Exposure of beta-tubulin regions defined by antibodies on an Arabidopsis thalianamicrotubule protofilament model and in the cells

Abstract

BackgroundThe function of the cortical microtubules, composed of αβ-tubulin heterodimers, is linked to their organizational state which is subject to spatial and temporal modulation by environmental cues. The role of tubulin posttranslational modifications in these processes is largely unknown. Although antibodies against small tubulin regions represent useful tool for studying molecular configuration of microtubules, data on the exposure of tubulin epitopes on plant microtubules are still limited.ResultsUsing homology modeling we have generated an Arabidopsis thaliana microtubule protofilament model that served for the prediction of surface exposure of five β-tubulin epitopes as well as tyrosine residues. Peptide scans newly disclosed the position of epitopes detected by antibodies 18D6 (β1-10), TUB2.1 (β426-435) and TU-14 (β436-445). Experimental verification of the results by immunofluorescence microscopy revealed that the exposure of epitopes depended on the mode of fixation. Moreover, homology modeling showed that only tyrosines in the C-terminal region of β-tubulins (behind β425) were exposed on the microtubule external side. Immunofluorescence microscopy revealed tyrosine phosphorylation of microtubules in plant cells, implying that β-tubulins could be one of the targets for tyrosine kinases.ConclusionsWe predicted surface exposure of five β-tubulin epitopes, as well as tyrosine residues, on the surface of A. thaliana microtubule protofilament model, and validated the obtained results by immunofluorescence microscopy on cortical microtubules in cells.The results suggest that prediction of epitope exposure on microtubules by means of homology modeling combined with site-directed antibodies can contribute to a better understanding of the interactions of plant microtubules with associated proteins.

Members of the Plant CRK Superfamily Are Capable of Trans- and Autophosphorylation of Tyrosine Residues

“…2D), suggesting that the two Tyr residues are major phosphorylation sites for CRK2, CRK3, and CRK8. Interestingly, Tyr phosphorylation of ␤-tubulins in Arabidopsis and tobacco plants has already been reported (12,30,31). Our finding suggests that the three CRKs are the responsible PKs for Tyr phosphorylation of ␤-tubulins.…”

“…Previous studies have already shown that the Tyr residues of ␣-and ␤-tubulin are phosphorylated in Arabidopsis and tobacco, but the phosphorylation site(s) and the kinases responsible for phosphorylation remain unknown (30,31). By pulldown assays using protein lysate from cultured cells and in vitro kinase assays, we identified the Tyr residues of TBB7 that are phosphorylated by CRK2, CRK3, and CRK8.…”

Effects of tyrosine kinase and phosphatase inhibitors on mitosis progression in synchronized tobacco BY-2 cells

“…The authors proposed that the degree of tyrosine phosphorylation of -tubulin after the cells' exposure to inhibitors of tyrosine kinases and tyrosine phosphatases is an important factor to determine the state of assembly of microtubules in animal cells [40]. Recently using double-label staining experiments on A. thaliana microtubules with a polyclonal anti-phosphotyrosine antibody (P-Tyr), a monoclonal anti--tubulin (TU-01) and an anti--tubulin antibody (TUB 2.1) it was shown that phosphotyrosine labeling correlated with cortical microtubules in interphase cells as well as with all types of mitotic microtubules [20]. It was also estimated that the signal detected with the anti-phosphotyrosine antibody was markedly enhanced after pretreating the cells with sodium orthovanadate, as compared with control (untreated) cells.…”

“…Recently it was established that both -and -tubulins undergo phosphorylation on tyrosine residues [19]. Immunofluorescence microscopy revealed that tyrosine phosphorylation of -tubulin could be one of the targets for tyrosine kinases [20]. In our previous paper we have provided data that tyrosine kinase inhibitors as herbimycin A, genistein and tyrphostin AG18 as well as an inhibitor of tyrosine phosphatases, sodium orthovanadate, affect microtubule organization in Arabidopsis thaliana root cells.…”

Signal processing by protein tyrosine phosphorylation in plants

“…124 Involvement of PTKs and PTPs in the organization of microtubules in primary root cells was shown using specific inhibitors. 46,125 Other components of cytoskeleton are also modulated by Tyr phosphorylation. For example, in Mimosa pudica, Tyr phosphorylation of actin is induced by stimulation of the pulvinus and its stimulation is abolished by PAO.…”