Dimer-specific immunoprecipitation of active caspase-2 identifies TRAF proteins as novel activators.

Caspase-2 has been shown to initiate apoptotic cell death in response to specific intracellular stressors such as DNA damage. However, the molecular mechanisms immediately upstream of its activation are still poorly understood. We combined a caspase-2 bimolecular fluorescence complementation (BiFC) system with fluorophore-specific immunoprecipitation to isolate and study the active caspase-2 dimer and its interactome. Using this technique, we found that tumor necrosis factor receptor-associated factor 2 (TRAF2), as well as TRAF1 and 3, directly binds to the active caspase-2 dimer. TRAF2 in particular is necessary for caspase-2 activation in response to apoptotic cell death stimuli. Furthermore, we found that dimerized caspase-2 is ubiquitylated in a TRAF2-dependent manner at K15, K152, and K153, which in turn stabilizes the active caspase-2 dimer complex, promotes its association with an insoluble cellular fraction, and enhances its activity to fully commit the cell to apoptosis. Together, these data indicate that TRAF2 positively regulates caspase-2 activation and consequent cell death by driving its activation through dimer-stabilizing ubiquitylation.

A grafted ovarian fragment rescues host fertility after chemotherapy.

STUDY QUESTION: Can host fertility be rescued by grafting of a fragment of a healthy ovary soon after chemotherapy? SUMMARY ANSWER: We found that grafting a green fluorescent protein (GFP)-positive fragment from a healthy isogenic ovary to the left ovary of a chemo-treated host rescued function and fertility of the grafted host ovary, and resulted in the production of host-derived offspring as late as the sixth litter after chemotherapy (CTx) treatment, whereas none of the ungrafted controls produced a second litter. WHAT IS KNOWN ALREADY: In women and girls undergoing chemotherapy, infertility and premature ovarian failure are frequent outcomes. There are accumulating reports of improved endocrine function after autotransplantation of an ovarian fragment, raising the possibility that the transplant is beneficial to the endogenous ovary. STUDY DESIGN, SIZE, DURATION: We first established a CTx treatment regimen that resulted in the permanent loss of fertility in 100% of female mice of the FVB inbred strain. We grafted an isogenic ovary fragment from a healthy female homozygous for a GFP transgene to the left ovary of 100 CTx-treated hosts, and compared fertility to 39 ungrafted controls in 6 months of continuous matings, using GFP to distinguish offspring derived from the graft, and those derived from the host. PARTICIPANTS/MATERIALS, SETTING, METHODS: Immunofluoresece and western blot analysis of 39 treated ovaries during and 15 days after CTx treatment revealed elevated apoptosis, rapid loss of granulosa cells and an increased recruitment of growing follicles. Using immunofluorescence and confocal imaging, we tracked the outcome of the grafted tissue over 4 months and its effect on the adjacent and contralateral ovary of the host. MAIN RESULTS AND THE ROLE OF CHANCE: Fifty-three percent of grafted females produced a second litter whereas none of the ungrafted females produced a second litter. The likelihood that this could occur by chance is very low (P < 0.0001). LIMITATIONS, REASONS FOR CAUTION: These results are shown only in mice, and whether or how they might apply to chemotherapy patients subjected to different CTx regimens is not yet clear. WIDER IMPLICATIONS OF THE FINDINGS: Our experiments prove that rescue of a chemo-treated ovary is possible, and establish a system to investigate the mechanism of rescue and to identify the factors responsible with the long-term goal of developing therapies for preservation of ovarian endocrine function and fertility in women undergoing chemotherapy. LARGE SCALE DATA: No large datasets were produced. STUDY FUNDING/COMPETING INTERESTS: Duke University Medical Center Chancellor's Discovery Grant to BC; ESJ was supported by an NRSA 5F31CA165545; SK was supported by NIH RO1 GM08033; RWT was supported by the Duke University School of Medicine Ovarian Cancer Research Fellowship; XBM was supported by CONICYT. The authors have no conflicts of interest to declare.

Mitotic phosphatase activity is required for MCC maintenance during the spindle checkpoint.

The spindle checkpoint prevents activation of the anaphase-promoting complex (APC/C) until all chromosomes are correctly attached to the mitotic spindle. Early in mitosis, the mitotic checkpoint complex (MCC) inactivates the APC/C by binding the APC/C activating protein CDC20 until the chromosomes are properly aligned and attached to the mitotic spindle, at which point MCC disassembly releases CDC20 to activate the APC/C. Once the APC/C is activated, it targets cyclin B and securin for degradation, and the cell progresses into anaphase. While phosphorylation is known to drive many of the events during the checkpoint, the precise molecular mechanisms regulating spindle checkpoint maintenance and inactivation are still poorly understood. We sought to determine the role of mitotic phosphatases during the spindle checkpoint. To address this question, we treated spindle checkpoint-arrested cells with various phosphatase inhibitors and examined the effect on the MCC and APC/C activation. Using this approach we found that 2 phosphatase inhibitors, calyculin A and okadaic acid (1 µM), caused MCC dissociation and APC/C activation leading to cyclin A and B degradation in spindle checkpoint-arrested cells. Although the cells were able to degrade cyclin B, they did not exit mitosis as evidenced by high levels of Cdk1 substrate phosphorylation and chromosome condensation. Our results provide the first evidence that phosphatases are essential for maintenance of the MCC during operation of the spindle checkpoint.

Automated, quantitative analysis of histopathological staining in nuclei.

Technological advances have allowed the generation of high-throughput imaging of tissue sections. However, the analysis of these samples is typically still performed manually by one or multiple pathologists. We present a novel statistical model for the automated, quantitative analysis of these images. Our approach requires minimal tuning and allows recapitulation of estimates of staining strength in the nuclei of tumor cells as estimated by the gold standard. Besides, it compares favorably to other quantitative approaches available in the public domain.

Suppression of DNA-damage checkpoint signaling by Rsk-mediated phosphorylation of Mre11.

Ataxia telangiectasia mutant (ATM) is an S/T-Q-directed kinase that is critical for the cellular response to double-stranded breaks (DSBs) in DNA. Following DNA damage, ATM is activated and recruited by the MRN protein complex [meiotic recombination 11 (Mre11)/DNA repair protein Rad50/Nijmegen breakage syndrome 1 proteins] to sites of DNA damage where ATM phosphorylates multiple substrates to trigger cell-cycle arrest. In cancer cells, this regulation may be faulty, and cell division may proceed even in the presence of damaged DNA. We show here that the ribosomal s6 kinase (Rsk), often elevated in cancers, can suppress DSB-induced ATM activation in both Xenopus egg extracts and human tumor cell lines. In analyzing each step in ATM activation, we have found that Rsk targets loading of MRN complex components onto DNA at DSB sites. Rsk can phosphorylate the Mre11 protein directly at S676 both in vitro and in intact cells and thereby can inhibit the binding of Mre11 to DNA with DSBs. Accordingly, mutation of S676 to Ala can reverse inhibition of the response to DSBs by Rsk. Collectively, these data point to Mre11 as an important locus of Rsk-mediated checkpoint inhibition acting upstream of ATM activation.

A network of substrates of the E3 ubiquitin ligases MDM2 and HUWE1 control apoptosis independently of p53.

In the intrinsic pathway of apoptosis, cell-damaging signals promote the release of cytochrome c from mitochondria, triggering activation of the Apaf-1 and caspase-9 apoptosome. The ubiquitin E3 ligase MDM2 decreases the stability of the proapoptotic factor p53. We show that it also coordinated apoptotic events in a p53-independent manner by ubiquitylating the apoptosome activator CAS and the ubiquitin E3 ligase HUWE1. HUWE1 ubiquitylates the antiapoptotic factor Mcl-1, and we found that HUWE1 also ubiquitylated PP5 (protein phosphatase 5), which indirectly inhibited apoptosome activation. Breast cancers that are positive for the tyrosine receptor kinase HER2 (human epidermal growth factor receptor 2) tend to be highly aggressive. In HER2-positive breast cancer cells treated with the HER2 tyrosine kinase inhibitor lapatinib, MDM2 was degraded and HUWE1 was stabilized. In contrast, in breast cancer cells that acquired resistance to lapatinib, the abundance of MDM2 was not decreased and HUWE1 was degraded, which inhibited apoptosis, regardless of p53 status. MDM2 inhibition overcame lapatinib resistance in cells with either wild-type or mutant p53 and in xenograft models. These findings demonstrate broader, p53-independent roles for MDM2 and HUWE1 in apoptosis and specifically suggest the potential for therapy directed against MDM2 to overcome lapatinib resistance.

The tangled circuitry of metabolism and apoptosis.

For single-cell organisms, nutrient uptake and metabolism are central to the fundamental decision of whether to grow or divide. In metazoans, cell fate decisions are more complex: organismal homeostasis must be strictly maintained by balancing cell proliferation and death. Despite this increased complexity, cell fate within multicellular organisms is also influenced by metabolism; recent studies, triggered in part by an interest in tumor metabolism, are beginning to illuminate the mechanisms through which proliferation, death, and metabolism are intertwined. In particular, work on Bcl-2 family proteins suggests that the signaling pathways governing metabolism and apoptosis are inextricably linked. Here we review the crosstalk between these pathways, emphasizing recent work that illustrates the emerging dual nature of several core apoptotic proteins in regulating both metabolism and cell death.

Engineering a BCR-ABL-activated caspase for the selective elimination of leukemic cells.

Increased understanding of the precise molecular mechanisms involved in cell survival and cell death signaling pathways offers the promise of harnessing these molecules to eliminate cancer cells without damaging normal cells. Tyrosine kinase oncoproteins promote the genesis of leukemias through both increased cell proliferation and inhibition of apoptotic cell death. Although tyrosine kinase inhibitors, such as the BCR-ABL inhibitor imatinib, have demonstrated remarkable efficacy in the clinic, drug-resistant leukemias emerge in some patients because of either the acquisition of point mutations or amplification of the tyrosine kinase, resulting in a poor long-term prognosis. Here, we exploit the molecular mechanisms of caspase activation and tyrosine kinase/adaptor protein signaling to forge a unique approach for selectively killing leukemic cells through the forcible induction of apoptosis. We have engineered caspase variants that can directly be activated in response to BCR-ABL. Because we harness, rather than inhibit, the activity of leukemogenic kinases to kill transformed cells, this approach selectively eliminates leukemic cells regardless of drug-resistant mutations.

Ubiquitylation of p53 by the APC/C inhibitor Trim39.

Tripartite motif 39 (Trim39) is a RING domain-containing E3 ubiquitin ligase able to inhibit the anaphase-promoting complex (APC/C) directly. Through analysis of Trim39 function in p53-positive and p53-negative cells, we have found, surprisingly, that p53-positive cells lacking Trim39 could not traverse the G1/S transition. This effect did not result from disinhibition of the APC/C. Moreover, although Trim39 loss inhibited etoposide-induced apoptosis in p53-negative cells, apoptosis was enhanced by Trim39 knockdown in p53-positive cells. Furthermore, we show here that the Trim39 can directly bind and ubiquitylate p53 in vitro and in vivo, leading to p53 degradation. Depletion of Trim39 significantly increased p53 protein levels and cell growth retardation in multiple cell lines. We found that the relative importance of Trim39 and the well-characterized p53-directed E3 ligase, murine double minute 2 (MDM2), varied between cell types. In cells that were relatively insensitive to the MDM2 inhibitor, nutlin-3a, apoptosis could be markedly enhanced by siRNA directed against Trim39. As such, Trim39 may serve as a potential therapeutic target in tumors with WT p53 when MDM2 inhibition is insufficient to elevate p53 levels and apoptosis.

Mcl-1 rescues a glitch in the matrix.

Bcl-2 family proteins are known to control cell death and influence mitochondrial function. The function of Mcl-1, an anti-apoptotic Bcl-2 protein, is now shown to depend on its subcellular localization. Mcl-1 at the mitochondrial outer membrane inhibits mitochondrial permeabilization to block apoptosis. However, a cleaved form of Mcl-1 localizes to the mitochondrial matrix and controls inner mitochondrial morphology and oxidative phosphorylation, without directly modulating apoptosis.

The Trim39 ubiquitin ligase inhibits APC/CCdh1-mediated degradation of the Bax activator MOAP-1.

Proapoptotic Bcl-2 family members, such as Bax, promote release of cytochrome c from mitochondria, leading to caspase activation and cell death. It was previously reported that modulator of apoptosis protein 1 (MOAP-1), an enhancer of Bax activation induced by DNA damage, is stabilized by Trim39, a protein of unknown function. In this paper, we show that MOAP-1 is a novel substrate of the anaphase-promoting complex (APC/C(Cdh1)) ubiquitin ligase. The influence of Trim39 on MOAP-1 levels stems from the ability of Trim39 (a RING domain E3 ligase) to directly inhibit APC/C(Cdh1)-mediated protein ubiquitylation. Accordingly, small interfering ribonucleic acid-mediated knockdown of Cdh1 stabilized MOAP-1, thereby enhancing etoposide-induced Bax activation and apoptosis. These data identify Trim39 as a novel APC/C regulator and provide an unexpected link between the APC/C and apoptotic regulation via MOAP-1.

Phosphatases driving mitosis: pushing the gas and lifting the brakes.

Entry into and progression through mitosis depends critically on the establishment and maintenance of protein phosphorylation. For this reason, studies on mitotic progression have focused heavily on the activation of MPF (M phase promoting factor), a cyclin-dependent kinase responsible for phosphorylating proteins that execute the dynamic events of mitosis. Recent work, however, has significantly expanded our understanding of mechanisms that allow accumulation of phosphoproteins at M phase, suggesting that mitotic entry relies not only on MPF activation but also on the inhibition of antimitotic phosphatases. It is now clear that there exists a separate, albeit equally important, signaling pathway for the inactivation of protein phosphatases at the G2/M transition. This pathway, which is governed by the kinase Greatwall is essential for both entry into and maintenance of M phase. This chapter will outline the molecular events regulating entry into mitosis, specifically highlighting the role that protein phosphorylation plays in triggering both MPF activation and the inhibition of phosphatase activity that would otherwise prevent accumulation of mitotic phosphoproteins. These intricate regulatory pathways are essential for maintaining normal cell division and preventing inappropriate cell proliferation, a central hallmark of cancer cells.

Rsk-mediated phosphorylation and 14-3-3ɛ binding of Apaf-1 suppresses cytochrome c-induced apoptosis.

Many pro-apoptotic signals trigger mitochondrial cytochrome c release, leading to caspase activation and ultimate cellular breakdown. Cell survival pathways, including the mitogen-activated protein kinase (MAPK) cascade, promote cell viability by impeding mitochondrial cytochrome c release and by inhibiting subsequent caspase activation. Here, we describe a mechanism for the inhibition of cytochrome c-induced caspase activation by MAPK signalling, identifying a novel mode of apoptotic regulation exerted through Apaf-1 phosphorylation by the 90-kDa ribosomal S6 kinase (Rsk). Recruitment of 14-3-3ɛ to phosphorylated Ser268 impedes the ability of cytochrome c to nucleate apoptosome formation and activate downstream caspases. High endogenous levels of Rsk in PC3 prostate cancer cells or Rsk activation in other cell types promoted 14-3-3ɛ binding to Apaf-1 and rendered the cells insensitive to cytochrome c, suggesting a potential role for Rsk signalling in apoptotic resistance of prostate cancers and other cancers with elevated Rsk activity. Collectively, these results identify a novel locus of apoptosomal regulation wherein MAPK signalling promotes Rsk-catalysed Apaf-1 phosphorylation and consequent binding of 14-3-3ɛ, resulting in decreased cellular responsiveness to cytochrome c.

A biotin switch-based proteomics approach identifies 14-3-3ζ as a target of Sirt1 in the metabolic regulation of caspase-2.

While lysine acetylation in the nucleus is well characterized, comparatively little is known about its significance in cytoplasmic signaling. Here we show that inhibition of the Sirt1 deacetylase, which is primarily cytoplasmic in cancer cell lines, sensitizes these cells to caspase-2-dependent death. To identify relevant Sirt1 substrates, we developed a proteomics strategy, enabling the identification of a range of putative substrates, including 14-3-3ζ, a known direct regulator of caspase-2. We show here that inhibition of Sirtuin activity accelerates caspase activation and overrides caspase-2 suppression by nutrient abundance. Furthermore, 14-3-3ζ is acetylated prior to caspase activation, and supplementation of Xenopus egg extract with glucose-6-phosphate, which promotes caspase-2/14-3-3ζ binding, enhances 14-3-3ζ-directed Sirtuin activity. Conversely, inhibiting Sirtuin activity promotes14-3-3ζ dissociation from caspase-2 in both egg extract and human cultured cells. These data reveal a role for Sirt1 in modulating apoptotic sensitivity, in response to metabolic changes, by antagonizing 14-3-3ζ acetylation.

Regulation of the ATM-activator protein Aven by CRM1-dependent nuclear export.

Aven is a regulator of apoptosis whose overexpression is associated with poor prognosis in several cancers, including childhood acute lymphoblastic leukemia and acute myeloid leukemia. We have recently shown that Aven serves as an activator and substrate of ATM, thereby modulating the DNA-damage response and G(2)/M cell cycle progression. Under physiological conditions, the cellular localization of Aven is mainly cytosolic, but a small fraction of the protein is present in the nucleus. Here, we show that treatment of cells with leptomycin B, an inhibitor of Exportin-1/CRM (chromosomal region maintenance) 1, resulted in nuclear accumulation of Aven. Furthermore, we identified a functional LR-NES between amino acid residues 282-292 of the human Aven protein, a sequence that is evolutionary conserved among a range of vertebrate species. Disruption of this LR-NES by site-directed mutagenesis resulted in enhanced nuclear localization of Aven, but did not alter the ability of the protein to induce G(2)/M cell cycle arrest in interphase Xenopus laevis extracts. However, elimination of the LR-NES sequence led to a reduction in the capacity of Aven to arrest Xenopus oocytes containing intact nuclei. Our results suggest that the regulation of nucleocytoplasmatic traffic of Aven could modulate its ability to influence cell cycle progression.

The engine driving the ship: metabolic steering of cell proliferation and death.

Metabolic activity is a crucial determinant of a cell's decision to proliferate or die. Although it is not fully understood how metabolic pathways such as glycolysis and the pentose phosphate pathway communicate to cell cycle and apoptotic effectors, it is clear that a complex network of signalling molecules is required to integrate metabolic inputs. D-type cyclins, cyclin-dependent kinases, the anaphase-promoting complex, p53, caspase 2 and B cell lymphoma 2 proteins, among others, have been shown to be regulated by metabolic crosstalk. Elucidating these pathways is of great importance, as metabolic aberrations and their downstream effects are known to contribute to the aetiology of cancer and degenerative disorders.

Metabolic regulation of Drosophila apoptosis through inhibitory phosphorylation of Dronc.

Apoptosis ensures tissue homeostasis in response to developmental cues or cellular damage. Recently reported genome-wide RNAi screens have suggested that several metabolic regulators can modulate caspase activation in Drosophila. Here, we establish a previously unrecognized link between metabolism and Drosophila apoptosis by showing that cellular NADPH levels modulate the initiator caspase Dronc through its phosphorylation at S130. Depletion of NADPH removed this inhibitory phosphorylation, resulting in the activation of Dronc and subsequent cell death. Conversely, upregulation of NADPH prevented Dronc-mediated apoptosis upon DIAP1 RNAi or cycloheximide treatment. Furthermore, this CaMKII-mediated phosphorylation of Dronc hindered Dronc activation, but not its catalytic activity. Blockade of NADPH production aggravated the death-inducing activity of Dronc in specific neurons, but not in the photoreceptor cells of the eyes of transgenic flies; similarly, non-phosphorylatable Dronc was more potent than wild type in triggering specific neuronal apoptosis. Our observations reveal a novel regulatory circuitry in Drosophila apoptosis, and, as NADPH levels are elevated in cancer cells, also provide a genetic model to understand aberrations in cancer cell apoptosis resulting from metabolic alterations.

Restraint of apoptosis during mitosis through interdomain phosphorylation of caspase-2.

The apoptotic initiator caspase-2 has been implicated in oocyte death, in DNA damage- and heat shock-induced death, and in mitotic catastrophe. We show here that the mitosis-promoting kinase, cdk1-cyclin B1, suppresses apoptosis upstream of mitochondrial cytochrome c release by phosphorylating caspase-2 within an evolutionarily conserved sequence at Ser 340. Phosphorylation of this residue, situated in the caspase-2 interdomain, prevents caspase-2 activation. S340 was susceptible to phosphatase 1 dephosphorylation, and an interaction between phosphatase 1 and caspase-2 detected during interphase was lost in mitosis. Expression of S340A non-phosphorylatable caspase-2 abrogated mitotic suppression of caspase-2 and apoptosis in various settings, including oocytes induced to undergo cdk1-dependent maturation. Moreover, U2OS cells treated with nocodazole were found to undergo mitotic catastrophe more readily when endogenous caspase-2 was replaced with the S340A mutant to lift mitotic inhibition. These data demonstrate that for apoptotic stimuli transduced by caspase-2, cell death is prevented during mitosis through the inhibitory phosphorylation of caspase-2 and suggest that under conditions of mitotic arrest, cdk1-cyclin B1 activity must be overcome for apoptosis to occur.