Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA.
Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA; Department of Chemistry; East Carolina University, Greenville, NC 27858, USA.
Cell Chem Biol. 2018 Jan 18;25(1):100-109.e8. doi: 10.1016/j.chembiol.2017.09.011. Epub 2017 Nov 5.
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.
虽然环腺苷酸依赖的蛋白激酶（PKA）广泛表达，但它被隔离在细胞内的特定亚细胞位置，从而导致细胞内信号的分隔，引发特定部位的行为表型。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 optoPKA 在光照下迁移到特定的细胞内位置。此外，我们设计了细胞内空间分隔的 PKA 活性报告器，并证实了 optoPKA 以光依赖的方式磷酸化这些报告器。最后，蛋白质组学实验表明，光激活 optoPKA 导致已知的内源性 PKA 底物以及潜在的新底物的磷酸化。
