TAK1 participates in c-Jun N-terminal kinase signaling during Drosophila development

Transforming growth factor beta (TGF-beta)-activated kinase 1 (TAK1) is a member of the MAPKKK superfamily and has been characterized as a component of the TGF-beta/bone morphogenetic protein signaling pathway. TAK1 function has been extensively studied in cultured cells, but its in vivo function is not fully understood. In this study, we isolated a Drosophila homolog of TAK1 (dTAK1) which contains an extensively conserved NH(2)-terminal kinase domain and a partially conserved COOH-terminal domain. To learn about possible endogenous roles of TAK1 during animal development, we generated transgenic flies which express dTAK1 or the mouse TAK1 (mTAK1) gene in the fly visual system. Ectopic activation of TAK1 signaling leads to a small eye phenotype, and genetic analysis reveals that this phenotype is a result of ectopically induced apoptosis. Genetic and biochemical analyses also indicate that the c-Jun amino-terminal kinase (JNK) signaling pathway is specifically activated by TAK1 signaling. Expression of a dominant negative form of dTAK during embryonic development resulted in various embryonic cuticle defects including dorsal open phenotypes. Our results strongly suggest that in Drosophila melanogaster, TAK1 functions as a MAPKKK in the JNK signaling pathway and participates in such diverse roles as control of cell shape and regulation of apoptosis.     

c-Jun N-terminal kinase pathways in diabetes

Type 2 diabetes develops from insulin resistance and has become a worldwide epidemic. The c-Jun N-terminal kinases have been considered as signaling molecules linking inflammation and insulin resistance. Genetic disruption of c-Jun N-terminal kinase-1 gene prevents the development of insulin resistance in obese and diabetic mice. Inhibition of c-Jun N-terminal kinases by a small cell-permeable peptide improves insulin sensitivity in mice. Hepatic inhibition of c-Jun N-terminal kinases using a dominant-negative protein or knockdown of c-Jun N-terminal kinase-1 gene by RNA interference reduces blood glucose and insulin levels and enhances hepatic insulin signaling in mice. Recent evidence demonstrates that the hepatic c-Jun N-terminal kinase pathway plays an important role in lipid and lipoprotein homeostasis in mice. This review discusses recent advances in our understanding of the role of c-Jun N-terminal kinase pathway in metabolic control and its potential as a target for the treatment of type 2 diabetes.     

Drosophila melanogaster myotropins have unique functions and signaling pathways

Drosophila melanogaster TDVDHVFLRFamide (DMS), SDNFMRFamide, and pEVRFRQCYFNPISCF (FLT) represent three structurally distinct peptide families. Each peptide decreases heart rate albeit with different magnitudes and time-dependent responses. DMS and FLT are expressed in the crop and decrease crop motility; however, SDNFMRFamide expression and effect on the crop has not been reported. These data suggest the peptides have different physiological roles. The peptides have non-overlapping expression patterns in neural tissue, which suggests different mechanisms regulate their synthesis and release. The structures, expression patterns, and activities of the myotropins suggest they have important but different roles in biology and different signaling pathways.     

Involvement of TRAF4 in oxidative activation of c-Jun N-terminal kinase

We previously found that the angiogenic factors TNFalpha and HIV-1 Tat activate an NAD(P)H oxidase in endothelial cells, which operates upstream of c-Jun N-terminal kinase (JNK), a MAPK involved in the determination of cell fate. To further understand oxidant-related signaling pathways, we screened lung and endothelial cell libraries for interaction partners of p47(phox) and recovered the orphan adapter TNF receptor-associated factor 4 (TRAF4). Domain analysis suggested a tail-to-tail interaction between the C terminus of p47(phox) and the conserved TRAF domain of TRAF4. In addition, TRAF4, like p47(phox), was recovered largely in the cytoskeleton/membrane fraction. Coexpression of p47(phox) and TRAF4 increased oxidant production and JNK activation, whereas each alone had minimal effect. In addition, a fusion between p47(phox) and the TRAF4 C terminus constitutively activated JNK, and this activation was decreased by the antioxidant N-acetyl cysteine. In contrast, overexpression of the p47(phox) binding domain of TRAF4 blocked endothelial cell JNK activation by TNFalpha and HIV-1 Tat, suggesting an uncoupling of p47(phox) from upstream signaling events. A secondary screen of endothelial cell proteins for TRAF4-interacting partners yielded a number of proteins known to control cell fate. We conclude that endothelial cell agonists such as TNFalpha and HIV-1 Tat initiate signals that enter basic signaling cassettes at the level of TRAF4 and an NAD(P)H oxidase. We speculate that endothelial cells may target endogenous oxidant production to specific sites critical to cytokine signaling as a mechanism for increasing signal specificity and decreasing toxicity of these reactive species.     

IL-2 deprivation triggers apoptosis which is mediated by c-Jun N-terminal kinase 1 activation and prevented by Bcl-2

A variety of environmental stresses, as well as inflammatory cytokines, induce activation of c-Jun N-terminal kinases. We describe here that IL-2 deprivation-induced apoptosis in TS1alphabeta cells does not modify c-Jun protein levels and correlates Bcl-2 downregulation and an increase in JNK1, but not JNK2, activity directly related to the induction of apoptosis. Indeed, downregulation of JNK1 expression using antisense oligonucleotides inhibits apoptosis induced by IL-2 withdrawal. Overexpression of Bcl-2 promotes cell survival and blocks JNK1 activation as well as apoptosis caused by IL-2 deprivation. This suggests that inhibition of the JNK1 signaling pathway may be a mechanism through which Bcl-2 promotes cell survival and prevents apoptosis triggered by growth factor withdrawal.     

Regulation of myostatin signaling by c-Jun N-terminal kinase in C2C12 cells

Myostatin, a member of the transforming growth factor beta (TGF-beta) superfamily, is a negative regulator of skeletal muscle growth. We found that myostatin could activate c-Jun N-terminal kinase (JNK) signaling pathway in both proliferating and differentiating C2C12 cells. Using small interfering RNA (siRNA) mediated activin receptor type IIB (ActRIIB) knockdown, the myostatin-induced JNK activation was significantly reduced, indicating that ActRIIB was required for JNK activation by myostatin. Transfection of C2C12 cells with TAK1-specific siRNA reduced myostatin-induced JNK activation. In addition, JNK could not be activated by myostatin when the expression of MKK4 was suppressed with MKK4-specific siRNA, suggesting that TAK1-MKK4 cascade was involved in myostatin-induced JNK activation. We also found that blocking JNK signaling pathway by pretreatment with JNK specific inhibitor SP600125, attenuated myostatin-induced upregulation of p21 and downregulation of the differentiation marker gene expression. Furthermore, it was also observed that the presence of SP600125 almost annulled the growth inhibitory role of myostatin. Our findings provide the first evidence to reveal the involvement of JNK signaling pathway in myostatin's function as a negative regulator of muscle growth.     

The Drosophila cell shape regulator c-Jun N-terminal kinase also functions as a stress-activated protein kinase.

Mammalian c-Jun N-terminal kinases (JNKs) are members of a group of stress-activated intracellular signalling molecules within the MAP kinase family. Molecular genetic analysis of a highly evolutionarily conserved Drosophila JNK homologue, DJNK, has demonstrated that this molecule plays an essential developmental role in cell shape regulation. However, it remains to be determined whether DJNK also responds to the broad range of cellular stresses and other stimuli that affect its mammalian counterpart. Here we demonstrate that c-Jun, a substrate for mammalian JNKs, is a specific substrate for DJNK and that an antiserum that cross-reacts with activated mammalian JNK at the conserved threonyl-prolyl-tyrosyl (TPY) motif within the activation loop also specifically recognises the activated form of DJNK. Using these two assays, we show that DJNK activity is stimulated in cultured cells by several treatments that activate mammalian JNKs, including addition of arsenite, vanadate and ceramide derivatives. It is therefore concluded that in addition to its essential developmental functions, DJNK plays an important role in stress responses that mirrors its mammalian counterpart.     

Tumor necrosis factor alpha-induced activation of c-jun N-terminal kinase is mediated by TRAF2.

Tumor necrosis factor alpha (TNF alpha) a pro-inflammatory cytokine is an endogenous mediator of septic shock, inflammation, anti-viral responses and apoptotic cell death. TNF alpha elicits its complex biological responses through the individual or cooperative action of two TNF receptors of mol. wt 55 kDa (TNF-RI) and mol. wt 75 kDa (TNF-RII). To determine signaling events specific for TNF-RII we fused the extracellular domain of the mouse CD4 antigen to the intracellular domain of TNF-RII. Crosslinking of the chimeric receptor using anti-CD4 antibodies initiates exclusively TNF-RII-mediated signals. Our findings show that: (i) TNF-RII is able to activate two members of the MAP kinase family: extracellular regulated kinase (ERK) and c-jun N-terminal kinase (JNK); (ii) TRAF2, a molecule that binds TNF-RII and associates indirectly with TNF-RI, is sufficient to activate JNK upon overexpression; (iii) dominant-negative TRAF2 blocks TNF alpha-mediated JNK activation and (iv) TRAF2 signals the activation of JNK and NF-kappaB through different pathways. Our findings suggest that TNF alpha-mediated JNK activation in fibroblasts is independent of the cell death pathway and that TRAF2 occupies a key role in TNF receptor signaling to JNK.     

AKT/mTOR and c-Jun N-terminal kinase signaling pathways are required for chrysotile asbestos-induced autophagy

Chrysotile asbestos is closely associated with excess mortality from pulmonary diseases such as lung cancer, mesothelioma, and asbestosis. Although multiple mechanisms in which chrysotile asbestos fibers induce pulmonary disease have been identified, the role of autophagy in human lung epithelial cells has not been examined. In this study, we evaluated whether chrysotile asbestos induces autophagy in A549 human lung epithelial cells and then analyzed the possible underlying molecular mechanism. Chrysotile asbestos induced autophagy in A549 cells based on a series of biochemical and microscopic autophagy markers. We observed that asbestos increased expression of A549 cell microtubule-associated protein 1 light chain 3 (LC3-II), an autophagy marker, in conjunction with dephosphorylation of phospho-AKT, phospho-mTOR, and phospho-p70S6K. Notably, AKT1/AKT2 double-knockout murine embryonic fibroblasts (MEFs) had negligible asbestos-induced LC3-II expression, supporting a crucial role for AKT signaling. Chrysotile asbestos also led to the phosphorylation/activation of Jun N-terminal kinase (JNK) and p38 MAPK. Pharmacologic inhibition of JNK, but not p38 MAPK, dramatically inhibited the protein expression of LC3-II. Moreover, JNK2^−/− MEFs but not JNK1^−/− MEFs blocked LC3-II levels induced by chrysotile asbestos. In addition, N-acetylcysteine, an antioxidant, attenuated chrysotile asbestos-induced dephosphorylation of P-AKT and completely abolished phosphorylation/activation of JNK. Finally, we demonstrated that chrysotile asbestos-induced apoptosis was not affected by the presence of the autophagy inhibitor 3-methyladenine or autophagy-related gene 5 siRNA, indicating that the chrysotile asbestos-induced autophagy may be adaptive rather than prosurvival. Our findings demonstrate that AKT/mTOR and JNK2 signaling pathways are required for chrysotile asbestos-induced autophagy. These data provide a mechanistic basis for possible future clinical applications targeting these signaling pathways in the management of asbestos-induced lung disease.     

A scaffold protein in the c-Jun NH2-terminal kinase signaling pathways suppresses the extracellular signal-regulated kinase signaling pathways

We previously reported that c-Jun NH(2)-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1) functions as a putative scaffold factor in the JNK mitogen-activated protein kinase (MAPK) cascades. In that study we also found MEK1 and Raf-1, which are involved in the extracellular signal-regulated kinase (ERK) MAPK cascades, bind to JSAP1. Here we have defined the regions of JSAP1 responsible for the interactions with MEK1 and Raf-1. Both of the binding regions were mapped to the COOH-terminal region (residues 1054-1305) of JSAP1. We next examined the effect of overexpressing JSAP1 on the activation of ERK by phorbol 12-myristate 13-acetate in transfected COS-7 cells and found that JSAP1 inhibits ERK's activation and that the COOH-terminal region of JSAP1 was required for the inhibition. Finally, we investigated the molecular mechanism of JSAP1's inhibitory function and showed that JSAP1 prevents MEK1 phosphorylation and activation by Raf-1, resulting in the suppression of the activation of ERK. Taken together, these results suggest that JSAP1 is involved both in the JNK cascades, as a scaffolding factor, and the ERK cascades, as a suppressor.     

Elucidation of the c-Jun N-terminal kinase pathway mediated by Estein-Barr virus-encoded latent membrane protein 1

Epstein-Barr virus (EBV) is associated with several human diseases including infectious mononucleosis and nasopharyngeal carcinoma. EBV-encoded latent membrane protein 1 (LMP1) is oncogenic and indispensable for cellular transformation caused by EBV. Expression of LMP1 in host cells constitutively activates both the c-Jun N-terminal kinase (JNK) and NF-kappaB pathways, which contributes to the oncogenic effect of LMP1. However, the underlying signaling mechanisms are not very well understood. Based mainly on overexpression studies with various dominant-negative constructs, LMP1 was generally thought to functionally mimic members of the tumor necrosis factor (TNF) receptor superfamily in signaling. In contrast to the prevailing paradigm, using embryonic fibroblasts from different knockout mice and the small interfering RNA technique, we find that the LMP1-mediated JNK pathway is distinct from those mediated by either TNF-alpha or interleukin-1. Moreover, we have further elucidated the LMP1-mediated JNK pathway by demonstrating that LMP1 selectively utilizes TNF receptor-associated factor 6, TAK1/TAB1, and c-Jun N-terminal kinase kinases 1 and 2 to activate JNK.     

Receptor activator of NF-kappaB recruits multiple TRAF family adaptors and activates c-Jun N-terminal kinase

Receptor activator of NF-kappaB (RANK) is a recently cloned member of the tumor necrosis factor receptor (TNFR) superfamily, and its function has been implicated in osteoclast differentiation and dendritic cell survival. Many of the TNFR family receptors recruit various members of the TNF receptor-associated factor (TRAF) family for transduction of their signals to NF-kappaB and c-Jun N-terminal kinase. In this study, the involvement of TRAF family members and the activation of the JNK pathway in signal transduction by RANK were investigated. TRAF1, 2, 3, 5, and 6 were found to bind RANK in vitro. Association of RANK with each of these TRAF proteins was also detected in vivo. Expression of RANK in cultured cells also induced the activation of JNK, which was blocked by a dominant-negative form of JNK. Furthermore, by employing various C-terminal deletion mutants of RANK, the regions responsible for TRAF interaction and JNK activation were identified. TRAF5 was determined to bind to the C-terminal 11 amino acids and the other TRAF members to a region N-terminal to the TRAF5 binding site. The domain responsible for JNK activation was localized to the same region where TRAF1, 2, 3, and 6 bound, which suggests that these TRAF molecules might mediate the RANK-induced JNK activation.     

Regulation of apoptotic c-Jun N-terminal kinase signaling by a stabilization-based feed-forward loop

A sequential kinase cascade culminating in activation of c-Jun N-terminal kinases (JNKs) plays a fundamental role in promoting apoptotic death in many cellular contexts. The mechanisms by which this pathway is engaged in response to apoptotic stimuli and suppressed in viable cells are largely unknown. Here, we show that apoptotic stimuli increase endogenous cellular levels of pathway components, including POSH, mixed lineage kinases (MLKs), and JNK interacting protein 1, and that this effect occurs through protein stabilization and requires the presence of POSH as well as activation of MLKs and JNKs. Our findings suggest a self-amplifying, feed-forward loop mechanism by which apoptotic stimuli promote the stabilization of JNK pathway components, thereby contributing to cell death.     

TRAF6 is a novel regulator of Notch signaling in Drosophila melanogaster

Notch signaling pathway unravels a fundamental cellular communication system that plays an elemental role in development. It is evident from different studies that the outcome of Notch signaling depends on signal strength, timing, cell type, and cellular context. Since Notch signaling affects a spectrum of cellular activity at various developmental stages by reorganizing itself in more than one way to produce different intensities in the signaling output, it is important to understand the context dependent complexity of Notch signaling and different routes of its regulation. We identified, TRAF6 (Drosophila homolog of mammalian TRAF6) as an interacting partner of Notch intracellular domain (Notch-ICD). TRAF6 genetically interacts with Notch pathway components in trans-heterozygous combinations. Immunocytochemical analysis shows that TRAF6 co-localizes with Notch in Drosophila third instar larval tissues. Our genetic interaction data suggests that the loss-of-function of TRAF6 leads to the rescue of previously identified Kurtz–Deltex mediated wing notching phenotype and enhances Notch protein survival. Co-expression of TRAF6 and Deltex results in depletion of Notch in the larval wing discs and down-regulates Notch targets, Wingless and Cut. Taken together, our results suggest that TRAF6 may function as a negative regulator of Notch signaling.     

Protein kinase PKN1 associates with TRAF2 and is involved in TRAF2-NF-kappaB signaling pathway

PKN1 is a fatty acid and Rho-activated serine/threonine protein kinase whose catalytic domain is highly homologous to protein kinase C (PKC) family. In yeast two-hybrid screening for PKN1 binding proteins, we identified tumor necrosis factor alpha (TNFalpha) receptor-associated factor 2 (TRAF2). TRAF2 is one of the major mediators of TNF receptor superfamily transducing TNF signal to various functional targets, including activation of NF-kappaB, JNK, and apoptosis. FLAG-tagged PKN1 was co-immunoprecipitated with endogenous TRAF2 from HEK293 cell lysate, and in vitro binding assay using the deletion mutants of TRAF2 showed that PKN1 directly binds to the TRAF domain of TRAF2. PKN1 has the TRAF2-binding consensus sequences PXQX (S/T) at amino acid residues 580-584 (PIQES), and P580AQ582A mutant was not co-immunoprecipitated with TRAF2. Furthermore, the reduced expression of PKN1 by RNA interference (RNAi) down-regulated TRAF2-induced NF-kappaB activation in HEK293T cells. These results suggest that PKN1 is involved in TRAF2-NF-kappaB signaling pathway.     

Noncanonical function of MEKK2 and MEK5 PB1 domains for coordinated extracellular signal-regulated kinase 5 and c-Jun N-terminal kinase signaling

MEKK2 and MEK5 encode Phox/Bem1p (PB1) domains that heterodimerize with one another. MEKK2, MEK5, and extracellular signal-related kinase 5 (ERK5) form a ternary complex through interactions involving the MEKK2 and MEK5 PB1 domains and a 34-amino-acid C-terminal extension of the MEK5 PB1 domain. This C-terminal extension encodes an ERK5 docking site required for MEK5 activation of ERK5. The PB1 domains bind in a front-to-back arrangement, with a cluster of basic amino acids in the front of the MEKK2 PB1 domain binding to the back-end acidic clusters of the MEK5 PB1 domain. The C-terminal moiety, including the acidic cluster of the MEKK2 PB1 domain, is not required for MEK5 binding and binds MKK7. Quiescent MEKK2 preferentially binds MEK5, and MEKK2 activation results in ERK5 activation. Activated MEKK2 binds and activates MKK7, leading to JNK activation. The findings define how the MEKK2 and MEK5 PB1 domains are uniquely used for differential binding of two mitogen-activated protein kinase kinases, MEK5 and MKK7, for the coordinated control of ERK5 and c-Jun N-terminal kinase activation.