CENP-F is a .ca 400 kDa kinetochore protein that exhibits a cell-cycle dependent localization. Cell Motil Cytoskeleton. 1993;26(3):214–226. doi: 10.1002/cm.970260305. [DOI] [PubMed] [Google Scholar]
  55. Rieder C. L., Salmon E. D. Motile kinetochores and polar ejection forces dictate chromosome position on the vertebrate mitotic spindle. J Cell Biol. 1994 Feb;124(3):223–233. doi: 10.1083/jcb.124.3.223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Schild D., Byers B. Diploid spore formation and other meiotic effects of two cell-division-cycle mutations of Saccharomyces cerevisiae. Genetics. 1980 Dec;96(4):859–876. doi: 10.1093/genetics/96.4.859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Schultz S. J., Nigg E. A. Identification of 21 novel human protein kinases, including 3 members of a family related to the cell cycle regulator nimA of Aspergillus nidulans. Cell Growth Differ. 1993 Oct;4(10):821–830. [PubMed] [Google Scholar]
  58. Sharon G., Simchen G. Mixed segregation of chromosomes during single-division meiosis of Saccharomyces cerevisiae. Genetics. 1990 Jul;125(3):475–485. doi: 10.1093/genetics/125.3.475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Simmons D. L., Neel B. G., Stevens R., Evett G., Erikson R. L. Identification of an early-growth-response gene encoding a novel putative protein kinase. Mol Cell Biol. 1992 Sep;12(9):4164–4169. doi: 10.1128/mcb.12.9.4164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Stone E. M., Yamano H., Kinoshita N., Yanagida M. Mitotic regulation of protein phosphatases by the fission yeast sds22 protein. Curr Biol. 1993 Jan;3(1):13–26. doi: 10.1016/0960-9822(93)90140-j. [DOI] [PubMed] [Google Scholar]
  61. Sunkel C. E., Glover D. M. polo, a mitotic mutant of Drosophila displaying abnormal spindle poles. J Cell Sci. 1988 Jan;89(Pt 1):25–38. doi: 10.1242/jcs.89.1.25. [DOI] [PubMed] [Google Scholar]
  62. Taylor S. S., Knighton D. R., Zheng J., Ten Eyck L. F., Sowadski J. M. Structural framework for the protein kinase family. Annu Rev Cell Biol. 1992;8:429–462. doi: 10.1146/annurev.cb.08.110192.002241. [DOI] [PubMed] [Google Scholar]
  63. Toyn J. H., Johnston L. H. The Dbf2 and Dbf20 protein kinases of budding yeast are activated after the metaphase to anaphase cell cycle transition. EMBO J. 1994 Mar 1;13(5):1103–1113. doi: 10.1002/j.1460-2075.1994.tb06359.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Vandre D. D., Davis F. M., Rao P. N., Borisy G. G. Phosphoproteins are components of mitotic microtubule organizing centers. Proc Natl Acad Sci U S A. 1984 Jul;81(14):4439–4443. doi: 10.1073/pnas.81.14.4439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Vandré D. D., Burry R. W. Immunoelectron microscopic localization of phosphoproteins associated with the mitotic spindle. J Histochem Cytochem. 1992 Dec;40(12):1837–1847. doi: 10.1177/40.12.1453002. [DOI] [PubMed] [Google Scholar]
  66. Verde F., Berrez J. M., Antony C., Karsenti E. Taxol-induced microtubule asters in mitotic extracts of Xenopus eggs: requirement for phosphorylated factors and cytoplasmic dynein. J Cell Biol. 1991 Mar;112(6):1177–1187. doi: 10.1083/jcb.112.6.1177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Verde F., Dogterom M., Stelzer E., Karsenti E., Leibler S. Control of microtubule dynamics and length by cyclin A- and cyclin B-dependent kinases in Xenopus egg extracts. J Cell Biol. 1992 Sep;118(5):1097–1108. doi: 10.1083/jcb.118.5.1097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Verde F., Labbé J. C., Dorée M., Karsenti E. Regulation of microtubule dynamics by cdc2 protein kinase in cell-free extracts of Xenopus eggs. Nature. 1990 Jan 18;343(6255):233–238. doi: 10.1038/343233a0. [DOI] [PubMed] [Google Scholar]
  69. Wadsworth P. Mitosis: spindle assembly and chromosome motion. Curr Opin Cell Biol. 1993 Feb;5(1):123–128. doi: 10.1016/s0955-0674(05)80017-1. [DOI] [PubMed] [Google Scholar]
  70. Yanagida M., Kinoshita N., Stone E. M., Yamano H. Protein phosphatases and cell division cycle control. Ciba Found Symp. 1992;170:130–146. doi: 10.1002/9780470514320.ch9. [DOI] [PubMed] [Google Scholar]
  71. Yen T. J., Compton D. A., Wise D., Zinkowski R. P., Brinkley B. R., Earnshaw W. C., Cleveland D. W. CENP-E, a novel human centromere-associated protein required for progression from metaphase to anaphase. EMBO J. 1991 May;10(5):1245–1254. doi: 10.1002/j.1460-2075.1991.tb08066.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES