The A. thaliana genome possesses 20 genes with similarity to TPX2
The EggNOG 4.5 database (20) was searched to discover proteins that contain typical TPX2- domains in Arabidopsis. In total, 20 proteins were identified, which can be classified into two groups based on their domain composition (Figure 1A, Additional File 1, 3). Group A comprises members with the TPX2_importin domain, while group B members contain the TPX2_Xklp2 domain.
We performed a maximum likelihood phylogenetic analysis with the identified proteins. In agreement with the domain composition, the phylogeny (Figure 1A; Additional File 3) revealed two major groups, A and B. Group A could be further divided into two clusters based on the absence (cluster I) or presence (cluster II) of a plant-specific KLEEK-motif. The KLEEK-motif is present in an already characterized group of WAVE-DAMPENED (WVD) and WAVE-DAMPENED-LIKE (WDL) proteins (21,22) and is typical for microtubule binding proteins (23) (Additional File 3).
The deepest clades (Additional File 2) comprised Arabidopsis orthologues of TPXL1, TPXL5, TPXL6 and TPXL7 (clades 1, 2 and 3 in (19)). It should be noted that TPXL7 formed a separate clade with only protein sequence from A. thaliana. Subsequently derived clade was composed of canonical TPX2 and TPXL2, TPXL3 and TPXL8. The canonical TPX2 clade (clade 3 after (19)) was subdivided into 5 groups representing metazoa, angiosperms, mosses, algae and fungi. Analyzed representatives of metazoa, algae and fungi contain a single copy of the canonical TPX2 (Additional File 3).
The group of plant-specific proteins with a KLEEK domain (WDLs) contains three major lineages. The first lineage consists of WDLs 5, 7, 9 and TPXL proteins. The second lineage contains WDLs 1, 2 and 3. Finally, the third lineage embraced WDLs 4, 5 and 6. Special attention should be paid to the moss Physcomitrella patens, which evolved a unique WDL paralog group (Additional File 3). Interestingly, the Aurora binding domain containing TPXL4 was grouped together with the WDL lineage (Additional File 3).
Each clade of plant TPX2 and TPXL proteins was divided into two sublades containing monocots and eudicots (Additional File 3), thus, there seems to be not a specific paralog for any of these groups.
Functional prediction of Arabidopsis Aurora binding domain
Activation of Aurora A kinases is a significant function of TPX2 proteins. Binding of TPX2 to human Aurora A activates the phosphorylation activity of the kinase and protects it from dephosphorylation by protein phosphatase 2A (6). Similarly, Aurora kinase binding domain of canonical Arabidopsis TPX2 is able to activate Aurora1 in vitro (15). Recently we showed that Aurora kinase binding domains of TPXL2 and TPLX3 also are able to activate Aurora1 in vitro (18). To characterize the Aurora binding domain of plant group A TPXL proteins, a multiple sequence alignment was performed with orthologues of Arabidopsis TPX2. Additionally to TPX2 the Arabidopsis genome contains four genes, TPXL2, TPXL3, TPXL4 and TPXL8 with both TPX2_importin and putative Aurora kinase binding domains (Figure 1).
The overall sequence conservation is very poor between human TPX2 and plant TPX2-like proteins, but the key residues important for binding of Aurora kinases are evolutionary conserved (Figure 2A, 2B). The Aurora binding domain of human TPX2 contains upstream and downstream helical stretches (6). Despite some amino acid substitutions in the upstream helical stretch of TPXL4/TPXL8 or TPXL2/TPXL3 compared to human TPX2, the hydrophobic side chain involved in stacking interactions is preserved (Figure 2A, aminoacid 8). Similarly, the amino acid residues important for Aurora kinase activation (Figure 2A, aminoacids 34, 35) (6) are conserved, further indicating the functional conservation of the TPX2/Aurora kinase complex. Importantly, MEME (Multiple Em for Motif Elicitation; (24)) analyses confirmed the presence of a similar Aurora binding domain with key conserved residues in 45 proteins from different plants species (Figure 2B, Additional File 4).
All Arabidopsis TPX2 family members possessing an Aurora kinase binding domain activate AUR1 in vitro
The Aurora kinase binding domains of canonical Arabidopsis TPX2, TPXL2 and TPXL3 were shown to activate Aurora1 in vitro and increase its phosphorylation activity towards histone H3 as a physiological substrate (15,18). To address the functionality of the Aurora kinase binding domains of Arabidopsis TPXL proteins, we performed in vitro kinase assays. The Aurora binding domains of TPXL2, TPXL3, TPXL4 and TPXL8 were expressed in E. coli, purified (Additional File 5A) and combined with recombinant AUR1 as enzyme and histone H3 as a substrate. An increase in histone H3 phosphorylation detected by incorporation of radioactive isotope P32 into histone H3 was used as a means to measure the activity of AUR1 (26). The in vitro kinase assay showed that Aurora binding domains of all TPXL proteins are able to activate AUR1 kinase (Figure 2C, Additional File 5B). TPXL3 has the highest activation potential with up to 5-fold increase compared to Aurora1 kinase alone, which was significantly higher than AUR1 activation by canonical TPX2 (Figure 2C).
The eight closest homologues of canonical TPX2 are differentially expressed during Arabidopsis development
In order to investigate the expression of TPXL genes, we analyzed publicly available RNA sequencing data from different developmental stages of Arabidopsis (25). To profile gene expression patterns of selected TPXL genes, we analyzed the expression across all developmental stages (Figure 3, Additional File 5). These heat maps illustrate distinct gene expression of TPXLs during development. TPXL2, TPXL3, TPXL5 and TPX2 seem to be the most widely expressed TPXLs. In general, expression of TPX2 was among the highest in all tissues. Strikingly, TPXL4 was considered as pseudogene (17); however, transcriptome analyses showed specific expression of TPXL4 in mature anthers. TPXL6 expression was restricted to siliques and TPXL7 was only detected in dry seeds. Taken together, our data confirmed the validity of our hypothesis that TPXL genes might have evolved different functions during plant development. Similar expression patterns for TPX2, TPXL2, TPXL3 and TPXL5 point to possible functional redundancy (18). Moreover, these four proteins are expressed in a similar pattern to Aurora1 and Aurora2 in agreement with the fact that they are physiological activators of the kinases.
TPXL proteins mostly localize on microtubules
The canonical TPX2 was shown to localize on microtubular arrays and the TPX2_Xklp2 domain is involved in microtubule binding (14,17). TPXL proteins of group A (Figure 1) contain a TPX2_importin domain important to interact with alpha importin (26) and an Aurora binding domain. Group B proteins contain a TPX2_Xklp2 domain with a kinesin-targeting signature. To uncover the localization patterns of the selected TPXL proteins, constructs to express fluorescently-labelled translational fusions were infiltrated into N. benthamiana leaves and visualized by confocal microscopy. Consistent with the presence of a TPX2_importin and an Aurora kinase binding domain, group A TPXL proteins showed strong microtubular labelling at the nuclei (Figure 4A). Moreover, the proteins also faintly labelled cortical microtubules (Figure 4A). It should be noted that two members group A TPXL2 and TPXL3 labelled microtubular fibers decorating the nuclear envelope like canonical TPX2 (Figure 4A). Group B TPXL proteins mainly localized with cortical microtubules, TPXL1, TPXL5 and TPXL6 showed very strong labelling resembling cytoskeletal filaments (Figure 4B). TPXL5 also labelled microtubules close to the nucleus (Figure 4B). Interestingly, TPXL7 showed a different localization pattern compared to all other TPXL proteins. TPXL7 lacks microtubular localization and mainly localized in the vicinity of the nuclear membrane (Figure 4B). The canonical TPX2 decorates cytoskeletal cables and bundles of microtubules around nuclei (Figure 4C). These results indicate that TPXL gene family has probably evolved differential targeting and different functions in the regulation of microtubule cytoskeletal dynamics.
TPXL proteins re-localize Aurora1 kinase by loading it on microtubular arrays
During interphase, AUR1 kinase is localized in in very low amounts in the nucleus, while the microtubular localization of AUR1 is a hallmark of cell division (5,8). To check whether TPXL proteins co-localize with AUR1, DNA constructs of fluorescently-labelled fusion variants of AUR1 and TPXL were co-infiltrated into N. benthamiana leaves. Normally, AUR1 shows diffuse nuclear and weak cytoplasmic labelling in infiltrated N. benthamiana epidermal cells (Figure 4D). Interestingly, after co-infiltration with TPX2 construct, AUR1-GFP is mostly localized on cortical microtubules (Figure 4G). Co-expression of TPXL proteins also re-localized AUR1. TPX2, TPXL1, TPXL2, TPXL3, TPXL4, TPXL6 and TPXL8 re-localize AUR to the nucleus as well as to cortical microtubules, while TPXL4 and TPXL5 relocalize it to microtubules (Figure 4E, 4F, 4G). Interestingly, co-expression of TPXL proteins with AUR1 not only changed the localization of the kinase, but also re-localized some TPXL proteins. The most striking redistribution was observed for TPXL7, which shared a strong nuclear localization with AUR1. These results indicate that co-localization of TPXL proteins with AUR1 is not only dependent on the presence of a functional Aurora binding domain, but other mechanisms must exist to regulate these proteins.