A dual parameter FRET probe for measuring PKC and PKA activity in living cells.
Cell function is regulated by complex and often interdependent networks of signaling molecules. To accurately describe these networks, it is important to monitor multiple signals in parallel. To this end, we have developed a genetically encoded, FRET-based probe that independently monitors both protein kinase A (PKA) and protein kinase C (PKC) activity in vivo. Artificial as well as physiological stimulants produced a negative or positive change in FRET efficiency following PKA or PKC activation, respectively. Mutations of the phosphate-accepting amino acids of the PKC substrate yielded a probe that was sensitive to PKA activation alone.
Brumbaugh
Justin
J
European Molecular Biology Institute (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany.
Schleifenbaum
Andreas
A
Gasch
Alexander
A
Sattler
Michael
M
Schultz
Carsten
C
D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism; D045744:Cell Line, Tumor; D016384:Consensus Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009447:Neuroblastoma; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D013329:Structure-Activity Relationship
D001798:Blood Proteins; D005456:Fluorescent Dyes; D009842:Oligopeptides; D010750:Phosphoproteins; C039077:platelet protein P47; C037642:kemptide; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C