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Background

The family of c-Jun NH2-terminal kinases (JNK) plays important roles in embryonic development and in cellular responses to stress. Toxic metals and their compounds are potent activators of JNK in mammalian cells. The mechanism of mammalian JNK activation by cadmium and sodium arsenite involves toxicant-induced oxidative stress. The study of mammalian signaling pathways to JNK is complicated by the significant degree of redundancy among upstream JNK regulators, especially at the level of JNK kinase kinases (JNKKK).

Results

Using Drosophila melanogaster S2 cells, we demonstrate here that cadmium and arsenite activate Drosophila JNK (D-JNK) via oxidative stress as well, thus providing a simpler model system to study JNK signaling. To elucidate the signaling pathways that lead to activation of D-JNK in response to cadmium or arsenite, we employed RNA interference (RNAi) to knock down thirteen upstream regulators of D-JNK, either singly or in combinations of up to seven at a time.

Conclusion

D-MEKK1, the fly orthologue of mammalian MEKK4/MTK1, and Hemipterous/D-MKK7 mediates the activation of D-JNK by cadmium and arsenite.

The c-Jun N-terminal kinase (JNK) signaling pathway is known to mediate both survival and apoptosis of tumor cells. Although JNK1 and JNK2 have been shown to differentially regulate the development of skin cancer, the underlying mechanistic basis remains unclear. Here, we demonstrate that JNK1, but not JNK2, interacts with and phosphorylates Myt1 ex vivo and in vitro. UVA induces substantial apoptosis in JNK wild-type (JNK+/+) or JNK2-deficient (JNK2−/−) mouse embryonic fibroblasts but has no effect on JNK1-deficient (JNK1−/−) cells. In addition, UVA-induced caspase-3 cleavage and DNA fragmentation were suppressed by the knockdown of human Myt1 in skin cancer cells. JNK1 deficiency results in suppressed Myt1 phosphorylation and caspase-3 cleavage in skin exposed to UVA irradiation. In contrast, the absence of JNK2 induces Myt1 phosphorylation and caspase-3 cleavage in skin exposed to UVA. The overexpression of JNK1 with Myt1 promotes cellular apoptosis during the early embryonic development of Xenopus laevis, whereas the presence of JNK2 reduces the phenotype of Myt1-induced apoptotic cell death. Most importantly, JNK1−/− mice developed more UVA-induced papillomas than either JNK+/+ or JNK2−/− mice, which was associated with suppressed Myt1 phosphorylation and decreased caspase-3 cleavage. Taken together, these data provide mechanistic insights into the distinct roles of the different JNK isoforms, specifically suggesting that the JNK1-mediated phosphorylation of Myt1 plays an important role in UVA-induced apoptosis and the prevention of skin carcinogenesis.

The c-Jun NH2-terminal protein kinase (JNK) is a member of the mitogen-activated protein kinase (MAPK) group and is an essential component of a signaling cascade that is activated by exposure of cells to environmental stress. JNK activation is regulated by phosphorylation on both Thr and Tyr residues by a dual-specificity MAPK kinase (MAPKK). Two MAPKKs, MKK4 and MKK7, have been identified as JNK activators. Genetic studies demonstrate that MKK4 and MKK7 serve nonredundant functions as activators of JNK in vivo. We report here the molecular cloning of the gene that encodes MKK7 and demonstrate that six isoforms are created by alternative splicing to generate a group of protein kinases with three different NH2 termini (α, β, and γ isoforms) and two different COOH termini (1 and 2 isoforms). The MKK7α isoforms lack an NH2-terminal extension that is present in the other MKK7 isoforms. This NH2-terminal extension binds directly to the MKK7 substrate JNK. Comparison of the activities of the MKK7 isoforms demonstrates that the MKK7α isoforms exhibit lower activity, but a higher level of inducible fold activation, than the corresponding MKK7β and MKK7γ isoforms. Immunofluorescence analysis demonstrates that these MKK7 isoforms are detected in both cytoplasmic and nuclear compartments of cultured cells. The presence of MKK7 in the nucleus was not, however, required for JNK activation in vivo. These data establish that the MKK4 and MKK7 genes encode a group of protein kinases with different biochemical properties that mediate activation of JNK in response to extracellular stimuli.

Mechanistic studies underlying dopaminergic neuron death may identify new drug targets for the treatment of Parkinson disease (PD). Epidemiological studies have linked pesticide exposure to increased risk for sporadic PD. Here, we investigated the role of c-Jun N-terminal kinase 3 (JNK3), a neural-specific JNK isoform, in dopaminergic neuron death induced by the pesticides rotenone and paraquat. The role of JNK3 was evaluated using RNA silencing and gene deletion to block JNK3 signaling. Using an antibody that recognizes all isoforms of activated JNKs, we found that paraquat and rotenone stimulate JNK phosphorylation in primary cultured dopaminergic neurons. In cultured neurons transfected with Jnk3-specific siRNA and in neurons from Jnk3−/− mice, JNK phosphorylation was nearly abolished, suggesting that JNK3 is the main JNK isoform activated in dopaminergic neurons by these pesticides. Paraquat- and rotenone-induced death of dopaminergic neurons was also significantly reduced by Jnk3 siRNA or Jnk3 gene deletion and deletion of the Jnk3 gene completely attenuated paraquat-induced dopaminergic neuron death and motor-deficits in vivo. Our data identify JNK3 as a common and critical mediator of dopaminergic neuron death induced by paraquat and rotenone, suggesting that it is a potential drug target for PD treatment.

The classical concept of linear pathways is being increasingly challenged by network representations, which emphasize the importance of interactions between components of a biological system, and motivates for adopting a system‐level approach in biology. We have developed a dynamical system that integrates quantitative, dynamic and topological representation of network of ERK5 (Extracellular signal‐regulated kinases 5), JNK(c‐Jun N‐terminal kinases) and P38 kinase cascades. We have observered that, the transient activation of ERK5, JNK1 and P38β kinase, and the persistent activation of JNK2, JNK3 and P38 δ kinase does not get affected due to the cross‐talks between ERK5, JNK and P38 kinase cascades. But it is due to the cross ‐ talks, the transiently activated P38α kinase become inactivated, and the transiently activated P38γ kinase become persistently activated. The impacts of one‐way cross‐talks between the cascades are insignificant and differ from the impact of two‐way cross‐talks. We generate a hypothesis that, signaling pathways should be studied as a system by considering the cross‐talks between the two adjacent cascades.

Background

The JNK pathway is a mitogen-activated protein (MAP) kinase pathway involved in the regulation of numerous physiological processes during development and in response to environmental stress. JNK activity is controlled by two MAPK kinases (MAPKK), Mkk4 and Mkk7. Mkk7 plays a prominent role upon Tumor Necrosis Factor (TNF) stimulation. Eiger, the unique TNF-superfamily ligand in Drosophila, potently activates JNK signaling through the activation of the MAPKKK Tak1.

Methodology/Principal Findings

In a dominant suppressor screen for new components of the Eiger/JNK-pathway in Drosophila, we have identified an allelic series of the Mkk4 gene. Our genetic and biochemical results demonstrate that Mkk4 is dispensable for normal development and host resistance to systemic bacterial infection but plays a non-redundant role as a MAPKK acting in parallel to Hemipterous/Mkk7 in dTAK1-mediated JNK activation upon Eiger and Imd pathway activation.

Conclusions/Significance

In contrast to mammals, it seems that in Drosophila both MAPKKs, Hep/Mkk7 and Mkk4, are required to induce JNK upon TNF or pro-inflammatory stimulation.

DLK is part of a specialized JNK signaling complex in axons that promotes apoptosis via c-Jun but axon degeneration via distinct JNK substrates.

The c-Jun N-terminal kinase (JNK) signaling pathway is essential for neuronal degeneration in multiple contexts but also regulates neuronal homeostasis. It remains unclear how neurons are able to dissociate proapoptotic JNK signaling from physiological JNK activity. In this paper, we show that the mixed lineage kinase dual leucine zipper kinase (DLK) selectively regulates the JNK-based stress response pathway to mediate axon degeneration and neuronal apoptosis without influencing other aspects of JNK signaling. This specificity is dependent on interaction of DLK with the scaffolding protein JIP3 to form a specialized JNK signaling complex. Local activation of DLK-based signaling in the axon results in phosphorylation of c-Jun and apoptosis after redistribution of JNK to the cell body. In contrast, regulation of axon degeneration by DLK is c-Jun independent and mediated by distinct JNK substrates. DLK-null mice displayed reduced apoptosis in multiple neuronal populations during development, demonstrating that prodegenerative DLK signaling is required in vivo.

To further understand how the mitogen-activated protein kinase (MAPK) signaling pathways regulate AP-1 activity, we have elucidated the physiological role of these cascades in the regulation of c-jun gene expression. c-Jun is a crucial component of AP-1 complexes and has been shown in vitro to be a point of integration of numerous signals that can differentially affect its expression as well as its transcriptional activity. Our strategy was based on the use of (i) genetically modified fibroblasts deficient in components of the MAPK cascades and (ii) pharmacological reagents. The results demonstrate that c-Jun NH2-terminal protein kinase (JNK) is essential for a basal level of c-Jun expression and for c-Jun phosphorylation in response to stress. In addition to JNK, p38 MAPK or ERK1/2 and ERK5 are required for mediating UV radiation- or epidermal growth factor (EGF)-induced c-Jun expression, respectively. Further studies indicate that p38 MAPK inhibits the activation of JNK in response to EGF, causing a down-regulation of c-Jun. Overall, these data provide important insights into the mechanisms that ultimately determine the function of c-Jun as a regulator of cell fate.

The c-Jun NH2-terminal kinase (JNK) is activated by the cytokine tumor necrosis factor (TNF). This pathway is implicated in the regulation of AP-1-dependent gene expression by TNF. To examine the role of the JNK signaling pathway, we compared the effects of TNF on wild-type and Jnk1−/− Jnk2−/− murine embryo fibroblasts. We show that JNK is required for the normal regulation of AP-1 by TNF. The JNK-deficient cells exhibited decreased expression of c-Jun, JunD, c-Fos, Fra1, and Fra2; decreased phosphorylation of c-Jun and JunD; and decreased AP-1 DNA binding activity. The JNK-deficient cells also exhibited defects in the regulation of the AP-1-related transcription factor ATF2. These changes were associated with marked defects in TNF-regulated gene expression. The JNK signal transduction pathway is therefore essential for AP-1 transcription factor regulation in cells exposed to TNF.

The c-Jun NH2-terminal kinases (JNKs) are a group of mitogen-activated protein (MAP) kinases that participate in signal transduction events mediating specific cellular functions. Activation of JNK is regulated by phosphorylation in response to cellular stress and inflammatory cytokines. Here, we demonstrate that JNK is regulated by a second, novel mechanism. Induction of Jnk gene expression is required in specific tissues before activation of this signaling pathway. The in vivo and in vitro ligation of the T cell receptor (TCR) leads to induction of JNK gene and protein expression. TCR signals are sufficient to induce JNK expression, whereas JNK phosphorylation also requires CD28-mediated costimulatory signals. Therefore, both expression and activation contribute to the regulation of the JNK pathway to ensure proper control during the course of an immune response.

Nickel (Ni) is a known carcinogen, although the mechanism of its carcinogenicity is not clear. Here, we provide evidence that Ni can induce phosphorylation of histone H3 at its serine 10 residue in a c-jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK)-dependent manner. Ni induces the phosphorylation of JNK, with no effect on the phosphorylation states of the extracellular signal-regulated kinase (ERK) or p38 mitogen-activated protein kinases. An inhibitor of JNK eliminated the Ni-initiated JNK-mediated induction of histone H3 phosphorylation at serine 10, whereas inhibitors specific for ERK or p38 kinases had no effect on the phosphorylation levels of histone H3 at serine 10 (P-H3S10) in Ni-treated cells. A complete loss of Ni ion-induced phosphorylation of H3S10 was observed when JNK was specifically knocked down with RNAi. These results are the first to show the specific JNK-mediated phosphorylation of histone H3 at its serine 10 residue. We show that addition of Ni to an in vitro P-H3S10 dephosphorylation reaction does not change the loss of phosphorylation in the reaction, supporting the notion that Ni causes H3S10 phosphorylation via the JNK/SAPK pathway. It is likely that modification of H3S10 is one of a growing number of epigenetic changes believed to be involved in the carcinogenesis caused by Ni.

Nickel (Ni) is a known carcinogen, although the mechanism of its carcinogenicity is not clear. Here, we provide evidence that Ni can induce phosphorylation of histone H3 at its serine 10 residue in a c-jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK)-dependent manner. Ni induces the phosphorylation of JNK, with no effect on the phosphorylation states of the extracellular signal-regulated kinase (ERK) or p38 mitogen-activated protein kinases. An inhibitor of JNK eliminated the Ni-initiated JNK-mediated induction of histone H3 phosphorylation at serine 10, whereas inhibitors specific for ERK or p38 kinases had no effect on the phosphorylation levels of histone H3 at serine 10 (P-H3S10) in Ni-treated cells. A complete loss of Ni ion-induced phosphorylation of H3S10 was observed when JNK was specifically knocked down with RNAi. These results are the first to show the specific JNK-mediated phosphorylation of histone H3 at its serine 10 residue. We show that addition of Ni to an in vitro P-H3S10 dephosphorylation reaction does not change the loss of phosphorylation in the reaction, supporting the notion that Ni causes H3S10 phosphorylation via the JNK/SAPK pathway. It is likely that modification of H3S10 is one of a growing number of epigenetic changes believed to be involved in the carcinogenesis caused by Ni.

The c-Jun N-terminal kinase (JNK) is a stress-activated member of MAP kinase family. JNK activation has been strongly implicated in inflammatory responses, neurodegeneration, and apoptosis. Recent evidence shows that JNK pathway is also transiently activated in primary sensory neurons after tissue or nerve injury, which is required for the development of hyperalgesia and allodynia. In particular, JNK is persistently activated in astrocytes of the spinal cord after nerve injury, and this activation can maintain central sensitization and mechanical allodynia. In this mini-review, we will provide evidence for the involvement of JNK pathway in regulating persistent pain sensitization. We will also discuss possible upstream signaling mechanisms that cause JNK activation and downstream signaling mechanisms by which JNK modulates pain sensitivity. Thus, targeting JNK pathway might be a useful strategy to treat both neurodegeneration and chronic pain.

The oncoprotein c-Jun is a component of the activator protein-1 (AP-1) transcription factor complex, which is involved in cellular proliferation, transformation and death. The stabilization of c-Jun is critically important for its function. Phosphorylation of c-Jun by c-Jun N-terminal kinase 1 (JNK1) and extracellular signal-regulated protein kinases (ERKs) reduces c-Jun ubiquitination resulting in increased stabilization of c-Jun. In this report, we showed that C-terminal Src kinase (CSK) binds with and phosphorylates c-Jun at Y26 and Y170. Phosphorylation of c-Jun by CSK, in opposition to JNK1 and ERKs, promoted c-Jun degradation and reduced stability. By promoting c-Jun degradation, CSK helps to maintain a low steady-state level of c-Jun, thereby inhibiting AP-1 activity and cell transformation caused by c-Jun. These results indicated that this function of CSK controls cell proliferation under normal growth conditions and may have implications for CSK loss of function in carcinogenesis.

In the nervous system, cell death by apoptosis plays a critical role during normal development and pathological neurodegeneration. Jun N-terminal kinases (JNKs) are essential regulators of neuronal apoptosis. The AP-1 transcription factor c-Jun is phosphorylated at multiple sites within its transactivation domain by the JNKs, and c-Jun phosphorylation is required for JNK-induced neurotoxicity. While the importance of c-Jun as a mediator of apoptotic JNK signaling in neurons is firmly established, the molecular mechanism underlying the requirement for c-Jun N-terminal phosphorylation is enigmatic. Here we identify the multifunctional protein Bag1-L as a coactivator of phosphorylated c-Jun. Bag1-L preferentially interacts with N-terminally phosphorylated c-Jun, and Bag1-L greatly augments transcriptional activation by phosphorylated c-Jun. Chromatin immunoprecipitation experiments revealed binding of Bag1-L to the promoters of proapoptotic AP-1 target genes, and overexpression of Bag1-L augmented cell death in primary neurons. Therefore, Bag1-L functions as a coactivator regulating neurotoxicity mediated by phosphorylated c-Jun.

The major components of the mitogen-activated protein kinase (MAPK) cascades are MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). Recent rapid progress in identifying members of MAPK cascades suggests that a number of such signaling pathways exist in cells. To date, however, how the specificity and efficiency of the MAPK cascades is maintained is poorly understood. Here, we have identified a novel mouse protein, termed Jun N-terminal protein kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), by a yeast two-hybrid screen, using JNK3 MAPK as the bait. Of the mammalian MAPKs tested (JNK1, JNK2, JNK3, ERK2, and p38α), JSAP1 preferentially coprecipitated with the JNKs in cotransfected COS-7 cells. JNK3 showed a higher binding affinity for JSAP1, compared with JNK1 and JNK2. In similar cotransfection studies, JSAP1 also interacted with SEK1 MAPKK and MEKK1 MAPKKK, which are involved in the JNK cascades. The regions of JSAP1 that bound JNK, SEK1, and MEKK1 were distinct from one another. JNK and MEKK1 also bound JSAP1 in vitro, suggesting that these interactions are direct. In contrast, only the activated form of SEK1 associated with JSAP1 in cotransfected COS-7 cells. The unstimulated SEK1 bound to MEKK1; thus, SEK1 might indirectly associate with JSAP1 through MEKK1. Although JSAP1 coprecipitated with MEK1 MAPKK and Raf-1 MAPKKK, and not MKK6 or MKK7 MAPKK, in cotransfected COS-7 cells, MEK1 and Raf-1 do not interfere with the binding of SEK1 and MEKK1 to JSAP1, respectively. Overexpression of full-length JSAP1 in COS-7 cells led to a considerable enhancement of JNK3 activation, and modest enhancement of JNK1 and JNK2 activation, by the MEKK1-SEK1 pathway. Deletion of the JNK- or MEKK1-binding regions resulted in a significant reduction in the enhancement of the JNK3 activation in COS-7 cells. These results suggest that JSAP1 functions as a scaffold protein in the JNK3 cascade. We also discuss a scaffolding role for JSAP1 in the JNK1 and JNK2 cascades.

The c-jun N-terminal kinases (JNKs) are responsive to stress stimuli leading to activation of proapoptotic proteins and transcription. Additionally, JNK mitochondrial localization has been reported. To selectively target mitochondrial JNK signaling, we exploited JNKs interaction with its mitochondrial scaffold, Sab, using small interfering RNAs (siRNAs) and a cell permeable peptide corresponding to the KIM1 domain of Sab. Gene silencing and peptide interference of this interaction disrupted JNK translocation to the mitochondria and reduced phosphorylation of Bcl-2 without significant impact on c-Jun phosphorylation or AP-1 transcription. In contrast, the JNK inhibitory peptide (TI-JIP1) prevented these three functions. Tat-SabKIM1 selectivity was also demonstrated in anisomycin-stressed HeLa cells where Tat-SabKIM1 prevented Bcl-2 phosphorylation, cell death, loss of mitochondrial membrane potential, and superoxide generation, but not c-Jun phosphorylation. Conversely, TI-JIP1 prevented all aforementioned stress-induced events. This probe introduces a means to evaluate JNK-mediated events on the mitochondria without intervening in nuclear functions of JNK.

Signaling through the heterotrimeric G protein, G12, via Rho induces a striking increase in breast cancer cell invasion. In this study, evidence is provided that the c-Jun NH2-terminal kinase (JNK) is a key downstream effector of G12 on this pathway. Expression of constitutively-active Gα12 or activation of G12 signaling by thrombin leads to increased JNK and c-Jun phosphorylation. Pharmacologic inhibition of JNK or knockdown of JNK expression by siRNA significantly decreases G12-induced JNK activation as well as the ability of breast cancer cells to invade a reconstituted basement membrane. Furthermore, expression of dominant-negative Rho or treatment of cells with an inhibitor of the Rho kinase, ROCK, reduces G12-induced JNK and c-Jun activation, and ROCK inhibitor treatment also inhibits G12-induced cellular invasion. JNK knockdown or ROCK inhibitor treatment has no effect on activation of Rho by G12. Taken together, our data indicate that JNK activation is required for G12-induced invasion of breast cancer cells and that JNK is downstream of Rho and ROCK on this pathway. This study implicates a G12-stimulated mitogen-activated protein kinase cascade in cancer cell invasion, and supports a role for JNK in cancer progression.

The cJun NH2-terminal kinase isoform JNK1 is implicated in the mechanism of obesity-induced insulin resistance. Feeding a high fat diet causes activation of the JNK1 signaling pathway, insulin resistance, and obesity in mice. Germ-line ablation of Jnk1 prevents both diet-induced obesity and insulin resistance. Genetic analysis indicates that the effects of JNK1 on insulin resistance can be separated from effects of JNK1 on obesity. Emerging research indicates that JNK1 plays multiple roles in the regulation of insulin resistance, including altered gene expression, hormone/cytokine production, and lipid metabolism. Together, these studies establish JNK1 as a potential pharmacological target for the development drugs that might be useful for the treatment of insulin resistance, metabolic syndrome, and type 2 diabetes.

JNK (c-Jun N-terminal kinase) is part of a MAPK (mitogen-activated protein kinase) signalling cascade. Scaffold proteins simultaneously associate with various components of the MAPK signalling pathway and play a crucial role in signal transmission and MAPK regulation. WDR62 (WD repeat domain 62) is a JNK scaffold protein. Recessive mutations within WDR62 result in severe cerebral cortical malformation. In the present study we demonstrate the association of WDR62 with endogenous and overexpressed proteins of both JNK2 and the JNK2-activating kinase MKK7 (MAPK kinase 7). Association of WDR62 with JNK2 and MKK7 occurs via direct protein–protein interactions. We mapped the docking domain of WDR62 responsible for the association with JNK. WDR62 interacts with all JNK isoforms through a D domain motif located at the C-terminus. A WDR62 mutant lacking the putative JNK-binding domain fails to activate and recruit JNK to cellular granules. Furthermore, a synthetic peptide composed of the WDR62 docking domain inhibits JNK2 activity in vitro. WDR62 association with JNK2 requires both the JNK CD and ED domains, and the binding requisite is distinct from that of the previously described JNK2 association with JIP1 (JNK-interacting protein 1). Next, we characterized the association between WDR62 and MKK7. WDR62 associates directly with the MKK7β1 isoform independently of JNK binding, but fails to interact with MKK7α1. Furthermore, MKK7β1 recruits a protein phosphatase that dephosphorylates WDR62. Interestingly, a premature termination mutation in WDR62 that results in severe brain developmental defects does not abrogate WDR62 association with either JNK or MKK7. Therefore such mutations represent a loss of WDR62 function independent of JNK signalling.

SUMMARY

Members of the c-Jun NH2-terminal kinase (JNK) family play crucial roles in cell activation, differentiation, and apoptosis. Although many studies have indicated that JNK1 and JNK2 have functional differences and redundancy, the upstream signaling pathway that selectively activates JNK1 or JNK2 remains unknown. In this study, we have revealed a novel regulatory mechanism of JNK activation, in which JNK2, but not JNK1, is regulated by CARMA1, a scaffold molecule, following stimulation of the T cell receptor (TCR). This CARMA1-dependent regulation of JNK2 is through Bcl10 that inducibly associates with JNK2 and serves as a JNK-interacting protein (JIP)-like scaffold to assemble JNK2, MKK7, and TAK1. Finally, we show that CARMA1- and Bcl10-mediated JNK2 activation plays a critical role in regulating the level of c-Jun protein. Together, our studies provide the first genetic evidence that JNK1 and JNK2 are differentially regulated in the TCR signaling pathway, and play different functions.

c-Jun NH2-terminal kinases (JNKs) are essential during brain development, when they regulate morphogenic changes involving cell movement and migration. In the adult, JNK determines neuronal cytoarchitecture. To help uncover the molecular effectors for JNKs in these events, we affinity purified JNK-interacting proteins from brain. This revealed that the stathmin family microtubule-destabilizing proteins SCG10, SCLIP, RB3, and RB3′ interact tightly with JNK. Furthermore, SCG10 is also phosphorylated by JNK in vivo on sites that regulate its microtubule depolymerizing activity, serines 62 and 73. SCG10-S73 phosphorylation is significantly decreased in JNK1−/− cortex, indicating that JNK1 phosphorylates SCG10 in developing forebrain. JNK phosphorylation of SCG10 determines axodendritic length in cerebrocortical cultures, and JNK site–phosphorylated SCG10 colocalizes with active JNK in embryonic brain regions undergoing neurite elongation and migration. We demonstrate that inhibition of cytoplasmic JNK and expression of SCG10-62A/73A both inhibited fluorescent tubulin recovery after photobleaching. These data suggest that JNK1 is responsible for regulation of SCG10 depolymerizing activity and neurite elongation during brain development.

Abstract

Tumor cell invasion and metastasis require precise coordination of adherence to extracellular matrix (ECM) and controlled degradation of its components. Invasive cells secrete proteolytic enzymes known as matrix metalloproteinases (MMPs) which degrade specific basement membrane molecules. Expression of these enzymes is regulated by multiple signaling mechanisms, including the mitogen-activated protein kinase (MAPK) pathway. One of the terminal effectors of this signaling cascade is jun N-terminal kinase 1 (JNK1) which phosphorylates the transcription factor c-jun, a component of the AP-1 complex. MMP-9 expression is regulated by two well-characterized AP-1 sites in the promoter of this gene. To determine how JNK1 activity regulated MMP-9 expression in human squamous cell carcinoma lines, we overexpressed this kinase in SCC25 cells. JNK1 overexpression induced MMP-9 protein levels and activity in this cell line. Elevated MMP-9 expression correlated with increased invasion of reconstituted basement membranes by JNK1-overexpressing clones. Site-directed mutagenesis of the MMP-9 promoter revealed that JNK1 cooperated with its transcription factor target c-jun to increase MMP-9 expression at the transcriptional level via the proximal AP-1 site. These results suggest that elevated JNK1 expression may contribute to increased MMP-9 activity and ECM invasion by tumor cells.

Jun N-terminal kinases (JNKs) are implicated in various neuropathological conditions. However, physiological roles for JNKs in neurons remain largely unknown, despite the high expression level of JNKs in brain. Here, using bioinformatic and biochemical approaches, we identify the AMPA receptor GluR2L and GluR4 subunits as novel physiological JNK substrates in vitro, in heterologous cells and in neurons. Consistent with this finding, GluR2L and GluR4 associate with specific JNK signaling components in the brain. Moreover, the modulation of the novel JNK sites in GluR2L and GluR4 is dynamic and bi-directional, such that phosphorylation and de-phosphorylation are triggered within minutes following decreases and increases in neuronal activity, respectively. Using live-imaging techniques to address the functional consequence of these activity-dependent changes we demonstrate that the novel JNK site in GluR2L controls reinsertion of internalized GluR2L back to the cell surface following NMDA treatment, without affecting basal GluR2L trafficking. Taken together, our results demonstrate that JNK directly regulates AMPA-R trafficking following changes in neuronal activity in a rapid and bi-directional manner.

The c-Jun N-terminal kinases (JNKs) are members of a larger group of serine/threonine (Ser/Thr) protein kinases from the mitogen-activated protein kinase family. JNKs were originally identified as stress-activated protein kinases in the livers of cycloheximide-challenged rats. Their subsequent purification, cloning, and naming as JNKs have emphasized their ability to phosphorylate and activate the transcription factor c-Jun. Studies of c-Jun and related transcription factor substrates have provided clues about both the preferred substrate phosphorylation sequences and additional docking domains recognized by JNK. There are now more than 50 proteins shown to be substrates for JNK. These include a range of nuclear substrates, including transcription factors and nuclear hormone receptors, heterogeneous nuclear ribonucleoprotein K, and the Pol I-specific transcription factor TIF-IA, which regulates ribosome synthesis. Many nonnuclear substrates have also been characterized, and these are involved in protein degradation (e.g., the E3 ligase Itch), signal transduction (e.g., adaptor and scaffold proteins and protein kinases), apoptotic cell death (e.g., mitochondrial Bcl2 family members), and cell movement (e.g., paxillin, DCX, microtubule-associated proteins, the stathmin family member SCG10, and the intermediate filament protein keratin 8). The range of JNK actions in the cell is therefore likely to be complex. Further characterization of the substrates of JNK should provide clearer explanations of the intracellular actions of the JNKs and may allow new avenues for targeting the JNK pathways with therapeutic agents downstream of JNK itself.