DNA damage-induced Bcl-x(L) deamidation is mediated by NHE-1 antiport regulated intracellular pH

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-Asp (52)/iso-Asp(66) 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.