Activated Ha-ras induces apoptosis by association with phosphorylated Bcl-2 in a mitogen-activated protein kinase-independent manner

Serum deprivation of Ha-ras-transformed brown adipocyte cell line resulted in a dramatic apoptotic cell death, as detected either by DNA laddering or by an increase in the percentage of hypodiploid cells or by nuclei condensa tion and fragmentation, as compared with immortalized cell line or primary fetal brown adipocytes, Moreover, transient transfection of immortalized br own adipocytes with a constitutively active ras gene (Ha-ras(lys12)) mimics the high rate of apoptosis detected in the transformed cell line. On the o ther hand, transient transfection of the dominant-negative construct of raf -l rescued serum-deprived Ha-ras-transformed brown adipocytes from apoptosi s, decreasing the percentage of hypodiploid cells, the external display of phosphatidylserine, and the DNA laddering. However, inhibition of mitogen-a ctivated protein kinase with PD098059 did not preclude apoptosis and in fac t increased the rate of apoptosis observed in serum-deprived Ha-ras-transfo rmed cells, indicating that the Ras/Raf-1 pathway induced apoptosis through out a mitogen-activated protein kinase kinase 1 (MEK-1)-independent pathway . Furthermore, apoptosis in Ha-ras-transformed brown adipocytes is concurre nt with an up-regulation in the expression of the pro-apoptotic protein Bcl -xS, the expression of the anti-apoptotic protein Bcl-2 being down-regulate d. Finally, an association of Ras and Raf with phosphorylated Bcl-2 protein was demonstrated in immunoprecipitates from apoptotic cells. Thus, we prop ose a mechanism of apoptosis in Ha-ras-transformed adipocytes under serum d eprivation involving Raf-1 association with phosphorylated Bcl-2, down-regu lation of Bcl-2 expression, and up-regulation of Bcl-xS expression.