Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering

doi:10.1073/pnas.211566798

Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering

Departments of ^*Pharmacology and ^†Chemistry and Biochemistry, and ^‡Howard Hughes Medical Institute, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093

Contributed by Susan S. Taylor

Abstract

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.

Footnotes

↵ § To whom reprint requests should be addressed at: 310 CMM-W 0647, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0647. E-mail: rtsien{at}ucsd.edu.
Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AF440230).
Abbreviations:

AKAP,

A-kinase anchor protein;

AKAR,

A-kinase activity reporter;

Bt2cAMP,

N6,2′-O-dibutyryl cAMP;

DMNB-cAMP,

P-(4,5-dimethoxy-2-nitrobenzyl) cAMP;

ECFP,

enhanced cyan fluorescent protein;

FRET,

fluorescence resonance energy transfer;

FsK,

forskolin;

GFP,

green fluorescent protein;

PKA,

protein kinase A;

PKC,

protein kinase C;

PKG,

protein kinase G;

YFP,

yellow fluorescent protein