title: α-tubulin is rapidly phosphorylated in response to hyperosmotic stress in rice and Arabidopsis.
abstract: By using high-resolution two-dimensional PAGE followed by phosphoprotein-specific staining and peptide mass fingerprint analysis along with other assays, we found that α-tubulin is phosphorylated in response to hyperosmotic stress in rice and Arabidopsis. The onset of the phosphorylation response was as early as 2 min after hyperosmotic stress treatment, and a major proportion of α-tubulin was phosphorylated after 60 min in root tissues. However, the phosphorylated form of α-tubulin was readily dephosphorylated upon stress removal. The phosphorylation site was identified as Thr349 by comprehensive mutagenesis of serine/threonine residues in a rice α-tubulin isoform followed by evaluation in cultured cell protoplasts. This residue is located at the surface for the interaction with β-tubulin in polymerized α-β tubulin dimers and has been proposed to be directly involved in this interaction. Thus, α-tubulin phosphorylation was considered to occur on free tubulin dimers in response to hyperosmotic stress. The incorporation of green fluorescent protein (GFP)-α-tubulin into cortical microtubules was completely inhibited in transgenic Arabidopsis when Thr349 was substituted with glutamate or aspartate. Using transgenic Arabidopsis plants expressing GFP-α-tubulin, we found that hyperosmotic stress causes extensive cortical microtubule depolymerization. Microtubule-destabilizing treatments such as propyzamide or oryzalin and temperature stresses resulted in α-tubulin phosphorylation, whereas hyperosmotic stress-induced α-tubulin phosphorylation was partially inhibited by taxol, which stabilizes microtubules. These results and the three-dimensional location of the phosphorylation site suggested that microtubules are depolymerized in response to hyperosmotic stress via α-tubulin phosphorylation. Together, the results of the present study reveal a novel mechanism that globally regulates the microtubule polymerization.
authors: Bioscience and Biotechnology Center, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8601, Japan.
authors: Yoshinori
authors: Y
authors: Ban
authors: Yuhko
authors: Y
authors: Kobayashi
authors: Tomomi
authors: T
authors: Hara
authors: Takahiro
authors: T
authors: Hamada
authors: Takashi
authors: T
authors: Hashimoto
authors: Shin
authors: S
authors: Takeda
authors: Tsukaho
authors: T
authors: Hattori
mesh_terms: D017360:Arabidopsis / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D008870:Microtubules / Q000378:metabolism; D012275:Oryza / Q000166:cytology / Q000378:metabolism*; D009997:Osmotic Pressure; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism*; D058105:Polymerization; D055503:Protein Multimerization; D040901:Proteomics; D011523:Protoplasts / Q000378:metabolism; D036226:Seedlings / Q000378:metabolism; D013312:Stress, Physiological; D013696:Temperature; D014404:Tubulin / Q000378:metabolism*
chemical_list: D010750:Phosphoproteins; D010940:Plant Proteins; D014404:Tubulin; D010769:Phosphothreonine
keywords: Arabidopsis thaliana; Dephosphorylation; Microtubule; Oryza sativa; Osmotic stress; Phosphorylation