The Prize:
The 1992 Nobel Prize in Physiology or Medicine went to Edmond H. Fischer and Edwin G. Krebs for their discoveries of “reversible protein phosphorylation as a biological regulatory mechanism.”
The Science:
Most critical processes in the cell, including cell division, metabolism, and general housekeeping, are carried out by proteins. Proteins perform the daily activities central to life. But how does the cell control when and where these reactions take place? There needs to be a mechanism to control cellular processes both in space (i.e. during cell division, the cell should split only through the middle and not just anywhere) and in time (i.e. muscle cells should contract only when stimulated by a motor neuron). To be useful, this control should be quick and reversible.
In the 1940s, it was known that the breakdown of glycogen (the body’s major form of glucose storage) required the action of an enzyme called phosphorylase. Phosphorylase catalyzes glycogen breakdown by removing a phosphate from an ATP molecule (the phosphate-rich energy currency of the cell) and transferring it to the glucose molecule it releases. This process is important to muscle cells between meals as it ensures that there’s always enough glucose to provide the energy needed to move our bodies. Just after meals, however, the cell must be able to shut the phosphorylase off so that the muscle cell can take up and store (as glycogen) the sugars that the digestive system has deposited into the blood. This balance must therefore be tightly regulated.
Phosphorylase was one of the first known examples of an enzyme that could exist in both an active an inactive state. The transition between the two states was one that scientists knew how to control in a test tube. In the cell, however, they had to rely on hormones. They knew that adrenaline could somehow turn off phosphorylase activity, but how?
Edwin Krebs and Edmond Fischer worked diligently on this problem and eventually discovered that ATP was required for the conversion of the inactive to active form. A “conversion enzyme,” now called phosphorylase kinase, transfers a phosphate from ATP to phosphorylase making it active and in turn, allowing phosphorylase to add a phosphate group to an exiting glucose.
Phosphorylase activity can be shut down just as easily as it’s turned on by removing the phosphate group via another conversion enzyme called phosphatase. This means that between meals you find phosphorylase decorated with a phosphate group, actively releasing glucose from glycogen, and just after a meal there’s a “nude” phosphorylase in an inactive state allowing glycogen stores to build up.
The truly ground-breaking result came when it was recognized that resting muscle (which is actively building glycogen stores for its next burst of activity) has more inactive phosphorylase and active muscle (which is breaking down glycogen for fuel) has more active phosphorylase. These two activity levels can be connected to nerve stimulation. The difference between a resting and active muscle is the result of a nerve impulse. An electric impulse from a motor neuron releases stored calcium into the muscle cell, thus activating phosphorylase kinase, releasing glucose, and providing the muscle with the energy it needs to contract.
Further work demonstrated that phosphorylase kinase activity, the “activator” of phosphorylase, is controlled not only by incoming calcium, but also by levels of cyclic-AMP, a molecule that accumulates in starving cells. Interestingly, cyclic-AMP is also built up by the cell when it is exposed to adrenaline, tying together decades’ worth of research.
This was the first example of what is now called a “kinase cascade” (because of its likeness to a waterfall), a ladder of activation (or deactivation) steps that each result in the transfer of a phosphate group from ATP to a target protein to modulate its function. The cascade is simple and elegant, allowing for easy regulation of proteins with two activity states.
The Significance:
As with many discoveries in biology, the principle of reversible protein phosphorylation is not unique to glucose metabolism. Almost every cellular pathway that requires temporal or spatial control makes use of reversible phosphorylation. These pathways control such diverse functions as metabolism, neurotransmitter release, cell-to-cell communication, cell division, and cell mortality. All of these processes require a kinase enzyme to transfer the phosphate group, and a phosphatase enzyme to remove it.
Reversible phosphorylation is only one of many ways the cell can fine-tune a protein’s function. Many proteins can also be tagged with other small proteins to further control cellular function. These discoveries (one of which led to the 2004 Nobel Prize in Chemistry) may not have been possible without the knowledge of reversible modification of a protein.
Abl kinase is a kinase that is an important regulator of cell growth and has shown to be important for the development of chronic myelogenous leukemia. In the late 1990s, the pharmaceutical company Novartis targeted Abl kinase and designed an inhibitor of Abl, called Gleevec, into a very effective treatment for leukemia. Gleevec is still in use today as an anti-leukemia drug and is the poster-child for rational drug design.
