Umbrasaite Julija, Schweighofer Alois, Meskiene Irute
Max F. Perutz Laboratories, University of Vienna, Wien, Austria.
Methods Mol Biol. 2011;779:149-61. doi: 10.1007/978-1-61779-264-9_8.
Protein phosphorylation by protein kinases can be reversed by the action of protein phosphatases. In plants, the Ser/Thr-specific phosphatases dominate among the protein phosphatase families with the type 2C protein phosphatases (PP2Cs) being the most abundant among them. PP2Cs are monomeric enzymes that require metal cations for their activity and are insensitive to known phosphatase inhibitors. PP2Cs were shown to counteract the mitogen-activated protein kinase (MAP kinase/MAPK) activities in plants and to regulate developmental and stress signaling pathways. Studies of PP2C activities can be performed in vitro using recombinant proteins. The potential substrates of PP2Cs can be tested for dephosphorylation by the phosphatase in vitro. We have found that the stress-induced PP2Cs from alfalfa and Arabidopsis interact with stress-activated MAPKs in yeast two-hybrid (Y2H) screens. Consequently, recombinant MAPKs were employed as substrates for dephosphorylation by selected PP2Cs from different family clusters. The members of the PP2C phosphatase family demonstrated specificity toward the substrate already in vitro, supporting the notion that protein phosphatases are specific enzymes. The PP2C from Arabidopsis thaliana cluster B, Arabidopsis PP2C-type phosphatase (AP2C1), and its homolog from Medicago sativa, Medicago PP2C-type phosphatase (MP2C), were able to dephosphorylate and inactivate MAPKs, whereas the ABSCISIC ACID (ABA)-INSENSITIVE 2 (ABI2) and HOMOLOGY TO ABI1 (HAB1) PP2Cs from the distinct Arabidopsis cluster A were not able to do so. The method described here can be used for the determination of PP2C protein activity and for studying the effect of mutations introduced into their catalytic domains.
蛋白激酶介导的蛋白质磷酸化作用可被蛋白磷酸酶逆转。在植物中，丝氨酸/苏氨酸特异性磷酸酶在蛋白磷酸酶家族中占主导地位，其中2C型蛋白磷酸酶（PP2C）最为丰富。PP2C是单体酶，其活性需要金属阳离子，且对已知的磷酸酶抑制剂不敏感。研究表明，PP2C可抵消植物中丝裂原活化蛋白激酶（MAP激酶/MAPK）的活性，并调节发育和胁迫信号通路。PP2C活性的研究可使用重组蛋白在体外进行。PP2C的潜在底物可在体外通过磷酸酶进行去磷酸化测试。我们发现在酵母双杂交（Y2H）筛选中，来自苜蓿和拟南芥的胁迫诱导型PP2C与胁迫激活的MAPK相互作用。因此，重组MAPK被用作来自不同家族簇的选定PP2C进行去磷酸化的底物。PP2C磷酸酶家族成员在体外就已表现出对底物的特异性，这支持了蛋白磷酸酶是特异性酶的观点。来自拟南芥B簇的PP2C、拟南芥PP2C型磷酸酶（AP2C1）及其来自苜蓿的同源物苜蓿PP2C型磷酸酶（MP2C）能够使MAPK去磷酸化并使其失活，而来自拟南芥不同A簇的脱落酸（ABA）不敏感2（ABI2）和与ABI1同源（HAB1）的PP2C则不能。本文所述方法可用于测定PP2C蛋白活性，以及研究引入其催化结构域的突变的影响。
