Genetically Encoded Reporters of Protein Kinase A Activity Reveal Impact of Substrate Tethering

The complexity and specificity of many forms of signal transduction are widely suspected to require spatial microcompartmentation of protein kinase and phosphatase activities, yet current relevant imaging methods such as phosphorylation-specific antibodies or fluorescent peptide substrates require f...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2001-12, Vol.98 (26), p.14997-15002
Main Authors: Zhang, Jin, Ma, Yuliang, Taylor, Susan S., Tsien, Roger Y.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Antibodies
Biological Sciences
Cell Line
cyan fluorescent protein
Cyclic AMP-Dependent Protein Kinases - genetics
Cyclic AMP-Dependent Protein Kinases - metabolism
Energy Transfer
Fluorescence
fluorescence resonance energy transfer
Genes, Reporter
Genetic code
Genetics
HEK293 cells
HeLa cells
Humans
Kinetics
Molecular Sequence Data
Molecules
Phosphatases
Phosphorylation
Proteins
Scientific imaging
Substrate Specificity
yellow fluorescent protein
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Summary:The complexity and specificity of many forms of signal transduction are widely suspected to require spatial microcompartmentation of protein kinase and phosphatase activities, yet current relevant imaging methods such as phosphorylation-specific antibodies or fluorescent peptide substrates require fixation or microinjection and lack temporal or spatial resolution. We present a genetically encoded fluorescent reporter for protein kinase A (PKA) consisting of fusions of cyan fluorescent protein, a phosphoamino acid binding domain (14-3-3τ), a consensus substrate for PKA, and yellow fluorescent protein, cAMP elevations cause 25-50% changes in the ratios of yellow to cyan emissions in live cells caused by phosphorylation-induced changes in fluorescence resonance energy transfer. The reporter response was accelerated by tethering to PKA holoenzyme and slowed by localization to the nucleus. We demonstrate that deliberate redistribution of a substrate or colocalizing a substrate and PKA can modulate its susceptibility to phosphorylation by the kinase. The successful design of a fluorescent reporter of PKA activity and its application for studying compartmentalized and dynamic modulation of kinases lays a foundation for studying targeting and compartmentation of PKA and other kinases and phosphatases.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.211566798