Schweighofer Alois, Ayatollahi Zahra, Meskiene Irute
Max F. Perutz Laboratories, University of Vienna, Vienna, Austria.
Methods Mol Biol. 2009;479:247-60. doi: 10.1007/978-1-59745-289-2_16.
Protein phosphatases act to reverse phosphorylation-related modifications induced by protein kinases. Type 2C protein phosphatases (PP2C) are monomeric Ser/Thr phosphatases that require a metal for their activity and are abundant in prokaryotes and eukaryotes. In plants, such as Medicago and Arabidopsis PP2Cs control several essential processes, including ABA signaling, development, and wound-induced mitogen-activated protein kinase (MAPK) pathways. In vitro assays with recombinant proteins and yeast two-hybrid systems usually provide initial information about putative PP2C substrates; however, these observations have to be verified in vivo. Therefore, a method for transient expression in isolated Arabidopsis suspension cell protoplasts was developed to assay PP2C action in living cells. This system has proven to be very useful in producing active enzymes and their substrates and in performing enzymatic reactions in vivo. Transient gene expression in isolated cells enabled assembly of functional protein kinase cascades and the creation of phosphorylated targets for PP2Cs. The method is based on the co-transformation and transient co-expression of different PP2C proteins with MAPK. It shows that epitope-tagged PP2C and MAPK proteins exhibit high enzymatic activities and produce substantial protein amounts easily monitored by Western blot analysis. Additionally, PP2C phosphatase activities can be directly tested in protein extracts from protoplasts, suggesting a possibility for analysis of activities of new PP2C family members.
蛋白磷酸酶的作用是逆转由蛋白激酶诱导的磷酸化相关修饰。2C型蛋白磷酸酶（PP2C）是单体丝氨酸/苏氨酸磷酸酶，其活性需要金属，在原核生物和真核生物中都很丰富。在植物中，如苜蓿和拟南芥，PP2C控制着几个重要过程，包括脱落酸信号传导、发育以及伤口诱导的丝裂原活化蛋白激酶（MAPK）途径。使用重组蛋白的体外试验和酵母双杂交系统通常能提供有关假定的PP2C底物的初步信息；然而，这些观察结果必须在体内进行验证。因此，开发了一种在分离的拟南芥悬浮细胞原生质体中瞬时表达的方法，以检测PP2C在活细胞中的作用。该系统已被证明在产生活性酶及其底物以及在体内进行酶促反应方面非常有用。分离细胞中的瞬时基因表达能够组装功能性蛋白激酶级联反应，并为PP2C创造磷酸化靶点。该方法基于不同PP2C蛋白与MAPK的共转化和瞬时共表达。结果表明，表位标记的PP2C和MAPK蛋白表现出高酶活性，并且产生大量易于通过蛋白质印迹分析监测的蛋白质。此外，PP2C磷酸酶活性可以在原生质体的蛋白质提取物中直接检测，这为分析新的PP2C家族成员的活性提供了可能性。
