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Human homologue of S. pombe Rad9 interacts with BCL-2/BCL-xL and promotes apoptosis

Abstract

DNA damage induces apoptosis through a signalling pathway that can be suppressed by the BCL-2 protein, but the mechanism by which DNA damage does this is unknown. Here, using yeast two-hybrid and co-immunoprecipitation studies, we show that RAD9, a human protein involved in the control of a cell-cycle checkpoint, interacts with the anti-apoptotic Bcl-2-family proteins BCL-2 and BCL-xL, but not with the pro-apoptotic BAX and BAD. When overexpressed in mammalian cells, RAD9 induces apoptosis that can be blocked by BCL-2 or BCL-xL. Conversely, antisense RAD9 RNA suppresses cell death induced by methyl methanesulphonate. These findings indicate that RAD9 may have a new role in regulating apoptosis after DNA damage, in addition to its previously described checkpoint-control and other radioresistance-promoting functions.

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Figure 1: Alignment of amino-acid residues in the BH3-homology regions of Bcl-2-family members and human RAD9.
Figure 2: RAD9 selectively interacts with Bcl-2-family proteins.
Figure 3: A DNA-damaging agent induces perinuclear localization of RAD9 and co-localization of RAD9 with BCL-2.
Figure 4: Effects of RAD9 on survival of FL5.12 cells.
Figure 5: RAD9 promotes apoptosis in a BH3-dependent manner.
Figure 6: Antisense RAD9 RNA suppresses cell death induced by MMS.

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References

  1. Oltvai, Z. N. & Korsmeyer, S. J. Checkpoints of dueling dimers foil death wishes. Cell 79, 189– 192 (1994).

    Article  CAS  Google Scholar 

  2. Kelekar, A. & Thompson, C. B. Bcl-2-family proteins: the role of the BH3 domain in apoptosis. Trends Cell Biol. 8 , 324–330 (1998).

    Article  CAS  Google Scholar 

  3. Adams, J. M. & Cory, S. The Bcl-2 protein family: arbiters of cell survival. Science 281, 1322– 1326 (1998).

    Article  CAS  Google Scholar 

  4. Russell, P. Checkpoints on the road to mitosis. Trends Biochem. Sci. 23, 399–402 (1998).

    Article  CAS  Google Scholar 

  5. Paulovich, A. G., Toczyski, D. P. & Hartwell, L. H. When checkpoints fail. Cell 88, 315–321 (1997).

    Article  CAS  Google Scholar 

  6. al-Khodairy, F. & Carr, A. M. DNA repair mutants defining G2 checkpoint pathways in Schizosaccharomyces pombe. EMBO J. 11, 1343–1350 ( 1992).

    Article  CAS  Google Scholar 

  7. Enoch, T., Carr, A. M. & Nurse, P. Fission yeast genes involved in coupling mitosis to completion of DNA replication. Genes Dev. 6, 2035–2046 (1992).

    Article  CAS  Google Scholar 

  8. Rowley, R., Subramani, S. & Young, P. G. Checkpoint controls in Schizosaccharomyces pombe: rad1. EMBO J. 11, 1335–1342 (1992).

    Article  CAS  Google Scholar 

  9. al-Khodairy, F. et al. Identification and characterization of new elements involved in checkpoint and feedback controls in fission yeast. Mol. Biol Cell. 5, 147–160 ( 1994).

    Article  CAS  Google Scholar 

  10. Zha, H., Aime-Sempe, C., Sato, T. & Reed, J. C. Pro-apoptotic protein Bax heterodimerizes with Bcl-2 and homodimerizes with Bax via a novel domain (BH3) distinct from BH1 and BH2. J. Biol. Chem. 271, 7440–7444 (1996).

    Article  CAS  Google Scholar 

  11. Chittenden, T. et al. A conserved domain in Bak, distinct from BH1 and BH2, mediates cell death and protein binding functions. EMBO J. 14 , 5589–5596 (1995).

    Article  CAS  Google Scholar 

  12. Sattler, M. et al. Structure of Bcl-xL–Bak peptide complex: recognition between regulators of apoptosis. Science 275, 983–986 (1997).

    Article  CAS  Google Scholar 

  13. Zha, J. et al. BH3 domain of BAD is required for heterodimerization with BCL-XL and pro-apoptotic activity. J. Biol. Chem. 272, 24101–24104 (1997).

    Article  CAS  Google Scholar 

  14. Inohara, N., Ding, L., Chen, S. & Nunez, G. Harakiri, a novel regulator of cell death, encodes a protein that activates apoptosis and interacts selectively with survival-promoting proteins Bcl-2 and Bcl-X L. EMBO J. 16, 1686–1694 (1997).

    Google Scholar 

  15. Wang, K., Yin, W.-M., Chao, D. T., Milliman, C. L. & Korsmeyer, S. J. BID: a novel BH3 domain-only death agonist. Genes Dev. 10, 2859–2869 (1996).

    Article  CAS  Google Scholar 

  16. Conradt, B. & Horvitz, H. R. The C. elegans protein EGL-1 is required for programmed cell death and interacts with the Bcl-2-like protein CED-9. Cell 93, 519–529 (1998).

    Article  CAS  Google Scholar 

  17. Diaz, J.-L. et al. A common binding site mediates heterodimerization and homodimerization of Bcl-2 family members. J. Biol. Chem. 272, 11350–11355 (1997).

    Article  CAS  Google Scholar 

  18. Monaghan, P. et al. Ultrastructural localization of bcl-2 protein. J. Histochem. Cytochem. 40, 1819–1825 (1992).

    Article  CAS  Google Scholar 

  19. Krajewski, S. et al. Investigation of the subcellular distribution of the bcl-2 oncoprotein: residence in the nuclear envelope, endoplasmic reticulum, and outer mitochondrial membranes. Cancer Res. 53, 4701–4714 (1993).

    CAS  PubMed  Google Scholar 

  20. Lieberman, H. B., Hopkins, K. M., Nass, M., Demetrick, D. & Davey, S. A human homolog of the Schizosaccharomyces pombe rad9 + checkpoint control gene. Proc. Natl Acad. Sci. USA 93, 13890–13895 (1996).

    Article  CAS  Google Scholar 

  21. Davey, S. et al. Fission yeast rad12+ regulates cell cycle checkpoint control and is homologous to the Bloom’s syndrome disease gene. Mol. Cell Biol. 18, 2721– 2728 (1998).

    Article  CAS  Google Scholar 

  22. Volkmer, E. & Karnitz, L. M. Human homologs of Schizosaccharomyces pombe rad1, hus1, and rad9 form a DNA damage-responsive protein complex. J. Biol. Chem. 274, 567–570 (1999).

    Article  CAS  Google Scholar 

  23. Onge, R. P., Udell, C. M., Casselman, R. & Davey, S. The human G2 checkpoint control protein hRAD9 is a nuclear phosphoprotein that forms complexes with hRAD1 and hHUS1. Mol. Biol. Cell 10, 1985–1995 (1999).

    Article  Google Scholar 

  24. Murray, J. M., Carr, A. M., Lehmann, A. R. & Watts, F. Z. Cloning and characterisation of the rad9 DNA repair gene from Schizosaccharomyces pombe. Nucleic Acids Res. 19, 3525– 3531 (1991).

    Article  CAS  Google Scholar 

  25. Lieberman, H. B., Hopkins, K. M., Laverty, M. & Chu, H. M. Molecular cloning and analysis of Schizosaccharomyces pombe rad9, a gene involved in DNA repair and mutagenesis. Mol. Gen. Genet. 232 , 367–376 (1992).

    Article  CAS  Google Scholar 

  26. Lieberman, H. B. Extragenic suppressors of Schizosaccharomyces pombe rad9 mutations uncouple radioresistance and hydroxyurea sensitivity from cell cycle checkpoint control . Genetics 141, 107–117 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Kastan, M. B., Onyekwere, O., Sidransky, D., Vogelstein, B. & Craig, R. W. Participation of p53 protein in the cellular response to DNA damage. Cancer Res. 51 , 6304–6311 (1991).

    CAS  PubMed  Google Scholar 

  28. Kastan, M. B. et al. A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell 71, 587–597 (1992).

    Article  CAS  Google Scholar 

  29. Takagi, M. et al. Defective control of apoptosis, radiosensitivity, and spindle checkpoint in ataxia telangiectasia. Cancer Res 58, 4923–4929 (1998).

    CAS  PubMed  Google Scholar 

  30. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. & Pease, L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59 (1989).

    Article  CAS  Google Scholar 

  31. Wang, H.-G., Takayama, S., Rapp, U. R. & Reed, J. C. Bcl-2 interacting protein, BAG-1, binds to and activates the kinase Raf-1 . Proc. Natl Acad. Sci. USA 93, 7063– 7068 (1996).

    Article  CAS  Google Scholar 

  32. Wang, H.-G., Rapp, U. R. & Reed, J. C. Bcl-2 targets the protein kinase Raf-1 to mitochondria . Cell 87, 629–638 (1996).

    Article  CAS  Google Scholar 

  33. Wang, H.-G. et al. Ca2+-induced apoptosis through calcineurin dephosphorylation of BAD. Science 284, 339 –343 (1999).

    Article  CAS  Google Scholar 

  34. Estojak, J., Brent, R. & Golemis, E. A. Correlation of two-hybrid affinity data with in vitro measurements. Mol. Cell Biol. 15, 5820– 5829 (1995).

    Article  CAS  Google Scholar 

  35. Sato, T. et al. Interactions among members of the bcl-2 protein family analyzed with a yeast two-hybrid system. Proc. Natl Acad. Sci.USA 91, 9238–9242 (1994).

    Article  CAS  Google Scholar 

  36. Wang, H.-G. et al. Apoptosis regulation by interaction of bcl-2 protein and Raf-1 kinase. Oncogene 9, 2751– 2756 (1994).

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank J. C. Reed and R. Jove for comments on the manuscript; S. Dalton for technical assistance; and the Molecular Biology, Flow Cytometry, and Molecular Imaging core facilities of Moffitt Cancer Center. This work was partially supported by grants from the ACS (IRG032) and NIH (CA82197-01) to H.-G.W., and grants from the NIH (GM52493 and CA68446) and DOE (DE-FG07-96ER62309) to H.B.L.

Correspondence and requests for materials should be addressed to H.-G.W.

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Correspondence to Hong-Gang Wang.

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Komatsu, K., Miyashita, T., Hang, H. et al. Human homologue of S. pombe Rad9 interacts with BCL-2/BCL-xL and promotes apoptosis. Nat Cell Biol 2, 1–6 (2000). https://doi.org/10.1038/71316

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