Expression of constitutively active Raf-1 in the mitochondria restores antiapoptotic and leukemogenic potential of a transformation-deficient BCR/ABL mutant.
The oncogenic BCR/ABL protein protects hematopoietic cells from apoptosis induced by growth factor deprivation, but the mechanisms are only partially understood. A BCR/ABL mutant lacking amino acids 176-426 in the BCR domain (p185DeltaBCR) failed to protect interleukin 3-deprived 32Dcl3 myeloid precursor cells from apoptosis, although it possessed tyrosine kinase activity and was capable of activating the Ras-Raf-MAP kinase pathway. Compared to p185 wild-type transfectants, p185DeltaBCR-transfected cells showed markedly reduced levels of Bcl-2 and expressed the hypophosphorylated, proapoptotic form of BAD. Bcl-2 expression in the mitochondrial fraction of p185DeltaBCR cells was also markedly diminished and mitochondrial RAF was undetectable. In p185DeltaBCR cells transfected with a mitochondria-targeted, constitutively active RAF (M-Raf) BAD was expressed in the hyperphosphorylated form and released from the mitochondria into the cytosol. p185DeltaBCR/M-Raf-transfected cells were completely resistant to apoptosis induced by growth factor deprivation in vitro. Moreover, constitutive expression of dominant-negative M-Raf (K375W) enhanced the susceptibility of 32Dcl3 cells expressing wild-type BCR/ABL to apoptosis. In severe combined immunodeficiency (SCID) mice, p185DeltaBCR/M-Raf double transfectants were leukemogenic, whereas cells expressing only p185DeltaBCR showed no leukemogenic potential. Together, these data support the existence of a BCR/ABL-dependent pathway that leads to expression of an active RAF in the mitochondria and promotes antiapoptotic and leukemia-inducing effects of BCR/ABL.
Salomoni
P
P
Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Wasik
M A
MA
Riedel
R F
RF
Reiss
K
K
Choi
J K
JK
Skorski
T
T
Calabretta
B
B
D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002451:Cell Compartmentation; D016044:Fusion Proteins, bcr-abl / Q000235:genetics*; D006412:Hematopoietic Stem Cells; D007377:Interleukin-3 / Q000172:deficiency; D007942:Leukemia, Experimental / Q000235:genetics*; D051379:Mice; D008928:Mitochondria / Q000201:enzymology*; D009154:Mutation; D009857:Oncogenes / Q000235:genetics*; D019908:Proto-Oncogene Proteins c-raf / Q000096:biosynthesis* / Q000235:genetics; D015398:Signal Transduction; D018631:ras Proteins / Q000378:metabolism
D007377:Interleukin-3; D016044:Fusion Proteins, bcr-abl; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D018631:ras Proteins