Florian J. Bock

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.

The enigma of caspase-2: the laymen's view.

Proteolysis of cellular substrates by caspases (cysteine-dependent aspartate-specific proteases) is one of the hallmarks of apoptotic cell death. Although the activation of apoptotic caspases is considered a ‘late-stage’ event in apoptosis signaling, past the commitment stage, one caspase family member, caspase-2, splits the cell death community into half – those searching for evidence of an apical initiator function of this molecule and those considering it as an amplifier of the apoptotic caspase cascade, at best, if relevant for apoptosis at all. This review screens past and present biochemical as well as genetic evidence for caspase-2 function in cell death signaling and beyond.

DNA-PKcs-PIDDosome: a nuclear caspase-2-activating complex with role in G2/M checkpoint maintenance.

A reciprocating piston type compressor having a cylinder block, a plurality of cylinder bores, and at least a housing closing an end of the cylinder block. The housing contains a suction chamber for a refrigerant gas to be compressed and a discharge chamber for the compressed refrigerant gas discharged from the cylinder bores in response to reciprocation of a plurality of pistons. The compressed gas is discharged through discharge ports closed by a discharge valve element having a plurality of integral discharge reed-valves movable between a closed positions and a predetermined open positions. The open position is defined by a stop unit integrally formed in an inner wall of the housing. The stop unit has a plurality of flat stop faces formed on the inner wall to permit free ends of the discharge reed-valves to come into contact engagement therewith, when the discharge reed-valves are moved from the closed positions to the open positions.Caspase-2 is unique among all the mammalian caspases in that it is the only caspase that is present constitutively in the cell nucleus, in addition to other cellular compartments. However, the functional significance of this nuclear localization is unknown. Here we show that DNA damage induced by gamma-radiation triggers the phosphorylation of nuclear caspase-2 at the S122 site within its prodomain, leading to its cleavage and activation. This phosphorylation is carried out by the nuclear serine/threonine protein kinase DNA-PKcs and promoted by the p53-inducible death-domain-containing protein PIDD within a large nuclear protein complex consisting of DNA-PKcs, PIDD, and caspase-2, which we have named the DNA-PKcs-PIDDosome. This phosphorylation and the catalytic activity of caspase-2 are involved in the maintenance of a G2/M DNA damage checkpoint and DNA repair mediated by the nonhomologous end-joining (NHEJ) pathway. The DNA-PKcs-PIDDosome thus represents a protein complex that impacts mammalian G2/M DNA damage checkpoint and NHEJ.

Caspase-2 at a glance.

Despite two decades of research, the role of caspase-2 in physiology and disease is still poorly understood and controversial. This Cell Science at a Glance article provides an overview of the proposed functions and possible modes of action and regulation of caspase-2. In addition, we will highlight

PIDDosome-independent tumor suppression by Caspase-2

The PIDDosome, a multiprotein complex constituted of the ‘p53-induced protein with a death domain (PIDD), ‘receptor-interacting protein (RIP)-associated ICH-1/CED-3 homologous protein with a death domain’ (RAIDD) and pro-Caspase-2 has been defined as an activating platform for this apoptosis-related protease. PIDD has been implicated in p53-mediated cell death in response to DNA damage but also in DNA repair and nuclear factor kappa-light-chain enhancer (NF-κB) activation upon genotoxic stress, together with RIP-1 kinase and Nemo/IKKγ. As all these cellular responses are critical for tumor suppression and deregulated expression of individual PIDDosome components has been noted in human cancer, we investigated their role in oncogenesis induced by DNA damage or oncogenic stress in gene-ablated mice. We observed that Pidd or Caspase-2 failed to suppress lymphoma formation triggered by γ-irradiation or 3-methylcholanthrene-driven fibrosarcoma development. In contrast, Caspase-2 showed tumor suppressive capacity in response to aberrant c-Myc expression, which did not rely on PIDD, the BH3-only protein Bid (BH3 interacting domain death agonist) or the death receptor ligand Trail (TNF-related apoptosis-inducing ligand), but associated with reduced rates of p53 loss and increased extranodal dissemination of tumor cells. In contrast, Pidd deficiency associated with abnormal M-phase progression and delayed disease onset, indicating that both proteins are differentially engaged upon oncogenic stress triggered by c-Myc, leading to opposing effects on tumor-free survival.

Death of p53-defective cells triggered by forced mitotic entry in the presence of DNA damage is not uniquely dependent on Caspase-2 or the PIDDosome.

Much effort has been put in the discovery of ways to selectively kill p53-deficient tumor cells and targeting cell cycle checkpoint pathways has revealed promising candidates. Studies in zebrafish and human cell lines suggested that the DNA damage response kinase, checkpoint kinase 1 (Chk1), not only regulates onset of mitosis but also cell death in response to DNA damage in the absence of p53. This effect reportedly relies on ataxia telangiectasia mutated (ATM)-dependent and PIDDosome-mediated activation of Caspase-2. However, we show that genetic ablation of PIDDosome components in mice does not affect cell death in response to γ-irradiation. Furthermore, Chk1 inhibition largely failed to sensitize normal and malignant cells from p53−/− mice toward DNA damaging agents, and p53 status did not affect the death-inducing activity of DNA damage after Chk1 inhibition in human cancer cells. These observations argue against cross-species conservation of a Chk1-controlled cell survival pathway demanding further investigation of the molecular machinery responsible for cell death elicited by forced mitotic entry in the presence of DNA damage in different cell types and model organisms.

Haematopoietic stem cell survival and transplantation efficacy is limited by the BH3‐only proteins Bim and Bmf

Anti‐apoptotic Bcl‐2 family members are critical for the regulation of haematopoietic stem and progenitor cell (HSPC) survival. Little is known about the role of their pro‐apoptotic antagonists, i.e. ‘BH3‐only’ proteins, in this cell compartment. Based on the analysis of cytokine deprivation‐induced changes in mRNA expression levels of Bcl‐2 family proteins, we determined the consequences of BH3‐only protein depletion on HSPC survival in culture and, for selected candidates, on engraftment in vivo. Thereby, we revealed a critical role for Bim and Bmf as regulators of HSPC dynamics both during early engraftment and long‐term reconstitution. HSPCs derived from wild‐type donors were readily displaced by Bim‐ or Bmf‐deficient or Bcl‐2‐overexpressing HSPCs as early as 10 days after engraftment. Moreover, in the absence of Bim, significantly lower numbers of transplanted HSPCs were able to fully engraft radio‐depleted recipients. Finally, we provide proof of principle that RNAi‐based reduction of BIM or BMF, or overexpression of BCL‐2 in human CD34+ cord blood cells may be an attractive therapeutic option to increase stem cell survival and transplantation efficacy.

P53-induced protein with a death domain (PIDD): master of puppets?

P53-induced protein with a death domain (PIDD) has been described as primary p53 target gene, induced upon DNA damage. More than 10 years after its discovery, its physiological role in the DNA damage response remains enigmatic, as it seems to be able to execute life–death decisions in vitro, yet genetic ablation in mice failed to reveal an obvious phenotype. Nonetheless, evidence is accumulating that it contributes to the fine-tuning of the DNA-damage response by orchestrating critical processes such as caspase activation or nuclear factor κB translocation and can also exert additional nuclear functions, for example, the modulation of translesion synthesis. In this review, we aim to integrate these observations and propose possible unexplored functions of PIDD.

Loss of PIDD limits NF-κB activation and cytokine production but not cell survival or transformation after DNA damage.

Activation of NF-κB (nuclear factor of kappa light chain gene enhancer in B cells) in response to DNA damage is considered to contribute to repair of genetic lesions, increased cell survival and cytokine release. The molecular mechanisms orchestrating this cytoplasmic event involve core components of the nuclear DNA damage response machinery, including ATM-kinase (ataxia telangiectasia mutated kinase) and PARP-1 (poly (ADP-ribose) polymerase 1). The physiological consequences of defective NF-κB activation in this context, however, remain poorly investigated. Here we report on the role of the ‘p53-induced protein with a death domain’, PIDD, which appears rate limiting in this process, as is PARP-1. Despite impaired NF-κB activation, DNA damage did not increase cell death or reduce clonal survival of various cell types lacking PIDD, such as mouse embryonic fibroblasts or stem and progenitor cells of the hematopoietic system. Furthermore, lymphomagenesis induced by γ-irradiation (IR) was unaffected by deficiency for PIDD or PARP-1, indicating that loss of DNA damage-triggered NF-κB signalling does not affect IR-driven tumorigenesis. However, loss of either gene compromised cytokine release after acute IR injury. Hence, we propose that NF-κB’s most notable function after DNA damage in primary cells is related to the release of cytokines, thereby contributing to sterile inflammation.

The p53 binding protein PDCD5 is not rate-limiting in DNA damage induced cell death

The tumour suppressor p53 is an important mediator of cell cycle arrest and apoptosis in response to DNA damage, acting mainly by transcriptional regulation of specific target genes. The exact details how p53 modulates this decision on a molecular basis is still incompletely understood. One mechanism of regulation is acetylation of p53 on lysine K120 by the histone-acetyltransferase Tip60, resulting in preferential transcription of proapoptotic target genes. PDCD5, a protein with reported pro-apoptotic function, has recently been identified as regulator of Tip60-dependent p53-acetylation. In an effort to clarify the role of PDCD5 upon DNA damage, we generated cell lines in which PDCD5 expression was conditionally ablated by shRNAs and investigated their response to genotoxic stress. Surprisingly, we failed to note a rate-limiting role of PDCD5 in the DNA damage response. PDCD5 was dispensable for DNA damage induced apoptosis and cell cycle arrest and we observed no significant changes in p53 target gene transcription. While we were able to confirm interaction of PDCD5 with p53, we failed to do so for Tip60. Altogether, our results suggest a role of PDCD5 in the regulation of p53 function but unrelated to cell cycle arrest or apoptosis, at least in the cell types investigated.