THE PROTOONCOGENE CRK-II ENHANCES APOPTOSIS BY A RAS-DEPENDENT, RAF-1MAP KINASE-INDEPENDENT PATHWAY/

Human embryonic kidney 293 cells and 293 cells overexpressing differen t amounts of the adaptor protein Crk-II (ranging from 3- to 10-fold hi gher levels than the parental cell line) were examined for their abili ty to undergo apoptosis when maintained in control and serum-free (SF) medium. Parental 293 cells undergo apoptosis only when deprived of se rum for prolonged periods of time (24-48 h). On the other hand, 293 ce lls overexpressing different levels of Crk-II present detectable level s 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 domina nt-negative Pas construct (N17-Ras). Compared to cells transfected wit h control vectors, the cells overexpressing N17-Ras presented lower le vels of apoptosis when maintained in SF-medium. On the other hand, tra nsient transfection of a dominant-negative Raf-1 construct (K375W-Raf- 1) did not decrease apoptosis; slightly increasing DNA fragmentation l evels were seen. Similar results were obtained when the cells were inc ubated in the presence of a MEK1 inhibitor. The results presented here suggest that overexpression of Crk-II induces apoptosis via a Ras-dep endent, Raf-1/MEK1/ERK-independent pathway. (C) 1997 Academic Press.