DNA Damage–Induced Bcl-xL Deamidation Is Mediated by NHE-1 Antiport Regulated Intracellular pH

Abstract

The pro-survival protein Bcl-x_L is critical for the resistance of tumour cells to DNA damage. We have previously demonstrated, using a mouse cancer model, that oncogenic tyrosine kinase inhibition of DNA damage–induced Bcl-x_L deamidation tightly correlates with T cell transformation in vivo, although the pathway to Bcl-x_L deamidation remains unknown and its functional consequences unclear. We show here that rBcl-x_L deamidation generates an iso-Asp⁵²/iso-Asp⁶⁶ species that is unable to sequester pro-apoptotic BH3-only proteins such as Bim and Puma. DNA damage in thymocytes results in increased expression of the NHE-1 Na/H antiport, an event both necessary and sufficient for subsequent intracellular alkalinisation, Bcl-x_L deamidation, and apoptosis. In murine thymocytes and tumour cells expressing an oncogenic tyrosine kinase, this DNA damage–induced cascade is blocked. Enforced intracellular alkalinisation mimics the effects of DNA damage in murine tumour cells and human B-lineage chronic lymphocytic leukaemia cells, thereby causing Bcl-x_L deamidation and increased apoptosis. Our results define a signalling pathway leading from DNA damage to up-regulation of the NHE-1 antiport, to intracellular alkalanisation to Bcl-x_L deamidation, to apoptosis, representing the first example, to our knowledge, of how deamidation of internal asparagine residues can be regulated in a protein in vivo. Our findings also suggest novel approaches to cancer therapy. Cell survival and cell death (apoptosis) are controlled by a finely tuned ensemble of pro-survival and pro-apoptotic proteins. When the two types of protein are balanced, cells survive. But if the pro-survival proteins dominate, there is a danger that cells with damaged DNA will stay alive, leading to malignancy. One of the key pro-survival proteins, Bcl-x_L, acts by blocking the actions of pro-apoptotic proteins. We show here that DNA damage results in an important modification of Bcl-x_L. Specifically, when the amide groups are removed from two critical asparagine (amino acid) residues, Bcl-x_L can no longer block pro-apoptotic proteins, leading to cell death. Surprisingly, Bcl-x_L deamidation is catalysed not by an enzyme, but by increased pH inside the cell due to the up-regulation of an NHE-1 transporter that moves positive ions across the cell membrane. Indeed, artificially increasing pH causes Bcl-x_L deamidation and apoptosis in the absence of initial DNA damage. Exploring this novel pathway may ultimately suggest approaches to cancer therapy, especially when malignant cells are resistant to chemotherapy or radiotherapy.