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

ACC合成酶（ACC synthase， 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也受翻译后调控。为了阐明ACS是如何在翻译后水平调控的，我们使用抗LE-ACS2抗体对番茄果实（Lycopersicon esculentum L.）中ACS家族的伤口诱导同工酶LE-ACS2蛋白进行了修饰。研究人员从灌注[^<32>P]无机磷酸盐的受伤番茄果实提取物中，用微量沉淀法在55-kDa下检测到磷酸化的LE-ACS2。LE-ACS2的磷酸化氨基酸分析表明丝氨酸残基被磷酸化。以重组LE-ACS2为底物的定点诱变体外磷酸化分析表明，位于ACS c端区的460丝氨酸被磷酸化。LE-ACS2的磷酸化/去磷酸化不影响酶的活性。为了阐明LE-ACS2的磷酸化状态，我们利用合成的磷酸化肽作为抗原制备了抗磷酸化LE-ACS2抗体。用抗LE-ACS2抗体和抗磷酸化LE-ACS2抗体进行Western blot分析表明，LE-ACS2在LE-ACS2翻译后立即被磷酸化。用花青素A或冈田酸处理损伤比单独损伤积累更多的LE-ACS2蛋白（对照）。相比之下，与对照组相比，施陶孢素或k252a治疗损伤后积累的LE-ACS2蛋白较少。这些结果表明，LE-ACS2的半衰期受磷酸化控制。为了确定LE-ACS2的半衰期，我们在蛋白磷酸酶激酶抑制剂存在/不存在的情况下进行了脉冲追踪实验。结果表明，在对照组中，LE-ACS2的半衰期为60 min，而在calyculin A和k252a作用下，LE-ACS2的半衰期分别为100 min和45 min。基于这些结果，我们提出了以下调控机制：LE-ACS2在细胞内以磷酸化形式起作用，去磷酸化导致LE-ACS2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