Sci. STKE, 23 January 2007
Biochemistry Bcl-xL Deamidation a Trigger for Apoptosis
Nancy R. Gough
Science's STKE, AAAS, Washington, DC 20005, USA
Using a transgenic mouse as a model for T cell lymphoma, Zhao et al. provide evidence that defective deamidation of Bcl-xL contributes to the apoptosis resistance and oncogenesis of thymocytes in these animals. These mice (CD45/lckF505) are deficient in the phosphatase CD45 and have a mutant form of the kinase Lck. DNA damage does not cause apoptosis in the thymocytes of these mice despite a normal p53 response. Bcl-xL sequesters the proapoptotic BH3 domain proteins, such as Bim and Puma, and deamidation, which converts Asn52 and Asn66 to isoaspartate (Iso-Asp), has been suggested to prevent this interaction, allowing apoptosis to proceed. Zhao et al. show that Bcl-xL deamidation (monitored by retardation on native gel electrophoresis) was not prevented by a pan-caspase inhibitor or by knockdown of Bax and Bak (two proteins required for mitochondrial-mediated apoptosis). Thus, Bcl-xL deamidation appears to occur before caspase activation and not as a consequence of caspase activation. Coimmunoprecipitation experiments confirmed that Bcl-xL did not bind Bim or Puma and that the interaction between Bcl-xL and these two proteins was not lost in response to DNA damage in the cells from the CD45/lckF505 mice. Anion-exchange chromatography was used to separate native from deamidated Bcl-xL, and the lack of interaction between Bim and the deamidated forms was confirmed. The authors noted that deamidation occurs more readily at higher pH and found that DNA damage raises the intracellular pH of wild-type thymocytes. When the pH of wild-type cells was artificially elevated, Bcl-xL deamidation occurred to a greater extent than at neutral pH, and when DNA damage was induced under conditions that prevented the elevation in pH, Bcl-xL deamidation was prevented, and the number of apoptotic cells was reduced. The resistance to apoptosis in response to DNA damage was overcome in the CD45/lckF505 thymocytes when the pH was increased to 7.55 or above. DNA damage triggers an increase in the abundance of the Na+/H+ exchanger 1 (NHE-1) through increased gene expression in wild-type cells, and this is not observed in the CD45/lckF505 cells. Overexpression of NHE-1 in the CD45/lckF505 and wild-type cells caused Bcl-xL deamidation and triggered apoptosis; whereas pharmacological inhibition of NHE-1 activity blocked alkalinization, Bcl-xL deamidation, and apoptosis. Using cells from patients with chronic lymphocytic leukemia, the authors showed that enforced alkalinization caused an increase in Bcl-xL deamidation and apoptosis.
R. Zhao, D. Oxley, T. S. Smith, G. A. Follows, A. R. Green, D. R. Alexander, DNA damage-induced Bcl-xL deamidation is mediated by NHE-1 antiport regulated intracellular pH. PLoS Biol. 5, e1 (2007). [PubMed]
Citation: N. R. Gough, Bcl-xL Deamidation a Trigger for Apoptosis. Sci. STKE 2007, tw30 (2007).
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