Apoptosis of hematopoietic cells induced by growth factor withdrawal is associated with caspase-9 mediated cleavage of Raf-1.
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.
Cornelis
Sigrid
S
Unit of Molecular Signal Transduction in Inflammation, Department for Molecular Biomedical Research, VIB-Ghent University, Technologiepark 927, B-9052 Ghent-Zwijnaarde, Belgium.
Bruynooghe
Yanik
Y
Van Loo
Geert
G
Saelens
Xavier
X
Vandenabeele
Peter
P
Beyaert
Rudi
R
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000502:physiology; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D003600:Cytosol / Q000648:ultrastructure; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D005354:Fibrosarcoma; D006133:Growth Substances / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology; D051379:Mice; D008928:Mitochondria / Q000473:pathology; D008969:Molecular Sequence Data; D011003:Ploidies; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D015183:Restriction Mapping
D017931:DNA Primers; D018076:DNA, Complementary; D006133:Growth Substances; D007376:Interleukin-2; D019908:Proto-Oncogene Proteins c-raf; C506433:CASP9 protein, human; C506434:Casp9 protein, mouse; D053453:Caspase 9; D020169:Caspases