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

ACC合成酶（ACS）是乙烯生物合成途径的限速酶，各ACS同工酶在不同的刺激下表达。近年来的研究表明，ACS不仅受到转录调控，而且还受到转录后调控。我们发现LeACS2是番茄（Lycopersicon esculentum Mill.）在C-末端区域的Ser-460翻译后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起作用。此外，激酶和磷酸酶抑制剂的处理结果表明，磷酸化的LeACS 2的半衰期比非磷酸化的LeACS 2的半衰期长。这些结果表明，磷酸化/去磷酸化调节细胞中LeACS2蛋白的周转，LeACS2的去磷酸化导致降解。乙烯过量生产（eto）突变体的分析也支持了我们的推测。因此，我们试图确定蛋白磷酸酶参与LeACS2营业额。合成基于LeACS 2的Ser-460附近序列的生物素标记的磷酸肽（生物素基-KNNLRL（pS）FSKRMYD-CHO）。将该肽与受伤的番茄果实组织的提取物一起孵育。通过链霉亲和素结合的Dynabeads捕获与肽结合的蛋白质，并通过SDS洗脱。对该酶进行SDS-PAGE和印迹，并用HRP结合的链霉亲和素检测。结果，检测到32 kDa蛋白。32 kDa蛋白质的量取决于加入反应混合物中的磷酸肽的浓度。基于分子量，我们推测32 kDa的蛋白质是一个催化亚基的丝氨酸/苏氨酸蛋白磷酸酶，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.