alpha-Synuclein protects against oxidative stress via inactivation of the c-Jun N-terminal kinase stress-signaling pathway in neuronal cells.

The expression of alpha-synuclein, a synaptic molecule implicated in the pathogenesis of neurodegenerative disorders such as Parkinson's disease and Lewy body disease is increased upon injury to the nervous system, indicating that it might play a role in regeneration and plasticity; however, the mechanisms are unclear. Because c-Jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinase family, plays an important role in stress response, the main objective of the present study was to better understand the involvement of this pathway in the signaling responses associated with resistance to injury in cells expressing alpha-synuclein. For this purpose, the JNK-signaling pathway was investigated in alpha-synuclein-transfected neuronal cell line glucose transporter (GT) 1-7 under oxidative stress conditions. Although hydrogen peroxide challenge resulted in JNK activation and cell death in cells transfected with vector control or beta-synuclein, alpha-synuclein-transfected cells were resistant to hydrogen peroxide, and JNK was not activated. The inactivation of JNK in the alpha-synuclein-transfected cells was associated with increased expression and activity of JNK-interacting protein (JIP)-1b/islet-brain (IB)1, the scaffold protein for the JNK pathway. Similarly, cells transfected with JIP-1b/IB1 were resistant to hydrogen peroxide associated with inactivation of the JNK pathway. In these cells, expression of endogenous alpha-synuclein was significantly increased at the protein level. Furthermore, alpha-synuclein was co-localized with JIP-1b/IB1 in the growth cones. Taken together, these results suggest that increased alpha-synuclein expression might protect cells from oxidative stress by inactivation of JNK via increased expression of JIP-1b/IB1. Furthermore, interactions between alpha-synuclein and JIP-1b/IB1 may play a mutual role in the neuronal response to injury and neurodegeneration.     

Role of the JNK pathway in NMDA-mediated excitotoxicity of cortical neurons

Excitotoxic insults induce c-Jun N-terminal kinase (JNK) activation, which leads to neuronal death and contributes to many neurological conditions such as cerebral ischemia and neurodegenerative disorders. The action of JNK can be inhibited by the D-retro-inverso form of JNK inhibitor peptide (D-JNKI1), which totally prevents death induced by N-methyl-D-aspartate (NMDA) in vitro and strongly protects against different in vivo paradigms of excitotoxicity. To obtain optimal neuroprotection, it is imperative to elucidate the prosurvival action of D-JNKI1 and the death pathways that it inhibits. In cortical neuronal cultures, we first investigate the pathways by which NMDA induces JNK activation and show a rapid and selective phosphorylation of mitogen-activated protein kinase kinase 7 (MKK7), whereas the only other known JNK activator, mitogen-activated protein kinase kinase 4 (MKK4), was unaffected. We then analyze the action of D-JNKI1 on four JNK targets containing a JNK-binding domain: MAPK-activating death domain-containing protein/differentially expressed in normal and neoplastic cells (MADD/DENN), MKK7, MKK4 and JNK-interacting protein-1 (IB1/JIP-1).     

Interaction of c-Jun amino-terminal kinase interacting protein-1 with p190 rhoGEF and its localization in differentiated neurons

c-Jun amino-terminal kinase (JNK) interacting protein-1 (JIP-1) was originally identified as a cytoplasmic inhibitor of JNK. More recently, JIP-1 was proposed to function as a scaffold protein by complexing specific components of the JNK signaling pathway, namely JNK, mitogen-activated protein kinase kinase 7, and mixed lineage kinase 3. We have identified the human homologue of JIP-1 that contains a phosphotyrosine binding (PTB) domain in addition to a JNK binding domain and an Src homology 3 domain. To identify binding targets for the hJIP-1 PTB domain, a mouse embryo cDNA library was screened using the yeast two-hybrid system. One clone encoded a 191-amino acid region of the neuronal protein rhoGEF, an exchange factor for rhoA. Overexpression of rhoGEF promotes cytoskeletal rearrangement and cell rounding in NIE-115 neuronal cells. The interaction of JIP-1 with rhoGEF was confirmed by coimmunoprecipitation of these proteins from lysates of transiently transfected HEK 293 cells. Using glutathione S-transferase rhoGEF fusion proteins containing deletion or point mutations, we identified a putative PTB binding site within rhoGEF. This binding site does not contain tyrosine, indicating that the JIP PTB domain, like that of Xll alpha and Numb, binds independently of phosphotyrosine. Several forms of endogenous JIP-1 protein can be detected in neuronal cell lines. Indirect immunofluorescence analysis localized endogenous JIP-1 to the tip of the neurites in differentiated NIE-115 and PC12 cells. The interaction of JIP-1 with rhoGEF and its subcellular localization suggests that JIP-1 may function to specifically localize a signaling complex in neuronal cells.     

The JNK-interacting protein-1 scaffold protein targets MAPK phosphatase-7 to dephosphorylate JNK

The c-Jun N-terminal kinase (JNK) group of mitogen-activated protein kinases (MAPKs) are activated by pleiotropic signals including environmental stresses, growth factors, and hormones. A subset of JNK can bind to distinct scaffold proteins that also bind upstream kinases of the JNK pathway, allowing sequential kinase activation within a signaling module. The JNK-interacting protein-1 (JIP-1) scaffold protein specifically binds JNK, MAP kinase kinase 7, and members of the MLK family and is essential for stress-mediated JNK activation in neurones. Here we report that JIP-1 also binds the dual-specificity phosphatases MKP7 and M3/6 via a region independent of its JNK binding domain. The C-terminal region of MKP7, homologous to that of M3/6 but not other DSPs, is required for interaction with JIP-1. When MKP7 is bound to JIP-1 it reduces JNK activation leading to reduced phosphorylation of the JNK target c-Jun. These results indicate that the JIP-1 scaffold protein modulates JNK signaling via association with both protein kinases and protein phosphatases that target JNK.     

Docking interactions in the c-Jun N-terminal kinase pathway

The c-Jun N-terminal kinase (JNK) signaling pathway is a major mediator of stress responses in cells. Similar to other mitogen-activated protein kinases (MAPKs), JNK activity is controlled by a cascade of protein kinases and by protein phosphatases, including dual-specificity MAPK phosphatases. Components of the JNK pathway associate with scaffold proteins that modulate their activities and cellular localization. The JNK-interacting protein-1 (JIP-1) scaffold protein specifically binds JNK, MAPK kinase 7 (MKK7), and members of the mixed lineage kinase (MLK) family, and regulates JNK activation in neurons. In this study we demonstrate that distinct regions within the N termini of MKK7 and the MLK family member dual leucine zipper kinase (DLK) mediate their binding to JIP-1. We have also identified amino acids in JNK required for: (a) binding to JIP-1 and for JIP-1-mediated JNK activation, (b) docking to MAPK kinase 4 (MKK4) and efficient phosphorylation by MKK4, and (c) docking to its substrate c-Jun and efficient c-Jun phosphorylation. None of the amino acids identified were essential for JNK docking to MKK7 or the dual-specificity phosphatase MAPK phosphatase 7 (MKP7). These findings uncover molecular determinants of JIP-1 scaffold complex assembly and demonstrate that there are overlapping, but also distinct, binding determinants within JNK that mediate interactions with scaffold proteins, activators, phosphatases, and substrates.     

A unique set of SH3-SH3 interactions controls IB1 homodimerization

Islet-brain 1 (IB1 or JIP-1) is a scaffold protein that interacts with components of the c-Jun N-terminal kinase (JNK) signal-transduction pathway. IB1 is expressed at high levels in neurons and in pancreatic beta-cells, where it controls expression of several insulin-secretory components and secretion. IB1 has been shown to homodimerize, but neither the molecular mechanisms nor the function of dimerization have yet been characterized. Here, we show that IB1 homodimerizes through a novel and unique set of Src homology 3 (SH3)-SH3 interactions. X-ray crystallography studies show that the dimer interface covers a region usually engaged in PxxP-mediated ligand recognition, even though the IB1 SH3 domain lacks this motif. The highly stable IB1 homodimer can be significantly destabilized in vitro by three individual point mutations directed against key residues involved in dimerization. Each mutation reduces IB1-dependent basal JNK activity in 293T cells. Impaired dimerization also results in a reduction in glucose transporter type 2 expression and in glucose-dependent insulin secretion in pancreatic beta-cells. Taken together, these results indicate that IB1 homodimerization through its SH3 domain has pleiotropic effects including regulation of the insulin secretion process.     

cDNA cloning and mapping of a novel islet-brain/JNK-interacting protein

IB1/JIP-1 is a scaffold protein that regulates the c-Jun NH(2)-terminal kinase (JNK) signaling pathway, which is activated by environmental stresses and/or by treatment with proinflammatory cytokines including IL-1beta and TNF-alpha. The JNKs play an essential role in many biological processes, including the maturation and differentiation of immune cells and the apoptosis of cell targets of the immune system. IB1 is expressed predominantly in brain and pancreatic beta-cells where it protects cells from proapoptotic programs. Recently, a mutation in the amino-terminus of IB1 was associated with diabetes. A novel isoform, IB2, was cloned and characterized. Overall, both IB1 and IB2 proteins share a very similar organization, with a JNK-binding domain, a Src homology 3 domain, a phosphotyrosine-interacting domain, and polyacidic and polyproline stretches located at similar positions. The IB2 gene (HGMW-approved symbol MAPK8IP2) maps to human chromosome 22q13 and contains 10 coding exons. Northern and RT-PCR analyses indicate that IB2 is expressed in brain and in pancreatic cells, including insulin-secreting cells. IB2 interacts with both JNK and the JNK-kinase MKK7. In addition, ectopic expression of the JNK-binding domain of IB2 decreases IL-1beta-induced pancreatic beta-cell death. These data establish IB2 as a novel scaffold protein that regulates the JNK signaling pathway in brain and pancreatic beta-cells and indicate that IB2 represents a novel candidate gene for diabetes.     

Genomic Organization,Fine-Mapping,and Expression of the HumanIslet-Brain 1 (IB1)/C-Jun-Amino-Terminal Kinase Interacting Protein-1 (JIP-1)Gene

Islet-brain 1 (IB1), a regulator of the pancreatic β-cell function in the rat, is homologous to JIP-1, a murine inhibitor of c-Jun amino-terminal kinase (JNK). Whether IB1 and JIP-1 are present in humans was not known. We report the sequence of the 2133-bp human IB1 cDNA, the expression, structure, and fine-mapping of the humanIB1gene, and the characterization of anIB1pseudogene. Human IB1 is 94% identical to rat IB1. The tissue-specific expression of IB1 in human is similar to that observed in rodent. TheIB1gene contains 12 exons and maps to chromosome 11 (11p11.2–p12), a region that is deleted in DEFECT-11 syndrome. Apart from anIB1pseudogene on chromosome 17 (17q21), no additionalIB1-related gene was found in the human genome. Our data indicate that the sequence and expression pattern of IB1 are highly conserved between rodent and human and provide the necessary tools to investigate whether IB1 is involved in human diseases.     

Reverse two-hybrid screening identifies residues of JNK required for interaction with the kinase interaction motif of JNK-interacting protein-1

The development of specific inhibitors for the c-Jun N-terminal kinase (JNK) family of mitogen-activated protein kinases (MAPKs) has been a recent research focus because of the association of JNK with cell death in conditions such as stroke and neurodegeneration. We have demonstrated previously the presence of critical inhibitory residues within an 11-mer peptide (TI-JIP) based on the sequence of JNK-interacting protein-1 (JIP-1). However, the corresponding region of JNK bound by this JIP-1-based peptide was unknown. To identify this region, we used a novel reverse two-hybrid approach with TI-JIP as bait. We screened a library of JNK1 mutants that had been generated by random PCR mutagenesis and found three mutants of JNK1 that failed to interact with TI-JIP. The mutations in JNK1 were L131R, R309W, and Y320H. Of these mutated residues, Leu-131 and Tyr-320 were located on a common face of the JNK protein close to other residues implicated previously in the interactions of MAPKs with substrates, phosphatases, and scaffolds. To test whether these JNK1 mutants were thus affected in their regulation, we evaluated their activation in mammalian cells in response to hyperosmolarity or cotransfection with a constitutively active upstream kinase or their direct phosphorylation by either MAPK kinase (MKK)4 or MKK7. In each situation, all three JNK mutants were not activated or phosphorylated to the same level as wild-type JNK. Therefore, the results of our unbiased reverse two-hybrid screening approach have identified residues of JNK responsible for binding JIP-1-based peptides as well as MKK4 or MKK7.     

Identification of ASK1, MKK4, JNK, c-Jun, and caspase-3 as a signaling cascade involved in cadmium-induced neuronal cell apoptosis

Cd induces oxidative stress and apoptosis in various cells by activating mitogen-activated protein kinases (MAPKs), but the precise signaling components of the MAPK cascade and their role in neuronal apoptosis are still unclear. Here, we report that Cd treatment of SH-SY5Y cells caused apoptosis through sequential phosphorylation of the apoptosis signal regulating kinase 1, MAPK kinase 4, c-Jun N-terminal kinase (JNK), and c-Jun as determined by overexpression of dominant negative (DN) constructs of these genes or using a specific JNK inhibitor SP600125. Both Cd-induced JNK and c-Jun phosphorylation and apoptosis were inhibited dramatically by N-acetyl-L-cysteine, a free radical scavenger. In addition, caspase inhibitors, zDEVD and zVAD, reduced apoptosis but not JNK and c-Jun phosphorylation induced by Cd, while overexpression of DN JNK1 inhibited caspase-3 activity. Taken together, our data suggested that the JNK/c-Jun signaling cascade plays a crucial role in Cd-induced neuronal cell apoptosis and provides a molecular linkage between oxidative stress and neuronal apoptosis.     

Activation of the c-Jun N-terminal kinase (JNK) signaling pathway is essential during PBOX-6-induced apoptosis in chronic myelogenous leukemia (CML) cells

The mitogen-activated protein (MAP) kinase family is activated in response to a wide variety of external stress signals such as UV irradiation, heat shock, and many chemotherapeutic drugs and leads to the induction of apoptosis. A novel series of pyrrolo-1,5-benzoxazepines have been shown to potently induce apoptosis in chronic myelogenous leukemia (CML) cells, which are resistant to many chemotherapeutic agents. In this study we have delineated part of the mechanism by which a representative compound known as PBOX-6 induces apoptosis. We have investigated whether PBOX-6 induces activation of MAP kinase signaling pathways in CML cells. Treatment of K562 cells with PBOX-6 resulted in the transient activation of two JNK isoforms, JNK1 and JNK2. In contrast, PBOX-6 did not activate the extracellular signal-regulated kinase (ERK) or p38. Apoptosis was found to occur independently of the small GTPases Ras, Rac, and Cdc42 but involved phosphorylation of the JNK substrates, c-Jun and ATF-2. Pretreatment of K562 cells with the JNK inhibitor, dicoumarol, abolished PBOX-6-induced phosphorylation of c-Jun and ATF-2 and inhibited the induced apoptosis, suggesting that JNK activation is an essential component of the apoptotic pathway induced by PBOX-6. Consistent with this finding, transfection of K562 cells with the JNK scaffold protein, JIP-1, inhibited JNK activity and apoptosis induced by PBOX-6. JIP-1 specifically scaffolds JNK, MKK7, and members of the mixed-lineage kinase (MLK) family, implicating these kinases upstream of JNK in the apoptotic pathway induced by PBOX-6 in K562 cells.     

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.     

Inhibition of JNK by overexpression of the JNL binding domain of JIP-1 prevents apoptosis in sympathetic neurons

Studies in non-neuronal cells show that c-Jun N-terminal kinases (JNK) play a key role in apoptotic cell death. In some neurons JNK is also thought to initiate cell death by the activation of c-Jun. JNK inhibition has been achieved pharmacologically by inhibiting upstream kinases, but there has been no direct demonstration that inhibition of JNK can prevent neuronal death. We have therefore examined whether the JNK binding domain (JBD) of JNK-interacting protein-1 (JIP-1, a scaffold protein and specific inhibitor of JNK) can inhibit c-Jun phosphorylation and support the survival of sympathetic neurons deprived of NGF. We show that expression of the JBD in >80% of neurons was sufficient to prevent the phosphorylation of c-Jun and its nuclear accumulation as well as abrogate neuronal cell death induced by NGF deprivation. JBD expression also preserved the capacity of mitochondria to reduce MTT. Interestingly, although the PTB domain of JIP was reported to interact with rhoGEF, expression of the JBD domain was sufficient to localize the protein to the membrane cortex and growth cones. Hence, JNK activation is a key event in apoptotic death induced by NGF withdrawal, where its point of action lies upstream of mitochondrial dysfunction.     

Mixed lineage kinase LZK forms a functional signaling complex with JIP-1, a scaffold protein of the c-Jun NH(2)-terminal kinase pathway.

Leucine zipper-bearing kinase (LZK) is a novel member of the mixed lineage kinase (MLK) protein family, the cDNA of which was first cloned from a human brain cDNA library [Sakuma, H., Ikeda, A., Oka, S., Kozutsumi, Y., Zanetta, J.-P., and Kawasaki, T. (1997) J. Biol. Chem. 272, 28622-28629]. Several MLK family proteins have been proposed to function as MAP kinase kinase kinases in the c-Jun NH(2) terminal kinase (JNK)/stress-activated protein kinase (SAPK) pathway. In the present study, we demonstrated that, like other MLKs, LZK activated the JNK/SAPK pathway but not the ERK pathway. LZK directly phosphorylated and activated MKK7, one of the two MAPKKs in the JNK/SAPK pathway, to a comparable extent to a constitutive active form of MEKK1 (MEKK1DeltaN), suggesting a biological role of LZK as a MAPKKK in the JNK/SAPK pathway. Recent studies have revealed the essential roles of scaffold proteins in intracellular signaling pathways including MAP kinase pathways. JIP-1, one of the scaffold proteins, has been shown to be associated with MLKs, MKK7, and JNK [Whitmarsh, A.J., Cavanagh, J., Tournier, C., Yasuda, J., and Davis, R.J. (1998) Science 281, 1671-1674], suggesting the presence of a selective signaling pathway including LZK, MKK7, and JNK. Consistent with this hypothesis, we provided evidence that LZK is associated with the C-terminal region of JIP-1 through its kinase catalytic domain. In addition, LZK-induced JNK activation was markedly enhanced when LZK and JNK were co-expressed with JIP-1. These results constituted important clues for understanding the molecular mechanisms regulating the signaling specificities of various JNK activators under different cellular conditions.     

Activation of the JNK pathway is important for cardiomyocyte death in response to simulated ischemia

Multiple signaling pathways, including the c-Jun N-terminal kinase (JNK) pathway, are activated in myocardial ischemia and reperfusion (MI/R) and correlate with cell death. However, the role of the JNK pathway in MI/R-induced cell death is poorly understood. In a rabbit model, we found that ischemia followed by reperfusion resulted in JNK activation which could be detected in cytosol as well as in mitochondria. To address the functional role of the JNK activation, we examined the consequences of blockade of JNK activation in isolated cardiomyocytes under conditions of simulated ischemia. The JNK activity was stimulated approximately sixfold by simulated ischemia and reperfusion (simulated MI). When a dominant negative mutant of JNK kinase-2 (dnJNKK2), an upstream regulator of JNK, and JNK-interacting protein-1 (JIP-1) were expressed in myocytes by recombinant adenovirus, the activation of JNK by simulated MI was reduced 53%. Furthermore, the TNFalpha-activated JNK activity in H9c2 cells was completely abolished by dnJNKK2 and JIP-1. In correlation, when dnJNKK2 and JIP-1 were expressed in cardiomyocytes, both constructs significantly reduced cell death after simulated MI compared to vector controls. We conclude that activation of the JNK cascade is important for cardiomyocyte death in response to simulated ischemia.