Scientists at the University of Illinois at Chicago (UIC) say they have developed a novel method to determine the effects of turning protein kinases on and off, calculate their downstream effects, and uncover new drug targets.
The team's study (“Mimicking Transient Activation of Protein Kinases in Living Cells”) is reported in the Proceedings of the National Academy of Sciences.
Several hundred different kinases work to phosphorylate various proteins and drive the entire range of cellular activities. Understanding precisely how kinases work would reveal cellular pathways that drive cells to become cancerous and identify novel drug targets.
Until now, it was difficult to study cellular processes triggered by specific kinases that turn on only briefly, says Andrei Karginov, Ph.D., assistant professor of pharmacology in the UIC College of Medicine.
“Previously, we had the ability to artificially turn on a kinase, but we didn't know how to turn it off. So what we could see was only the result of that kinase being on for a long time,” notes Dr. Karginov. “That approach has proved useful to study the prolonged cancer-causing effects of some kinases. But in living cells, kinases are often turned only for a set period of time, and the duration of this activation often dictates what will happen to the cell. To mimic this transient action we had come up with a new strategy.”
The new procedure allows scientists to turn kinases on, and then off, providing a tool for studying the effects of kinases as they actually work in the body.
The researchers used two protein-engineering techniques to switch on and switch off one kinase (tyrosine kinase c-Src). They were able to observe a series of structural changes when they turned the enzyme on for various periods of time.
The first wave of changes, where the cell seemed to expand and then contract, was driven by kinase activation and inactivation. Interestingly, a second expansion sometimes occurred after the kinase had been switched off, depending on how long it had been left on.
The experiment demonstrated that even short-term activation of a kinase can have a long-lasting effect on cell behavior, and allowed researchers to identify the molecular mechanism driving these events.