Abstract
The Raf-1 serine/threonine kinase is a key protein that is implicated in the transmission of many growth and cell survival signals. In the present study we demonstrate that apoptosis of hematopoietic cells induced by IL-3-deprivation is associated with the cleavage of Raf-1, resulting in the separation of the N-terminal regulatory domain and the C-terminal kinase domain. Raf-1 cleavage specifically occurs upon triggering of the mitochondrial death pathway, and coincides with the activation of specific caspases. Moreover, Bcl-2 overexpression or treatment with the caspase inhibitor z-VAD.fmk completely prevented Raf-1 cleavage, whereas caspase inhibition by treatment of cells with Ac-DEVD.fmk or z-IETD.fmk, or CrmA overexpression had no effect. Furthermore, in vitro cleavage studies indicate that caspase-9, which is the apical protease in the mitochondrial death pathway, is able to cleave Raf-1 at position D279. Cell fractionation studies showed that the Raf-1 C-terminal fragment that is generated upon IL-3 withdrawal is localized predominantly to the mitochondria. In addition, constitutive expression of this C-terminal Raf-1 fragment fused to a mitochondrial targeting sequence in Ba/F3 pre-B cells significantly delays apoptosis induced by IL-3 withdrawal. These results suggest an important role for caspase-9 mediated cleavage of Raf-1 in the negative feedback regulation of hematopoietic cell apoptosis induced by growth factor withdrawal.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 50 print issues and online access
$259.00 per year
only $5.18 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Alavi A, Hood JD, Frausto R, Stupack DG and Cheresh DA . (2003). Science, 301, 94–96.
Boatright KM, Renatus M, Scott FL, Sperandio S, Shin H, Pedersen IM, Ricci JE, Edris WA, Sutherlin DP, Green DR and Salvesen GS . (2003). Mol. Cell, 11, 529–541.
Boatright KM and Salvesen GS . (2003). Curr. Opin. Cell Biol., 15, 725–731.
Bojes HK, Feng X, Kehrer JP and Cohen GM . (1999). Cell Death Differ., 6, 61–70.
Bokoch GM . (1998). Cell Death Differ., 5, 637–645.
Boldin MP, Goncharov TM, Goltsev YV and Wallach D . (1996). Cell, 85, 803–815.
Chen J, Fujii K, Zhang L, Roberts T and Fu H . (2001). Proc. Natl. Acad. Sci. USA, 98, 7783–7788.
Chong H and Guan KL . (2003). J. Biol. Chem., 278, 36269–36276.
Chong H, Vikis HG and Guan KL . (2003). Cell. Signal., 15, 463–469.
Cleveland JL, Troppmair J, Packham G, Askew DS, Lloyd P, Gonzalez-Garcia M, Nunez G, Ihle JN and Rapp UR . (1994). Oncogene, 9, 2217–2226.
Cornelis S, Bruynooghe Y, Denecker G, Van Huffel S, Tinton S and Beyaert R . (2000). Mol. Cell, 5, 597–605.
Costantini P, Bruey JM, Castedo M, Metivier D, Loeffler M, Susin SA, Ravagnan L, Zamzami N, Garrido C and Kroemer G . (2002). Cell Death Differ., 9, 82–88.
Cutler Jr RE, Stephens RM, Saracino MR and Morrison DK . (1998). Proc. Natl. Acad. Sci. USA, 95, 9214–9219.
Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y and Greenberg ME . (1997). Cell, 91, 231–241.
Davis RJ . (1994). Trends Biochem. Sci., 19, 470–473.
Denecker G, Vercammen D, Steemans M, Vanden Berghe T, Brouckaert G, Van Loo G, Zhivotovsky B, Fiers W, Grooten J, Declercq W and Vandenabeele P . (2001). Cell Death Differ., 8, 829–840.
Earnshaw WC, Martins LM and Kaufmann SH . (1999). Annu. Rev. Biochem., 68, 383–424.
Emoto Y, Manome Y, Meinhardt G, Kisaki H, Kharbanda S, Robertson M, Ghayur T, Wong WW, Kamen R and Weichselbaum R . (1995). EMBO J., 14, 6148–6156.
Garcia-Calvo M, Peterson EP, Rasper DM, Vaillancourt JP, Zamboni R, Nicholson DW and Thornberry NA . (1999). Cell Death Differ., 6, 362–369.
Hapel AJ, Warren HS and Hume DA . (1984). Blood, 64, 786–790.
Hase T, Muller U, Riezman H and Schatz G . (1984). EMBO J., 3, 3157–3164.
Heidecker G, Huleihel M, Cleveland JL, Kolch W, Beck TW, Lloyd P, Pawson T and Rapp UR . (1990). Mol. Cell. Biol., 10, 2503–2512.
Huser M, Luckett J, Chiloeches A, Mercer K, Iwobi M, Giblett S, Sun XM, Brown J, Marais R and Pritchard C . (2001). EMBO J., 20, 1940–1951.
Jesenberger V, Procyk KJ, Ruth J, Schreiber M, Theussl HC, Wagner EF and Baccarini M . (2001). J. Exp. Med., 193, 353–364.
Krajewski S, Krajewska M, Ellerby LM, Welsh K, Xie Z, Deveraux QL, Salvesen GS, Bredesen DE, Rosenthal RE, Fiskum G and Reed JC . (1999). Proc. Natl. Acad. Sci. USA, 96, 5752–5757.
Le Mellay V, Troppmair J, Benz R and Rapp UR . (2002). BMC Cell Biol., 3, 14.
Li H, Zhu H, Xu CJ and Yuan J . (1998). Cell, 94, 491–501.
Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES and Wang X . (1997). Cell, 91, 479–489.
Luo X, Budihardjo I, Zou H, Slaughter C and Wang X . (1998). Cell, 94, 481–490.
Majewski M, Nieborowska-Skorska M, Salomoni P, Slupianek A, Reiss K, Trotta R, Calabretta B and Skorski T . (1999). Cancer Res., 59, 2815–2819.
Mikula M, Schreiber M, Husak Z, Kucerova L, Ruth J, Wieser R, Zatloukal K, Beug H, Wagner EF and Baccarini M . (2001). EMBO J., 20, 1952–1962.
Muzio M, Chinnaiyan AM, Kischkel FC, O’Rourke K, Shevchenko A, Ni J, Scaffidi C, Bretz JD, Zhang M, Gentz R, Mann M, Krammer PH, Peter ME and Dixit VM . (1996). Cell, 85, 817–827.
Palacios R and Steinmetz M . (1985). Cell, 41, 727–734.
Peruzzi F, Prisco M, Dews M, Salomoni P, Grassilli E, Romano G, Calabretta B and Baserga R . (1999). Mol. Cell. Biol., 19, 7203–7215.
Potokar M, Milisav I, Kreft M, Stenovec M and Zorec R . (2003). FEBS Lett., 544, 153–159.
Robinson MJ and Cobb MH . (1997). Curr. Opin. Cell Biol., 9, 180–186.
Salomoni P, Wasik MA, Riedel RF, Reiss K, Choi JK, Skorski T and Calabretta B . (1998). J. Exp. Med., 187, 1995–2007.
Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, Debatin KM, Krammer PH and Peter ME . (1998). EMBO J., 17, 1675–1687.
Stanton Jr VP and Cooper GM . (1987). Mol. Cell. Biol., 7, 1171–1179.
Stanton Jr VP, Nichols DW, Laudano AP and Cooper GM . (1989). Mol. Cell. Biol., 9, 639–647.
Stennicke HR, Deveraux QL, Humke EW, Reed JC, Dixit VM and Salvesen GS . (1999). J. Biol. Chem., 274, 8359–8362.
Susin SA, Lorenzo HK, Zamzami N, Marzo I, Brenner C, Larochette N, Prevost MC, Alzari PM and Kroemer G . (1999). J. Exp. Med., 189, 381–394.
Thornberry NA, Rano TA, Peterson EP, Rasper DM, Timkey T, Garcia-Calvo M, Houtzager VM, Nordstrom PA, Roy S, Vaillancourt JP, Chapman KT and Nicholson DW . (1997). J. Biol. Chem., 272, 17907–17911.
Tran NH and Frost JA . (2003). J. Biol. Chem., 278, 11221–11226.
Troppmair J and Rapp UR . (2003). Biochem. Pharmacol., 66, 1341–1345.
van Loo G, Saelens X, Matthijssens F, Schotte P, Beyaert R, Declercq W and Vandenabeele P . (2002). Cell Death Differ., 9, 1207–1211.
Vander Heiden MG, Chandel NS, Williamson EK, Schumacker PT and Thompson CB . (1997). Cell, 91, 627–637.
Vercammen D, Brouckaert G, Denecker G, Van de Craen M, Declercq W, Fiers W and Vandenabeele P . (1998). J. Exp. Med., 188, 919–930.
Wang HG, Miyashita T, Takayama S, Sato T, Torigoe T, Krajewski S, Tanaka S, Hovey III L, Troppmair J, Rapp UR and Reed YC . (1994). Oncogene, 9, 2751–2756.
Wang HG, Rapp UR and Reed JC . (1996). Cell, 87, 629–638.
Wojnowski L, Stancato LF, Zimmer AM, Hahn H, Beck TW, Larner AC, Rapp UR and Zimmer A . (1998). Mech. Dev., 76, 141–149.
Zha J, Harada H, Yang E, Jockel J and Korsmeyer SJ . (1996). Cell, 87, 619–628.
Acknowledgements
The technical assistance of A Meeuws and W Burm is gratefully acknowledged. This work was supported by the ‘Belgian Federation against Cancer’, the ‘Interuniversity Attraction Poles’ (IUAP-V), and the ‘Fund for Scientific Research-Flanders’ (FWO-Vlaanderen). SC is a postdoctoral research associate with the FWO-Vlaanderen.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Cornelis, S., Bruynooghe, Y., Van Loo, G. et al. Apoptosis of hematopoietic cells induced by growth factor withdrawal is associated with caspase-9 mediated cleavage of Raf-1. Oncogene 24, 1552–1562 (2005). https://doi.org/10.1038/sj.onc.1208401
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/sj.onc.1208401
Keywords
This article is cited by
-
Analysis of erythrocyte signalling pathways during Plasmodium falciparum infection identifies targets for host-directed antimalarial intervention
Nature Communications (2020)
-
The mitochondrial serine protease HtrA2/Omi cleaves RIP1 during apoptosis of Ba/F3 cells induced by growth factor withdrawal
Cell Research (2010)
-
Caspase-mediated cleavage of Beclin-1 inactivates Beclin-1-induced autophagy and enhances apoptosis by promoting the release of proapoptotic factors from mitochondria
Cell Death & Disease (2010)
-
Functional implications of caspase-mediated RhoGDI2 processing during apoptosis of HL60 and K562 leukemia cells
Apoptosis (2007)
-
Differential effects of NF-κB on apoptosis induced by DNA-damaging agents: the type of DNA damage determines the final outcome
Oncogene (2006)