AKAR2-AKAP12 fusion protein "biosenses" dynamic phosphorylation and localization of a GPCR-based scaffold.

Q2 Biochemistry, Genetics and Molecular Biology

Journal of Molecular Signaling Pub Date : 2010-04-22 DOI:10.1186/1750-2187-5-3

Jiangchuan Tao, Hsien-Yu Wang, Craig C Malbon

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引用次数: 10

Abstract

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.

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AKAR2-AKAP12融合蛋白“生物感官”动态磷酸化和基于gpcr的支架定位。

背景:camp依赖性蛋白激酶A (PKA)在几乎所有细胞中都起着关键作用，在细胞信号传导中有许多重要的靶分子是PKA的底物。通过智能生物传感器的发展，PKA在活细胞中激活的时空动态已经可以实现，这些传感器可以产生FRET信号来响应PKA的磷酸化。AKAR2是基因编码的荧光探针，作为PKA激活的生物传感器。AKAP12是一个支架，连接PKA、g蛋白偶联受体、细胞膜带负电荷的磷脂，并催化受体的再敏化和再循环。在目前的工作中，我们将AKAR2生物传感器融合到AKAP12的n端，以评估其功能能力，并报道AKAP12支架的动态磷酸化。结果:AKAR2-AKAP12在哺乳动物细胞中表达，功能完备，揭示了在人表皮样癌A431细胞中，被PKA磷酸化的AKAP12响应β -肾上腺素能激活的时空激活。结论:AKAR2-AKAP12“生物感知”的AKAP12的动态磷酸化揭示了支架与细胞膜的关联，被PKA快速磷酸化。磷酸化支架的核周细胞质积累反映了AKAP12的磷酸化，pka活化形式，它催化脱敏的内化g蛋白偶联受体的再敏和再循环。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Journal of Molecular Signaling Biochemistry, Genetics and Molecular Biology-Biochemistry

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期刊介绍： Journal of Molecular Signaling is an open access, peer-reviewed online journal that encompasses all aspects of molecular signaling. Molecular signaling is an exponentially growing field that encompasses different molecular aspects of cell signaling underlying normal and pathological conditions. Specifically, the research area of the journal is on the normal or aberrant molecular mechanisms involving receptors, G-proteins, kinases, phosphatases, and transcription factors in regulating cell proliferation, differentiation, apoptosis, and oncogenesis in mammalian cells. This area also covers the genetic and epigenetic changes that modulate the signaling properties of cells and the resultant physiological conditions.