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) (original) (raw)

Abstract

Programmed cell death is essential in organ development and tissue homeostasis and its deregulation is associated with the development of several diseases in mice and humans. The precise mechanisms that control cell death have not been elucidated fully, but it is well established that this form of cellular demise is regulated by a genetic program which is activated in the dying cell. Here we report the identification, cloning and characterization of harakiri, a novel gene that regulates apoptosis. The product of harakiri, Hrk, physically interacts with the death-repressor proteins Bcl-2 and Bcl-X(L), but not with death-promoting homologs, Bax or Bak. Hrk lacks conserved BH1 and BH2 regions and significant homology to Bcl-2 family members or any other protein, except for a stretch of eight amino acids that exhibits high homology with BH3 regions. Expression of Hrk induces cell death which is inhibited by Bcl-2 and Bcl-X(L). Deletion of 16 amino acids including the conserved BH3 region abolished the ability of Hrk to interact with Bcl-2 and Bcl-X(L) in mammalian cells. Moreover, the killing activity of this mutant form of Hrk (Hrk deltaBH3) was eliminated or dramatically reduced, suggesting that Hrk activates cell death at least in part by interacting with and inhibiting the protection afforded by Bcl-2 and Bcl-X(L). Because Hrk lacks conserved BH1 and BH2 domains that define Bcl-2 family members, we propose that Hrk and Bik/Nbk, another BH3-containing protein that activates apoptosis, represent a novel class of proteins that regulate apoptosis by interacting selectively with survival-promoting Bcl-2 and Bcl-X(L).

Full Text

The Full Text of this article is available as a PDF (589.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Askew D. S., Ashmun R. A., Simmons B. C., Cleveland J. L. Constitutive c-myc expression in an IL-3-dependent myeloid cell line suppresses cell cycle arrest and accelerates apoptosis. Oncogene. 1991 Oct;6(10):1915–1922. [PubMed] [Google Scholar]
  2. Boyd J. M., Malstrom S., Subramanian T., Venkatesh L. K., Schaeper U., Elangovan B., D'Sa-Eipper C., Chinnadurai G. Adenovirus E1B 19 kDa and Bcl-2 proteins interact with a common set of cellular proteins. Cell. 1994 Oct 21;79(2):341–351. doi: 10.1016/0092-8674(94)90202-x. [DOI] [PubMed] [Google Scholar]
  3. Chinnaiyan A. M., Orth K., O'Rourke K., Duan H., Poirier G. G., Dixit V. M. Molecular ordering of the cell death pathway. Bcl-2 and Bcl-xL function upstream of the CED-3-like apoptotic proteases. J Biol Chem. 1996 Mar 1;271(9):4573–4576. doi: 10.1074/jbc.271.9.4573. [DOI] [PubMed] [Google Scholar]
  4. Chittenden T., Harrington E. A., O'Connor R., Flemington C., Lutz R. J., Evan G. I., Guild B. C. Induction of apoptosis by the Bcl-2 homologue Bak. Nature. 1995 Apr 20;374(6524):733–736. doi: 10.1038/374733a0. [DOI] [PubMed] [Google Scholar]
  5. Clarke M. F., Apel I. J., Benedict M. A., Eipers P. G., Sumantran V., González-García M., Doedens M., Fukunaga N., Davidson B., Dick J. E. A recombinant bcl-x s adenovirus selectively induces apoptosis in cancer cells but not in normal bone marrow cells. Proc Natl Acad Sci U S A. 1995 Nov 21;92(24):11024–11028. doi: 10.1073/pnas.92.24.11024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Evan G. I., Wyllie A. H., Gilbert C. S., Littlewood T. D., Land H., Brooks M., Waters C. M., Penn L. Z., Hancock D. C. Induction of apoptosis in fibroblasts by c-myc protein. Cell. 1992 Apr 3;69(1):119–128. doi: 10.1016/0092-8674(92)90123-t. [DOI] [PubMed] [Google Scholar]
  7. Feilotter H. E., Hannon G. J., Ruddell C. J., Beach D. Construction of an improved host strain for two hybrid screening. Nucleic Acids Res. 1994 Apr 25;22(8):1502–1503. doi: 10.1093/nar/22.8.1502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. González-García M., Pérez-Ballestero R., Ding L., Duan L., Boise L. H., Thompson C. B., Núez G. bcl-XL is the major bcl-x mRNA form expressed during murine development and its product localizes to mitochondria. Development. 1994 Oct;120(10):3033–3042. doi: 10.1242/dev.120.10.3033. [DOI] [PubMed] [Google Scholar]
  9. Han J., Sabbatini P., Perez D., Rao L., Modha D., White E. The E1B 19K protein blocks apoptosis by interacting with and inhibiting the p53-inducible and death-promoting Bax protein. Genes Dev. 1996 Feb 15;10(4):461–477. doi: 10.1101/gad.10.4.461. [DOI] [PubMed] [Google Scholar]
  10. Han J., Sabbatini P., White E. Induction of apoptosis by human Nbk/Bik, a BH3-containing protein that interacts with E1B 19K. Mol Cell Biol. 1996 Oct;16(10):5857–5864. doi: 10.1128/mcb.16.10.5857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hannon G. J., Demetrick D., Beach D. Isolation of the Rb-related p130 through its interaction with CDK2 and cyclins. Genes Dev. 1993 Dec;7(12A):2378–2391. doi: 10.1101/gad.7.12a.2378. [DOI] [PubMed] [Google Scholar]
  12. Hockenbery D., Nuñez G., Milliman C., Schreiber R. D., Korsmeyer S. J. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature. 1990 Nov 22;348(6299):334–336. doi: 10.1038/348334a0. [DOI] [PubMed] [Google Scholar]
  13. Krajewski S., Tanaka S., Takayama S., Schibler M. J., Fenton W., Reed J. C. Investigation of the subcellular distribution of the bcl-2 oncoprotein: residence in the nuclear envelope, endoplasmic reticulum, and outer mitochondrial membranes. Cancer Res. 1993 Oct 1;53(19):4701–4714. [PubMed] [Google Scholar]
  14. Merino R., Grillot D. A., Simonian P. L., Muthukkumar S., Fanslow W. C., Bondada S., Núez G. Modulation of anti-IgM-induced B cell apoptosis by Bcl-xL and CD40 in WEHI-231 cells. Dissociation from cell cycle arrest and dependence on the avidity of the antibody-IgM receptor interaction. J Immunol. 1995 Oct 15;155(8):3830–3838. [PubMed] [Google Scholar]
  15. Motoyama N., Wang F., Roth K. A., Sawa H., Nakayama K., Nakayama K., Negishi I., Senju S., Zhang Q., Fujii S. Massive cell death of immature hematopoietic cells and neurons in Bcl-x-deficient mice. Science. 1995 Mar 10;267(5203):1506–1510. doi: 10.1126/science.7878471. [DOI] [PubMed] [Google Scholar]
  16. Nuñez G., London L., Hockenbery D., Alexander M., McKearn J. P., Korsmeyer S. J. Deregulated Bcl-2 gene expression selectively prolongs survival of growth factor-deprived hemopoietic cell lines. J Immunol. 1990 May 1;144(9):3602–3610. [PubMed] [Google Scholar]
  17. Oltvai Z. N., Milliman C. L., Korsmeyer S. J. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell. 1993 Aug 27;74(4):609–619. doi: 10.1016/0092-8674(93)90509-o. [DOI] [PubMed] [Google Scholar]
  18. Shaw G., Kamen R. A conserved AU sequence from the 3' untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell. 1986 Aug 29;46(5):659–667. doi: 10.1016/0092-8674(86)90341-7. [DOI] [PubMed] [Google Scholar]
  19. Simonian P. L., Grillot D. A., Merino R., Nuñez G. Bax can antagonize Bcl-XL during etoposide and cisplatin-induced cell death independently of its heterodimerization with Bcl-XL. J Biol Chem. 1996 Sep 13;271(37):22764–22772. doi: 10.1074/jbc.271.37.22764. [DOI] [PubMed] [Google Scholar]
  20. Swaroop A., Xu J. cDNA libraries from human tissues and cell lines. Cytogenet Cell Genet. 1993;64(3-4):292–294. doi: 10.1159/000133595. [DOI] [PubMed] [Google Scholar]
  21. Takayama S., Sato T., Krajewski S., Kochel K., Irie S., Millan J. A., Reed J. C. Cloning and functional analysis of BAG-1: a novel Bcl-2-binding protein with anti-cell death activity. Cell. 1995 Jan 27;80(2):279–284. doi: 10.1016/0092-8674(95)90410-7. [DOI] [PubMed] [Google Scholar]
  22. Veis D. J., Sorenson C. M., Shutter J. R., Korsmeyer S. J. Bcl-2-deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair. Cell. 1993 Oct 22;75(2):229–240. doi: 10.1016/0092-8674(93)80065-m. [DOI] [PubMed] [Google Scholar]
  23. 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 U S A. 1996 Jul 9;93(14):7063–7068. doi: 10.1073/pnas.93.14.7063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Yamagata K., Sanders L. K., Kaufmann W. E., Yee W., Barnes C. A., Nathans D., Worley P. F. rheb, a growth factor- and synaptic activity-regulated gene, encodes a novel Ras-related protein. J Biol Chem. 1994 Jun 10;269(23):16333–16339. [PubMed] [Google Scholar]
  25. Yang E., Zha J., Jockel J., Boise L. H., Thompson C. B., Korsmeyer S. J. Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death. Cell. 1995 Jan 27;80(2):285–291. doi: 10.1016/0092-8674(95)90411-5. [DOI] [PubMed] [Google Scholar]
  26. Zha H., Aimé-Sempé C., Sato T., Reed J. C. Proapoptotic protein Bax heterodimerizes with Bcl-2 and homodimerizes with Bax via a novel domain (BH3) distinct from BH1 and BH2. J Biol Chem. 1996 Mar 29;271(13):7440–7444. doi: 10.1074/jbc.271.13.7440. [DOI] [PubMed] [Google Scholar]