The proto-oncogene Crk-II enhances apoptosis by a Ras-dependent, Raf-1/MAP kinase-independent pathway.
Human embryonic kidney 293 cells and 293 cells overexpressing different amounts of the adaptor protein Crk-II (ranging from 3- to 10-fold higher levels than the parental cell line) were examined for their ability to undergo apoptosis when maintained in control and serum-free (SF) medium. Parental 293 cells undergo apoptosis only when deprived of serum for prolonged periods of time (24-48 h). On the other hand, 293 cells overexpressing different levels of Crk-II present detectable levels of apoptosis as measured by DNA fragmentation when grown in control medium, with a marked increase when they are deprived of serum for 12-48 h. To determine the pathways involved in Crk-II-induced apoptosis, Crk-II overexpressing cells were transiently transfected with a dominant-negative Ras construct (N17-Ras). Compared to cells transfected with control vectors, the cells overexpressing N17-Ras presented lower levels of apoptosis when maintained in SF-medium. On the other hand, transient transfection of a dominant-negative Raf-1 construct (K375W-Raf-1) did not decrease apoptosis; slightly increasing DNA fragmentation levels were seen. Similar results were obtained when the cells were incubated in the presence of a MEK1 inhibitor. The results presented here suggest that overexpression of Crk-II induces apoptosis via a Ras-dependent, Raf-1/MEK1/ERK-independent pathway.
Parrizas
M
M
Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-1770, USA.
Blakesley
V A
VA
Beitner-Johnson
D
D
Le Roith
D
D
D017209:Apoptosis / Q000235:genetics*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002460:Cell Line; D016895:Culture Media, Serum-Free; D005799:Genes, Dominant; D006801:Humans; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism*; D050737:Proto-Oncogene Proteins c-crk; D019908:Proto-Oncogene Proteins c-raf
D016895:Culture Media, Serum-Free; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases