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


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.


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



We thank Oda Weiss for the excellent technical support. This research was supported by grants from the Czech Science Foundation (14-28443S) and the National Program of Sustainability I (LO1204); Internal Grant Agency of Palacky University, Olomouc (IGA_PrF_2014001, PrF-2013-003) and Interdoc (OPVK-CZ.1.07/2.4.00/17.0008) for ET and HJ. DD and AH are supported by the DFG (SFB 648). Grant from the Czech Science Foundation P501-12-2333 for PB. We thank Dr. H. P. Mock for helpful discussion about protein purification.

Supplementary material

11105_2015_890_MOESM1_ESM.pdf (14.8 mb)
Figure S1 Analyses 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 S2 Analyses 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 S3 Alignment 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 S4 Analyses 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 S5 Analyses 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)


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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

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