组成图示
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传感器类型
荧光生物传感器
检测对象
AKAP12磷酸化（phosphorylation of AKAP12 by PKA）；样品基质：人表皮样癌细胞A431活细胞
检测原理
β2肾上腺素受体（β2AR）被异丙肾上腺素激活后，经Gs-腺苷酸环化酶-cAMP通路激活PKA。AKAR2-AKAP12中AKAR2模块含有PKA底物序列、FHA磷酸氨基酸结合域、CFP和citrine YFP。PKA磷酸化AKAR2底物序列后，FHA域与磷酸化序列紧密结合，使CFP与citrine YFP距离缩短，产生FRET，表现为CFP荧光下降、YFP荧光上升；磷酸酶去磷酸化则FRET减弱。AKAP12模块通过PCD靶向膜、RBD结合β2AR、RII-BD锚定PKA，使报告域定位于支架所在微区。因此FRET强度随局部磷酸化AKAP12比例变化，并可反映膜区与核周胞质区的时空动态。
检测灵敏度
原文未报告LOD、线性范围、灵敏度斜率或R^2。
效应效果
AKAR2-AKAP12在A431细胞中表达后保留AKAP12功能，可挽救AKAP12敲低细胞中β2AR再敏化与回收缺陷。FRET信号具PKA依赖性：KT5720消除异丙肾上腺素诱导的FRET；AKAR2 T/P突变无FRET；HT-31肽阻断PKA RII-AKAP结合后FRET消失，HT-31p不阻断。β激动刺激使膜AKAP12增加3-5倍，膜区FRET 5 min内上升并稳态，核周胞质FRET 30 min内渐进增加。morpholino敲低内源AKAP12>85%（部分>90%）。未报告RSD和加标回收率，作者认为其可活细胞时空监测AKAP12磷酸化与GPCR再敏化/回收。
传感器的构成
- 融合蛋白主体：AKAR2-AKAP12，N端AKAR2与AKAP12融合，作为PKA磷酸化报告器
- AKAR2报告模块：CFP、PKA底物序列、FHA磷酸氨基酸结合域、citrine YFP，磷酸化后产生CFP-YFP FRET
- AKAP12靶向模块：PCD、RBD、RII-BD，锚定PKA、结合β2AR并靶向细胞膜
- 表达载体：pcDNA3 hygro质粒，携带AKAR2-AKAP12 cDNA，用于A431细胞表达
- 细胞基质：人表皮样癌细胞A431，提供β2AR、PKA和cAMP信号环境
- 刺激配体：异丙肾上腺素（isoproterenol），激活β2AR-PKA通路
- 读出系统：Zeiss LSM 510共聚焦显微镜，CFP 473-495 nm与YFP/citrine 527-591 nm检测FRET
中文摘要
蛋白激酶A（PKA）在几乎所有细胞信号转导中起核心作用，但其在活细胞中的时空激活动态难以直接观察。遗传编码荧光探针AKAR2可响应PKA磷酸化并产生荧光共振能量转移（FRET）信号，是PKA活性的生物传感器。AKAP12是一种支架蛋白，可锚定PKA、G蛋白偶联受体（GPCR）和膜磷脂，并催化受体再敏化与回收。本研究将AKAR2融合至AKAP12 N端，构建AKAR2-AKAP12融合蛋白，以评估其能否在保留AKAP12功能的同时报告该支架的动态磷酸化。结果显示，AKAR2-AKAP12可在哺乳动物细胞中表达并完全保留功能，在人表皮样癌细胞A431中响应β肾上腺素能刺激，揭示AKAP12被PKA磷酸化的时空激活过程。结论表明，该融合蛋白可“生物感知”AKAP12磷酸化：支架与细胞膜结合并快速被PKA磷酸化，核周胞质中磷酸化支架的积累反映PKA激活形式的AKAP12，其催化失敏内化GPCR的再敏化与回收。
英文摘要
BACKGROUND: The cAMP-dependent protein kinase A (PKA) plays a pivotal role in virtually all cells, there being a multitude of important target molecules that are substrates for PKA in cell signaling. The spatial-temporal dynamics of PKA activation in living cells has been made accessible by the development of clever biosensors that yield a FRET signal in response to the phosphorylation by PKA. AKAR2 is genetically encoded fluorescent probe that acts as a biosensor for PKA activation. AKAP12 is a scaffold that docks PKA, G-protein-coupled receptors, cell membrane negatively-charged phospholipids, and catalyzes receptor resensitization and recycling. In the current work, the AKAR2 biosensor was fused to the N-terminus of AKAP12 to evaluate its ability to function and report on dynamic phosphorylation of the AKAP12 scaffold.
RESULTS: AKAR2-AKAP12 can be expressed in mammalian cells, is fully functional, and reveals the spatial-temporal activation of AKAP12 undergoing phosphorylation by PKA in response to beta-adrenergic activation in human epidermoid carcinoma A431 cells.
CONCLUSION: The dynamic phosphorylation of AKAP12 "biosensed" by AKAR2-AKAP12 reveals the scaffold in association with the cell membrane, undergoing rapid phosphorylation by PKA. The perinuclear, cytoplasmic accumulation of phosphorylated scaffold reflects the phosphorylated, PKA-activated form of AKAP12, which catalyzes the resensitization and recycling of desensitized, internalized G-protein-coupled receptors.