Tumor Necrosis Factor Alpha-Induced Apoptosis Requires p73 and c-ABL Activation Downstream of RB Degradation

Tumor necrosis factor alpha (TNF-α) is an inflammatory cytokine that coordinates the systemic response to infection and injury. TNF-α interacts with two types of cell surface receptors (types I and II) to regulate various cell-type-specific responses (8). Binding of TNF-α to the type I receptor (TNFRI) can activate the NF-κB survival pathway (23) or caspase-dependent cell death (24, 38). Activated TNFRI recruits TRADD (TNFR-associated death domain) (25), which in turn recruits TRAFs (TNFR-associated factors) and RIP to activate NF-κB. TNFRI also stimulates the formation of a cytoplasmic TRADD complex, containing FADD (FAS-associated death domain) and pro-caspase 8, leading to the activation of caspase 8 and the initiation of an apoptotic signaling cascade (24, 25, 38). Activated caspase 8 can directly cleave and activate executioner caspases (11). Caspase 8 also cleaves a BH3-only protein, BID (32, 35). The truncated BID, in collaboration with BAX and BAK, causes the release of apoptogenic factors from mitochondria to activate the apoptosome and downstream caspases (58). Cells derived from Bid−/− mice show delayed caspase activation and reduced apoptosis in response to TNF-α (63). Thus, TNFRI-induced apoptosis is mediated through mitochondrion-independent and mitochondrion-dependent mechanisms. It was previously shown that degradation of the retinoblastoma tumor suppressor protein (RB) is required for TNF-α to activate mitochondrion-dependent apoptosis (6). Upon the activation of TNFRI, RB is cleaved by caspase and then degraded. We have created in the mouse germ line an Rb-MI allele, which contains two substitutions at a C-terminal caspase cleavage site (DEADG to DEAAE) (6). Rb-MI is not degraded upon the activation of TNFRI; cells expressing Rb-MI maintain their mitochondrial integrity and display a protracted death response to TNF-α (6). Rb-MI is sensitive to degradation when TNFRI and TNFRII are both activated (6). As a result, cells expressing Rb-MI are sensitive to death induced by the costimulation of TNFRI and TNFRII (6). These and other results strongly suggest that RB has an apoptosis suppression function (7). It has been previously shown that RB binds to nuclear c-ABL tyrosine kinase and inhibits its activity (59, 60). The activation of nuclear c-ABL tyrosine kinase by DNA damage is associated with the induction of apoptosis (57). In addition to DNA damage, nuclear c-ABL kinase could also be activated by TNF-α (12, 57), and STI-571, a chemical inhibitor of c-ABL kinase, reduces the apoptotic response to TNF-α (12). It was found that z-VAD, a general inhibitor of caspase, blocks TNF-α-induced activation of c-ABL (12). While caspase can cleave c-ABL protein, cleavage does not activate the tyrosine kinase activity but instead promotes the nuclear accumulation of c-ABL (3). In this study, we show that caspase-dependent removal of RB is required for TNF-α to activate the nuclear c-ABL tyrosine kinase, thus explaining the inhibitory effect of z-VAD on c-ABL activation. c-ABL tyrosine kinase regulates the function of p53 and p73, which play essential roles in DNA damage-induced apoptosis (17). The human c-ABL protein may interact directly with p53 to activate its function (18). Overproduction of c-ABL can also interfere with MDM2-mediated degradation of p53 (19, 46). However, activated nuclear c-ABL kinase can induce apoptosis in p53-deficient cells (52, 53). By contrast, activated nuclear c-ABL kinase does not induce apoptosis in p73-deficient cells (52). The c-ABL kinase can directly phosphorylate p73 (1, 64), increase the half-life of p73 (21), and promote the acetylation of p73 by p300 (10). Coexpression of activated nuclear c-ABL kinase with p73 synergistically activates cell death (52). Thus, c-ABL and p73 cooperate to promote apoptosis. In this study, we show p73 to also be required for TNF-α-induced cell death and RB to be a suppressor of the p73 apoptotic function.