Abstract
TPX2 (Targeting Protein for Xklp2) is an evolutionary conserved microtubule-associated protein important for microtubule nucleation and mitotic spindle assembly. The protein was described as an activator of the mitotic kinase Aurora A in humans and the Arabidopsis AURORA1 (AUR1) kinase. In contrast to animal genomes that encode only one TPX2 gene, higher plant genomes encode a family with several TPX2-LIKE gene members (TPXL). TPXL genes of Arabidopsis can be divided into two groups. Group A proteins (TPXL2, 3, 4, and 8) contain Aurora binding and TPX2_importin domains, while group B proteins (TPXL1, 5, 6, and 7) harbor an Xklp2 domain. Canonical TPX2 contains all the above-mentioned domains. We confirmed using in vitro kinase assays that the group A proteins contain a functional Aurora kinase binding domain. Transient expression of Arabidopsis TPX2-like proteins in Nicotiana benthamiana revealed preferential localization to microtubules and nuclei. Co-expression of AUR1 together with TPX2-like proteins changed the localization of AUR1, indicating that these proteins serve as targeting factors for Aurora kinases. Taken together, we visualize the various localizations of the TPX2-LIKE family in Arabidopsis as a proxy to their functional divergence and provide evidence of their role in the targeted regulation of AUR1 kinase activity.
Highlights
Cell cycle progression and timing of events are crucial for cell division and viability
The Aurora kinase family of Arabidopsis thaliana contains three members that are subdivided into two groups: α (AURORA1 and AURORA2) and β (AURORA3). α-Aurora kinases are localized at spindle microtubules during mitosis and cell division plate in cytokinesis, while β-Aurora is localized at centromeres during metaphase [3,4,5]
The in vitro kinase assay showed that Aurora binding domains of all Targeting Protein for Xklp2 (TPX2)-LIKE gene members (TPXL) proteins can activate AUR1 kinase (Figure 2C, Figure S3B)
Summary
Cell cycle progression and timing of events are crucial for cell division and viability. Similar to the human TPX2, Arabidopsis canonical TPX2 contains three conserved domains—the Aurora binding domain responsible for binding and activation of Aurora kinase, the TPX2_importin domain involved in the transfer of protein to the cell nuclei, and the TPX2_Xklp motif for binding to microtubules. The first members of the TPX2-LIKE PROTEIN (TPXL) gene family in Arabidopsis, TPXL2 and TPXL3, were functionally characterized only recently [18]. Based on in silico analyses we identified a group of TPXL proteins with a predicted Aurora kinase binding domain. In vitro kinase assays demonstrated that the Aurora binding domains of Arabidopsis TPXL homologs can activate recombinant AUR1. Each clade of plant TPX2 and TPXL proteins were divided into two subclades containing monocots and eudicots (Figure S2), there seems to be not a specific paralog for any of these groups
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