Engineered kinases as a tool for phosphorylation of selected targets in vivo.
Engineered kinases as a tool for phosphorylation of selected targets in vivo.
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DOI：
10.1083/jcb.202106179
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发表时间：
2022-10-03
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--
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Lepeta等人设计了一种新的方法，使用基于纳米抗体的蛋白质结合剂，以组织特异性方式在体内通过定义的激酶磷酸化选定的GFP融合蛋白靶标。他们展示了基于ROCK和Src的两种工程激酶的详细表征和验证。可逆的蛋白质磷酸化激酶控制着多细胞生物体的正常发育和体内平衡所必需的大量过程。研究确定的激酶-底物相互作用的作用的一个主要障碍是激酶形成复杂的信号传导网络，并且最经常磷酸化参与各种细胞过程的多种底物。近年来，已经开发了几种新的方法来控制给定激酶的活性。然而，它们中的大多数不能调节单个蛋白质靶点，可能通过多效性效应隐藏了独特的激酶-底物相互作用的效果。为了克服这一限制，我们已经创建了基于蛋白结合剂的工程化激酶，其允许荧光融合蛋白在体内的直接的、稳健的和组织特异性的磷酸化。我们展示了基于Rho相关蛋白激酶（ROCK）和Src的两种工程化激酶的详细表征。合成激酶在发育中的苍蝇胚胎中的表达导致其各自的GFP融合靶点的磷酸化，首次提供了一种将磷酸化导向体内所选择和标记的靶点的方法。我们推测，经过仔细的优化，我们在这里描述的新方法可以适用于其他激酶和各种真核遗传系统中的目标，以调节特定的下游效应子。
Lepeta et al. design a novel approach, using nanobody-based protein binders, to phosphorylate a chosen GFP fusion protein target by a defined kinase in vivo in a tissue-specific manner. They show detailed characterization and validation of two engineered kinases, based on ROCK and Src. Reversible protein phosphorylation by kinases controls a plethora of processes essential for the proper development and homeostasis of multicellular organisms. One main obstacle in studying the role of a defined kinase–substrate interaction is that kinases form complex signaling networks and most often phosphorylate multiple substrates involved in various cellular processes. In recent years, several new approaches have been developed to control the activity of a given kinase. However, most of them fail to regulate a single protein target, likely hiding the effect of a unique kinase–substrate interaction by pleiotropic effects. To overcome this limitation, we have created protein binder-based engineered kinases that permit a direct, robust, and tissue-specific phosphorylation of fluorescent fusion proteins in vivo. We show the detailed characterization of two engineered kinases based on Rho-associated protein kinase (ROCK) and Src. Expression of synthetic kinases in the developing fly embryo resulted in phosphorylation of their respective GFP-fusion targets, providing for the first time a means to direct the phosphorylation to a chosen and tagged target in vivo. We presume that after careful optimization, the novel approach we describe here can be adapted to other kinases and targets in various eukaryotic genetic systems to regulate specific downstream effectors.
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