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

ACC合成酶（ACC synthase， ACS）是乙烯生物合成途径的限速酶，每个ACS同工酶在各种刺激下表达。近年来的研究表明，ACS不仅受转录调控，还受翻译后调控。我们发现，番茄（Lycopersicon esculentum Mill.）中伤口诱导的ACS LeACS2在c端Ser-460位点被钙依赖性蛋白激酶（CDPK）翻译后立即磷酸化，然后以磷酸化形式在细胞中起作用。此外，激酶和磷酸酶抑制剂处理的结果表明，磷酸化的LeACS2的半衰期比未磷酸化的LeACS2长。这些结果表明，磷酸化/去磷酸化调节LeACS2蛋白在细胞中的周转，LeACS2的去磷酸化引起降解。对乙烯过量生产者（eto）突变体的分析也支持了我们的推测。因此，我们试图确定参与LeACS2转换的蛋白磷酸酶。以LeACS2 Ser-460邻近序列为基础合成生物素标记磷酸肽（Biotinyl-KNNLRL(pS)FSKRMYD-CHO）。该肽与番茄损伤果实组织提取物孵育。结合到肽上的蛋白用链霉亲和素结合的dynabead捕获，用SDS洗脱。洗脱液进行SDS-PAGE和印迹，用HRP结合的链亲和素检测。结果检测到32 kDa蛋白。32 kDa蛋白的数量取决于加入到反应混合物中的磷酸肽的浓度。根据分子质量，我们推测32 kDa蛋白是丝氨酸/苏氨酸蛋白磷酸酶的催化亚基，参与LeACS2的去磷酸化。

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.