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：

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亚细胞靶向光遗传性cAMP依赖性蛋白激酶的设计和分析。

尽管普遍存在cAMP依赖性蛋白激酶（PKA），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。我们开发了一个三步工程策略来构建光遗传学PKA（optoPKA），并证明了光照后optoPKA迁移到指定的细胞内位点。此外，我们设计了细胞内PKA活性的空间隔离报道基因，并确认optoPKA以光依赖性方式磷酸化了这些报道基因。最后，蛋白质组学实验表明optoPKA的光活化导致已知内源性PKA底物以及潜在的新型底物的磷酸化。