Caspase-mediated cleavage of JNK during stress-induced apoptosis

The c-Jun N-terminal kinases (JNKs) are a subfamily of the mitogen-activated protein kinases (MAPKs). The JNKs are encoded by three separate genes (jnk1, jnk2, and jnk3), which are spliced alternatively to create 10 JNK isoforms that are either p46 or p54 in size. In this study, we found that the p52 form of JNK emerged in human leukemia MOLT-4 or U937 cells following X-irradiation or heat treatment. The accumulation of p52 coincided with the reduction of p54 JNK. On the other hand, the amounts of p46 JNK did not change by X-irradiation. Induction of the p52 form of JNK also paralleled the appearance of the active form of caspase-3 and was suppressed by a caspase-specific inhibitor, Ac-DEVD-CHO, but not by Ac-YVAD-CHO. In vitro cleavage assays indicated that recombinant human JNK1beta2 and JNK2beta2 were cleaved by caspase-3, and that the mutation of aspartic acid at position 413 of JNK1beta2 or 410 of JNK2beta2 to alanine abolished the cleavage. Altogether, our results demonstrated that p54 JNKs, at least JNK1beta2 and JNK2beta2, were new selective targets of caspases in JNK splicing variants, and suggested that the p52 form could serve as a marker of apoptosis.     

Tumor necrosis factor-induced toxic liver injury results from JNK2-dependent activation of caspase-8 and the mitochondrial death pathway

In vitro studies of hepatocytes have implicated over-activation of c-Jun N-terminal kinase (JNK) signaling as a mechanism of tumor necrosis factor-alpha (TNF)-induced apoptosis. However, the functional significance of JNK activation and the role of specific JNK isoforms in TNF-induced hepatic apoptosis in vivo remain unclear. JNK1 and JNK2 function was, therefore, investigated in the TNF-dependent, galactosamine/lipopolysaccharide (GalN/LPS) model of liver injury. The toxin GalN converted LPS-induced JNK signaling from a transient to prolonged activation. Liver injury and mortality from GalN/LPS was equivalent in wild-type and jnk1-/- mice but markedly decreased in jnk2-/- mice. This effect was not secondary to down-regulation of TNF receptor 1 expression or TNF production. In the absence of jnk2, the caspase-dependent, TNF death pathway was blocked, as reflected by the failure of caspase-3 and -7 and poly(ADP-ribose) polymerase cleavage to occur. JNK2 was critical for activation of the mitochondrial death pathway, as in jnk2-/- mice Bid cleavage and mitochondrial translocation and cytochrome c release were markedly decreased. This effect was secondary to the failure of jnk2-/- mice to activate caspase-8. Liver injury and caspase activation were similarly decreased in jnk2 null mice after GalN/TNF treatment. Ablation of jnk2 did not inhibit GalN/LPS-induced c-Jun kinase activity, although activity was completely blocked in jnk1-/- mice. Toxic liver injury is, therefore, associated with JNK over-activation and mediated by JNK2 promotion of caspase-8 activation and the TNF mitochondrial death pathway through a mechanism independent of c-Jun kinase activity.     

Genetic inhibition or activation of JNK1/2 protects the myocardium from ischemia-reperfusion-induced cell death in vivo

The c-Jun NH2-terminal kinase (JNK) branch of the mitogen-activated protein kinase signaling cascade has been implicated in the regulation of apoptosis in a variety of mammalian cell types. In the heart, disagreement persists concerning the role that JNKs may play in regulating apoptosis, since both pro- and antiapoptotic regulatory functions have been reported in cultured cardiomyocytes. Here we report the first analysis of cardiomyocyte cell death due to JNK inhibition or activation in vivo using genetically modified mice. Three separate mouse models with selective JNK inhibition were assessed for ventricular damage and apoptosis levels following ischemia-reperfusion injury. jnk1-/-, jnk2-/-, and transgenic mice expressing dominant negative JNK1/2 within the heart were each shown to have less JNK activity in the heart and less injury and cellular apoptosis in vivo following ischemia-reperfusion injury. To potentially address the reciprocal gain-of-function phenotype associated with sustained JNK activation, transgenic mice were generated that express MKK7 in the heart. These transgenic mice displayed elevated cardiac c-Jun kinase activity but, ironically, were also significantly protected from ischemia-reperfusion. Mechanistically, JNK-inhibited mice showed increased phosphorylation of the proapoptotic factor Bad at position 112, whereas MKK7 transgenic mice showed decreased phosphorylation of this site. Collectively, these results underscore the complexity associated with JNK signaling in regulating apoptosis, such that sustained inhibition or activation both elicit cellular protection in vivo, although probably through different mechanisms.     

c-Jun N-terminal kinase (JNK) signaling: Recent advances and challenges

c-Jun N-terminal kinases (JNKs), first characterized as stress-activated members of the mitogen-activated protein kinase (MAPK) family, have become a focus of inhibitor screening strategies following studies that have shown their critical roles in the development of a number of diseases, such as diabetes, neurodegeneration and liver disease. We discuss recent advances in the discovery and development of ATP-competitive and ATP-noncompetitive JNK inhibitors. Because understanding the modes of actions of these inhibitors and improving their properties will rely on a better understanding of JNK structure, JNK catalytic mechanisms and substrates, recent advances in these areas of JNK biochemistry are also considered. In addition, the use of JNK gene knockout animals is continuing to reveal in vivo functions for these kinases, with tissue-specific roles now being dissected with tissue-specific knockouts. These latest advances highlight the many challenges now faced, particularly in the directed targeting of the JNK isoforms in specific tissues.     

Defective neural tube morphogenesis and altered apoptosis in the absence of both JNK1 and JNK2

Mice lacking both c-Jun-NH(2)-terminal kinases (JNK1 and JNK2) were generated to define their roles in development. Jnk1/jnk2 double mutant fetuses die around embryonic day 11 (E11) and were found to display an open neural tube (exencephaly) at the hindbrain level with reduced apoptosis in the hindbrain neuroepithelium at E9.25. In contrast, a dramatic increase in cell death was observed one day later at E10.5 in both the hindbrain and forebrain regions. Moreover, about 25% of jnk1-/-jnk2+/- fetuses display exencephaly probably due to reduced levels of JNK proteins, whereas jnk1+/-jnk2-/- mice are viable. These results assign both pro- and anti-apoptotic functions for JNK1 and JNK2 in the development of the fetal brain.     

c-Jun N-terminal kinase 2 (JNK2) enhances cell migration through epidermal growth factor substrate 8 (EPS8)

Membrane-bound receptors induce biochemical signals to remodel the actin cytoskeleton and mediate cell motility. In association with receptor tyrosine kinases, several downstream mitogen-induced kinases facilitate cell migration. Here, we show a role for c-Jun N-terminal kinase 2 (JNK2) in promoting mammary cancer cell migration through inhibition of epidermal growth factor substrate 8 (EPS8) expression, a key regulator of EGF receptor (R) signaling and trafficking. Using jnk2(-/-) mice, we found that EPS8 expression is higher in polyoma middle T antigen (PyVMT)jnk2(-/-) mammary tumors and jnk2(-/-) mammary glands compared with the respective jnk2(+/+) controls. The inverse relationship between the jnk2 and eps8 expression was also associated with cancer progression in that patients with basal-type breast tumors expressing high jnk2 and low eps8 experienced poor disease-free survival. In mammary tumor cell lines, the absence of jnk2 greatly reduces cell migration that is rescued by EPS8 knockdown. Subsequent studies show that JNK2 enhances formation of the EPS8-Abi-1-Sos-1 complex to augment EGFR activation of Akt and ERK, whereas the absence of JNK2 promotes ESP8/RN-Tre association to inhibit endocytotic trafficking of the EGFR. Together, these studies unveil a critical role for JNK2 and EPS8 in receptor tyrosine kinase signaling and trafficking to convey distinctly different effects on cell migration.     

Differential gene regulation by specific gain-of-function JNK1 proteins expressed in Swiss 3T3 fibroblasts

The c-Jun N-terminal kinases (JNKs) are encoded by three genes that yield 10 isoforms through alternative mRNA splicing. The roles of each JNK isoform in the many putative biological responses where the JNK pathway is activated are still unclear. To examine the cellular responses mediated by different JNK isoforms, gain-of-function JNK1 polypeptides were generated by fusing the upstream mitogen-activated protein kinase kinase, MKK7, with p46JNK1alpha or p46JNK1beta. The MKK7-JNK fusion proteins, which exhibited constitutive activity in 293T cells, were stably expressed in Swiss 3T3 fibroblasts using retrovirus-mediated gene transfer. Swiss 3T3 cells expressing either of the MKK7-JNK polypeptides were equally sensitized to induction of cell death following serum withdrawal. To search for other cellular responses that may be selectively regulated by the JNK1 isoforms, the gene expression profiles of Swiss 3T3 cells expressing MKK7-JNK1alpha or MKK7-JNK1beta were compared with empty vector-transfected control cells. Affymetrix Genechips identified 46 genes for which expression was increased in MKK7-JNK-expressing cells relative to vector control cells. Twenty genes including those for c-Jun, MKP-7, interluekin-1 receptor family member ST2L/ST2, and c-Jun-binding protein were induced similarly by MKK7-JNK1alpha and MKK7-JNK1beta proteins, whereas 13 genes were selectively increased by MKK7-JNK1alpha and 13 genes were selectively increased by MKK7-JNK1beta. The set of genes selectively induced by MKK7-JNK1beta included a number of known interferon-stimulated genes (ISG12, ISG15, IGTP, and GTPI). Consistent with these gene expression changes, Swiss 3T3 cells expressing MKK7-JNK1beta exhibited increased resistance to vesicular stomatitis virus-induced cell death. These findings reveal evidence for JNK isoform-selective gene regulation and support a role for distinct JNK isoforms in specific cellular responses.     

Coordination of JNK1 and JNK2 is critical for GADD45alpha induction and its mediated cell apoptosis in arsenite responses

Arsenite is a well documented environmental pathogen, whereas it has also been applied as medication to treat various neoplasmas. The pathogenic and therapeutic effects of arsenite are associated with cellular apoptotic responses. However, the molecular mechanisms of arsenite-induced apoptosis are not very well understood. Our previous study has shown that arsenite exposure is able to activate JNKs, which subsequently mediate the apoptotic outcome. The present study further revealed that the coordination of JNK1 and JNK2 was critical for the arsenite-induced expression of GADD45alpha (growth arrest and DNA damage 45alpha), which in turn mediated the cellular apoptosis. The arsenite-induced apoptosis and GADD45alpha expression were significantly impaired in mouse embryonic fibroblasts deficient in either jnk1 (JNK1-/-) or jnk2 (JNK2-/-). Knockdown of GADD45alpha by its specific small interfering RNA also dramatically reduced the apoptotic responses, and overexpression of GADD45alpha in either JNK1-/- or JNK2-/- mouse embryonic fibroblasts partially resensitized the cell death. Furthermore, it was found that the regulation of GADD45alpha by JNK1 and JNK2 was achieved through mediating the activation of c-Jun, since in the JNK1-/- and JNK2-/- cells the c-Jun activation was impaired, and overexpression of the dominant negative mutant of c-Jun (TAM67) in wild type cells could also block GADD45alpha induction as well as cellular apoptosis. Our results demonstrate that the coordination of JNK1 and JNK2 is critical for c-Jun/GADD45alpha-mediated cellular apoptosis induced by arsenite.     

Distinct roles for JNK1 and JNK2 in regulating JNK activity and c-Jun-dependent cell proliferation

Different c-Jun N-terminal kinases (JNKs) are activated by a plethora of signals and phosphorylate substrates such as c-Jun, which is required for efficient cell cycle progression. Although JNK1 and JNK2 were shown to differentially regulate fibroblast proliferation, the underlying mechanistic basis remains unclear. We found that Jnk2-/- fibroblasts exit G1 and enter S phase earlier than wild-type counterparts, while Jnk1-/- cells show the inverse phenotype. Moreover, Jnk2-/- erythroblasts also exhibit a proliferative advantage. JNK2 deficiency results in elevated c-Jun phosphorylation and stability, whereas the absence of JNK1 reduces c-Jun phosphorylation and stability. Re-expression of JNK2 in Jnk2-/- cells reverses the JNK2 null phenotype, whereas ectopic expression of JNK1 augments it. JNK2 is preferentially bound to c-Jun in unstimulated cells, thereby contributing to c-Jun degradation. In contrast, JNK1 becomes the major c-Jun interacting kinase after cell stimulation. These data provide mechanistic insights into the distinct roles of different JNK isoforms.     

JNK2 and JNK3 combined are essential for apoptosis in dopamine neurons of the substantia nigra,but are not required for axon degeneration

Activation of c‐jun N‐terminal kinase (JNK) by the mitogen‐activated protein kinase cascade has been shown to play an important role in the death of dopamine neurons of the substantia nigra, one of the principal neuronal populations affected in Parkinson’s disease. However, it has remained unknown whether the JNK2 and JNK3 isoforms, either singly or in combination, are essential for apoptotic death, and, if so, the mechanisms involved. In addition, it has been unclear whether they play a role in axonal degeneration of these neurons in disease models. To address these issues we have examined the effect of single and double jnk2 and jnk3 null mutations on apoptosis in a highly destructive neurotoxin model, that induced by intrastriatal 6‐hydroxydopamine. We find that homozygous jnk2/3 double null mutations result in a complete abrogation of apoptosis and a prolonged survival of the entire population of dopamine neurons. In spite of this complete protection at the cell soma level, there was no protection of axons. These studies provide a striking demonstration of the distinctiveness of the mechanisms that mediate cell soma and axon degeneration, and they illustrate the need to identify and target pathways of axon degeneration in the development of neuroprotective therapeutics.     

All JNKs can kill, but nuclear localization is critical for neuronal death

JNKs are implicated in a range of brain pathologies and receive considerable attention as potential therapeutic targets. However, JNKs also regulate physiological and homeostatic processes. An attractive hypothesis from the drug development perspective is that distinct JNK isoforms mediate "physiological" and "pathological" responses. However, this lacks experimental evaluation. Here we investigate the isoforms, subcellular pools, and c-Jun/ATF2 targets of JNK in death of central nervous system neurons following withdrawal of trophic support. We use gene knockouts, gene silencing, subcellularly targeted dominant negative constructs, and pharmacological inhibitors. Combined small interfering RNA knockdown of all JNKs 1, 2, and 3, provides substantial neuroprotection. In contrast, knockdown or knock-out of individual JNKs or two JNKs together does not protect. This explains why the evidence for JNK in neuronal death has to date been largely pharmacological. Complete knockdown of c-Jun and ATF2 using small interfering RNA also fails to protect, casting doubt on c-Jun as a critical effector of JNK in neuronal death. Nonetheless, the death requires nuclear but not cytosolic JNK activity as nuclear dominant negative inhibitors of JNK protect, whereas cytosolic inhibitors only block physiological JNK function. Thus any one of the three JNKs is capable of mediating apoptosis and inhibition of nuclear JNK is protective.     

Isoforms of Jun kinase are differentially expressed and activated in human monocyte/macrophage (THP-1) cells

Ten isoforms of c-jun N-terminal kinase (JNK) have been described that arise by differential mRNA splicing of three genes. In that the relative expression and function of these different JNK proteins in human monocytic cells is not known, we have examined the JNK isoforms in THP-1 monocyte/macrophage cells. Differentiation of THP-1 cells by exposure to 10(-8) M PMA for 42-48 h enhances cellular responses to LPS, including enhanced activation of total JNK activity and increased phosphorylation of p54 JNK as well as p46 JNK. Examination of JNK proteins on Western blots reveals a predominance of p46 JNK1 and p54 JNK2 proteins. Clearing of lysates by immunoprecipitation of JNK1(99% effective) removes 46% of the JNK enzymatic activity (p < 0.01), whereas clearing of JNK1 plus JNK2 (70% effective) depletes the sample of 72% of the JNK activity (p < 0.01). Further analysis, undertaken with real-time RT-PCR, revealed that 98% of the JNK messages code for three isoforms: JNK1beta1, JNK2alpha1, and JNK2alpha2. The p54 JNK that is phosphorylated in LPS-stimulated, PMA-differentiated THP-1 cells is most likely JNK2alpha2 because 97% of the p54 JNK-encoding messages code for JNK2alpha2. By analogous reasoning, the p46 JNKs that are not heavily phosphorylated, but account for approximately half of the N-terminal c-jun kinase enzymatic activity, are most likely either JNK1beta1 or JNK2alpha1 because they account for 98% of the messages that can code for 46kDa JNKS:     

Designed Ankyrin Repeat Proteins (DARPins) as Novel Isoform-Specific Intracellular Inhibitors of c-Jun N-Terminal Kinases

The c-Jun N-terminal kinases (JNKs) are involved in many biological processes such as proliferation, differentiation, apoptosis, and inflammation and occur in highly similar isoforms in eukaryotic cells. Isoform-specific functions and diseases have been reported for individual JNK isoforms mainly from gene-knockout studies in mice. There is, however, a high demand for intracellular inhibitors with high selectivity to improve the understanding of isoform-specific mechanisms and for use as therapeutic tools. The commonly used JNK inhibitors are based on small molecules or peptides that often target the conserved ATP binding site or docking sites and thus show only moderate selectivity. To target novel binding epitopes, we used designed ankyrin repeat proteins (DARPins) to generate alternative intracellular JNK inhibitors that discriminate two very similar isoforms, JNK1 and JNK2. DARPins are small binding proteins that are well expressed, stable, and cysteine-free, which makes them ideal candidates for applications in the reducing intracellular environment. We performed ribosome display selections against JNK1α1 and JNK2α1 using highly diverse combinatorial libraries of DARPins. The selected binders specifically recognize either JNK1 or JNK2 or both isoforms in vitro and in mammalian cells. All analyzed DARPins show affinities in the low nanomolar range and isoform-specific inhibition of JNK activation in vitro at physiological ATP concentrations. Importantly, DARPins that selectively inhibit JNK activation in human cells were also identified. These results emphasize the great potential of DARPins as a novel class of highly specific intracellular inhibitors of distinct enzyme isoforms for use in biological studies and as possible therapeutic leads.     

Physiological roles of MKK4 and MKK7: insights from animal models

c-Jun NH2-terminal protein kinase (JNK) is a mitogen-activated protein kinase (MAPK) involved in the regulation of numerous physiological processes during development and in response to stress. Its activity is increased upon phosphorylation by the MAPK kinases, MKK4 and MKK7. Similar to the early embryonic death of mice caused by the targeted deletion of the jnk genes, mice lacking mkk4 or mkk7 die before birth. The inability of MKK4 and MKK7 to compensate for each other's functions in vivo is consistent with their synergistic effect in mediating JNK activation. However, the phenotypic analysis of the mutant mouse embryos indicates that MKK4 and MKK7 have specific roles that may be due to their selective regulation by extracellular stimuli and their distinct tissue distribution. MKK4 and MKK7 also have different biochemical properties. For example, whereas MKK4 can activate p38 MAPK, MKK7 functions as a specific activator of JNK. Here we summarize the studies that have shed light on the mechanism of activation of MKK4 and MKK7 and on their physiological functions.