Hidenori Ichijo

Mammalian thioredoxin is a direct inhibitor of apoptosis signal‐regulating kinase (ASK) 1

Apoptosis signal‐regulating kinase (ASK) 1 was recently identified as a mitogen‐activated protein (MAP) kinase kinase kinase which activates the c‐Jun N‐terminal kinase (JNK) and p38 MAP kinase pathways and is required for tumor necrosis factor (TNF)‐α‐induced apoptosis; however, the mechanism regulating ASK1 activity is unknown. Through genetic screening for ASK1‐binding proteins, thioredoxin (Trx), a reduction/oxidation (redox)‐regulatory protein thought to have anti‐apoptotic effects, was identified as an interacting partner of ASK1. Trx associated with the N‐terminal portion of ASK1 in vitro and in vivo. Expression of Trx inhibited ASK1 kinase activity and the subsequent ASK1‐dependent apoptosis. Treatment of cells with N‐acetyl‐L‐cysteine also inhibited serum withdrawal‐, TNF‐α‐ and hydrogen peroxide‐induced activation of ASK1 as well as apoptosis. The interaction between Trx and ASK1 was found to be highly dependent on the redox status of Trx. Moreover, inhibition of Trx resulted in activation of endogenous ASK1 activity, suggesting that Trx is a physiological inhibitor of ASK1. The evidence that Trx is a negative regulator of ASK1 suggests possible mechanisms for redox regulation of the apoptosis signal transduction pathway as well as the effects of antioxidants against cytokine‐ and stress‐induced apoptosis.

ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis.

Apoptosis signal‐regulating kinase (ASK) 1 is activated in response to various cytotoxic stresses including TNF, Fas and reactive oxygen species (ROS) such as H2O2, and activates c‐Jun NH2‐terminal kinase (JNK) and p38. However, the roles of JNK and p38 signaling pathways during apoptosis have been controversial. Here we show that by deleting ASK1 in mice, TNF‐ and H2O2‐induced sustained activations of JNK and p38 are lost in ASK1−/− embryonic fibroblasts, and that ASK1−/− cells are resistant to TNF‐ and H2O2‐induced apoptosis. TNF‐ but not Fas‐induced apoptosis requires ROS‐dependent activation of ASK1–JNK/p38 pathways. Thus, ASK1 is selectively required for TNF‐ and oxidative stress‐induced sustained activations of JNK/p38 and apoptosis.

BCL-2 IS PHOSPHORYLATED AND INACTIVATED BY AN ASK1/JUN N-TERMINAL PROTEIN KINASE PATHWAY NORMALLY ACTIVATED AT G2/M

ABSTRACT Multiple signal transduction pathways are capable of modifying BCL-2 family members to reset susceptibility to apoptosis. We used two-dimensional peptide mapping and sequencing to identify three residues (Ser70, Ser87, and Thr69) within the unstructured loop of BCL-2 that were phosphorylated in response to microtubule-damaging agents, which also arrest cells at G2/M. Changing these sites to alanine conferred more antiapoptotic activity on BCL-2 following physiologic death signals as well as paclitaxel, indicating that phosphorylation is inactivating. An examination of cycling cells enriched by elutriation for distinct phases of the cell cycle revealed that BCL-2 was phosphorylated at the G2/M phase of the cell cycle. G2/M-phase cells proved more susceptible to death signals, and phosphorylation of BCL-2 appeared to be responsible, as a Ser70Ala substitution restored resistance to apoptosis. We noted that ASK1 and JNK1 were normally activated at G2/M phase, and JNK was capable of phosphorylating BCL-2. Expression of a series of wild-type and dominant-negative kinases indicated an ASK1/Jun N-terminal protein kinase 1 (JNK1) pathway phosphorylated BCL-2 in vivo. Moreover, the combination of dominant negative ASK1, (dnASK1), dnMKK7, and dnJNK1 inhibited paclitaxel-induced BCL-2 phosphorylation. Thus, stress response kinases phosphorylate BCL-2 during cell cycle progression as a normal physiologic process to inactivate BCL-2 at G2/M.

Cloning of a TGFβ type I receptor that forms a heteromeric complex with the TGFβ type II receptor

A cDNA clone encoding a 53 kd serine/threonine kinase receptor with an overall structure similar to that of the type II receptor for transforming growth factor beta (TGF beta) was obtained. 125I-TGF beta 1 bound to porcine endothelial cells transfected with the cDNA and formed a cross-linked complex of 70 kd, characteristic of a TGF beta type I receptor. Immunoprecipitation of the cross-linked complexes by antibodies against the cloned receptor revealed the 70 kd complex as well as a 94 kd TGF beta type II receptor complex. The immunoprecipitated novel serine/threonine kinase receptor had biochemical properties of the TGF beta type I receptor and was observed in different cell types. Transfection of the cloned cDNA into TGF beta type I receptor-deficient cells restored TGF beta-induced plasminogen activator inhibitor 1 production. These results suggest that signal transduction by TGF beta involves the formation of a heteromeric complex of two different serine/threonine kinase receptors.

ASK1 Is Essential for JNK/SAPK Activation by TRAF2

Tumor necrosis factor (TNF)-induced activation of the c-jun N-terminal kinase (JNK, also known as SAPK; stress-activated protein kinase) requires TNF receptor-associated factor 2 (TRAF2). The apoptosis signal-regulating kinase 1 (ASK1) is activated by TNF and stimulates JNK activation. Here we show that ASK1 interacts with members of the TRAF family and is activated by TRAF2, TRAF5, and TRAF6 overexpression. A truncated derivative of TRAF2, which inhibits JNK activation by TNF, blocks TNF-induced ASK1 activation. A catalytically inactive mutant of ASK1 is a dominant-negative inhibitor of TNF- and TRAF2-induced JNK activation. In untransfected mammalian cells, ASK1 rapidly associates with TRAF2 in a TNF-dependent manner. Thus, ASK1 is a mediator of TRAF2-induced JNK activation.

Apoptosis inhibitory activity of cytoplasmic p21Cip1/WAF1 in monocytic differentiation

p21Cip1/WAF1 inhibits cell‐cycle progression by binding to G1 cyclin/CDK complexes and proliferating cell nuclear antigen (PCNA) through its N‐ and C‐terminal domains, respectively. The cell‐cycle inhibitory activity of p21Cip1/WAF1 is correlated with its nuclear localization. Here, we report a novel cytoplasmic localization of p21Cip1/WAF1 in peripheral blood monocytes (PBMs) and in U937 cells undergoing monocytic differentiation by in vitro treatment with vitamin D3 or ectopic expression of p21Cip1/WAF1, and analyze the biological consequences of this cytoplasmic expression. U937 cells which exhibit nuclear p21Cip1/WAF1 demonstrated G1 cell‐cycle arrest and subsequently differentiated into monocytes. The latter event was associated with a cytoplasmic expression of nuclear p21Cip1/WAF1, concomitantly with a resistance to various apoptogenic stimuli. Biochemical analysis showed that cytoplasmic p21Cip1/WAF1 forms a complex with the apoptosis signal‐regulating kinase 1 (ASK1) and inhibits stress‐activated MAP kinase cascade. Expression of a deletion mutant of p21Cip1/WAF1 lacking the nuclear localization signal (ΔNLS‐p21) did not induce cell cycle arrest nor monocytic differentiation, but led to an apoptosis‐resistant phenotype, mediated by binding to and inhibition of the stress‐activated ASK1 activity. Thus, cytoplasmic p21Cip1/WAF1 itself acted as an inhibitor of apoptosis. Our findings highlight the different functional roles of p21Cip1/WAF1, which are determined by its intracellular distribution and are dependent on the stage of differentiation.

From receptors to stress-activated MAP kinases.

The cell signaling pathways that culminate in activation of a family of stress-activated MAP kinases are beginning to be defined. Determination of cell life and cell death is known to largely depend on the balance of intrinsic life and death signals within cells. Recently, two representative mammalian stress-activated kinases, the JNK and p38 MAP kinases, have been implicated in determination of cell fate by modifying the life, death and differentiation signals. However, the molecular mechanisms by which extracellular signals are transmitted from membrane receptors to the most upstream kinases in the JNK and p38 signaling modules are not fully understood. This review will provide an overview of current knowledge of molecular links between inflammatory cyokine receptors and stress-activated MAP kinase cascades.

ROS-dependent activation of the TRAF6-ASK1-p38 pathway is selectively required for TLR4-mediated innate immunity

Apoptosis signal–regulating kinase 1 (ASK1) is an evolutionarily conserved mitogen-activated protein 3-kinase that activates both Jnk and p38 mitogen-activated protein kinases. Here we used ASK1-deficient mice to show that ASK1 was selectively required for lipopolysaccharide-induced activation of p38 but not of Jnk or the transcription factor NF-κB. ASK1 was required for the induction of proinflammatory cytokines dependent on Toll-like receptor 4 (TLR4) but not TLR2 or other TLRs. Consistent with this, ASK1-deficient mice were resistant to lipopolysaccharide-induced septic shock. Lipopolysaccharide induced the production of intracellular reactive oxygen species, which was required for the formation of a complex of the adaptor molecule TRAF6 and ASK1 and subsequent activation of the ASK1-p38 pathway. Our data demonstrate that the reactive oxygen species–dependent TRAF6-ASK1-p38 axis is crucial for TLR4-mediated mammalian innate immunity.

Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes

Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.

Activation of apoptosis signal-regulating kinase 1 (ASK1) by tumor necrosis factor receptor-associated factor 2 requires prior dissociation of the ASK1 inhibitor thioredoxin.

ABSTRACT The stress-activated protein kinases (SAPKs, also called c-Jun NH2-terminal kinases) and the p38s, two mitogen-activated protein kinase (MAPK) subgroups activated by cytokines of the tumor necrosis factor (TNF) family, are pivotal to the de novo gene expression elicited as part of the inflammatory response. Apoptosis signal-regulating kinase 1 (ASK1) is a MAPK kinase kinase (MAP3K) that activates both the SAPKs and p38s in vivo. Here we show that TNF receptor (TNFR) associated factor 2 (TRAF2), an adapter protein that couples TNFRs to the SAPKs and p38s, can activate ASK1 in vivo and can interact in vivo with the amino- and carboxyl-terminal noncatalytic domains of the ASK1 polypeptide. Expression of the amino-terminal noncatalytic domain of ASK1 can inhibit TNF and TRAF2 activation of SAPK. TNF can stimulate the production of reactive oxygen species (ROS), and the redox-sensing enzyme thioredoxin (Trx) is an endogenous inhibitor of ASK1. We also show that expression of TRAF2 fosters the production of ROS in transfected cells. We demonstrate that Trx significantly inhibits TRAF2 activation of SAPK and blocks the ASK1-TRAF2 interaction in a reaction reversed by oxidants. Finally, the mechanism of ASK1 activation involves, in part, homo-oligomerization. We show that expression of ASK1 with TRAF2 enhances in vivo ASK1 homo-oligomerization in a manner dependent, in part, upon the TRAF2 RING effector domain and the generation of ROS. Thus, activation of ASK1 by TNF requires the ROS-mediated dissociation of Trx possibly followed by the binding of TRAF2 and consequent ASK1 homo-oligomerization.