Design and Profiling of a Subcellular Targeted Optogenetic cAMP-Dependent Protein Kinase,Cell Chemical Biology

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Design and Profiling of a Subcellular Targeted Optogenetic cAMP-Dependent Protein Kinase
Cell Chemical Biology ( IF 6.6 ) Pub Date : 2017-11-02 , DOI: 10.1016/j.chembiol.2017.09.011
Colin P O'Banion ₁ , Melanie A Priestman ₁ , Robert M Hughes ₂ , Laura E Herring ₃ , Stephen J Capuzzi ₁ , David S Lawrence ₄

Affiliation

Although the cAMP-dependent protein kinase (PKA) is ubiquitously expressed, it is sequestered at specific subcellular locations throughout the cell, thereby resulting in compartmentalized cellular signaling that triggers site-specific behavioral phenotypes. We developed a three-step engineering strategy to construct an optogenetic PKA (optoPKA) and demonstrated that, upon illumination, optoPKA migrates to specified intracellular sites. Furthermore, we designed intracellular spatially segregated reporters of PKA activity and confirmed that optoPKA phosphorylates these reporters in a light-dependent fashion. Finally, proteomics experiments reveal that light activation of optoPKA results in the phosphorylation of known endogenous PKA substrates as well as potential novel substrates.

中文翻译：

亚细胞靶向光遗传学 cAMP 依赖性蛋白激酶的设计和分析

尽管 cAMP 依赖性蛋白激酶 (PKA) 普遍表达，但它被隔离在整个细胞的特定亚细胞位置，从而导致触发位点特异性行为表型的区室化细胞信号传导。我们开发了一种三步工程策略来构建光遗传学 PKA (optoPKA)，并证明，在光照下，optoPKA 会迁移到特定的细胞内位点。此外，我们设计了 PKA 活性的细胞内空间分离报告基因，并证实 optoPKA 以光依赖性方式磷酸化这些报告基因。最后，蛋白质组学实验表明，optoPKA 的光激活会导致已知内源 PKA 底物以及潜在的新型底物的磷酸化。

更新日期：2018-01-18

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