Caspase-2 activation in the absence of PIDDosome formation

PIDD (p53-induced protein with a death domain [DD]), together with the bipartite adapter protein RAIDD (receptor-interacting protein-associated ICH-1/CED-3 homologous protein with a DD), is implicated in the activation of pro–caspase-2 in a high molecular weight complex called the PIDDosome during apoptosis induction after DNA damage. To investigate the role of PIDD in cell death initiation, we generated PIDD-deficient mice. Processing of caspase-2 is readily detected in the absence of PIDDosome formation in primary lymphocytes. Although caspase-2 processing is delayed in simian virus 40–immortalized pidd^−/− mouse embryonic fibroblasts, it still depends on loss of mitochondrial integrity and effector caspase activation. Consistently, apoptosis occurs normally in all cell types analyzed, suggesting alternative biological roles for caspase-2 after DNA damage. Because loss of either PIDD or its adapter molecule RAIDD did not affect subcellular localization, nuclear translocation, or caspase-2 activation in high molecular weight complexes, we suggest that at least one alternative PIDDosome-independent mechanism of caspase-2 activation exists in mammals in response to DNA damage.     

PIDD mediates NF-kappaB activation in response to DNA damage

Activation of NF-kappaB following genotoxic stress allows time for DNA-damage repair and ensures cell survival accounting for acquired chemoresistance, an impediment to effective cancer therapy. Despite this clinical relevance, little is known about pathways that enable genotoxic-stress-induced NF-kappaB induction. Previously, we reported a role for the p53-inducible death-domain-containing protein, PIDD, in caspase-2 activation and apoptosis in response to DNA damage. We now demonstrate that PIDD plays a critical role in DNA-damage-induced NF-kappaB activation. Upon genotoxic stress, a complex between PIDD, the kinase RIP1, and a component of the NF-kappaB-activating kinase complex, NEMO, is formed. PIDD expression enhances genotoxic-stress-induced NF-kappaB activation through augmented sumoylation and ubiquitination of NEMO. Depletion of PIDD and RIP1, but not caspase-2, abrogates DNA-damage-induced NEMO modification and NF-kappaB activation. We propose that PIDD acts as a molecular switch, controlling the balance between life and death upon DNA damage.     

Deregulation of DNA damage signal transduction by herpesvirus latency-associated M2

Infected cells recognize viral replication as a DNA damage stress and elicit a DNA damage response that ultimately induces apoptosis as part of host immune surveillance. Here, we demonstrate a novel mechanism where the murine gamma herpesvirus 68 (gammaHV68) latency-associated, anti-interferon M2 protein inhibits DNA damage-induced apoptosis by interacting with the DDB1/COP9/cullin repair complex and the ATM DNA damage signal transducer. M2 expression constitutively induced DDB1 nuclear localization and ATM kinase activation in the absence of DNA damage. Activated ATM subsequently induced Chk activation and p53 phosphorylation and stabilization without eliciting H2AX phosphorylation and MRN recruitment to foci upon DNA damage. Consequently, M2 expression inhibited DNA repair, rendered cells resistant to DNA damage-induced apoptosis, and induced a G(1) cell cycle arrest. Our results suggest that gammaHV68 M2 blocks apoptosis-mediated intracellular innate immunity, which might ultimately contribute to its role in latent infection.     

Autoproteolysis of PIDD marks the bifurcation between pro-death caspase-2 and pro-survival NF-kappaB pathway

Upon DNA damage, a complex called the PIDDosome is formed and either signals NF-kappaB activation and thus cell survival or alternatively triggers caspase-2 activation and apoptosis. PIDD (p53-induced protein with a death domain) is constitutively processed giving rise to a 48-kDa N-terminal fragment containing the leucine-rich repeats (LRRs, PIDD-N) and a 51-kDa C-terminal fragment containing the death domain (DD, PIDD-C). The latter undergoes further cleavage resulting in a 37-kDa fragment (PIDD-CC). Here we show that processing occurs at S446 (generating PIDD-C) and S588 (generating PIDD-CC) by an auto-processing mechanism similar to that found in the nuclear pore protein Nup98/96 and inteins. Auto-cleavage of PIDD determines the outcome of the downstream signaling events. Whereas initially formed PIDD-C mediates the activation of NF-kappaB via the recruitment of RIP1 and NEMO, subsequent formation of PIDD-CC causes caspase-2 activation and thus cell death. A non-cleavable PIDD mutant is unable to translocate from the cytoplasm to the nucleus and loses both activities. In this way, auto-proteolysis of PIDD might participate in the orchestration of the DNA damage-induced life and death signaling pathways.     

c-Abl tyrosine kinase regulates caspase-9 autocleavage in the apoptotic response to DNA damage

Activation of the initiator caspase-9 is essential for induction of apoptosis by developmental signals, oncogenic transformation, and genotoxic stress. The c-Abl tyrosine kinase is also involved in the apoptotic response to DNA damage. The present results demonstrate that c-Abl binds directly to caspase-9. We show that c-Abl phosphorylates caspase-9 on Tyr-153 in vitro and in cells treated with DNA damaging agents. Moreover, inhibition of c-Abl with STI571 blocked DNA damage-induced autoprocessing of caspase-9 to the p35 subunit and activation of caspase-3. Caspase-9(Y153F) also attenuated DNA damage-induced processing of caspase-9 to p35, activation of caspase-3, and apoptosis. These findings indicate that caspase-9 autoprocessing is regulated by c-Abl in the apoptotic response to genotoxic stress.     

The plant isoflavenoid genistein activates p53 and Chk2 in an ATM-dependent manner

Genistein is an isoflavenoid that is abundant in soy beans. Genistein has been reported to have a wide range of biological activities and to play a role in the diminished incidence of breast cancer in populations that consume a soy-rich diet. Genistein was originally identified as an inhibitor of tyrosine kinases; however, it also inhibits topoisomerase II by stabilizing the covalent DNA cleavage complex, an event predicted to cause DNA damage. The topoisomerase II inhibitor etoposide acts in a similar manner. Here we show that genistein induces the up-regulation of p53 protein, phosphorylation of p53 at serine 15, activation of the sequence-specific DNA binding properties of p53, and phosphorylation of the hCds1/Chk2 protein kinase at threonine 68. Phosphorylation and activation of p53 and phosphorylation of Chk2 were not observed in ATM-deficient cells. In contrast, the topoisomerase II inhibitor etoposide induced phosphorylation of p53 and Chk2 in ATM-positive and ATM-deficient cells. In addition, genistein-treated ATM-deficient cells were significantly more susceptible to genistein-induced killing than were ATM-positive cells. Together our data suggest that ATM is required for activation of a DNA damage-induced pathway that activates p53 and Chk2 in response to genistein.     

The molecular origin of the MMR-dependent apoptosis pathway from dynamics analysis of MutSα-DNA complexes

The cellular response to DNA damage signaling by mismatch-repair (MMR) proteins is incompletely understood. It is generally accepted that MMR-dependent apoptosis pathway in response to DNA damage detection is independent of MMR's DNA repair function. In this study, we investigate correlated motions in response to the binding of mismatched and platinum cross-linked DNA fragments by MutSα, as derived from 50 ns molecular dynamics simulations. The protein dynamics in response to the mismatched and damaged DNA recognition suggests that MutSα signals their recognition through independent pathways providing evidence for the molecular origin of the MMR-dependent apoptosis. MSH2 subunit is indicated to play a key role in signaling both mismatched and damaged DNA recognition; localized and collective motions within the protein allow identifying sites on the MSH2 surface possible involved in recruiting proteins responsible for downstream events. Unlike in the mismatch complex, predicted key communication sites specific for the damage recognition are on the list of known cancer-causing mutations or deletions. This confirms MSH2's role in signaling DNA damage-induced apoptosis and suggests that defects in MMR alone is sufficient to trigger tumorigenesis, supporting the experimental evidence that MMR-damage response function could protect from the early occurrence of tumors. Identifying these particular communication sites may have implications for the treatment of cancers that are not defective for MMR, but are unable to function optimally for MMR-dependent responses following DNA damage such as the case of resistance to cisplatin.     

The human REV1 gene codes for a DNA template-dependent dCMP transferase.

DNA is frequently damaged by various physical and chemical agents. DNA damage can lead to mutations during replication. In the yeast Saccharomyces cerevisiae, the damage-induced mutagenesis pathway requires the Rev1 protein. We have isolated a human cDNA homologous to the yeast REV1 gene. The human REV1 cDNA consists of 4255 bp and codes for a protein of 1251 amino acid residues with a calculated molecular weight of 138 248 Da. The human REV1 gene is localized between 2q11.1 and 2q11.2. We show that the human REV1 protein is a dCMP transferase that specifically inserts a dCMP residue opposite a DNA template G. In addition, the human REV1 transferase is able to efficiently and specifically insert a dCMP opposite a DNA template apurinic/apyrimidinic (AP) site or a uracil residue. These results suggest that the REV1 transferase may play a critical role during mutagenic translesion DNA synthesis bypassing a template AP site in human cells. Consistent with its role as a fundamental mutagenic protein, the REV1 gene is ubiquitously expressed in various human tissues.     

BMI1 is recruited to DNA breaks and contributes to DNA damage-induced H2A ubiquitination and repair

DNA damage activates signaling pathways that lead to modification of local chromatin and recruitment of DNA repair proteins. Multiple DNA repair proteins having ubiquitin ligase activity are recruited to sites of DNA damage, where they ubiquitinate histones and other substrates. This DNA damage-induced histone ubiquitination is thought to play a critical role in mediating the DNA damage response. We now report that the polycomb protein BMI1 is rapidly recruited to sites of DNA damage, where it persists for more than 8 h. The sustained localization of BMI1 to damage sites is dependent on intact ATM and ATR and requires H2AX phosphorylation and recruitment of RNF8. BMI1 is required for DNA damage-induced ubiquitination of histone H2A at lysine 119. Loss of BMI1 leads to impaired repair of DNA double-strand breaks by homologous recombination and the accumulation of cells in G(2)/M. These data support a crucial role for BMI1 in the cellular response to DNA damage.     

Regulation of PIDD auto-proteolysis and activity by the molecular chaperone Hsp90

In response to DNA damage, p53-induced protein with a death domain (PIDD) forms a complex called the PIDDosome, which either consists of PIDD, RIP-associated protein with a death domain and caspase-2, forming a platform for the activation of caspase-2, or contains PIDD, RIP1 and NEMO, important for NF-κB activation. PIDDosome activation is dependent on auto-processing of PIDD at two different sites, generating the fragments PIDD-C and PIDD-CC. Despite constitutive cleavage, endogenous PIDD remains inactive. In this study, we screened for novel PIDD regulators and identified heat shock protein 90 (Hsp90) as a major effector in both PIDD protein maturation and activation. Hsp90, together with p23, binds PIDD and inhibition of Hsp90 activity with geldanamycin efficiently disrupts this association and impairs PIDD auto-processing. Consequently, both PIDD-mediated NF-κB and caspase-2 activation are abrogated. Interestingly, PIDDosome formation itself is associated with Hsp90 release. Characterisation of cytoplasmic and nuclear pools of PIDD showed that active PIDD accumulates in the nucleus and that only cytoplasmic PIDD is bound to Hsp90. Finally, heat shock induces Hsp90 release from PIDD and PIDD nuclear translocation. Thus, Hsp90 has a major role in controlling PIDD functional activity.     

Role of SHP-2 tyrosine phosphatase in the DNA damage-induced cell death response

SHP-2, a ubiquitously expressed Src hmology 2 (SH2) domain-containing tyrosine phosphatase, plays a critical role in the regulation of growth factor and cytokine signal transduction. Here we report a novel function of this phosphatase in DNA damage-induced cellular responses. Mutant embryonic fibroblast cells lacking functional SHP-2 showed significantly decreased apoptosis in response to DNA damage. Following cisplatin treatment, induction of p73 and its downstream effector p21(Cip1) was essentially blocked in SHP-2 mutant cells. Further investigation revealed that activation of the nuclear tyrosine kinase c-Abl, an essential mediator in DNA damage induction of p73, was impaired in the mutant cells, suggesting a functional requirement of SHP-2 in c-Abl activation. Consistent with this observation, the effect of overexpression of c-Abl kinase in SHP-2 mutant cells on sensitizing the cells to DNA damage-induced death was abolished. Additionally, we found that in embryonic fibroblast cells 30-40% of SHP-2 was localized in the nuclei, and that a fraction of nuclear SHP-2 was constitutively associated with c-Abl via its SH3 domain. Phosphatase activity of nuclear but not cytoplasmic SHP-2 was significantly enhanced in response to DNA damage. These results together suggest a novel nuclear function for SHP-2 phosphatase in the regulation of DNA damage-induced apoptotic responses.     

Tip60 and p400 are both required for UV-induced apoptosis but play antagonistic roles in cell cycle progression

The histone acetyl transferase Tip60 (HTATIP) belongs to a multimolecular complex involved in the cellular response to DNA damage. Tip60 participates in cell cycle arrest following DNA damage by allowing p53 to activate p21CIP (p21) expression. We show here that Tip60 and the E1A-associated p400 protein (EP400), which belongs to the Tip60 complex, are also required for DNA damage-induced apoptosis. Tip60 favours the expression of some proapoptotic p53 target genes most likely through the stimulation of p53 DNA binding activity. In contrast, p400 represses p21 expression in unstressed cells, thereby allowing cell cycle progression and DNA damage-induced apoptosis. Tip60 and p400 have thus opposite effects on p21 expression in the absence of DNA damage. We further found that this antagonism relies on the inhibition of Tip60 function by p400, a property that is abolished following DNA damage. Therefore, taken together, our results indicate that Tip60 and p400 play distinct roles in DNA damage-induced apoptosis and underline the importance of the Tip60 complex and its regulation in the proper control of cell fate.     

Activation of ubiquitin-dependent DNA damage bypass is mediated by replication protein a

Replicative DNA damage bypass, mediated by the ubiquitylation of the sliding clamp protein PCNA, facilitates the survival of a cell in the presence of genotoxic agents, but it can also promote genomic instability by damage-induced mutagenesis. We show here that PCNA ubiquitylation in budding yeast is activated independently of the replication-dependent S phase checkpoint but by similar conditions involving the accumulation of single-stranded DNA at stalled replication intermediates. The ssDNA-binding replication protein A (RPA), an essential complex involved in most DNA transactions, is required for damage-induced PCNA ubiquitylation. We found that RPA directly interacts with the ubiquitin ligase responsible for the modification of PCNA, Rad18, both in yeast and in mammalian cells. Association of the ligase with chromatin is detected where RPA is most abundant, and purified RPA can recruit Rad18 to ssDNA in vitro. Our results therefore implicate the RPA complex in the activation of DNA damage tolerance.     

Involvement of Exo1b in DNA damage-induced apoptosis

Apoptosis is essential for the maintenance of inherited genomic integrity. During DNA damage-induced apoptosis, mechanisms of cell survival, such as DNA repair are inactivated to allow cell death to proceed. Here, we describe a role for the mammalian DNA repair enzyme Exonuclease 1 (Exo1) in DNA damage-induced apoptosis. Depletion of Exo1 in human fibroblasts, or mouse embryonic fibroblasts led to a delay in DNA damage-induced apoptosis. Furthermore, we show that Exo1 acts upstream of caspase-3, DNA fragmentation and cytochrome c release. In addition, induction of apoptosis with DNA-damaging agents led to cleavage of both isoforms of Exo1. The cleavage of Exo1 was mapped to Asp514, and shown to be mediated by caspase-3. Expression of a caspase-3 cleavage site mutant form of Exo1, Asp514Ala, prevented formation of the previously observed fragment without any affect on the onset of apoptosis. We conclude that Exo1 has a role in the timely induction of apoptosis and that it is subsequently cleaved and degraded during apoptosis, potentially inhibiting DNA damage repair.     

DNA damage-induced G2/M checkpoint in SV40 large T antigen-immortalized embryonic fibroblast cells requires SHP-2 tyrosine phosphatase

DNA damage induced by radiation or DNA-damaging agents leads to apoptosis and cell cycle arrest. However, DNA damage-triggered signal transduction involved in these cellular responses is not well understood. We previously demonstrated an important role for SHP-2, a ubiquitously expressed SH2 domain-containing tyrosine phosphatase, in the DNA damage-induced apoptotic response. Here we report a potential role for SHP-2 in a DNA damage-activated cell cycle checkpoint. Cell cycle analysis and the mitotic index assay showed that following DNA damage induced by cisplatin or gamma-irradiation, the G2 (but not S) arrest response was diminished in SV40 large T antigen-immortalized embryonic fibroblast cells lacking functional SHP-2. Notably, reintroduction of wild-type SHP-2 into the mutant cells fully restored the DNA damage-induced G2 arrest response, suggesting a direct role of SHP-2 in the G2/M checkpoint. Further biochemical analysis revealed that SHP-2 constitutively associated with 14-3-3beta, and that Cdc25C cytoplasmic translocation induced by DNA damage was essentially blocked in SHP-2 mutant cells. Additionally, we showed that following DNA damage, activation of p38 kinase was significantly elevated, while Erk kinase activation was decreased in mutant cells, and treatment of SHP-2 mutant cells with SB203580, a selective inhibitor for p38 kinase, partially restored the DNA damage-induced G2 arrest response. These results together provide the first evidence that SHP-2 tyrosine phosphatase enhances the DNA damage G2/M checkpoint in SV40 large T antigen immortalized murine embryonic fibroblast cells.     

Delayed activation of Bax by DNA damage in embryonic stem cells with knock-in mutations of the Abl nuclear localization signals

The non-receptor tyrosine kinase Abl contains nuclear localization (NLS) and nuclear export signals that drive its nucleo-cytoplasmic shuttling. The nuclear Abl tyrosine kinase is activated by DNA damage through ataxia telangiectasia mutated (ATM). Previous studies have suggested nuclear Abl to have proapoptotic activity. To determine the requirement for Abl nuclear import in DNA damage-induced apoptosis, we took a genetic approach by mutating the three NLS (muNLS) of abl1 in mouse embryonic stem (ES) cells through homologous recombination. Exposure of ES cells to genotoxins caused an ATM-dependent nuclear accumulation of Abl but not Abl muNLS. ES cells expressing Abl muNLS exhibited delayed Bax activation, reduced cytochrome c release and decreased caspase-9 activity in response to DNA damage. These results provide a genetic proof that Abl nuclear entry contributes to DNA damage-induced activation of the intrinsic apoptotic pathway.