The therapeutic value of DNA-damaging antineoplastic agents is dependent upon their ability to induce tumor cell apoptosis while sparing most normal tissues. We recently found that a component of the apoptotic response to these agents in several different types of tumor cells is the deamidation of two asparagines in the unstructured loop of Bcl-xL, and we found that deamidation of these asparagines imparts susceptibility to apoptosis by disrupting the ability of Bcl-xL to block the proapoptotic activity of BH3 domain-only proteins. Conversely, we found that Bcl-xL deamidation is actively suppressed in fibroblasts, and that suppression of deamidation is an essential component of their resistance to DNA damage-induced apoptosis. Finally, we found that at least in some cells, the retinoblastoma protein mediates the suppression of Bcl-xL deamidation. We have begun to define the mechanism by which deamidation of Bcl-XL is regulated. Our data provides evidence that deamidation of Bcl-xL is induced by an upward shift in the cytosolic pH that is induced by cisplatin treatment and that the retinoblastoma protein suppresses Bcl-xL deamidation because it suppresses the increase in pH. Importantly, our data strongly suggests that the increase in pH does not directly cause deamidation of Bcl-xL. Instead, our findings support a mechanism in which the cytosolic pH increase induces deamidation of Bcl-xL by activating an autocatalytic """"""""deamidase"""""""" function of Bcl-xL. We propose that this mechanism affords the cell tighter control of Bcl-xL deamidation and allows deamidation to occur at a lower pH and more rapidly than if deamidation were regulated directly by pH. We speculate that a similar mechanism has an important role in the regulation of other proteins. We now propose to (1) characterize the autocatalytic deamidase activity by performing a structure-function analysis of Bcl-xL with respect to this activity; (2) delineate signal transduction pathways that mediate cisplatin-induced cytosolic alkalinization; and (3) examine the potential role of dysregulation of cisplatin-induced cytosolic alkalinization and Bcl-xL deamidation in tumor cell resistance to cisplatin. These studies may lead to the identification of cellular targets that allow for the development of more efficacious and less toxic antineoplastic therapies. ? ?
