From the Institute of Molecular, Cell, and Systems Biology, College of Medical, Veterinary, and Life Sciences, Bower Building, University of Glasgow, Glasgow G12 8QQ, United Kingdom and.
RNA Biology and Molecular Physiology, Faculty of Biology, Bielefeld University, 33615 Bielefeld, Germany.
J Biol Chem. 2018 Apr 13;293(15):5613-5623. doi: 10.1074/jbc.RA118.001834. Epub 2018 Feb 23.
Protein kinases (PKs) control many aspects of plant physiology by regulating signaling networks through protein phosphorylation. Phototropins (phots) are plasma membrane-associated serine/threonine PKs that control a range of physiological processes that collectively serve to optimize photosynthetic efficiency in plants. These include phototropism, leaf positioning and flattening, chloroplast movement, and stomatal opening. Despite their identification over two decades ago, only a handful of substrates have been identified for these PKs. Progress in this area has been hampered by the lack of a convenient means to confirm the identity of potential substrate candidates. Here we demonstrate that the kinase domain of phot1 and phot2 can be successfully engineered to accommodate non-natural ATP analogues by substituting the bulky gatekeeper residue threonine for glycine. This approach circumvents the need for radioactivity to track phot kinase activity and follow light-induced receptor autophosphorylation by incorporating thiophosphate from -benzyl-ATPγS. Consequently, thiophosphorylation of phot substrate candidates can be readily monitored when added or co-expressed with phots Furthermore, gatekeeper-modified phot1 retained its functionality and its ability to accommodate -benzyl-ATPγS as a phosphodonor when expressed in We therefore anticipate that this chemical genetic approach will provide new opportunities for labeling and identifying substrates for phots and other related AGC kinases under and near-native conditions.
蛋白激酶（PKs）通过蛋白磷酸化调控信号网络，从而控制植物生理学的许多方面。光受体（phots）是一种质膜相关的丝氨酸/苏氨酸 PK，可控制一系列生理过程，这些过程共同优化植物的光合作用效率。这些过程包括向光性、叶片定位和平整、叶绿体运动和气孔开放。尽管这些 PK 被鉴定出来已经有二十多年了，但这些 PK 的底物却只有少数几种被鉴定出来。该领域的进展受到缺乏方便的方法来确认潜在底物候选物的身份的阻碍。在这里，我们证明 phot1 和 phot2 的激酶结构域可以通过将 bulky 门控残基苏氨酸替换为甘氨酸来成功地设计成能够容纳非天然 ATP 类似物。这种方法避免了使用放射性同位素来跟踪光激酶活性和通过掺入 -benzyl-ATPγS 来跟踪光受体自磷酸化的需要。因此，当添加或与 phots 共表达时，光底物候选物的硫代磷酸化可以很容易地被监测到。此外，当在酵母中表达时，经过门控修饰的 phot1 保留了其功能以及其作为磷酸供体容纳 -benzyl-ATPγS 的能力。因此，我们预计这种化学遗传方法将为在生理和接近生理条件下标记和鉴定 phot 和其他相关 AGC 激酶的底物提供新的机会。
