Kyle W. Cunningham
Department of Biology
The Johns Hopkins University
3400 N. Charles Street
Baltimore, MD 21218
1996 Searle Scholar
Calcium Signalling and Protein Phosphorylation in Signal Transduction
Calcium ions (Ca2+) serve as a key regulator of numerous processes that occur within cells. Sudden changes in Ca2+ concentration can trigger contraction of muscle cells, fertilization of egg cells, release of neurotransmitters from neurons, export of insulin into the blood, phototransduction in the eye, and many other responses in different cell types. Ca2+ also plays a crucial role in the activation of T-cells, causing these important cells of the immune system to become activated protectors against foreign agents in the body. Drugs that block the effects of Ca2+ in T-cells are now used widely as immunosuppressants to prevent donor tissue from rejection after transplantation.
While much is known about Ca2+ movements and functions in many cell types, several key questions remain unanswered. For example, the channels that allow Ca2+ into T-cells and many other non-excitable cells are not yet identified and their key regulators are also unknown. A better understanding of these early steps in the Ca2+ signaling mechanism might therefore lead to new or improved therapeutics.
Recently, researchers in my laboratory have helped to show that the molecular mechanism responsible for T-cell activation also occurs in bakers' yeast, a simple single-celled organism that is well known for its utility in biomedical research. Because of this finding, we can now apply rapid and powerful methods of yeast genetics and cell biology first toward identifying the missing components of the Ca2+ signaling mechanism and then toward determining how they all work together. Using the yeast mechanism as both a paradigm and a tool, it should be much easier to identify the corresponding factors that operate in T-cells and other cells and begin to examine how they function in the human body.
How will yeast genetics reveal the missing factors and their functions? It turns out that the levels of Ca2+ in the yeast cytoplasm is controlled by ion pumps and exchangers related to those found in mammalian cells. These Ca2+ transporters are regulated by calcineurin, the target of the immunosuppressants Cyclosporin A and FK506. In both yeast and T-cells, calcineurin is a protein phosphatase that becomes activated in response to high levels of Ca2+. Calcineurin then activates transcription factors which enter the cell nucleus and induce the expression of several specific genes. The key tool we use to study this mechanism in yeast is a hybrid gene we constructed in which the promoter of one gene that is induced by calcineurin is fused to the gene for an easy to measure enzyme termed beta-galactosidase. Thus when appropriately stained for beta-galactosidase activity, the engineered yeast strains turn blue only when calcineurin becomes activated. We can now use this strain to identify new genes whose products function in the Ca2+ signaling mechanism by searching for rare mutants that fail to turn blue. Once these mutants are collected, the mutated genes can be readily identified and characterized to determine their position and function in the overall mechanism. Moreover, we can also compare their structures with a with a databank of partial genes from all species to potentially find related factors in humans.
The most mysterious part of the Ca2+ signaling mechanism right now is the process of Ca2+ entry into the cell known as "capacitative Ca2+ entry". In many cells (including yeast), an unknown Ca2+ channel at the cell surface becomes activated by unknown factors which become activated in response to insufficient Ca2+ levels in an intracellular organelle termed the endoplasmic reticulum (ER). During T-cell activation, the depleted ER therefore sends a crucial signal enabling more Ca2+ entry and subsequent activation of calcineurin and its targets. We observed a similar process in yeast when an intracellular organelle is depleted of Ca2+ by mutating the gene encoding the Ca2+ pump there. In this mutant strain, a new Ca2+ channel is highly active, resulting in the activation of calcineurin and expression of beta-galactosidase. By implementing the blue-to-white mutant search described above, it should be possible to find some of the first genes required for the widespread process of capacitative Ca2+ entry.
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