Analysis of the mechanism of post-translational regulatory of ACO synthase by phosphorylation
| Project/Area Number |
14360020
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
園芸・造園学
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| Research Institution | Nagoya University |
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Principal Investigator |
MORI Hitoshi Nagoya University, Bloagricultural Sciences, Professor, 大学院・生命農学研究科, 教授 (20220014)
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| Co-Investigator(Kenkyū-buntansha) |
YAMAKI Shyohei Nagoya University, Bioagricultural Sciences, Professor, 大学院・生命農学研究科, 教授 (70210341)
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥14,700,000 (Direct Cost: ¥14,700,000)
Fiscal Year 2003: ¥5,800,000 (Direct Cost: ¥5,800,000)
Fiscal Year 2002: ¥8,900,000 (Direct Cost: ¥8,900,000)
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| Keywords | ethylene / ACC synthase / phosohorylation / CDPK / post-translational regulation / ACC合成酸素 / タンパク質の代謝回転 |
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| Research Abstract |
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
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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
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