Grim Stimulates Diap1 Poly-Ubiquitination by Binding to UbcD1

  • Yoo, Soon Ji (Department of Biology, Kyung Hee University)
  • Received : 2005.10.21
  • Accepted : 2005.10.24
  • Published : 2005.12.31


Diap1 is an essential Drosophila cell death regulator that binds to caspases and inhibits their activity. Reaper, Grim and Hid each antagonize Diap1 by binding to its BIR domain, activating the caspases and eventually causing cell death. Reaper and Hid induce cell death in a Ring-dependent manner by stimulating Diap1 auto-ubiquitination and degradation. It was not clear that how Grim causes the ubiquitination and degradation of Diap1 in Grim-dependent cell death. We found that Grim stimulates poly-ubiquitination of Diap1 in the presence of UbcD1 and that it binds to UbcD1 in a GST pull-down assay, so presumably promoting Diap1 degradation. The possibility that dBruce is another E2 interacting with Diap1 was examined. The UBC domain of dBruce slightly stimulated poly-ubiquitination of Diap1 in Drosophila extracts but not in the reconstitution assay. However Grim did not stimulate Diap1 poly-ubiquitination in the presence of the UBC domain of dBruce. Taken together, these results suggest that Grim stimulates the poly-ubiquitination and presumably degradation of Diap1 in a novel way by binding to UbcD1 but not to the UBC domain of dBruce as an E2.




Supported by : Kyung Hee University


  1. Bartke, T., Pohl, C., Pyrowolakis, G., and Jentsch, S. (2004) Dual role of BRUCE as an antiapoptotic IAP and a chimeric E2/E3 ubiquitin ligase. Mol. Cell 14, 801-811
  2. Claveria, C., Albar, J. P., Serrano, A., Buesa, J. M., Barvero, J. L., et al. (1998) Drosophila grim induces apoptosis in mammalian cells. EMBO J. 17, 7199-7208
  3. Hay, B. A., Wassarman, D. A., and Rubin, G. M. (1995) Drosophila homologs of baculovirus inhibitor of apoptosis proteins function to block cell death. Cell 83, 1253-1262
  4. Lasi, S., Mazzon, I., and White, K. (2000) Diverse domains of THREAD/DIAP1 are required to inhibit apoptosis induced by REAPER and HID in Drosophila. Genetics 154, 669-678
  5. Liu, M.-L., Liu, M.-J., Kim, J.-M., Kim, H.-J., Kim, J.-H., et al. (2005) htra2 interacts with A$\beta$ peptides but does not directly alter its production or degradation. Mol. Cells 20, 83-89
  6. McCarthy, J. V. and Dixit, V. M. (1998) Apoptosis induced by Drosophila reaper and grim in a human system. Attenuation by inhibitor of apoptosis proteins (cIAPs). J. Biol. Chem. 273, 24009-24015
  7. Vernooy, S. Y., Chow, V., Su, J., Verbrugghe, K., Yang, J., et al. (2002) Drosophila Bruce can potently suppress Rpr- and Grim-dependent but not Hid-dependent cell death. Curr. Biol. 12, 1164-1168
  8. Yoo, S. J., Huh, J. R., Muro, I., Yu, H., Wang, S. L., et al. (2002) Hid, Reaper and Grim negatively regulate DIAP1 levels through distinct mechanisms. Nat. Cell Biol. 4, 414-424
  9. Holley, C. L., Olson, M. R., Colon-Ramos, D. A., and Kornbluth, S. (2002) Reaper eliminates IAP proteins through stimulated IAP degradation and generalized translational inhibition. Nat. Cell Biol. 4, 439-444
  10. Wyllie, A. H., Kerr, J. F., and Currie, A. R. (1980) Cell death: the significance of apoptosis. Int. Rev. Cytol. 68, 251-306
  11. Ditzel, M., Wilson, R., Tenev, T., Zachariou, A., Paul, A., et al. (2003) Degradation of Diap1 by the N-end rule pathway is essential for regulating apoptosis. Nat. Cell Biol. 5, 467-473
  12. Hashizume, R., Fukuda, M., Maeda, I., Nishkawa, H., Oyake, D., et al. (2001) The RING heterodimer BRCA1-BARD1 is an ubiquitin ligase inactivated by a breast cancer-derived mutation. J. Biol. Chem. 276, 14537-14540
  13. Wang, S. L., Hawkins, C. J., Yoo, S. J., Muller, H. A., and Hay, B. A. (1999) The Drosophila caspase inhibitor DIAP1 is essential for cell survival and is negatively regulated by HID. Cell 98, 453-463
  14. Arama, E., Agapite, J., and Steller, H. (2003) Caspase activity and a specific cytochrome C are required for sperm differentiation in Drosophila. Dev. Cell 4, 687-697
  15. Shi, Y. (2002) Mechanisms of caspase activation and inhibition during apoptosis. Mol. Cell 9, 459-470
  16. Zimmermann, K. C., Ricci, J.-E., Droin, N. M., and Green, D. R. (2002) The role of ARK in stress-induced apoptosis in Drosophila cells. J. Cell Biol. 156, 1077-1087
  17. Hao, Y., Skine, K., Kawabata, A., Nakamura, H., Ishioka, T., et al. (2004) Apollon ubiquitinates Smac and caspase-9, and has an essential cytoprotection function. Nat. Cell Biol. 6, 849-860
  18. Ryoo, H. D., Bergmann, A., Gonen, H., Ciechanover, A., and Steller, H. (2002) Regulation of Drosophila IAP1 degradation and apoptosis by reaper and ubcD1. Nat. Cell Biol. 4, 432-438
  19. Dohi, T., Okada, K., Xia, F., Wilford, C. E., Samuel, T., et al. (2004) An IAP-IAP complex inhibits apoptosis. J. Biol. Chem. 279, 34087-34090
  20. Sekine, K., Hao, Y., Suzuki, Y., Takahashi, R., Tsuruo, T., et al. (2005) HtrA2 cleaves Apollon and induces cell death by IAPbinding motif in Apollon-deficient cells. Biochem. Biophys. Res. Commun. 330, 279-285
  21. Silke, J., Kratina, T., Ekert, P. G., Pakusch, M., and Vaux, D. L. (2004) Unlike Diablo/smac, Grim promotes global ubiquitination and specific degradation of X chromosome-linked inhibitor of apoptosis (XIAP) and neither cause apoptosis. J. Biol. Chem. 279, 4313-4321
  22. Nagata, S. (1997) Apoptosis by death factor. Cell 88, 355-365
  23. Reed, J. C. (1999) Dysregulation of apoptosis in cancer. J. Clin. Oncol. 17, 2941-2953
  24. White, K., Grether, M. E., Abrams, J. M., Young, L., Farrell, K., et al. (1994) Genetic control of programmed cell death in Drosophila. Science 264, 677-683
  25. Hauser, H.-P., Bardroff, M., Pyrowolakis, G., and Jentsch, S. (1998) A giant ubiquitin-conjugating enzyme related to IAP apoptosis inhibitors. J. Cell. Biol. 141, 1415-1422
  26. Hengartner, M. O. (2000) The biochemistry of apoptosis. Nature 407, 770-776
  27. Yuan, J., Lipinski, M., and Degterev, A. (2003) Diversity in the mechanisms of neuronal cell death. Neuron 40, 401-413
  28. Wu, J.-W., Cocina, A. E., Chai, J., Hay, B. A., and Shi, Y. (2001) Structural analysis of a functional DIAP1 fragment bound to Grim and Hid peptides. Mol. Cell 8, 95-104
  29. Silke, J., Hawkins, C. J., Ekert, P. G., Chew, J., Day, C. L., et al. (2002) The anti-apoptotic activity of XIAP is retained upon mutation of both the caspase 3- and caspase 9- interacting sites. J. Cell Biol. 157, 115-124
  30. Yang, Y., Fang, S., Jensen, J. P., Weissman, A. M., and Ashwell, J. D. (2000) Ubiquitination protein ligase activity of IAPs and their degradation in proteasomes in response to apoptotic stimuli. Science 288, 874-877
  31. Olson, M. R., Holley, C. L., Yoo, S. J., Huh, J. R., Hay, B. A., et al. (2003) Reaper is regulated by IAP-mediated ubiquitination. J. Biol. Chem. 278, 4028-4034
  32. Vaux, D. L. and Silke, J. (2005) IAPs, RINGs and ubiquitylation. Nat. Rev. Mol. Cell. Biol. 6, 287-297