Cyclic-AMP-dependent protein kinase A regulates apoptosis by stabilizing the BH3-only protein Bim

The proapoptotic Bcl2 homology domain 3(BH3)-only protein Bim is controlled by stringent post-translational regulation, predominantly through alterations in phosphorylation status. To identify new kinases involved in its regulation, we carried out a yeast two-hybrid screen using a non-spliceable variant of the predominant isoform--Bim(EL)--as the bait and identified the regulatory subunit of cyclic-AMP-dependent protein kinase A--PRKAR1A--as an interacting partner. We also show that protein kinase A (PKA) is a Bim(EL) isoform-specific kinase that promotes its stabilization. Inhibition of PKA or mutation of the PKA phosphorylation site within Bim(EL) resulted in its accelerated proteasome-dependent degradation. These results might have implications for human diseases that are characterized by abnormally increased PKA activity, such as the Carney complex and dilated cardiomyopathy.     

BH3-only proteins are tail-anchored in the outer mitochondrial membrane and can initiate the activation of Bax

During mitochondrial apoptosis, pro-apoptotic BH3-only proteins cause the translocation of cytosolic Bcl-2-associated X protein (Bax) to the outer mitochondrial membrane (OMM) where it is activated to release cytochrome c from the mitochondrial intermembrane space, but the mechanism is under dispute. We show that most BH3-only proteins are mitochondrial proteins that are imported into the OMM via a C-terminal tail-anchor domain in isolated yeast mitochondria, independently of binding to anti-apoptotic Bcl-2 proteins. This C-terminal domain acted as a classical mitochondrial targeting signal and was sufficient to direct green fluorescent protein to mitochondria in human cells. When expressed in mouse fibroblasts, these BH3-only proteins localised to mitochondria and were inserted in the OMM. The BH3-only proteins Bcl-2-interacting mediator of cell death (Bim), tBid and p53-upregulated modulator of apoptosis sensitised isolated mitochondria from Bax/Bcl-2 homologous antagonist/killer-deficient fibroblasts to cytochrome c-release by recombinant, extramitochondrial Bax. For Bim, this activity is shown to require the C-terminal-targeting signal and to be independent of binding capacity to and presence of anti-apoptotic Bcl-2 proteins. Bim further enhanced Bax-dependent killing in yeast. A model is proposed where OMM-tail-anchored BH3-only proteins permit passive 'recruitment' and catalysis-like activation of extra-mitochondrial Bax. The recognition of C-terminal membrane-insertion of BH3-only proteins will permit the development of a more detailed concept of the initiation of mitochondrial apoptosis.     

大田软海绵酸对FL细胞DNA的损伤及凋亡相关蛋白表达的影响

本文旨在探讨大田软海绵酸对人羊膜细胞DNA的损伤及凋亡相关蛋白表达的影响。实验用0、20、40、608、0、100 nmol/L OA诱导FL细胞4h后,检测DNA损伤程度的彗星实验表明,OA对FL细胞DNA的损伤随染毒浓度的升高而增加。蛋白免疫印迹法显示凋亡相关蛋白Bcl-2、Bax和p53的表达与染毒浓度呈负相关;用100 nmol/L OA分别诱导2h、4h、8h后发现,三种蛋白的表达与染毒时间也呈负相关。由此可知在OA诱导的FL细胞凋亡中,损伤DNA,降低Bcl-2蛋白的表达可能参与了凋亡的部分作用,而Bax和p53蛋白则可能与OA诱导的细胞增殖有关。     

?Np73 is capable of inducing apoptosis by co-ordinately activating several BH3-only proteins

Inactivation of p53 is one of the most relevant events in human cancer, since it allows transformed cells to escape their own proliferation control and leave them irresponsive to drugs that aim to damage their DNA. When p53 falls, other members of its family may become targets to attack tumoural cells. p73 has shown capacity to mediate these attacks. However, its N-terminal truncated isoforms have been associated with oncogenesis due to their capacity to act as dominant negatives of p53 and the transactivation (TA) isoforms of p73. We previously found a relationship between the overexpression of N-terminus-truncated p73 isoform (∆Np73) and that of the proapoptotic gene Bcl-2-interacting killer (BIK). In the present report we demonstrate that ∆Np73-α has the capacity to induce apoptosis through the co-ordinated activation of a group of genes harbouring GC-rich elements in their regulatory regions. ∆Np73-α synergizes with specificity protein (Sp1) on these elements but the overall response of these genes probably depends on the additional presence of consensus p53 elements. We explore the domains of ∆Np73-α involved in this transactivation capacity and found divergences with the previously described functions for them. Moreover, we found that the transforming mutation V12 of HRas impairs this transactivation capacity of ∆Np73-α, further supporting the anti-tumoural function of this later. Our data add complexity to the action of p73 on the induction of apoptosis and tumourogenesis, opening new interpretations to the expression profile of p73 isoforms in different human neoplasias.     

The BH3-only protein bid is dispensable for DNA damage- and replicative stress-induced apoptosis or cell-cycle arrest

Bid, a caspase-activated proapoptotic BH3-only protein, is essential for Fas-induced hepatocyte destruction. Recent studies published in Cell produced conflicting results, indicating that loss of Bid either protects or enhances apoptosis induced by DNA damage or replicative stress. To resolve this controversy, we generated novel Bid-deficient mice on an inbred C57BL/6 background and removed the drug-selection cassette from the targeted locus. Nine distinct cell types from these Bid-deficient mice underwent cell-cycle arrest and apoptosis in a manner indistinguishable from control WT cells in response to DNA damage or replicative stress. Moreover, we found that even cells from the original Bid-deficient mice responded normally to these stimuli, indicating that differences in genetic background or the presence of a strong promoter within the targeted locus are unlikely to explain the differences between our results and those reported previously. We conclude that Bid has no role in DNA damage- or replicative stress-induced apoptosis or cell-cycle arrest.     

The deubiquitinase Usp27x stabilizes the BH3‐only protein Bim and enhances apoptosis

Bim is a pro‐apoptotic Bcl‐2 family member of the BH3‐only protein subgroup. Expression levels of Bim determine apoptosis susceptibility in non‐malignant and in tumour cells. Bim protein expression is downregulated by proteasomal degradation following ERK‐dependent phosphorylation and ubiquitination. Here, we report the identification of a deubiquitinase, Usp27x, that binds Bim upon its ERK‐dependent phosphorylation and can upregulate its expression levels. Overexpression of Usp27x reduces ERK‐dependent Bim ubiquitination, stabilizes phosphorylated Bim, and induces apoptosis in PMA‐stimulated cells, as well as in tumour cells with a constitutively active Raf/ERK pathway. Loss of endogenous Usp27x enhances the Bim‐degrading activity of oncogenic Raf. Overexpression of Usp27x induces low levels of apoptosis in melanoma and non‐small cell lung cancer (NSCLC) cells and substantially enhances apoptosis induced in these cells by the inhibition of ERK signalling. Finally, deletion of Usp27x reduces apoptosis in NSCLC cells treated with an EGFR inhibitor. Thus, Usp27x can trigger via its proteolytic activity the deubiquitination of Bim and enhance its levels, counteracting the anti‐apoptotic effects of ERK activity, and therefore acts as a tumour suppressor.     

Apoptosome dependent caspase-3 activation pathway is non-redundant and necessary for apoptosis in sympathetic neurons

Although sympathetic neurons are a well-studied model for neuronal apoptosis, the role of the apoptosome in activating caspases in these neurons remains debated. We find that the ability of sympathetic neurons to undergo apoptosis in response to nerve growth factor (NGF) deprivation is completely dependent on having an intact apoptosome pathway. Genetic deletion of Apaf-1, caspase-9, or caspase-3 prevents apoptosis after NGF deprivation, and importantly, allows these neurons to recover and survive long-term following readdition of NGF. The inability of caspase-3 deficient sympathetic neurons to undergo apoptosis is particularly striking, as apoptosis in dermal fibroblasts and cortical neurons proceeds even in the absence of caspase-3. Our results show that in contrast to dermal fibroblasts and cortical neurons, sympathetic neurons express no detectable levels of caspase-7. The strict requirement for an intact apoptosome, coupled with a lack of effector caspase redundancy, provides sympathetic neurons with a markedly increased control over their apoptotic pathway.     

The avian retroviral IN protein is both necessary and sufficient for integrative recombination in vitro

The integration of viral DNA into the host cell chromosome is an essential feature of the retroviral life cycle. The integration reaction requires cis-acting sequences at the ends of linear viral DNA and a trans-acting product of the pol gene, the integration protein (IN). Previously, we demonstrated that avian sarcoma-leukosis virus (ASLV) IN is able to carry out the first step in the integration process in vitro: nicking of the ends of linear viral DNA. In this paper, using two independent assays, we demonstrate that IN, alone, is sufficient to carry out the second step: cleavage and joining to the target DNA. These results demonstrate that the retroviral IN protein is an integrase.     

JNK and NFκB dependence of apoptosis induced by vinblastine in human acute promyelocytic leukaemia cells

The relationship between the mitogen‐activated protein kinase response, nuclear factor‐κB (NFκB) expression and the apoptosis in human acute promyelocytic leukaemia NB4 cells treated with vinblastine was investigated in this work. Cell viability, subdiploid DNA and cell cycle were analysed by propidium iodide permeability and flow cytometry analyses. Apoptosis was determined by annexin V‐Fluorescein isothiocyanate assays. Western‐blot analysis was used for determination of expression levels of apoptotic factors (p53, Bax and Bcl2), intracellular kinases [serine/threonine‐specific protein kinase, extracellular signal‐regulated kinase and c‐Jun N‐terminal kinase (JNK)], NFκB factor and caspases. Electrophoretic mobility shift assay was usefully applied to study DNA‐NFκB interaction. In NB4 cells, vinblastine produces alteration of p53 and DNA fragmentation. Vinblastine treatment had an antiproliferative effect via the induction of apoptosis producing Bax/Bcl‐2 imbalance. Vinblastine treatment suppressed NFκB expression and depressed NFκB‐DNA binding activity while maintaining JNK activation that subsequently resulted in apoptotic response through caspase‐dependent pathway. Our study provides a possible anti‐cancer mechanism of vinblastine action on NB4 cells by deregulation of the intracellular signalling cascade affecting to JNK activation and NFκB expression. Moreover, JNK activation and NFκB depression can be very significant factors in apoptosis induction by vinblastine. Copyright © 2015 John Wiley & Sons, Ltd.     

Nucleolar protein CSIG is required for p33ING1 function in UV-induced apoptosis

Cellular senescence-inhibited gene (CSIG) protein, a nucleolar protein with a ribosomal L1 domain in its N-terminus, can exert non-ribosomal functions to regulate biological processes, such as cellular senescence. Here, we describe a previously unknown function for CSIG: promotion of apoptosis in response to ultraviolet (UV) irradiation-induced CSIG upregulation. We identified p33ING1 as a binding partner that interacts with CSIG. After UV irradiation, p33ING1 increases its protein expression, translocates into the nucleolus and binds CSIG. p33ING1 requires its nucleolar targeting sequence region to interact with CSIG and enhance CSIG protein stability, which is essential for activation of downstream effectors, Bcl-2-associated X protein, to promote apoptosis. Thus, our data imply that p33ING1–CSIG axis functions as a novel pro-apoptotic regulator in response to DNA damage.     

Signal transduction pathways leading to cell cycle arrest and apoptosis induced by DNA topoisomerase poisons

Summary In this overview, I have summarized the important pathways of stress-induced signal transduction: stabilization and activation of p53 playing a central role in stress-induced cell cycle checkpoint and apoptosis, and activation of ASK1-JNK/p38 pathway often induced by a variety of stress stimuli, which appears to be essentially required for apoptosis to follow.