Calretinin (CR) is a calcium binding protein that we have isolated and purified. It is a neuron-specific protein found in a variety of interesting regions in the brain. Calretinin is over-expressed in mesothelioma cells and is used as a diagnostic marker for this type of cancer. Mesothelioma is a cancer that occurs in the tissues lining the outside of the lungs (pleural tissue). We have preliminary data showing that calretinin might function as a susceptibility gene by modulating the activity of the oncogene protein kinase CK2. We have demonstrated that CR inhibits the autophosphorylation of the oncogene protein kinase CK2. Furthermore, a point mutant of CR (A110T) discovered from the pleural cells of mesothelioma patients enhances autophosphorylation. The exact nature of this interaction and the identity of the other proteins interacting with CR are not known. We have also demonstrated, using fluorescence spectroscopy, that the mutation affects the calcium binding affinity of the protein as well as the confirmation of the protein. We have evidence that CR and the mutant CR forms a multimer in the presence of calcium, a property that may be critical in its interaction with protein kinase CK2. Phosphorylation experiments were carried out with the elongation factor as the substrate for protein kinase CK2 in the presence of CR. The results from this experiment showed that differential phosphorylation of elongation factor takes place with protein kinase CK2. This suggests that CR can be involved in the regulation of a protein that it is involved in cell proliferation The mutation also resulted in a restriction site change and hence it can be detected from the genomic DNA or total RNA using PCR and restriction digest with the enzyme Hae II. We developed this PCR/restriction digested method in order to detect this mutation in the genomic DNA isolated from the whole blood. The genetic susceptibility in mesothelioma can be established by screening for the A110T mutant and other mutations in the CR gene using samples from normal and mesothelioma groups of patients.
