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

ACC合成酶（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也受到术后调节。为了阐明ACS是如何在翻译后水平上被调控的，我们分析了番茄果实（Lycopersicon esculentum L.）使用抗LE-ACS 2抗体。我们用免疫沉淀法从喂饲无机磷酸盐的受伤番茄果实提取物中检测到55 kDa的磷酸化LE-ACS 2<32>。对LE-ACS 2的磷酸化氨基酸的分析表明丝氨酸残基被磷酸化。以重组LE-ACS 2为底物进行体外磷酸化分析，证实ACS C端丝氨酸460 kcated被磷酸化。LE-ACS 2的磷酸化/去磷酸化不影响酶活性。为了阐明磷酰基 关于我们 为了检测LE-ACS 2的磷酸化状态，我们使用磷酸化的合成肽作为抗原制备了抗磷酸化的LE-ACS 2抗体。用抗LE-ACS 2和抗磷酸化的LE-ACS 2抗体进行的蛋白质印迹分析表明，LE-ACS 2在LE-ACS 2翻译后立即被磷酸化。用calyculin A或冈田酸处理的创伤比单独创伤（对照）积累更多的LE-ACS 2蛋白。相反，较少的LE-ACS 2蛋白积累的创伤与星形孢菌素或k252 a治疗比对照组。这些结果表明LE-ACS 2的半衰期受磷酸化控制。为了确定LE-ACS 2的半衰期，我们在存在/不存在蛋白质磷酸酶激酶的情况下进行脉冲追踪实验。结果表明，LE-ACS 2在contiols中的半衰期为60 min，而在calyculin A和k252 a损伤时，其半衰期分别为100 min和45 min。基于这些结果，我们提出以下调节机制：LE-ACS 2在细胞中以磷酸化形式起作用，去磷酸化导致LE-ACS 2降解。少

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