P. A. Karpov

Three BUB1 and BUBR1/MAD3-related spindle assembly checkpoint proteins are required for accurate mitosis in Arabidopsis.

The spindle assembly checkpoint (SAC) is a refined surveillance mechanism which ensures that chromosomes undergoing mitosis do not segregate until they are properly attached to the spindle microtubules (MT). The SAC has been extensively studied in metazoans and yeast, but little is known about its role in plants. We identified proteins interacting with a MT-associated protein MAP65-3, which plays a critical role in organising mitotic MT arrays, and carried out a functional analysis of previously and newly identified SAC components. We show that Arabidopsis SAC proteins BUB3.1, MAD2, BUBR1/MAD3s and BRK1 interact with each other and with MAP65-3. We found that two BUBR1/MAD3s interacted specifically at centromeres. When stably expressed in Arabidopsis, BRK1 localised to the kinetochores during all stages of the mitotic cell cycle. Early in mitosis, BUB3.1 and BUBR1/MAD3.1 localise to the mitotic spindle, where MAP65-3 organises spindle MTs. A double-knockout mad3.1 mad3.2 mutant presented spindle MT abnormalities, chromosome misalignments on the metaphase plate and the production of lagging chromosomes and micronuclei during mitosis. We conclude that BRK1 and BUBR1/MAD3-related proteins play a key role in ensuring faithful chromosome segregation during mitosis and that their interaction with MAP65-3 may be important for the regulation of MT-chromosome attachment.

Bioinformatic Search For Plant Homologues Of Animal Structural Maps In The Arabidopsis Thaliana Genome

Although various plant microtubule-associated proteins (MAPs) have been described because of their role in modulating microtubule functions, the full complement of these important sequences has not yet been described. Taking into account the high level of homology between animal and plant tubulins, and the potential conservation of their MAP binding domains, we carried out a bioinformatic investigation of the MAP1, MAP2, tau protein, and MAP4 families from animals and humans. The gene-identification strategy, utilizing site-based and comparative methods, allowed us to identify amino acid motifs that are conserved within each MAP family. According to these amino acid motifs we designed corresponding nucleotide motifs in IUPAC code, accommodating potential codon polymorphism by applying the universal symbols representing all possible substitutions. Scanning the complete sequences of all five Arabidopsis thaliana chromosomes, we identified candidate coding sequences for plant homologues of animal structural MAPs. Furthermore, we identified 200 loci (i.e., 178 of MAP1 and 22 of MAP2 and tau) that are of interest as potential chromosomal regions for plant structural MAPs. Interestingly, consensus regions for MAP4 were not identified in A. thaliana. Chromosomal locations for three previously known plant homologues of MAP1 (i.e., AtEB1a, AtEB1b and AtEB1c) were also identified in the A. thaliana genome.

Docking small ligands to molecule of the plant FtsZ protein: Application of the CUDA technology for faster computations

The opportunities to apply the CUDA technology for faster computations in structural biology and bioinformatics are reviewed and analyzed. Using HEX 6.1 software, we performed a comparative analysis of the efficiency and the increase in quality after CUDA application. The work was conducted on the example of rigid docking of low-molecular-weight compounds of different classes on the surface of the Arabidopsis thaliana FtsZ. Several potential binding sites of benzimidazoles to the plant FtsZ were identified.

Results of the clusterization of human microtubule and cell-cycle related serine/threonine protein kinases and their plant homologues

We identified 191 plant homologues of human protein kinases involved in the phosphorylation of microtubular proteins and cell-cycle regulation. Using neighbor-joining, the similarity of plant protein kinases was analyzed.

Bioinformatic Search for Plant Homologs of the Protein Kinase Bub1—a Key Component of the Mitotic Spindle Assembly Checkpoint

The bioinformatic search identified 14 plant homologs of the mitotic spindle assembly checkpoint protein kinase Bub1 of animals, yeasts, and myxomycetes. It was demonstrated that the closest plant homologs of the protein kinase Bub1 were proteins with previously unknown features: XP_002274770.1 (CBI21878.1) from Vitis vinifera, EEC82122.1 from Oryza sativa Indica, EEE67244.1 from O. sativa Japonica, EEF44403.1 from Ricinus communis, and CAL57156.1 from Ostreococcus tauri. The simulation and analysis of spatial structures of the catalytic domains of the EEC82122.1, EEE67244.1, and XP_002274770.1 (CBI21878.1) proteins confirmed their conformity with the spindle assembly checkpoint protein kinases Bub1.

Bioinformatic search of plant protein kinases involved in the phosphorylation of microtubular proteins and the regulation of the cell cycle

The bioinformatic search of the plant homologues of human protein kinases SLK, PAK6, PAK7, MARK1, MAST2, TTBK1, TTBK2, AURKA, PLK1, PLK2, and PASK, involved in the phosphorylation of microtubular proteins and regulation of cell division, was carried out. The plant homologues of protein kinases SLK, MAST2, and AURKA were identified. It was found that the closest homologue of human protein kinase AURKA is a protein A7PY12_VITVI (STALK, Serine-Threonine Aurora-Like Kinase) from grapes (Vitis vinifera), whose function is still unknown. The reconstruction and analysis of the 3D-structure of the STALK protein confirmed its relation to the group of AURKA-like protein kinases.

Bioinformatic comparison of human and higher plant phosphatomes

The study presents the results of bioinformatic comparison of protein phosphatases from higher plants and human phosphatome (150 proteins). Based on sequence and profile comparison with known catalytic domains, 204 plant homologues were selected from Physcomitrella patens and Arabidopsis thaliana. Clustering of joint group of plant and animal protein phosphatases revealed fundamental differences in plant and human phosphatomes. At the same time, significant differences in the sets of protein phosphatases in P. patens, A. thaliana, Orysa sativa, and Zea mays were shown.

Identification of Plant Homologues of Dual Specificity Yak1-Related Kinases

Currently, Dual Specificity YAK1-Related Kinases (MNB/DYRK) were found in slime molds, protista, fungi, and animals, but the existence of plant homologues is still unclear. In the present study, we have identified 14 potential plant homologues with the previously unknown functions, based on the strong sequence similarity. The results of bioinformatics analysis revealed their correspondence to DYRK1A, DYRK1B, DYRK3, and DYRK4. For two plant homologues of animal DYRK1A from Physcomitrella patens and Arabidopsis thaliana spatial structures of catalytic domains were predicted, as well as their complexes with ADP and selective inhibitor d15. Comparative analysis of 3D-structures of the human DYRK1A and plant homologues, their complexes with the specific inhibitors, and results of molecular dynamics confirm their structural and functional similarity with high probability. Preliminary data indicate the presence of potential MNB/DYRK specific phosphorylation sites in such proteins associated with plant cytoskeleton as plant microtubule-associated proteins WVD2 and WDL1, and FH5 and SCAR2 involved in the organization and polarity of the actin cytoskeleton and some kinesin-like microtubule motor proteins.

Is Casein Kinase 2 Able to Phosphorylate Plant α-Tubulin?

Results of classical and structural bioinformatical research allow to predict casein kinase 2 dependent phosphorylation of conservative residues of Ser94 and Ser419 in Trypanosoma and Arabidopsis α-tubulin. Location of these residues in the region of internal contact of α-/β-tubulin heterodimer has been demonstrated. It is hypothesized that phosphorylation of Ser94 can affect dimerization of α-/β-tubulin in Trypanosoma and Arabidopsis. Most likely, potential phosphorylation of Ser419 does not have a direct effect on microtubule structure but is related to interaction with associated proteins, in particular with kinesins.

MAST-like protein kinase IREH1 from Arabidopsis thaliana co-localizes with the centrosome when expressed in animal cells

AbstractMain conclusionThe similarity of IREH1 (Incomplete Root Hair Elongation 1) and animal MAST kinases was confirmed; IREH1cDNA was cloned while expressing in cultured animal cells co-localized with the centrosome. In mammals and fruit flies, microtubule-associated serine/threonine-protein kinases (MAST) are strongly involved in the regulation of the microtubule system. Higher plants also possess protein kinases homologous to MASTs, but their function and interaction with the cytoskeleton remain unclear. Here, we confirmed the sequence and structural similarity of MAST-related putative protein kinase IREH1 (At3g17850) and known animal MAST kinases. We report the first cloning of full-length cDNA of the IREH1 from Arabidopsis thaliana. Recombinant GFP-IREH1 protein was expressed in different cultured animal cells. It revealed co-localization with the centrosome without influencing cell morphology and microtubule arrangement. Structural N-terminal region of the IREH1 molecule co-localized with centrosome as well.