Pharmacologists Discover Linkage Of Key Cell Switches

July 01, 1998

DURHAM, N.C. -- Two critical molecular on and off switches that govern cell processes are intricately bound together in a single control unit, cancer pharmacologists at Vanderbilt University School of Medicine and the Duke University Medical Center have reported.

Their finding, like discovering that the accelerator and brakes of a car are mounted together, represents an important basic insight into the machinery that controls living cells. The discovery also offers the potential for a new strategy for developing drugs that would manipulate one or both of the linked switches to kill cancer cells or bacteria that invade the body, or to exert therapeutic control over cells.

In the May 22 issue of Science, the researchers reported discovering that the linkage of an enzyme called a kinase -- which turns on cell processes by chemically adding a phosphate to another protein -- itself is regulated by another enzyme called a phosphatase that is attached to it. Phosphatases are enzymes that remove phosphates from other enzymes.

Thus, the scientists found that the kinase, called "Calcium-calmodulin dependent protein kinase IV" (CaMKIV) would continue to stimulate cell activity uncontrolled, except that it is quickly shut down by the attached phosphatase, called "Protein Phosphatase 2A" (PP2A).

Reporting the discovery were Ryan Westphal and Brian Wadzinsky of the Vanderbilt department of pharmacology, and Anthony Means and Kristin Anderson of the Duke department of pharmacology and cancer biology. Their research is sponsored by the National Institutes of Health, the Keck Foundation, and the Vanderbilt Diabetes Research and Training Center, Cancer Center and Center for Molecular Neuroscience.

"It's long been known that many cell reactions are driven by phosphorylation by kinases," Means said. "And there's a large amount of evidence that kinase activity is tightly regulated. This new finding provides an important insight into how that regulation takes place."

The CaMKIV kinase that the researchers studied plays a critical part in activating T-cells, the warrior cells of the immune system that patrol the body seeking invaders such as viruses and bacteria. Specifically, CaMKIV switches on a complex of other proteins in the cell nucleus that triggers the T-cell to activate genes, a process called transcription. That ultimately leads to rapid proliferation of the T-cells.

CaMKIV is triggered to action when an outside signal unleashes a flood of calcium into the T-cell. What puzzled the researchers about this process was how the kinase quickly turned off, even in the continued presence of high levels of calcium.

"We found that after stimulation the CaMKIV rose to 15 times the background amount after one minute, but plunged all the way to zero after five minutes, even though there was still enough calcium in the cell to activate it. There had to be some mechanism that turned off this enzyme, while still allowing calcium to carry out its other regulatory duties in the cell," Means said.

Previously, it was believed that such phosphatases might be free-floating in the cell, performing their catalytic task on a "hit-and-run" basis, he said. Reasoning, however, that the kinase might have a phosphatase closely attached to it, the researchers performed experiments revealing that the isolation of one enzyme carried the other along with it, as if they were linked. The scientists' tests also revealed that PP2A bound the CaMKIV in a one-to-one ratio.

Their studies also showed that the binding of the two enzymes together did not depend on the catalytic activity, indicating that their connection did not relate to their acting on each other in their role as enzymes. However, other experiments showed that PP2A did act enzymatically on CaMKIV to remove a phosphate, resulting in inactivation of the kinase.

Taken together, the results do indicate that the complex of the kinase and phosphatase is an important regulatory system in the cell, Means said. Still unclear, though, is how calcium manages to turn on the kinase at all when the phosphatase is poised to quickly turn it off, Means added. The kinase reaction may temporarily outpace the phosphatase's ability to switch it off, he said, or the phosphatase may somehow be temporarily inhibited by some cellular mechanism.

The therapeutic implications of the discovery of the complex could be important, Means said.

"This helps us identify ways of approaching a targeted inhibition of signaling pathways. If a protein kinase and a phosphatase are not just working on the same protein, but are sitting there hand-in-glove, then in theory you could block the entire pathway by inhibiting one or activating the other. This would change the balance of the reaction and lead to novel targets for therapeutic intervention."

According to Means, the scientists are now exploring further the complex action of this kinase-phosphatase complex in the cell. For example, there is evidence that specific versions of the phosphatase may render the combined kinase-phosphatase partnership a specific controller for specific targeted cell processes, he said.

The researchers also are exploring how the CaMKIV-PP2A complex appears to trigger other cell control mechanisms that activate gene transcription in the process of turning on the immune system's T-cells.

"We believe that we will find a general mechanism in cells that contain CaMKIV that will show this enzyme complex to be an integrator of regulated transcription involving many molecules that have to be orchestrated carefully for the cell to proliferate or to exhibit a differentiated function," Means said.

Duke University

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