α-Tubulin is Rapidly Phosphorylated in Response to Hyperosmotic Stress in Rice and Arabidopsis
α-Tubulin is Rapidly Phosphorylated in Response to Hyperosmotic Stress in Rice and Arabidopsis
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DOI：
10.1093/pcp/pct065
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发表时间：
2013-06-01
影响因子：
4.9
通讯作者：
Hattori, Tsukaho
中科院分区：
文献类型：
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作者：
Ban, Yoshinori;Kobayashi, Yuhko;Hattori, Tsukaho
通过使用高分辨率的双向PAGE、磷酸蛋白特异性染色和多肽质量指纹图谱分析以及其他方法，我们发现α-微管蛋白在水稻和拟南芥中被磷酸化，以响应高渗透胁迫。高渗胁迫处理后2min，α-微管蛋白开始磷酸化，60min后，大部分α-微管蛋白在根组织中被磷酸化。然而，在应力消除后，α-微管蛋白的磷酸化形式很容易被去磷酸化。通过对水稻α-微管蛋白亚型中丝氨酸/苏氨酸残基的综合诱变，以及在培养的细胞原生质体中的鉴定，确定了该磷酸化位点为Thr349。在聚合的α-β微管蛋白二聚体中，该残基位于与β-微管蛋白相互作用的表面，并被认为直接参与了这种相互作用。因此，α-微管蛋白的磷酸化被认为发生在游离型微管蛋白二聚体上，以响应高渗胁迫。当Thr349被谷氨酸或天冬氨酸取代时，转基因拟南芥中绿色荧光蛋白(GFP)-α-微管蛋白的掺入被完全抑制。利用表达GFP-α-微管蛋白的转基因拟南芥植株，我们发现高渗胁迫引起皮层微管的广泛解聚。温度胁迫和丙氮酰胺等破坏微管稳定的处理导致α-微管蛋白的磷酸化，而高渗应激诱导的α-微管蛋白磷酸化则被稳定微管的紫杉醇部分抑制。这些结果和磷酸化位点的三维定位表明，微管通过α-微管蛋白磷酸化来响应高渗胁迫而解聚。综上所述，本研究的结果揭示了一种全局调节微管聚合的新机制。
By using high-resolution two-dimensional PAGE followed by phosphoprotein-specific staining and peptide mass fingerprint analysis along with other assays, we found that alpha-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 alpha-tubulin was phosphorylated after 60 min in root tissues. However, the phosphorylated form of alpha-tubulin was readily dephosphorylated upon stress removal. The phosphorylation site was identified as Thr349 by comprehensive mutagenesis of serine/threonine residues in a rice alpha-tubulin isoform followed by evaluation in cultured cell protoplasts. This residue is located at the surface for the interaction with beta-tubulin in polymerized alpha-beta tubulin dimers and has been proposed to be directly involved in this interaction. Thus, alpha-tubulin phosphorylation was considered to occur on free tubulin dimers in response to hyperosmotic stress. The incorporation of green fluorescent protein (GFP)-alpha-tubulin into cortical microtubules was completely inhibited in transgenic Arabidopsis when Thr349 was substituted with glutamate or aspartate. Using transgenic Arabidopsis plants expressing GFP-alpha-tubulin, we found that hyperosmotic stress causes extensive cortical microtubule depolymerization. Microtubule-destabilizing treatments such as propyzamide or oryzalin and temperature stresses resulted in alpha-tubulin phosphorylation, whereas hyperosmotic stress-induced alpha-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 alpha-tubulin phosphorylation. Together, the results of the present study reveal a novel mechanism that globally regulates the microtubule polymerization.