Xu Xiaolu, Lemmens Lenne J M, den Hamer Anniek, Merkx Maarten, Ottmann Christian, Brunsveld Luc
Laboratory of Chemical Biology , Department of Biomedical Engineering , Institute for Complex Molecular Systems (ICMS) , Eindhoven University of Technology , Den Dolech 2 , 5612AZ , Eindhoven , the Netherlands . Email:
Chem Sci. 2020 May 12;11(21):5532-5536. doi: 10.1039/d0sc00074d. eCollection 2020 Jun 7.
Phosphorylation is a key regulation event in cellular signaling. Sensing the underlying kinase activity is of crucial importance for its fundamental understanding and for drug development. For this, modular kinase activity sensing concepts are urgently needed. We engineered modular serine kinase sensors based on complementation of split NanoBiT luciferase on protein assembly platforms generated from the scaffold protein 14-3-3. The bioengineered platforms are modular and easy adaptable as exemplary shown using novel sensors for the kinases PKA, PKB, and CHK1. Two designs were conceptualized, both relying on binding of defined mono- or bivalent kinase recognition motifs to the 14-3-3 platform upon phosphorylation, resulting in reconstitution of active split-luciferase. Especially the design based on double phosphorylation and bivalent 14-3-3 binding exhibits high efficiency for signal amplification (>1000-fold) and sensitivity to specific kinases, including in cellular lysates.
磷酸化是细胞信号传导中的关键调控事件。检测潜在的激酶活性对于深入理解其基本原理以及药物开发至关重要。为此，迫切需要模块化的激酶活性传感概念。我们基于在由支架蛋白14-3-3生成的蛋白质组装平台上对分裂型纳米荧光素酶进行互补，设计了模块化丝氨酸激酶传感器。这些生物工程平台具有模块化且易于适配，以用于蛋白激酶A（PKA）、蛋白激酶B（PKB）和细胞周期检测点激酶1（CHK1）的新型传感器为例进行了展示。我们构思了两种设计，二者均依赖于在磷酸化时特定的单价或二价激酶识别基序与14-3-3平台的结合，从而导致活性分裂荧光素酶的重组。特别是基于双磷酸化和二价14-3-3结合的设计，对信号放大表现出高效率（>1000倍），并且对特定激酶具有敏感性，包括在细胞裂解物中。
