Post-translational regulation of cytosolic glutamine synthetase by reversible phosphorylation and 14-3-3 protein interaction
Post-translational regulation of cytosolic glutamine synthetase by reversible phosphorylation and 14-3-3 protein interaction
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DOI：
10.1046/j.1365-313x.2000.00863.x
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发表时间：
2000-10-01
期刊：
影响因子：
7.2
通讯作者：
Schjoerring, JK
中科院分区：
文献类型：
--
作者：
Finnemann, J;Schjoerring, JK
研究了甘蓝型油菜叶片谷氨酰胺合成酶（GS（1）; EC 6.3.1.2）胞浆同工酶的调节。表达和免疫检测研究表明，GS（1）是衰老叶片中唯一有活性的GS同工酶。通过使用[γ-P-32]ATP，然后进行免疫检测，表明GS（1）是一种磷蛋白。GS（1）在细胞分裂后由蛋白激酶和微囊藻毒素敏感的丝氨酸/苏氨酸蛋白磷酸酶催化的可逆磷酸化调节。去磷酸化GS（1）比磷酸化形式更容易降解。GS（1）的磷酸化状态在光/暗转换过程中发生变化，并取决于体外ATP/AMP比率。磷酸化GS（1）与14-3-3蛋白相互作用，如通过两种不同的方法验证的：与免疫检测相结合的His-标签14-3-3蛋白柱亲和方法，以及与14-3-3-GFP重叠的远Western方法。与14-3-3-蛋白的相互作用程度可以通过降低或增加GS的磷酸化状态在体外进行修改（1）。因此，这些结果表明，14-3-3蛋白是一个激活分子的胞质GS和提供了第一个证据的蛋白参与激活植物胞质GS。翻译后调节胞质GS和磷酸化的胞质GS和14-3-3蛋白在衰老叶片之间的相互作用的作用进行了讨论，氮再动员。
Regulation of the cytosolic isozyme of glutamine synthetase (GS(1); EC 6.3.1.2) was studied in leaves of Brassica napus L. Expression and immunodetection studies showed that GS(1) was the only active GS isozyme in senescing leaves. By use of [gamma-P-32]ATP followed by immunodetection, it was shown that GS(1) is a phospho-protein. GS(1) is regulated post-translationally by reversible phosphorylation catalysed by protein kinases and microcystin-sensitive serine/threonine protein phosphatases. Dephosphorylated GS(1) is much more susceptible to degradation than the phosphorylated form. The phosphorylation status of GS(1) changes during light/dark transitions and depends in vitro on the ATP/AMP ratio. Phosphorylated GS(1) interacts with 14-3-3 proteins as verified by two different methods: a His-tag 14-3-3 protein column affinity method combined with immunodetection, and a far-Western method with overlay of 14-3-3-GFP. The degree of interaction with 14-3-3-proteins could be modified in vitro by decreasing or increasing the phosphorylation status of GS(1). Thus, the results demonstrate that 14-3-3 protein is an activator molecule of cytosolic GS and provide the first evidence of a protein involved in the activation of plant cytosolic GS. The role of post-translational regulation of cytosolic GS and interactions between phosphorylated cytosolic GS and 14-3-3 proteins in senescing leaves is discussed in relation to nitrogen remobilization.