期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 101, 期 47, 页码 16513-16518出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0405973101
关键词
FRET; cAMP-dependent protein kinase
资金
- NIGMS NIH HHS [R01 GM066170, T32 GM008763, GM08763, GM066070] Funding Source: Medline
Second messenger cAMP regulates many cellular functions through its effectors, such as cAMP-dependent protein kinase (PKA) and Epac (exchange proteins directly activated by cAMP). Spatial and temporal control of cAMP signaling is crucial to differential regulation of cellular targets involved in various signaling cascades. To investigate the compartmentalized cAMP signaling, we constructed fluorescent indicators that report intracellular cAMP dynamics and Epac activation by sandwiching the full-length Epac1 between cyan and yellow mutants of GFP. Elevations of cAMP decreased FRET and increased the ratio of cyan-to-yellow emissions by 10-30% in living mammalian cells. This response can be reversed by removing cAMP-elevating agents and abolished by mutating the critical residue responsible for cAMP binding. Targeting of the reporter to the plasma membrane, where cAMP is produced in response to the activation of beta-adrenergic receptor, revealed a faster cAMP response at the membrane than in the cytoplasm and mitochondria. Simultaneous imaging with targeted cAMP indicator and PKA activity reporter allowed the detection of a much delayed PKA response in the nucleus after the rapid accumulation of cAMP at the plasma membrane of the same cell, despite the immediate presence of a pool of cAMP in the nucleus. Thus, cAMP dynamics and the activation of its effectors are precisely controlled spatiotemporally in vivo.
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