The protein p73 is a structural and functional homologue of the p53 tumour-suppressor protein but, unlike p53, it is not induced in response to DNA damage. The tyrosine kinase c-Abl is activated by certain DNA-damaging agents and contributes to the induction of programmed cell death (apoptosis) by p53-dependent and p53-independent mechanisms. Here we show that c-Abl binds to p73 in cells, interacting through its SH3 domain with the carboxy-terminal homo-oligomerization domain of p73. c-Abl phosphorylates p73 on a tyrosine residue at position 99 both in vitro and in cells that have been exposed to ionizing radiation. Our results show that c-Abl stimulates p73-mediated transactivation and apoptosis. This regulation of p73 by c-Abl in response to DNA damage is also demonstrated by a failure of ionizing-radiation-induced apoptosis after disruption of the c-Abl-p73 interaction. These findings show that p73 is regulated by a c-Abl-dependent mechanism and that p73 participates in the apoptotic response to DNA damage.

Apoptosis is induced by the release of cytochrome c from mitochondria to the cytoplasm. The present studies demonstrate that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induces translocation of protein kinase C (PKC) ␦ from the cytoplasm to mitochondria. The results also show that translocation of PKC␦ results in release of cytochrome c. The functional significance of this event is further supported by the demonstration that PKC␦ translocation is required for TPA-induced apoptosis. These findings demonstrate that translocation of PKC␦ to mitochondria is responsible, at least in part, for inducing cytochrome c release and apoptosis.

The ubiquitously expressed c-Abl protein tyrosine kinase localizes to both the nucleus and cytoplasm. The nuclear form of c-Abl is activated in the cellular response to genotoxic stress. Here we show that cytoplasmic c-Abl is activated by oxidative stress. The results also demonstrate that mitochondrial cytochrome c is released in the cellular response to H 2 O 2 and that this effect is mediated by a c-Abl-dependent mechanism. In concert with these results, we show that H 2 O 2 -induced apoptosis is attenuated in c-Abl-deficient cells. These findings demonstrate that cytoplasmic c-Abl is involved in the apoptotic response of cells to oxidative stress.Normal cellular metabolism is associated with the production of reactive oxygen species (ROS) 1 and, as a consequence, damage to DNA and proteins (1, 2). The generation of ROS is also known to induce apoptosis; however, the molecular mechanisms responsible for ROS-induced apoptosis are unclear. Studies have indicated that ROS induce activation of topoisomerase II-mediated cleavage of chromosomal DNA and thereby apoptosis (3). Other work has suggested that ROSinduced apoptosis is p53-dependent (4, 5) and that p53-induced apoptosis is mediated by . In addition, the p66 shc adaptor protein (5) and the p85 subunit of phosphatidylinositol 3-kinase (PI3K) (4) have been implicated in the apoptotic response to oxidative stress.The nuclear form of the c-Abl tyrosine kinase is activated in the cellular response to genotoxic stress (9). Nuclear c-Abl has been implicated in the apoptotic response to DNA damage by mechanisms in part dependent on p53 and its homolog, p73 (10 -14). c-Abl also functions as an upstream effector of the proapoptotic SAPK/JNK and p38 mitogen activated protein kinase (MAPK) pathways in the genotoxic stress response (9,15,16). Other studies have demonstrated that c-Abl phosphorylates p85 and thereby inhibits PI3K activity in the apoptotic response to DNA damage (17). Additional evidence supporting a role for c-Abl in apoptosis has been provided by the findings that cells deficient in c-Abl or expressing a dominant-negative c-Abl mutant exhibit an attenuated apoptotic response to genotoxic agents (18,19).Recent work has shown that c-Abl phosphorylates protein kinase C (PKC) ␦ in cells treated with H 2 O 2 (20). The present results demonstrate that the cytoplasmic, and not the nuclear, form of c-Abl is activated in the cellular response to H 2 O 2 . We also show that H 2 O 2 induces mitochondrial cytochrome c release and apoptosis by a c-Abl-dependent mechanism. MATERIALS AND METHODSCell Culture-COS7 cells and MEFs derived from wild-type and c-Abl Ϫ/Ϫ mice (21) were cultured in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin. DLD1 cells were grown as described (7). Cells were treated with H 2 O 2 (Sigma), 30 mM N-acetyl-L-cysteine (NAC; Sigma), or 10 M cis-platinum (Sigma).Analysis of Kinase Activity-Cell lysates were prepared in lysis buffer (10 mM Tr...