A chemical-genetic approach to elucidate protein kinase function in planta
A chemical-genetic approach to elucidate protein kinase function in planta
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DOI：
10.1007/s11103-007-9245-9
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发表时间：
2007-12-01
影响因子：
5.1
通讯作者：
Romeis, Tina
中科院分区：
文献类型：
--
作者：
Boehmer, Maik;Romeis, Tina
蛋白激酶研究的主要目标是鉴定生物过程，其中整合了单个酶。因此，蛋白激酶介导的信号传导通常通过基于单敲除突变或共抑制的反向遗传学方法来解决。然而，如果感兴趣的蛋白激酶是多基因家族的成员，则在形态学或生化参数中没有明显的表型改变可能变得明显，因为突变表型可以通过功能冗余或稳态来补偿。在这里，我们建立了一个化学遗传筛选相结合的ATP-类似物敏感（作为）激酶的变体和分子指纹技术，研究成员的植物钙依赖性蛋白激酶（CDPK）家族在体内。CDPKs参与了对外部非生物和生物胁迫刺激的快速信号传导反应。携带as突变的CDPKs没有显示出以ATP为底物的磷酸化动力学改变，但能够使用ATP类似物作为磷酸供体或激酶抑制剂。在植物中的功能表征，我们已经取代了拟南芥AtCPK 1的突变体线与各自的作为变体下的天然CPK 1启动子。用ATP类似物抑制剂1-NA-PP 1处理拟南芥野生型和AtCPK 1 as系的幼苗，并将其暴露于冷胁迫条件下。通过2D凝胶电泳和磷蛋白染色分析了快速冷诱导的磷酸化蛋白质组变化。野生型和AtCPK 1作为植物之间的比较之前和之后的抑制剂处理揭示了差异CPK 1依赖和冷应激诱导的磷蛋白信号。在这项研究中，我们建立了化学遗传学的方法作为一种工具，它允许在植物中的蛋白激酶的植物特异性类的调查，并促进激酶介导的信号网络中的分子生物标志物的快速变化的识别。
The major objective in protein kinase research is the identification of the biological process, in which an individual enzyme is integrated. Protein kinase-mediated signalling is thereby often addressed by single knock-out mutation- or co-suppression-based reverse genetics approaches. If a protein kinase of interest is a member of a multi gene family, however, no obvious phenotypic alteration in the morphology or in biochemical parameters may become evident because mutant phenotypes may be compensated by functional redundancy or homeostasis. Here we establish a chemical-genetic screen combining ATP-analogue sensitive (as) kinase variants and molecular fingerprinting techniques to study members of the plant calcium-dependent protein kinase (CDPK) family in vivo. CDPKs have been implicated in fast signalling responses upon external abiotic and biotic stress stimuli. CDPKs carrying the as-mutation did not show altered phosphorylation kinetics with ATP as substrate, but were able to use ATP analogues as phosphate donors or as kinase inhibitors. For functional characterization in planta, we have substituted an Arabidopsis thaliana mutant line of AtCPK1 with the respective as-variant under the native CPK1 promoter. Seedlings of Arabidopsis wild type and AtCPK1 as-lines were treated with the ATP analogue inhibitor 1-NA-PP1 and exposed to cold stress conditions. Rapid cold-induced changes in the phosphoproteome were analysed by 2D-gel-electrophoresis and phosphoprotein staining. The comparison between wild type and AtCPK1 as-plants before and after inhibitor treatment revealed differential CPK1-dependent and cold-stress-induced phosphoprotein signals. In this study, we established the chemical-genetic approach as a tool, which allows the investigation of plant-specific classes of protein kinases in planta and which facilitates the identification of rapid changes of molecular biomarkers in kinase-mediated signalling networks.