Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2001 Nov 15;360(Pt 1):49–56. doi: 10.1042/0264-6021:3600049

cDNA cloning and promoter analysis of rat caspase-9.

J Nishiyama 1, X Yi 1, M A Venkatachalam 1, Z Dong 1
PMCID: PMC1222201  PMID: 11695991

Abstract

Caspase-9 is the apex caspase of the mitochondrial pathway of apoptosis, which plays a critical role in apoptotic initiation and progression. However, gene regulation of caspase-9 is largely unknown. This is in part due to the lack of information on the gene promoter. Here we have cloned the full-length cDNA of rat caspase-9 and have isolated promoter regions of this gene. The rat caspase-9 cDNA of 2058 bp predicts a protein of 454 amino acids, which contains a caspase-recruitment domain ('CARD') at the N-terminus and enzymic domains at the C-terminus. The enzyme's active site, with a characteristic motif of QACGG, was also identified. Overall, rat and human caspase-9 have 71% identity. With the cDNA sequence, we subsequently isolated the proximal 5'-flanking regions of rat caspase-9 by the procedure of genomic walking. The 2270 bp genomic segment is 'TATA-less', but contains several GC boxes. Elements binding known transcription factors such as Sp-1, Pit-1, CCAAT-enhancer-binding protein (C/EBP), glucocorticoid receptor and hypoxia-inducible factor 1 (HIF-1) were also identified. When cloned into reporter gene vectors, the genomic segment showed significant promoter activity, indicating that the 5'-flanking regions isolated by genomic walking contain the gene promoter of rat caspase-9. Of significance is that the cloned promoter segments were activated by severe hypoxia, conditions inducing caspase-9 transcription. Thus, the genomic sequences reported here contain not only the basal promoter of rat caspase-9 but also regulatory elements responsive to pathophysiological stimuli including hypoxia.

Full Text

The Full Text of this article is available as a PDF (220.6 KB).

Selected References

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

  1. Alnemri E. S., Livingston D. J., Nicholson D. W., Salvesen G., Thornberry N. A., Wong W. W., Yuan J. Human ICE/CED-3 protease nomenclature. Cell. 1996 Oct 18;87(2):171–171. doi: 10.1016/s0092-8674(00)81334-3. [DOI] [PubMed] [Google Scholar]
  2. Asahi M., Hoshimaru M., Uemura Y., Tokime T., Kojima M., Ohtsuka T., Matsuura N., Aoki T., Shibahara K., Kikuchi H. Expression of interleukin-1 beta converting enzyme gene family and bcl-2 gene family in the rat brain following permanent occlusion of the middle cerebral artery. J Cereb Blood Flow Metab. 1997 Jan;17(1):11–18. doi: 10.1097/00004647-199701000-00003. [DOI] [PubMed] [Google Scholar]
  3. Bey L., Etienne J., Tse C., Brault D., Noé L., Raisonnier A., Arnault F., Hamilton M. T., Galibert F. Cloning, sequencing and structural analysis of 976 base pairs of the promoter sequence for the rat lipoprotein lipase gene. Comparison with the mouse and human sequences. Gene. 1998 Mar 16;209(1-2):31–38. doi: 10.1016/s0378-1119(98)00003-1. [DOI] [PubMed] [Google Scholar]
  4. Boone D. L., Tsang B. K. Caspase-3 in the rat ovary: localization and possible role in follicular atresia and luteal regression. Biol Reprod. 1998 Jun;58(6):1533–1539. doi: 10.1095/biolreprod58.6.1533. [DOI] [PubMed] [Google Scholar]
  5. Budihardjo I., Oliver H., Lutter M., Luo X., Wang X. Biochemical pathways of caspase activation during apoptosis. Annu Rev Cell Dev Biol. 1999;15:269–290. doi: 10.1146/annurev.cellbio.15.1.269. [DOI] [PubMed] [Google Scholar]
  6. Cardone M. H., Roy N., Stennicke H. R., Salvesen G. S., Franke T. F., Stanbridge E., Frisch S., Reed J. C. Regulation of cell death protease caspase-9 by phosphorylation. Science. 1998 Nov 13;282(5392):1318–1321. doi: 10.1126/science.282.5392.1318. [DOI] [PubMed] [Google Scholar]
  7. Chenchik A., Diachenko L., Moqadam F., Tarabykin V., Lukyanov S., Siebert P. D. Full-length cDNA cloning and determination of mRNA 5' and 3' ends by amplification of adaptor-ligated cDNA. Biotechniques. 1996 Sep;21(3):526–534. doi: 10.2144/96213pf02. [DOI] [PubMed] [Google Scholar]
  8. Cohen G. M. Caspases: the executioners of apoptosis. Biochem J. 1997 Aug 15;326(Pt 1):1–16. doi: 10.1042/bj3260001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cryns V., Yuan J. Proteases to die for. Genes Dev. 1998 Jun 1;12(11):1551–1570. doi: 10.1101/gad.12.11.1551. [DOI] [PubMed] [Google Scholar]
  10. Dong Z., Saikumar P., Patel Y., Weinberg J. M., Venkatachalam M. A. Serine protease inhibitors suppress cytochrome c-mediatedcaspase-9 activation and apoptosis during hypoxia-reoxygenation. Biochem J. 2000 May 1;347(Pt 3):669–677. [PMC free article] [PubMed] [Google Scholar]
  11. Dong Z., Venkatachalam M. A., Wang J., Patel Y., Saikumar P., Semenza G. L., Force T., Nishiyama J. Up-regulation of apoptosis inhibitory protein IAP-2 by hypoxia. Hif-1-independent mechanisms. J Biol Chem. 2001 Mar 12;276(22):18702–18709. doi: 10.1074/jbc.M011774200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Earnshaw W. C., Martins L. M., Kaufmann S. H. Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu Rev Biochem. 1999;68:383–424. doi: 10.1146/annurev.biochem.68.1.383. [DOI] [PubMed] [Google Scholar]
  13. Fujita E., Jinbo A., Matuzaki H., Konishi H., Kikkawa U., Momoi T. Akt phosphorylation site found in human caspase-9 is absent in mouse caspase-9. Biochem Biophys Res Commun. 1999 Oct 22;264(2):550–555. doi: 10.1006/bbrc.1999.1387. [DOI] [PubMed] [Google Scholar]
  14. Guarin E., Seuret P., Nef S., de Bilbao F., Nef P., Dubois-Dauphin M. cpp32 messenger RNA neosynthesis is induced by fatal axotomy and is not regulated by athanatal Bcl-2 over-expression. Neuroscience. 1999 May;90(2):653–664. doi: 10.1016/s0306-4522(98)00445-x. [DOI] [PubMed] [Google Scholar]
  15. Hofmann K., Bucher P., Tschopp J. The CARD domain: a new apoptotic signalling motif. Trends Biochem Sci. 1997 May;22(5):155–156. doi: 10.1016/s0968-0004(97)01043-8. [DOI] [PubMed] [Google Scholar]
  16. Kaushal G. P., Singh A. B., Shah S. V. Identification of gene family of caspases in rat kidney and altered expression in ischemia-reperfusion injury. Am J Physiol. 1998 Mar;274(3 Pt 2):F587–F595. doi: 10.1152/ajprenal.1998.274.3.F587. [DOI] [PubMed] [Google Scholar]
  17. Kone B. C., Higham S. C. A novel N-terminal splice variant of the rat H+-K+-ATPase alpha2 subunit. Cloning, functional expression, and renal adaptive response to chronic hypokalemia. J Biol Chem. 1998 Jan 30;273(5):2543–2552. doi: 10.1074/jbc.273.5.2543. [DOI] [PubMed] [Google Scholar]
  18. Krajewski S., Krajewska M., Ellerby L. M., Welsh K., Xie Z., Deveraux Q. L., Salvesen G. S., Bredesen D. E., Rosenthal R. E., Fiskum G. Release of caspase-9 from mitochondria during neuronal apoptosis and cerebral ischemia. Proc Natl Acad Sci U S A. 1999 May 11;96(10):5752–5757. doi: 10.1073/pnas.96.10.5752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kumar A., Commane M., Flickinger T. W., Horvath C. M., Stark G. R. Defective TNF-alpha-induced apoptosis in STAT1-null cells due to low constitutive levels of caspases. Science. 1997 Nov 28;278(5343):1630–1632. doi: 10.1126/science.278.5343.1630. [DOI] [PubMed] [Google Scholar]
  20. Li H., Zhu H., Xu C. J., Yuan J. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell. 1998 Aug 21;94(4):491–501. doi: 10.1016/s0092-8674(00)81590-1. [DOI] [PubMed] [Google Scholar]
  21. Li P., Nijhawan D., Budihardjo I., Srinivasula S. M., Ahmad M., Alnemri E. S., Wang X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell. 1997 Nov 14;91(4):479–489. doi: 10.1016/s0092-8674(00)80434-1. [DOI] [PubMed] [Google Scholar]
  22. Luo X., Budihardjo I., Zou H., Slaughter C., Wang X. Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell. 1998 Aug 21;94(4):481–490. doi: 10.1016/s0092-8674(00)81589-5. [DOI] [PubMed] [Google Scholar]
  23. O'Neill S., O'Neill A. J., Conroy E., Brady H. R., Fitzpatrick J. M., Watson R. W. Altered caspase expression results in delayed neutrophil apoptosis in acute pancreatitis. J Leukoc Biol. 2000 Jul;68(1):15–20. [PubMed] [Google Scholar]
  24. Ogbourne S., Antalis T. M. Transcriptional control and the role of silencers in transcriptional regulation in eukaryotes. Biochem J. 1998 Apr 1;331(Pt 1):1–14. doi: 10.1042/bj3310001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Prestridge D. S. Computer software for eukaryotic promoter analysis. Methods Mol Biol. 2000;130:265–295. doi: 10.1385/1-59259-686-x:265. [DOI] [PubMed] [Google Scholar]
  26. Prestridge D. S. Predicting Pol II promoter sequences using transcription factor binding sites. J Mol Biol. 1995 Jun 23;249(5):923–932. doi: 10.1006/jmbi.1995.0349. [DOI] [PubMed] [Google Scholar]
  27. Reed J. C. Mechanisms of apoptosis. Am J Pathol. 2000 Nov;157(5):1415–1430. doi: 10.1016/S0002-9440(10)64779-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Saikumar P., Dong Z., Patel Y., Hall K., Hopfer U., Weinberg J. M., Venkatachalam M. A. Role of hypoxia-induced Bax translocation and cytochrome c release in reoxygenation injury. Oncogene. 1998 Dec 31;17(26):3401–3415. doi: 10.1038/sj.onc.1202590. [DOI] [PubMed] [Google Scholar]
  29. Salvesen G. S., Dixit V. M. Caspases: intracellular signaling by proteolysis. Cell. 1997 Nov 14;91(4):443–446. doi: 10.1016/s0092-8674(00)80430-4. [DOI] [PubMed] [Google Scholar]
  30. Semenza G. L. Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1. Annu Rev Cell Dev Biol. 1999;15:551–578. doi: 10.1146/annurev.cellbio.15.1.551. [DOI] [PubMed] [Google Scholar]
  31. Siebert P. D., Chenchik A., Kellogg D. E., Lukyanov K. A., Lukyanov S. A. An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res. 1995 Mar 25;23(6):1087–1088. doi: 10.1093/nar/23.6.1087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Smale S. T., Baltimore D. The "initiator" as a transcription control element. Cell. 1989 Apr 7;57(1):103–113. doi: 10.1016/0092-8674(89)90176-1. [DOI] [PubMed] [Google Scholar]
  33. Smale S. T. Transcription initiation from TATA-less promoters within eukaryotic protein-coding genes. Biochim Biophys Acta. 1997 Mar 20;1351(1-2):73–88. doi: 10.1016/s0167-4781(96)00206-0. [DOI] [PubMed] [Google Scholar]
  34. Srinivasula S. M., Fernandes-Alnemri T., Zangrilli J., Robertson N., Armstrong R. C., Wang L., Trapani J. A., Tomaselli K. J., Litwack G., Alnemri E. S. The Ced-3/interleukin 1beta converting enzyme-like homolog Mch6 and the lamin-cleaving enzyme Mch2alpha are substrates for the apoptotic mediator CPP32. J Biol Chem. 1996 Oct 25;271(43):27099–27106. doi: 10.1074/jbc.271.43.27099. [DOI] [PubMed] [Google Scholar]
  35. Susin S. A., Lorenzo H. K., Zamzami N., Marzo I., Brenner C., Larochette N., Prévost M. C., Alzari P. M., Kroemer G. Mitochondrial release of caspase-2 and -9 during the apoptotic process. J Exp Med. 1999 Jan 18;189(2):381–394. doi: 10.1084/jem.189.2.381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Tamura T., Ishihara M., Lamphier M. S., Tanaka N., Oishi I., Aizawa S., Matsuyama T., Mak T. W., Taki S., Taniguchi T. An IRF-1-dependent pathway of DNA damage-induced apoptosis in mitogen-activated T lymphocytes. Nature. 1995 Aug 17;376(6541):596–599. doi: 10.1038/376596a0. [DOI] [PubMed] [Google Scholar]
  37. Thornberry N. A., Lazebnik Y. Caspases: enemies within. Science. 1998 Aug 28;281(5381):1312–1316. doi: 10.1126/science.281.5381.1312. [DOI] [PubMed] [Google Scholar]
  38. Wang K., Yin X. M., Chao D. T., Milliman C. L., Korsmeyer S. J. BID: a novel BH3 domain-only death agonist. Genes Dev. 1996 Nov 15;10(22):2859–2869. doi: 10.1101/gad.10.22.2859. [DOI] [PubMed] [Google Scholar]
  39. Wolf B. B., Green D. R. Suicidal tendencies: apoptotic cell death by caspase family proteinases. J Biol Chem. 1999 Jul 16;274(29):20049–20052. doi: 10.1074/jbc.274.29.20049. [DOI] [PubMed] [Google Scholar]
  40. Zhivotovsky B., Samali A., Gahm A., Orrenius S. Caspases: their intracellular localization and translocation during apoptosis. Cell Death Differ. 1999 Jul;6(7):644–651. doi: 10.1038/sj.cdd.4400536. [DOI] [PubMed] [Google Scholar]
  41. Zong W. X., Edelstein L. C., Chen C., Bash J., Gélinas C. The prosurvival Bcl-2 homolog Bfl-1/A1 is a direct transcriptional target of NF-kappaB that blocks TNFalpha-induced apoptosis. Genes Dev. 1999 Feb 15;13(4):382–387. doi: 10.1101/gad.13.4.382. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES