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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合成酶(ACC Synthase，ACS)是乙烯生物合成途径中的限速酶，其同工酶在不同的刺激条件下表达。最近的研究表明，ACS不仅在转录水平上受到调控，而且在翻译后也受到调控。我们发现，LeACS2是一种创伤诱导的番茄ACS基因，在C-末端的Ser-460处被钙依赖蛋白激酶(CDPK)翻译后立即被磷酸化，然后在细胞内以磷酸化的形式发挥作用。此外，激酶和磷酸酶抑制剂的处理结果表明，磷酸化的LeACS2的半衰期比非磷酸化的LeACS2的半衰期要长。这些结果表明，磷酸化/去磷酸化调节LeACS2蛋白在细胞内的周转，而去磷酸化LeACS2会导致降解。对乙烯过度生产型(ETO)突变体的分析也支持了我们的推测。因此，我们试图确定与LeACS2周转有关的蛋白磷酸酶。根据LeACS2 Ser-460附近的序列，合成了生物素标记的磷酸肽Biotinyl-KNNLRL(PS)FSKRMYD-CHO。将该多肽与受伤番茄果实组织的提取液孵育。结合多肽的蛋白质被链霉亲和素结合的动态链霉亲和素捕获，并用十二烷基硫酸钠洗脱。洗脱液经十二烷基硫酸钠-PAGE和印迹分析，用辣根过氧化物酶标记的链霉亲和素进行检测。结果检测到32 kDa的蛋白质。32 kDa蛋白的量依赖于加入反应混合物的磷酸多肽的浓度。根据分子质量，我们推测该32 kDa蛋白是参与LeACS2去磷酸化的丝氨酸/苏氨酸蛋白磷酸酶的催化亚基。