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Scientist Shines a Light on Molecular Signaling

With fluorescent biosensors, Jin Zhang is illuminating the cell’s intricate signaling pathways

October 2009-Signal transduction is something like the children’s game of telephone, in which a message is conveyed in whispers along a chain of players.

In the cell, the message announces an event outside of the cell—such as the presence of a protein that can stimulate the cell’s growth. This message is relayed inside the cell through a cascade of biochemical reactions, which end by eliciting a physiological response—the cell divides, for example.

Scientists have learned the identity of many of the molecular players in signal transduction. But that is not the same as seeing a molecule in action, says Jin Zhang, an associate professor of pharmacology and molecular sciences. So she is developing tools that make such intracellular voyeurism possible: fluorescent sensors that change color when a signaling event occurs.

Her research may do more than provide the curious a peephole into the cell, however. It is also yielding new information that may further inform our understanding of disease. As in the game of telephone, the message delivered in signal transduction can become garbled, and in the cell, such disruptions can contribute to cancer, diabetes, heart disease and a host of other diseases.

In recognition of the future potential of her studies, Zhang in September received an NIH Director’s Pioneer Award. The highly competitive five-year grant is awarded to “scientists of exceptional creativity who propose pioneering—and possibly transforming approaches—to major challenges in biomedical and behavioral research.”

To date, Zhang has focused on some of the key players in the signaling cascade, the family of protein kinase molecules. There are 518 different kinases in the human “kinome,” says Zhang, and all perform the same basic reaction: phosphorylating proteins, a biochemical step that changes the function of the target protein.

“We know the names of all 518 protein kinases, and previous studies, many of which have looked at these molecules in their purified forms, have taught us a lot about them,” says Zhang. “But there’s a lot we don’t know.” Scientists still do not know which molecules many kinases phosphorylate, or where or when they are active in the cell.

Such information is avidly sought by pharmaceutical researchers, notes Pharmacology Director Phil Cole. Several protein kinase inhibitors are on the market, including the cancer drug Gleevec, and several are in clinical trials. “Protein kinases are among the hottest new drug targets.”

Zhang first became interested in kinases when she was a postdoc at the University of California, San Diego. Until then, in order to study kinase activity, a scientist would have to lyse cells and use radioactive assays or antibodies, an approach, says Zhang, that “loses the live cell context.” Zhang had a better idea.

It involved using a unique molecular tool, green fluorescent protein (GFP), developed by Roger Tsien, her advisor. Tsien had developed GFP as a genetically encodable fluorescent tag that allows tracking the movements and actions of molecules inside living cells, work for which he received the Nobel Prize in Chemistry in 2008.

Also using GFP, Zhang constructed fluorescent biosensors that can be used to reveal kinase activity. Her basic approach involves using genetic engineering to attach two different fluorescent protein “reporter” molecules to a “molecular switch” consisting of a kinase’s target protein and a second protein that will bind to the target protein once it is phosphorylated. Phosphorylation causes a conformational change in the molecular switch, which in turn causes the reporter molecules to change color. The color change, which Zhang detects through a fluorescent microscope and charge-coupled device (CCD) camera, indicates that kinase activity is taking place.

Zhang has used her fluorescent biosensors to study a wide variety of protein kinases. The list includes a kinase called AKT, whose gene is frequently mutated and amplified in cancer, changes that apparently increase the kinase’s activity and contribute to the development of tumors.

Using her fluorescent sensors, Zhang recently discovered special “hot spots” of AKT activity along the plasma membrane—micro regions of the membrane where kinase activity intensifies. Such “hot spots” could serve as potential targets for anticancer therapies, says Zhang.

Using the sensors, she has also observed that kinase activity appears to oscillate—pulse on and off, on and off—in cells that secrete insulin. While the results are preliminary, they could have implications for improving insulin control in diabetes.

But her future studies, which garnered her a Pioneer Award, are what most excite Zhang. Until now, she says, she has used fluorescent biosensors to observe cells in their natural state. Now she wants to take a new approach, to see what happens when she perturbs signaling molecules.

Zhang plans to use genetic engineering to attach molecular “handles” onto particular signaling molecules. She will then use a method called magnetic tweezers to manipulate the molecules, for example, move them to different locations in the cell. By observing how cells behave under such abnormal conditions, Zhang hopes to gain insights into the normal function of various signaling molecules.

The idea, says Cole, is somewhat of a gamble. “It’s a very, very creative idea. It’s a wonderful plan. But it’s ambitious. It hasn’t been tested.”

To Zhang, the risk is worth it.

“You can learn a lot about cells just by observing them,” she says. “But I think by combining perturbation and visualization, you can learn even more.”

–­Melissa Hendricks

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