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Pharmacologists Discover Linkage of Key Cell Switches

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Duke Health News 919-660-1306

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.

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