Plant Molecular Biology Reporter

, Volume 33, Issue 6, pp 1988–1995 | Cite as

TPX2 Protein of Arabidopsis Activates Aurora Kinase 1, But Not Aurora Kinase 3 In Vitro

  • Eva Tomaštíková
  • Dmitri Demidov
  • Hana Jeřábková
  • Pavla Binarová
  • Andreas Houben
  • Jaroslav Doležel
  • Beáta Petrovská
Original Paper

Abstract

Aurora kinases are involved in various mitotic events, including chromosome segregation and bipolar mitotic spindle assembly. In animals, Aurora A is activated and protected by microtubule-associated protein TPX2. Such role in plants is not known. Here, we have assessed the ability of TPX2 of Arabidopsis to regulate Aurora family members in vitro. AtTPX2 acts as substrate as well as activator of AtAurora1, but not AtAurora3. Truncated version of AtTPX2 lacking the Aurora binding domain is unable to activate the kinases; however, it is still phosphorylated. AtTPX2-induced activation of AtAurora1 results in a dramatically increased phosphorylation level of downstream targets, particularly histone H3. The differences in the activation mechanism of AtAurora1 and 3 point to a specific regulation of both kinases, which may play an important role in cell cycle regulation and signaling cascade transduction in plants.

Keywords

Aurora kinase Targeting protein for Xklp2 In vitro kinase assay Kinase activation 

Supplementary material

11105_2015_890_MOESM1_ESM.pdf (14.8 mb)
Figure S1Analyses of putative TPX2-interaction motif in AtAurora1 plant homologues. Multiple sequence alignment revealed high level of similarity in the TPX2 interaction motif between AtAurora1 and its plant homologues. Black arrows indicate the conserved amino acids. Blue arrows indicate the amino acids involved in interaction with TPX2 which are not conserved. (PDF 15183 kb)
11105_2015_890_MOESM2_ESM.pdf (13 mb)
Figure S2Analyses of putative TPX2-interaction motif in AtAurora3 plant homologues. Multiple sequence alignment revealed high level of similarity in the TPX2 interaction motif between AtAurora3 and its plant homologues. Black arrows indicate the conserved amino acids. Blue arrows indicate the amino acids involved in interaction with TPX2 which are not conserved. Red frame indicated differences between AtAurora 1 and AtAurora3 homologues. (PDF 13295 kb)
11105_2015_890_MOESM3_ESM.pdf (3.2 mb)
Figure S3Alignment of Arabidopsis and human Aurora kinases. Multiple sequence alignment revealed high level of similarity in the TPX2 interaction motif between Arabidopsis and human Aurora kinase genes. Black arrows indicate conserved amino acids at potential TPX2-interaction region. Blue arrows indicate the amino acids which are not conserved. In the red frame are amino acids different between AtAurora1 and AtAurora3. (PDF 3245 kb)
11105_2015_890_MOESM4_ESM.pdf (7.6 mb)
Figure S4Analyses of putative AuroraA-binding sites in AtTPX2 plant homologues. Multiple sequence alignment revealed high level of similarity in the AuroraA binding site of AtTPX2 and its plant homologues. Black arrows indicate conserved amino acids for AuroraA binding region, based on analyses of non-plant TPX2 proteins. Green arrows indicate conserved amino acids exclusively in plant TPX2 Aurora1 binding region. (PDF 7743 kb)
11105_2015_890_MOESM5_ESM.pdf (3.8 mb)
Figure S5Analyses of putative AuroraA-binding sites in Arabidopsis TPX2-related proteins. Multiple sequence alignment revealed absence of AuroraA-interacting region in different splicing variants of TPX2-related proteins. (PDF 3924 kb)
11105_2015_890_Fig5_ESM.gif (60 kb)
Figure S6

Expression and purification of recombinant proteins. a – SDS-PAGE separation of purified MBP-tagged AtAurora3 (lane 1) and GST-tagged AtAurora1 (lane 2) after Commasie Brilliant Blue (CBB) staining. The positions and respective size of recombinant proteins are indicated. b – SDS-PAGE separation of purified His-tagged AtTPX2ΔN (truncated version of AtTPX2 without Aurora binding domain) and full length AtTPX2 protein. The positions and sizes of recombinant proteins are indicated. Degradation products of AtTPX2 are marked with asterisks. b – SDS-PAGE separation of purified His-tagged At histone H3 after CBB staining. (GIF 59 kb)

11105_2015_890_MOESM6_ESM.tif (1.2 mb)
High Resolution Image (GIF 171 kb) (TIFF 1228 kb)
11105_2015_890_Fig6_ESM.gif (171 kb)
Figure S7

In vitro activation of AtAurora1 by AtTPX2 causes increased phosphorylation of downstream targets. a – In vitro phosphorylation of histone H3 is markedly increased after activation of AtAurora1 by AtTPX2. Comparison of AtAurora1 activity without (lane 3) and with (lane 7) addition of AtTPX2 as kinase coactivator. b – In vitro phosphorylation of histone H3 is not increased after AtAurora3 incubation with AtTPX2. (GIF 171 kb)

11105_2015_890_MOESM7_ESM.tif (7.4 mb)
High Resolution Image (TIFF 7535 kb)

References

  1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410PubMedCrossRefGoogle Scholar
  2. Bayliss R, Sardon T, Vernos I, Conti E (2003) Structural basis of Aurora-A activation by TPX2 at the mitotic spindle. Mol Cell 12:851–862PubMedCrossRefGoogle Scholar
  3. Bibby RA, Tang C, Faisal A, Drosopoulos K, Lubbe S, Houlston R, Bayliss R, Linardopoulos S (2009) A cancer-associated Aurora A mutant is mislocalized and misregulated due to loss of interaction with TPX2. J Biol Chem 284:33177–33184PubMedPubMedCentralCrossRefGoogle Scholar
  4. Binarová P, Cenklová V, Procházková J, Doskočilová A, Volc J, Vrlík M, Bögre L (2006) Gamma-tubulin is essential for acentrosomal microtubule nucleation and coordination of late mitotic events in Arabidopsis. Plant Cell 18:1199–1212PubMedPubMedCentralCrossRefGoogle Scholar
  5. Demidov D, Van Damme D, Geelen D, Blattner FR, Houben A (2005) Identification and dynamics of two classes of aurora-like kinases in Arabidopsis and other plants. Plant Cell 17:836–848PubMedPubMedCentralCrossRefGoogle Scholar
  6. Demidov D, Hesse S, Tewes A, Rutten T, Fuchs J, Ashtiyani RK, Lein S, Fischer A, Reuter G, Houben A (2009) Aurora1 phosphorylation activity on histone H3 and its cross-talk with other post-translational histone modifications in Arabidopsis. Plant J 59:221–230PubMedCrossRefGoogle Scholar
  7. Evrard JL, Pieichot L, Vos JW, Vernos I, Schmit AC (2009) Plant TPX2 and related proteins. Plant Signal Behav 4:69–72PubMedPubMedCentralCrossRefGoogle Scholar
  8. Eyers PA, Erikson E, Chen LG, Maller JL (2003) A novel mechanism for activation of the protein kinase Aurora A. Curr Biol 13:691–697PubMedCrossRefGoogle Scholar
  9. Gruss OJ, Vernos I (2004) The mechanism of spindle assembly: functions of Ran and its target TPX2. J Cell Biol 166:949–955PubMedPubMedCentralCrossRefGoogle Scholar
  10. Iyer J, Tsai MY (2012) A novel role for TPX2 as a scaffold and co-activator protein of the chromosomal passenger complex. Cell Signal 2012(24):1677–1689CrossRefGoogle Scholar
  11. Karsenti E, Vernos I (2001) The mitotic spindle: a self-made machine. Science 294:543–547PubMedCrossRefGoogle Scholar
  12. Katayama H, Sasai K, Kloc M, Brinkley BR, Sen S (2008) Aurora kinase-A regulates kinetochore/chromatin associated mikrotubule assembly in human cells. Cell Cycle 7:2691–2704PubMedCrossRefGoogle Scholar
  13. Kawabe A, Matsunaga S, Nakagawa K, Kurihara D, Yoneda A, Hasezawa S, Uchiyama S, Fukui K (2005) Characterization of plant Aurora kinases during mitosis. Plant Mol Biol 58:1–13PubMedCrossRefGoogle Scholar
  14. Kufer TA, Silljé HH, Körner R, Gruss OJ, Meraldi P, Nigg EA (2002) Human TPX2 is required for targeting Aurora-A kinase to the spindle. J Cell Biol 158:617–623PubMedPubMedCentralCrossRefGoogle Scholar
  15. Kurihara D, Matsunaga S, Kawabe A, Fujimoto S, Noda M, Uchiyama S, Fukui K (2006) Aurora kinase is required for chromosome segregation in tobacco BY-2 cells. Plant J 48:572–580PubMedCrossRefGoogle Scholar
  16. Kurihara D, Sachihiro M, Uchiyama S, Fukui K (2008) Live cell imaging reveals plant aurora kinase has dual roles during mitosis. Plant Cell Physiol 49:1256–1261Google Scholar
  17. Marumoto T, Zhang D, Saya H (2005) Aurora A — a guardian of poles. Nat Rev Cancer 5:42–50PubMedCrossRefGoogle Scholar
  18. Murata T, Sonobe S, Baskin TI, Hyodo S, Hasezawa S, Nagata T, Horio T, Hasebe M (2005) Microtubule-dependent microtubule nucleation based on recruitment of gamma-tubulin in higher plants. Nat Cell Biol 7:961–968PubMedCrossRefGoogle Scholar
  19. Neumayer G, Helfricht A, Shim SY, Le HT, Lundin C, Belzil C, Chansard M, Yu Y, Lees-Miller SP, Gruss OJ, can Attikum H, Helleday T, Nguyen MD (2012) Targeting protein for xenopus kinesin-like protein 2 (TPX2) regulates gamma histone 2AX (gamma-H2AX) levels upon ionizing radiation. J Biol Chem 287:42206–42222PubMedPubMedCentralCrossRefGoogle Scholar
  20. Notredame C, Higgins DG, Heringa J (2000) T-Coffee: a novel method for fast and accurate multiple sequence alignment. J Mol Biol 302:205–217PubMedCrossRefGoogle Scholar
  21. Petrovská B, Cenklová V, Pochylová Ž, Kourová H, Doskočilová A, Plíhal O, Binarová L, Binarová P (2012) Plant Aurora kinases play a role in maintenance of primary meristems and control of endoreduplication. New Phytol 193:590–604PubMedCrossRefGoogle Scholar
  22. Petrovská B, Jeřábková H, Chamrád I, Vrána J, Lenobel R, Uřinovská J, Šebela M, Doležel J (2014) Proteomic analyses of barley cell nuclei purified by flow sorting. Cytogenet. Genome Res 143:78–86Google Scholar
  23. Petrovská B, Jeřábková H, Kohoutová L, Cenklová V, Pochylová Ž, Gelová Z, Kočárová G, Váchová L, Kurejová M, Tomaštíková E, Binarová P (2013) Overexpressed TPX2 causes ectopic formation of microtubular arrays in the nuclei of acentrosomal plant cells. J Exp Bot 64:4575–4587PubMedPubMedCentralCrossRefGoogle Scholar
  24. Stewart S, Fang G (2005) Anaphase promoting complex/cyclosome controls the stability of TPX2 during mitotic exit. Mol Cell Biol 25:10516–10527PubMedPubMedCentralCrossRefGoogle Scholar
  25. Tsai MY, Zheng Y (2005) Aurora A kinase-coated beads function as microtubule-organizing centers and enhance RanGTP-induced spindle assembly. Curr Biol 15:2156–2163PubMedCrossRefGoogle Scholar
  26. Van Damme D, De Rybel B, Gudesblat G, Demidov D, Grunewald W, De Smet I, Houben A, Beeckman T, Russinova E (2011) Arabidopsis alpha Aurora kinases function in formative cell division plane orientation. Plant Cell 23:4013–4024PubMedPubMedCentralCrossRefGoogle Scholar
  27. Vos JW, Pieuchot L, Evrard JL, Janski N, Bergdoll M, De Ronde D, Perez LH, Sardon T, Vernos I, Schmit AC (2008) The plant TPX2 protein regulates prospindle assembly before nuclear envelope breakdown. Plant Cell 20:2783–2797PubMedPubMedCentralCrossRefGoogle Scholar
  28. Walter AO, Seghezzi W, Korver W, Sheung J, Lees E (2000) The mitotic serine/threonine kinase Aurora2/AIK is regulated by phosphorylation and degradation. Oncogene 19:4906–4916PubMedCrossRefGoogle Scholar
  29. Wittmann T, Wilm M, Karsenti E, Vernos I (2000) TPX2, A novel xenopus MAP involved in spindle pole organization. J Cell Biol 149:1405–1418PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Eva Tomaštíková
    • 1
  • Dmitri Demidov
    • 2
  • Hana Jeřábková
    • 1
  • Pavla Binarová
    • 3
  • Andreas Houben
    • 2
  • Jaroslav Doležel
    • 1
  • Beáta Petrovská
    • 1
  1. 1.Centre of the Region Haná for Biotechnological and Agricultural ResearchInstitute of Experimental BotanyOlomoucCzech Republic
  2. 2.Leibniz Institute of Plant Genetics and Crop Plant ResearchGaterslebenGermany
  3. 3.Institute of MicrobiologyPrague 4Czech Republic

Personalised recommendations