Does tubulin phosphorylation correlate with cell death in plant cells?

Background Microtubules are necessary for a wide spectrum of cellular functions, which include cell division, intracellular transport, organelle positioning and generating of cell polarity. The major component of microtubules is tubulin heterodimeric protein which is consist of two subunits: αand β-tubulin. Both tubulin subunits can be extensively altered by post-translational modifications, including detyrosination/tyrosination, acetylation/deacetylation, polyglutamylation, polyglycylation, and phosphorylation. As for the different tubulin isotypes, the functionality of the post-translational modifications is still a matter of debate. Although, it is known that some of them are associated with stable/dynamic populations of microtubules, while others seem to influence the binding of motor proteins [1]. One of post-translational modifications, tubulin phosphorylation, is not a widely observed and its precise function is unknown both in animal and plant cells.


Background
Microtubules are necessary for a wide spectrum of cellular functions, which include cell division, intracellular transport, organelle positioning and generating of cell polarity. The major component of microtubules is tubulin heterodimeric protein which is consist of two subunits: αand β-tubulin. Both tubulin subunits can be extensively altered by post-translational modifications, including detyrosination/tyrosination, acetylation/deacetylation, polyglutamylation, polyglycylation, and phosphorylation. As for the different tubulin isotypes, the functionality of the post-translational modifications is still a matter of debate. Although, it is known that some of them are associated with stable/dynamic populations of microtubules, while others seem to influence the binding of motor proteins [1]. One of post-translational modifications, tubulin phosphorylation, is not a widely observed and its precise function is unknown both in animal and plant cells.
It was shown recently that animal tubulin can be phosphorylated by different systems of cyclic nucleotidedependent (cAMP-and cGMP-dependent) protein kinases, Ca 2+ -dependent protein kinases (including Ca 2+calmodulin-dependent and Ca 2+ -dependent, phospholipid-stimulated types of enzymes), casein kinases and tyrosine kinases, too [2,3]. The combined data demonstrate that plant tubulin can also undergo extensive phosphorylation by different types of protein kinases and that the phosphorylation on serine\threonine as well as at tyrosine residues can participate in the generation of high level of polymorphism of plant tubulin [4]. It is interesting to establish a functional role of this tubulin modification as phosphorylation is a universal post-translational modifi-cation which is typical for most of the proteins. The effects of different activators and inhibitors of protein kinases on microtubule dynamics and cell cycle progression in plan cells are present in this report.

Materials and methods
Two plant lines, Arabidopsis thaliana [5] and tobacco BY-2 cell culture [6] (kindly handed over by Prof. J.-P. Verbelen, University of Antwerp, Belgium) both expressing GFPtubulin as well as A. thaliana and Nicotiana tabacum wild types were used in this research. GFP-labeled microtubules in A. thaliana and BY-2 cells were analyzed by confocal laser scanning microscopy.
The root tips of 3-day old Allium cepa seedlings were also used in this study. The primary mouse monoclonal antibodies TU-01 (against α-tubulin) and TU-06 (against βtubulin) (kindly provided by Drs. V. Viklicky and P. Draber, Institute of Molecular Genetics, Prague, Czech Republic) were used for visualisation of microtubules in onion meristematic root tip cells by immunofluorescence microscopy. FITC-conjugated anti-mouse antibody (Sigma, USA) was used as a secondary one. The fixation and staining of microtubules by antibodies were performed as described by us early [7].

Results
For more detailed analysis of the functional role of tubulin phosphorylation in plant cells several specific inhibi- tors and activators of different types of protein kinases were used in our research. Dibutyryl-cAMP (10 µM in combination with 100 µM ATP) as an activator of cAMPdependent phosphorylation, polymyxin B (5 mM) as an inhibitors of the protein kinase C, trifluoperazine (5 mM), as an inhibitor of the Ca 2+ -calmodulin-dependent protein kinase, and okadaic acid (inhibitor of protein phosphatase type 2A, PP2A), in concentration 1-30 nM, were investigated with regard to their ability to affect microtubule dynamics and to induce structural changes of microtubules. The root tips of seedlings were treated with each of these compounds. The effects of these regulators of protein kinases on the structural reorganisation of interphase and mitotic microtubules were studied after exposure of plant material in the presence of activator or inhibitor during 6, 12 and 24 h.
Immunofluorescence analysis of microtubules showed that treatment by cAMP causes the disruption of both interphase and mitotic microtubules and accumulation of depolymerised tubulin around the nuclei in the cells. The treatment of onion cells by trifluoperazine caused the reorganization of microtubules and change of their spatial organization from a transverse to a longitudinal orientation and formation of thick longitudinal arrays. The treatment of A. cepa cells with polymyxin B caused the same effects on microtubular organisation as trifluoperazine.
Confocal laser scanning and light microscopy of A. thaliana and N. tabacum cells revealed that okadaic acid arrested cell growth, alter cell morphology, and affected the organization of microtubules.

Conclusion
It was reviewed by us that plant tubulin can undergo extensive phosphorylation by different types of protein kinases, and that tubulin phosphorylation participates in regulation of the plant cell cycle [4]. Many studies shown that different protein phosphatase inhibitors effect microtubules in animal and plant cells. For instance, it was shown that the treatment of Tradescantia stamen hair cells with okadaic acid and other protein phosphatase inhibitors caused changes of the metaphase transit times and the pattern of sister chromatid separation [8]. The treatment of Arabidopsis shoots with inhibitors of serine/threonine protein phosphatases (okadaic acid or calyculin A) provoked the destruction of root morphology, that can be explained by the influence of these compounds on cortical microtubules function [9]. The same authors later proved that phosphatase inhibitors as well as protein kinase inhibitors destroy not only root morphology but that cortical microtubules also become disorganized after exposure to some types of inhibitors [10]. In particular, these effects were characteristic of protein phosphatases such as calyculin A and cantaridin. The protein kinase inhibitor staurosporine also had similar effect in plant cells [11][12][13]. The disruption of microtubules was found recently after calyculin A and okadaic acid treatment in Lilium [14]. Thus, literature indicates that phosphorylation and dephosphorylation represent a part of the molecular mechanism responsible for both the organization of the cortical microtubular networks and of mitotic function.
Summarizing our data obtained we can conclude that the changes in the spatial organisation of microtubules after treatment by cAMP and the protein kinase inhibitors lead to disturbances of cell cycle progression and it is most likely to launch of the cell death program in plant cells.