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New tool highlights activity of key cellular signal

0s, biologists began harnessing this phenomenon to study molecular changes in cells. A team led by Roger Tsien, Ph.D., at the University of California San Diego created the first FRET probe of cyclic AMP activity by attaching small fluorescent molecules to the ends of a protein called PKA. Once activated by cyclic AMP, PKA breaks in two, separating its two fluorescent "caps." The first all-protein fluorescent version of PKA was developed a few years later.

But the PKA-based probes, which Zhang used as a postdoctoral fellow at the University of California San Diego, were difficult to use because PKA is made of four parts. For a simpler and easier to use probe, Lisa DiPilato, now a second-year graduate student in pharmacology, added fluorescent caps -- one cyan, the other yellow -- to a one-piece protein called Epac that is also activated by cyclic AMP.

By using a special microscope to measure how the fluorescence of the probe changed in response to cyclic AMP, DiPilato proved the probe's ability. Addition of a genetic "address label" then allowed her to direct the fluorescent probe to go to particular places in cells -- keeping it at the cell membrane, sending it to the nucleus, or directing it to the cell's power plant, the mitochondria.

"The probe has already provided new information," says Zhang, who arrived at Hopkins in September 2003. "The probe itself shows that Epac changes shape when it's activated, which had not been directly observed. And while some scientists had suggested cyclic AMP could get into the mitochondria, our studies are the first to show that it's there."

Stimulating cyclic AMP production in a human embryonic kidney cell line containing the targeted fluorescent Epac proteins caused an immediate build-up of cyclic AMP and activation of Epac at the cell membrane, where cyclic AMP is produced, the researchers report. Within seconds, build-up of cyclic AMP also began in the cytoplasm of the c
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Contact: Joanna Downer
jdowner1@jhmi.edu
410-614-5105
Johns Hopkins Medical Institutions
15-Nov-2004


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