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Study on mechanism of posttranslational regulation of ACC synthase by phosphorylation that involved in tomato fruit ripening

ACC合酶磷酸化翻译后调节参与番茄果实成熟的机制研究

基本信息

批准号：
17380020
负责人：
MORI Hitoshi
金额：
$ 10.11万
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：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

ACC synthase (ACS) is a rate-limiting enzyme of ethylene biosynthesis pathway and each ACS isozyme expresses in response to various each stimulus. Recent studies suggested that ACS was regulated not only transcriptionally but also post-translationally. We found that LeACS2, a wound-inducible ACS in tomato (Lycopersicon esculentum Mill.), is immediately phosphorylated after translation at Ser-460 in the C-terminal region by calcium-dependent protein kinase (CDPK), and then acts in the phosphorylated form in the cell. Moreover, the results of treatments of kinase and phosphatase inhibitors showed that the half-life of phosphorylated LeACS2 was longer than that of non-phosphorylated LeACS2. These results suggest that phosphorylation/dephosphorylation regulates the turnover of LeACS2 protein in the cell and that dephosphorylation of LeACS2 causes degradation. Analyses of ethylene-overproducer (eto) mutants also supported our speculation. Thus, we attempted to identify the protein phosphatase involved in LeACS2 turnover. Biotin-tagged phospho-peptide (Biotinyl-KNNLRL(pS)FSKRMYD-CHO) based on the sequence in the neighborhood of Ser-460 of LeACS2 was synthesized. This peptide was incubated with the extract of wounded tomato fruit tissue. Proteins bound to the peptide were captured by streptavidin-bound Dynabeads and eluted by SDS. The eluate was subjected to SDS-PAGE and blotting, and detected with HRP conjugated streptavidin. As the results, 32 kDa protein was detected. The amount of the 32 kDa protein was dependent on the concentration of the phospho-peptide that was added to reaction mixture. Based on the molecular mass, we speculate the 32 kDa protein is a catalytic subunit of Ser/Thr protein phosphatase that is involved in dephosphorylation of LeACS2.

ACC合酶（ACS）是乙烯生物合成途径的限速酶，每种ACS同工酶都会响应各种刺激而表达。最近的研究表明 ACS 不仅受到转录调控，而且还受到翻译后调控。我们发现LeACS2是番茄（Lycopersicon esculentum Mill.）中的一种伤口诱导型ACS，在翻译后C端区域的Ser-460处立即被钙依赖性蛋白激酶（CDPK）磷酸化，然后以磷酸化形式在细胞中发挥作用。此外，激酶和磷酸酶抑制剂处理的结果表明，磷酸化LeACS2的半衰期比非磷酸化LeACS2更长。这些结果表明磷酸化/去磷酸化调节细胞中LeACS2蛋白的周转并且LeACS2去磷酸化导致降解。对乙烯过度生产者（eto）突变体的分析也支持了我们的推测。因此，我们试图鉴定参与 LeACS2 周转的蛋白磷酸酶。合成了基于LeACS2的Ser-460附近的序列的生物素标记的磷酸肽(Biotinyl-KNNLRL(pS)FSKRMYD-CHO)。将该肽与受伤番茄果实组织的提取物一起孵育。与肽结合的蛋白质被链霉亲和素结合的 Dynabeads 捕获并通过 SDS 洗脱。将洗脱液进行 SDS-PAGE 和印迹，并用 HRP 缀合的链霉亲和素进行检测。结果，检测到32kDa的蛋白质。 32 kDa 蛋白质的量取决于添加到反应混合物中的磷酸肽的浓度。根据分子量，我们推测 32 kDa 蛋白是 Ser/Thr 蛋白磷酸酶的催化亚基，参与 LeACS2 的去磷酸化。