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Analysis of the mechanism of post-translational regulatory of ACO synthase by phosphorylation

ACO合酶磷酸化翻译后调控机制分析

基本信息

批准号：
14360020
负责人：
MORI Hitoshi
金额：
$ 9.41万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

ACC synthase (ACS) is a rate limiting enzyme of ethylene biosynthesis that is mainly regulated transcriptionally. Results from recent studies suggest that ACS is also regulated post-translationally. To elucidate how ACS is regulated at the post-translational level, we analyzed the modificalion of LE-ACS2 protein, a wound-inducible isozyme in the ACS family, in tomato fruit (Lycopersicon esculentum L.) using an anti-LE-ACS2 antibody. We detected a phosphorylated LE-ACS2 at 55-kDa using inmiunoprecipitation from an extract of wounded tomato fruit that were fed [^<32>P] inorganic phosphate. Analysis of the phosphoamino acids of LE-ACS2 indicated that serine residue(s) were phosphorylated. In vitro phosphorylation analyses using site-directed mutagenesis of recombinant LE-ACS2 as a substrate demonsirated that serine 460 kcated at the C-terminal region of ACS was phosphorylated. Phosphorylation/dephosphorylation of LE-ACS2 did not affect the enzymatic activities. To elucidate the phosphoryl … More ation state of LE-ACS2, we prepared an anti-phosphorylated LE-ACS2 antibody using the phosphorylated synthetic peptide as an antigen. Western blot analyses with the anti-LE-ACS2 and anti-phosphorylated LE-ACS2 antibodies suggested that LE-ACS2 was phosphorylated immediately after translation of LE-ACS2. More LE-ACS2 protein accumulated by wounding with calyculin A or okadaic acid treatment than by wounding alone (control). In contrast, less LE-ACS2 protein accumulated by wounding with staurosporine or k252a treatment than in controls. These results suggested that the half-life of LE-ACS2 was controlled by phosphorylation. To determine the half-life of LE-ACS2, we performed pulse-chase experiments in the presence/absence of protein phosphataseikinase inhibitora. The results indicated that the half-life of LE-ACS2 was 60 min in the contiols, whereas it was 100 min and 45 min when wounding with calyculin A and k252a, respectively. Based on these results, we propose the following regulatory mechanism : LE-ACS2 acts in the phosphorylated form in the cell and dephosphorylation causes degradation of LE-ACS2. Less

ACC合酶（ACS）是乙烯生物合成的限速酶，主要受转录调控。最近的研究结果表明 ACS 也受到翻译后调节。为了阐明 ACS 在翻译后水平如何受到调节，我们使用抗 LE-ACS2 抗体分析了番茄果实 (Lycopersicon esculentum L.) 中 LE-ACS2 蛋白（ACS 家族中的一种伤口诱导同工酶）的修饰。我们利用来自喂食[^ 32 P]无机磷酸盐的受伤番茄果实提取物的免疫沉淀，检测到55-kDa的磷酸化LE-ACS2。 LE-ACS2 的磷酸氨基酸分析表明丝氨酸残基被磷酸化。使用重组 LE-ACS2 定点诱变作为底物的体外磷酸化分析表明，ACS C 末端区域的丝氨酸 460 kcat 被磷酸化。 LE-ACS2 的磷酸化/去磷酸化不影响酶活性。为了阐明 LE-ACS2 的磷酰化状态，我们使用磷酸化合成肽作为抗原制备了抗磷酸化 LE-ACS2 抗体。使用抗 LE-ACS2 和抗磷酸化 LE-ACS2 抗体进行的蛋白质印迹分析表明，LE-ACS2 在 LE-ACS2 翻译后立即被磷酸化。用花萼蛋白 A 或冈田酸治疗造成的创伤比单独造成的创伤（对照）积累更多的 LE-ACS2 蛋白。相比之下，与对照组相比，用星形孢菌素或 k252a 治疗造成的损伤所积累的 LE-ACS2 蛋白较少。这些结果表明 LE-ACS2 的半衰期是由磷酸化控制的。为了确定 LE-ACS2 的半衰期，我们在存在/不存在蛋白磷酸酶抑制剂的情况下进行了脉冲追踪实验。结果表明，LE-ACS2 在对照组中的半衰期为 60 分钟，而用花萼蛋白 A 和 k252a 损伤时分别为 100 分钟和 45 分钟。基于这些结果，我们提出以下调节机制：LE-ACS2在细胞中以磷酸化形式起作用，去磷酸化导致LE-ACS2降解。较少的