Intracellular localization of TDAG51 modulates its effect on cell death

Our previously findings indicate that T‐cell death associated gene 51 (TDAG51) contributes to cell death in atherosclerotic lesions. To determine if these cell death effects are mediated by TDAG51 intracellular localization, we performed sequence analysis of the TDAG51 cDNA to identify novel cellular targeting domains. This analysis revealed two putative motifs; a nuclear exiting signal (NES) (70‐vasleppvkl‐80) and nuclear localization signal (NLS) (16‐krsdgllqlwkkk‐28). We hypothesized that these TDAG51 motifs play a critical role in its intracellular transport, thereby modulating its pro‐apoptotic effects. To investigate this phenomenon, VKL was mutated to AKA in NES to impair exiting of the TDAG51 protein from the nucleus to the cytoplasm and WKKK was mutated to WKAA in NLS to impair entrance of TDAG51 into the nucleus. Following transient transfection of the fused GFP constructs into HeLa cells, the cellular localization of these mutant TDAG51 proteins was assessed using confocal microscopy on fixed cells or visualized in living cells over a 2 to 48 hour time period. Our findings show that both motifs are involved in the intracellular transport of TDAG51. By determining cell viability, we were able to determine the predominant effects of nuclear TDAG51 on cell death. These studies provide a better understanding of the cellular motifs in TDAG51 that contribute to cell death. Supported by the CIHR (MOP‐74477).