Activation of the Cytoplasmic c-Abl Tyrosine Kinase by Reactive Oxygen Species

Abstract

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...

F-actin-binding domain of c-Abl regulates localized phosphorylation of C3G: role of C3G in c-Abl-mediated cell death

“…More recently, it has been reported that c-Abl activation induces the phosphorylation of Crk in the cytoplasmic compartment (Holcomb et al, 2006). H 2 O 2 is known to selectively activate cytoplasmic, but not nuclear Abl (Sun et al, 2000). Our results suggested that cytoplasmic Abl is responsible for phosphorylation of C3G and induction of apoptotic changes.…”

“…Inhibition of Rap1 activation in c-Abl-expressing cells by co-expression of GFPRap1GAP resulted in reduced apoptosis (Figure 8f). H 2 O 2 induces cell death dependent on c-Abl (Sun et al, 2000). Expression of WT C3G resulted in a significant increase in H 2 O 2 -induced apoptosis, but Y504F mutant, which inhibits signalling dependent on Y504 phosphorylation, reduced apoptosis by 59% (Figures 8g and h).…”

Cytoplasmic c-Abl provides a molecular ‘Rheostat’ controlling carcinoma cell survival and invasion

“…This, and the fact that nuclear p73 tyrosine phosphorylation was similar in viable and apoptotic cells, indicates that cytoplasmic and not nuclear Abl is responsible for serum deprivation-induced apoptosis (Yuan et al, 1997;Agami et al, 1999;Gong et al, 1999;Yuan et al, 1999). In support of these findings, treatment of Cos 7 and A/ A þ / þ cells with H 2 O 2 , which selectively activates cytoplasmic and not nuclear Abl activity (Sun et al, 2000), induced strong Crk phosphorylation and cell death (Figure 6a, b). In contrast, treatment of A/AÀ/À cells with H 2 O 2 failed to induce these responses (Figure 6c).…”

c-Abl and Arg tyrosine kinases regulate lysosomal degradation of the oncoprotein Galectin-3

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