In vivo quantitative analysis of PKA subunit interaction and cAMP level by dual color fluorescence cross correlation spectroscopy.
We employed dual color Fluorescence Cross Correlation Spectroscopy (FCCS) to measure the interaction between PKA regulatory (RII) and catalytic subunits (CAT) in living cells. Elevation of intracellular cAMP with forskolin decreased the cross-correlation amplitude between RFP-fused RII (RII-mRFP) and GFP-fused CAT (CAT-EGFP) by 50%, indicating that cAMP elevation leads to dissociation of RII-CAT complexes. Moreover, diffusion coefficient analysis showed that the diffusion rate of CAT-EGFP was significantly increased, suggesting that the decreased RII-CAT association caused by cAMP generated free CAT subunits. Our study demonstrates that in vivo FCCS measurements and their quantitative analysis permit one not only to directly quantify protein-protein interactions but also to estimate changes in the intracellular cAMP concentration.
Neurobiology Laboratory, Department of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul 151-747, Korea.
Hyungju
H
Park
Changi
C
Pack
Masataka
M
Kinjo
Bong-Kiun
BK
Kaang
D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D000242:Cyclic AMP / Q000378:metabolism*; D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit / Q000235:genetics / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D010957:Plasmids; D021122:Protein Subunits; D013050:Spectrometry, Fluorescence
D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit; D021122:Protein Subunits; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases