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. 1998 Jan 15;17(2):470–481. doi: 10.1093/emboj/17.2.470

A centrosomal function for the human Nek2 protein kinase, a member of the NIMA family of cell cycle regulators.

A M Fry 1, P Meraldi 1, E A Nigg 1
PMCID: PMC1170398  PMID: 9430639

Abstract

Nek2, a mammalian protein kinase of unknown function, is closely related to the mitotic regulator NIMA of Aspergillus nidulans. Here we show by both immunofluorescence microscopy and biochemical fractionation that human Nek2 localizes to the centrosome. Centrosome association occurs throughout the cell cycle, including all stages of mitosis, and is independent of microtubules. Overexpression of active Nek2 induces a striking splitting of centrosomes, whereas prolonged expression of either active or inactive Nek2 leads to dispersal of centrosomal material and loss of a focused microtubule-nucleating activity. Surprisingly, this does not prevent entry into mitosis, as judged by the accumulation of mitotically arrested cells induced by co-expression of a non-destructible B-type cyclin. These results bear on the dynamic function of centrosomes at the onset of mitosis. Moreover, they indicate that one function of mammalian Nek2 relates to the centrosome cycle and thus provide a new perspective on the role of NIMA-related kinases.

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Selected References

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  1. 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]
  2. Barton A. B., Davies C. J., Hutchison C. A., 3rd, Kaback D. B. Cloning of chromosome I DNA from Saccharomyces cerevisiae: analysis of the FUN52 gene, whose product has homology to protein kinases. Gene. 1992 Aug 1;117(1):137–140. doi: 10.1016/0378-1119(92)90502-g. [DOI] [PubMed] [Google Scholar]
  3. Blangy A., Lane H. A., d'Hérin P., Harper M., Kress M., Nigg E. A. Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo. Cell. 1995 Dec 29;83(7):1159–1169. doi: 10.1016/0092-8674(95)90142-6. [DOI] [PubMed] [Google Scholar]
  4. Boleti H., Karsenti E., Vernos I. Xklp2, a novel Xenopus centrosomal kinesin-like protein required for centrosome separation during mitosis. Cell. 1996 Jan 12;84(1):49–59. doi: 10.1016/s0092-8674(00)80992-7. [DOI] [PubMed] [Google Scholar]
  5. Bornens M., Paintrand M., Berges J., Marty M. C., Karsenti E. Structural and chemical characterization of isolated centrosomes. Cell Motil Cytoskeleton. 1987;8(3):238–249. doi: 10.1002/cm.970080305. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. De Brabander M., Geuens G., Nuydens R., Willebrords R., Aerts F., De Mey J. Microtubule dynamics during the cell cycle: the effects of taxol and nocodazole on the microtubule system of Pt K2 cells at different stages of the mitotic cycle. Int Rev Cytol. 1986;101:215–274. doi: 10.1016/s0074-7696(08)60250-8. [DOI] [PubMed] [Google Scholar]
  8. Doxsey S. J., Stein P., Evans L., Calarco P. D., Kirschner M. Pericentrin, a highly conserved centrosome protein involved in microtubule organization. Cell. 1994 Feb 25;76(4):639–650. doi: 10.1016/0092-8674(94)90504-5. [DOI] [PubMed] [Google Scholar]
  9. Fry A. M., Nigg E. A. Cell cycle. The NIMA kinase joins forces with Cdc2. Curr Biol. 1995 Oct 1;5(10):1122–1125. doi: 10.1016/s0960-9822(95)00227-2. [DOI] [PubMed] [Google Scholar]
  10. Fry A. M., Nigg E. A. Characterization of mammalian NIMA-related kinases. Methods Enzymol. 1997;283:270–282. doi: 10.1016/s0076-6879(97)83022-4. [DOI] [PubMed] [Google Scholar]
  11. Fry A. M., Schultz S. J., Bartek J., Nigg E. A. Substrate specificity and cell cycle regulation of the Nek2 protein kinase, a potential human homolog of the mitotic regulator NIMA of Aspergillus nidulans. J Biol Chem. 1995 May 26;270(21):12899–12905. doi: 10.1074/jbc.270.21.12899. [DOI] [PubMed] [Google Scholar]
  12. Félix M. A., Antony C., Wright M., Maro B. Centrosome assembly in vitro: role of gamma-tubulin recruitment in Xenopus sperm aster formation. J Cell Biol. 1994 Jan;124(1-2):19–31. doi: 10.1083/jcb.124.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gale M., Jr, Parsons M. A Trypanosoma brucei gene family encoding protein kinases with catalytic domains structurally related to Nek1 and NIMA. Mol Biochem Parasitol. 1993 May;59(1):111–121. doi: 10.1016/0166-6851(93)90012-m. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Glover D. M., Leibowitz M. H., McLean D. A., Parry H. Mutations in aurora prevent centrosome separation leading to the formation of monopolar spindles. Cell. 1995 Apr 7;81(1):95–105. doi: 10.1016/0092-8674(95)90374-7. [DOI] [PubMed] [Google Scholar]
  16. Hadjeb N., Berkowitz G. A. Preparation of T-over-hang vectors with high PCR product cloning efficiency. Biotechniques. 1996 Jan;20(1):20–22. doi: 10.2144/96201bm02. [DOI] [PubMed] [Google Scholar]
  17. Heald R., Tournebize R., Blank T., Sandaltzopoulos R., Becker P., Hyman A., Karsenti E. Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts. Nature. 1996 Aug 1;382(6590):420–425. doi: 10.1038/382420a0. [DOI] [PubMed] [Google Scholar]
  18. Heald R., Tournebize R., Habermann A., Karsenti E., Hyman A. Spindle assembly in Xenopus egg extracts: respective roles of centrosomes and microtubule self-organization. J Cell Biol. 1997 Aug 11;138(3):615–628. doi: 10.1083/jcb.138.3.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Horio T., Uzawa S., Jung M. K., Oakley B. R., Tanaka K., Yanagida M. The fission yeast gamma-tubulin is essential for mitosis and is localized at microtubule organizing centers. J Cell Sci. 1991 Aug;99(Pt 4):693–700. doi: 10.1242/jcs.99.4.693. [DOI] [PubMed] [Google Scholar]
  20. Jones D. G., Rosamond J. Isolation of a novel protein kinase-encoding gene from yeast by oligodeoxyribonucleotide probing. Gene. 1990 May 31;90(1):87–92. doi: 10.1016/0378-1119(90)90442-t. [DOI] [PubMed] [Google Scholar]
  21. Joshi H. C., Palacios M. J., McNamara L., Cleveland D. W. Gamma-tubulin is a centrosomal protein required for cell cycle-dependent microtubule nucleation. Nature. 1992 Mar 5;356(6364):80–83. doi: 10.1038/356080a0. [DOI] [PubMed] [Google Scholar]
  22. Karsenti E. Mitotic spindle morphogenesis in animal cells. Semin Cell Biol. 1991 Aug;2(4):251–260. [PubMed] [Google Scholar]
  23. Kellogg D. R., Moritz M., Alberts B. M. The centrosome and cellular organization. Annu Rev Biochem. 1994;63:639–674. doi: 10.1146/annurev.bi.63.070194.003231. [DOI] [PubMed] [Google Scholar]
  24. Kimble M., Kuriyama R. Functional components of microtubule-organizing centers. Int Rev Cytol. 1992;136:1–50. doi: 10.1016/s0074-7696(08)62049-5. [DOI] [PubMed] [Google Scholar]
  25. Kochanski R. S., Borisy G. G. Mode of centriole duplication and distribution. J Cell Biol. 1990 May;110(5):1599–1605. doi: 10.1083/jcb.110.5.1599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Lane H. A., Nigg E. A. Antibody microinjection reveals an essential role for human polo-like kinase 1 (Plk1) in the functional maturation of mitotic centrosomes. J Cell Biol. 1996 Dec;135(6 Pt 2):1701–1713. doi: 10.1083/jcb.135.6.1701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lauzé E., Stoelcker B., Luca F. C., Weiss E., Schutz A. R., Winey M. Yeast spindle pole body duplication gene MPS1 encodes an essential dual specificity protein kinase. EMBO J. 1995 Apr 18;14(8):1655–1663. doi: 10.1002/j.1460-2075.1995.tb07154.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lehner C. F., Kurer V., Eppenberger H. M., Nigg E. A. The nuclear lamin protein family in higher vertebrates. Identification of quantitatively minor lamin proteins by monoclonal antibodies. J Biol Chem. 1986 Oct 5;261(28):13293–13301. [PubMed] [Google Scholar]
  30. Letwin K., Mizzen L., Motro B., Ben-David Y., Bernstein A., Pawson T. A mammalian dual specificity protein kinase, Nek1, is related to the NIMA cell cycle regulator and highly expressed in meiotic germ cells. EMBO J. 1992 Oct;11(10):3521–3531. doi: 10.1002/j.1460-2075.1992.tb05435.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Levedakou E. N., He M., Baptist E. W., Craven R. J., Cance W. G., Welcsh P. L., Simmons A., Naylor S. L., Leach R. J., Lewis T. B. Two novel human serine/threonine kinases with homologies to the cell cycle regulating Xenopus MO15, and NIMA kinases: cloning and characterization of their expression pattern. Oncogene. 1994 Jul;9(7):1977–1988. [PubMed] [Google Scholar]
  32. Lu K. P., Hanes S. D., Hunter T. A human peptidyl-prolyl isomerase essential for regulation of mitosis. Nature. 1996 Apr 11;380(6574):544–547. doi: 10.1038/380544a0. [DOI] [PubMed] [Google Scholar]
  33. Lu K. P., Hunter T. Evidence for a NIMA-like mitotic pathway in vertebrate cells. Cell. 1995 May 5;81(3):413–424. doi: 10.1016/0092-8674(95)90394-1. [DOI] [PubMed] [Google Scholar]
  34. Lu K. P., Hunter T. The NIMA kinase: a mitotic regulator in Aspergillus nidulans and vertebrate cells. Prog Cell Cycle Res. 1995;1:187–205. doi: 10.1007/978-1-4615-1809-9_15. [DOI] [PubMed] [Google Scholar]
  35. Moritz M., Braunfeld M. B., Sedat J. W., Alberts B., Agard D. A. Microtubule nucleation by gamma-tubulin-containing rings in the centrosome. Nature. 1995 Dec 7;378(6557):638–640. doi: 10.1038/378638a0. [DOI] [PubMed] [Google Scholar]
  36. Morris N. R. Mitotic mutants of Aspergillus nidulans. Genet Res. 1975 Dec;26(3):237–254. doi: 10.1017/s0016672300016049. [DOI] [PubMed] [Google Scholar]
  37. Moudjou M., Bordes N., Paintrand M., Bornens M. gamma-Tubulin in mammalian cells: the centrosomal and the cytosolic forms. J Cell Sci. 1996 Apr;109(Pt 4):875–887. doi: 10.1242/jcs.109.4.875. [DOI] [PubMed] [Google Scholar]
  38. Moudjou M., Paintrand M., Vigues B., Bornens M. A human centrosomal protein is immunologically related to basal body-associated proteins from lower eucaryotes and is involved in the nucleation of microtubules. J Cell Biol. 1991 Oct;115(1):129–140. doi: 10.1083/jcb.115.1.129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. O'Connell M. J., Norbury C., Nurse P. Premature chromatin condensation upon accumulation of NIMA. EMBO J. 1994 Oct 17;13(20):4926–4937. doi: 10.1002/j.1460-2075.1994.tb06820.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Oakley B. R., Oakley C. E., Yoon Y., Jung M. K. Gamma-tubulin is a component of the spindle pole body that is essential for microtubule function in Aspergillus nidulans. Cell. 1990 Jun 29;61(7):1289–1301. doi: 10.1016/0092-8674(90)90693-9. [DOI] [PubMed] [Google Scholar]
  41. Oegema K., Whitfield W. G., Alberts B. The cell cycle-dependent localization of the CP190 centrosomal protein is determined by the coordinate action of two separable domains. J Cell Biol. 1995 Dec;131(5):1261–1273. doi: 10.1083/jcb.131.5.1261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Ohta Y., Ohba T., Miyamoto E. Ca2+/calmodulin-dependent protein kinase II: localization in the interphase nucleus and the mitotic apparatus of mammalian cells. Proc Natl Acad Sci U S A. 1990 Jul;87(14):5341–5345. doi: 10.1073/pnas.87.14.5341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Osmani A. H., McGuire S. L., Osmani S. A. Parallel activation of the NIMA and p34cdc2 cell cycle-regulated protein kinases is required to initiate mitosis in A. nidulans. Cell. 1991 Oct 18;67(2):283–291. doi: 10.1016/0092-8674(91)90180-7. [DOI] [PubMed] [Google Scholar]
  44. Osmani A. H., O'Donnell K., Pu R. T., Osmani S. A. Activation of the nimA protein kinase plays a unique role during mitosis that cannot be bypassed by absence of the bimE checkpoint. EMBO J. 1991 Sep;10(9):2669–2679. doi: 10.1002/j.1460-2075.1991.tb07810.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Osmani S. A., Pu R. T., Morris N. R. Mitotic induction and maintenance by overexpression of a G2-specific gene that encodes a potential protein kinase. Cell. 1988 Apr 22;53(2):237–244. doi: 10.1016/0092-8674(88)90385-6. [DOI] [PubMed] [Google Scholar]
  46. Osmani S. A., Ye X. S. Cell cycle regulation in Aspergillus by two protein kinases. Biochem J. 1996 Aug 1;317(Pt 3):633–641. doi: 10.1042/bj3170633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Paoletti A., Moudjou M., Paintrand M., Salisbury J. L., Bornens M. Most of centrin in animal cells is not centrosome-associated and centrosomal centrin is confined to the distal lumen of centrioles. J Cell Sci. 1996 Dec;109(Pt 13):3089–3102. doi: 10.1242/jcs.109.13.3089. [DOI] [PubMed] [Google Scholar]
  48. Pu R. T., Xu G., Wu L., Vierula J., O'Donnell K., Ye X. S., Osmani S. A. Isolation of a functional homolog of the cell cycle-specific NIMA protein kinase of Aspergillus nidulans and functional analysis of conserved residues. J Biol Chem. 1995 Jul 28;270(30):18110–18116. doi: 10.1074/jbc.270.30.18110. [DOI] [PubMed] [Google Scholar]
  49. Raff J. W., Kellogg D. R., Alberts B. M. Drosophila gamma-tubulin is part of a complex containing two previously identified centrosomal MAPs. J Cell Biol. 1993 May;121(4):823–835. doi: 10.1083/jcb.121.4.823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Rhee K., Wolgemuth D. J. The NIMA-related kinase 2, Nek2, is expressed in specific stages of the meiotic cell cycle and associates with meiotic chromosomes. Development. 1997 Jun;124(11):2167–2177. doi: 10.1242/dev.124.11.2167. [DOI] [PubMed] [Google Scholar]
  51. Sawin K. E., LeGuellec K., Philippe M., Mitchison T. J. Mitotic spindle organization by a plus-end-directed microtubule motor. Nature. 1992 Oct 8;359(6395):540–543. doi: 10.1038/359540a0. [DOI] [PubMed] [Google Scholar]
  52. Schliwa M., Pryzwansky K. B., Euteneuer U. Centrosome splitting in neutrophils: an unusual phenomenon related to cell activation and motility. Cell. 1982 Dec;31(3 Pt 2):705–717. doi: 10.1016/0092-8674(82)90325-7. [DOI] [PubMed] [Google Scholar]
  53. Schmidt-Zachmann M. S., Nigg E. A. Protein localization to the nucleolus: a search for targeting domains in nucleolin. J Cell Sci. 1993 Jul;105(Pt 3):799–806. doi: 10.1242/jcs.105.3.799. [DOI] [PubMed] [Google Scholar]
  54. Schultz S. J., Fry A. M., Sütterlin C., Ried T., Nigg E. A. Cell cycle-dependent expression of Nek2, a novel human protein kinase related to the NIMA mitotic regulator of Aspergillus nidulans. Cell Growth Differ. 1994 Jun;5(6):625–635. [PubMed] [Google Scholar]
  55. 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]
  56. Schweitzer B., Philippsen P. NPK1, a nonessential protein kinase gene in Saccharomyces cerevisiae with similarity to Aspergillus nidulans nimA. Mol Gen Genet. 1992 Jul;234(1):164–167. doi: 10.1007/BF00272358. [DOI] [PubMed] [Google Scholar]
  57. Sherline P., Mascardo R. Epidermal growth factor-induced centrosomal separation: mechanism and relationship to mitogenesis. J Cell Biol. 1982 Oct;95(1):316–322. doi: 10.1083/jcb.95.1.316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Spiegelman B. M., Lopata M. A., Kirschner M. W. Multiple sites for the initiation of microtubule assembly in mammalian cells. Cell. 1979 Feb;16(2):239–252. doi: 10.1016/0092-8674(79)90002-3. [DOI] [PubMed] [Google Scholar]
  59. Stearns T., Evans L., Kirschner M. Gamma-tubulin is a highly conserved component of the centrosome. Cell. 1991 May 31;65(5):825–836. doi: 10.1016/0092-8674(91)90390-k. [DOI] [PubMed] [Google Scholar]
  60. Stearns T., Kirschner M. In vitro reconstitution of centrosome assembly and function: the central role of gamma-tubulin. Cell. 1994 Feb 25;76(4):623–637. doi: 10.1016/0092-8674(94)90503-7. [DOI] [PubMed] [Google Scholar]
  61. Tassan J. P., Schultz S. J., Bartek J., Nigg E. A. Cell cycle analysis of the activity, subcellular localization, and subunit composition of human CAK (CDK-activating kinase). J Cell Biol. 1994 Oct;127(2):467–478. doi: 10.1083/jcb.127.2.467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. 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]
  63. 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]
  64. Walczak C. E., Mitchison T. J. Kinesin-related proteins at mitotic spindle poles: function and regulation. Cell. 1996 Jun 28;85(7):943–946. doi: 10.1016/s0092-8674(00)81295-7. [DOI] [PubMed] [Google Scholar]
  65. Winey M., Byers B. Assembly and functions of the spindle pole body in budding yeast. Trends Genet. 1993 Sep;9(9):300–304. doi: 10.1016/0168-9525(93)90247-f. [DOI] [PubMed] [Google Scholar]
  66. Ye X. S., Xu G., Pu R. T., Fincher R. R., McGuire S. L., Osmani A. H., Osmani S. A. The NIMA protein kinase is hyperphosphorylated and activated downstream of p34cdc2/cyclin B: coordination of two mitosis promoting kinases. EMBO J. 1995 Mar 1;14(5):986–994. doi: 10.1002/j.1460-2075.1995.tb07079.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Zheng Y., Jung M. K., Oakley B. R. Gamma-tubulin is present in Drosophila melanogaster and Homo sapiens and is associated with the centrosome. Cell. 1991 May 31;65(5):817–823. doi: 10.1016/0092-8674(91)90389-g. [DOI] [PubMed] [Google Scholar]
  68. Zheng Y., Wong M. L., Alberts B., Mitchison T. Nucleation of microtubule assembly by a gamma-tubulin-containing ring complex. Nature. 1995 Dec 7;378(6557):578–583. doi: 10.1038/378578a0. [DOI] [PubMed] [Google Scholar]
  69. van den Heuvel S., Harlow E. Distinct roles for cyclin-dependent kinases in cell cycle control. Science. 1993 Dec 24;262(5142):2050–2054. doi: 10.1126/science.8266103. [DOI] [PubMed] [Google Scholar]

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