DNA binding activity of the Arabidopsis G-box binding factor GBF1 is stimulated by phosphorylation by casein kinase II from broccoli.
DNA binding activity of the Arabidopsis G-box binding factor GBF1 is stimulated by phosphorylation by casein kinase II from broccoli.
复制标题
西兰花酪蛋白激酶 II 的磷酸化可刺激拟南芥 G-box 结合因子 GBF1 的 DNA 结合活性。
DOI：
10.1105/tpc.4.1.87
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
Cashmore,AR
中科院分区：
文献类型：
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作者：
Klimczak,LJ;Schindler,U;Cashmore,AR
为了研究拟南芥G-box结合因子（GBF 1）的磷酸化，我们通过在大肠杆菌中表达获得了大量的该蛋白。细菌GBF 1被证明是非常有效地磷酸化的核提取物从花椰菜。通过肝素-琼脂糖凝胶和DEAE-纤维素层析法部分纯化磷酸化活性，并进行表征。它显示了酪蛋白激酶II活性的基本特征：除了ATP作为磷酸供体之外还利用GTP，肝素的强烈抑制，对酸性蛋白质底物的偏好，盐诱导的与磷酸纤维素的结合，以及盐依赖性解聚。GBF 1的Km值非常低（220 nM，而酪蛋白约为10 μ M），在酪蛋白激酶II的鉴定生理底物中观察到的范围内。GBF 1的磷酸化导致G盒结合活性的刺激和较慢迁移的蛋白质-DNA复合物的形成。该刺激反应的条件完全对应于酪蛋白激酶II的性质，特别是其对已知磷酸供体的依赖性。内源性植物GBF的DNA结合活性被证明是减少与小牛碱性磷酸酶治疗，这种减少通过添加氟化物和磷酸盐或孵育中存在的酪蛋白激酶II和ATP。
To study the phosphorylation of one of the G-box binding factors from Arabidopsis (GBF1), we have obtained large amounts of this protein by expression in Escherichia coli. Bacterial GBF1 was shown to be phosphorylated very efficiently by nuclear extracts from broccoli. The phosphorylation activity was partially purified by chromatography on heparin-Sepharose and DEAE-cellulose and was characterized. It showed the essential features of casein kinase II activity: utilization of GTP in addition to ATP as a phosphate donor, strong inhibition by heparin, preference for acidic protein substrates, salt-induced binding to phosphocellulose, and salt-dependent deaggregation. The very low Km value for GBF1 (220 nM compared to approximately 10 microM for casein) was in the range observed for identified physiological substrates of casein kinase II. Phosphorylation of GBF1 resulted in stimulation of the G-box binding activity and formation of a slower migrating protein-DNA complex. The conditions of this stimulatory reaction fully corresponded to the properties of casein kinase II, in particular its dependence on the known phosphate donors. The DNA binding activity of the endogenous plant GBF was shown to be reduced by treatment with calf alkaline phosphatase; this reduction was diminished by addition of fluoride and phosphate or incubation in the presence of casein kinase II and ATP.