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.     

Dynamic interaction between the dual specificity phosphatase MKP7 and the JNK3 scaffold protein beta-arrestin 2

JNK scaffold proteins bind JNK and upstream kinases to activate subsets of JNK and localize activated JNK to specific subcellular sites. We previously demonstrated that the dual specificity phosphatases (DSPs) MKP7 and M3/6 bind the scaffold JNK-interacting protein-1 (JIP-1) and inactivate the bound subset of JNK (1). The G protein-coupled receptor (GPCR) adaptor beta-arrestin 2 is also a JNK3 scaffold. It binds the upstream kinases ASK1 and MKK4 and couples stimulation of the angiotensin II receptor AT1aR to activation of a cytoplasmic pool of JNK3. Here we report that MKP7 also binds beta-arrestin 2 via amino acids 394-443 of MKP7, the same region that interacts with JIP-1. This region of MKP7 interacts with beta-arrestin 2 at a central region near the JNK binding domain. MKP7 dephosphorylates JNK3 bound to beta-arrestin 2, either following activation by ASK1 overexpression or following AT1aR stimulation. Initial AT1aR stimulation causes a rapid (within 5 min) dissociation of MKP7 from beta-arrestin 2. MKP7 then reassociates with beta-arrestin 2 on endocytic vesicles 30-60 min after initial receptor stimulation. This dynamic interaction between phosphatase and scaffold permits signal transduction through a module that binds both positive and negative regulators.     

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.     

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.     

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.     

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.     

Axin utilizes distinct regions for competitive MEKK1 and MEKK4 binding and JNK activation

Axin is a multidomain protein that plays a critical role in Wnt signaling, serving as a scaffold for down-regulation of beta-catenin. It also activates the JNK mitogen-activated protein kinase by binding to MEKK1. However, it is intriguing that Axin requires several additional elements for JNK activation, including a requirement for homodimerization, sumoylation at the extreme C-terminal sites, and a region in the protein phosphatase 2A-binding domain. In our present study, we have shown that another MEKK family member, MEKK4, also binds to Axin in vivo and mediates Axin-induced JNK activation. Surprisingly MEKK4 binds to a region distinct from the MEKK1-binding site. Dominant negative mutant of MEKK4 attenuates the JNK activation by Axin. Activation of JNK by Axin in MEKK1-/- mouse embryonic fibroblast cells supports the idea that another MEKK can mediate Axin-induced JNK activation. Expression of specific small interfering RNA against MEKK4 effectively attenuates JNK activation by the MEKK1 binding-defective Axin mutant in 293T cells and inhibits JNK activation by wild-type Axin in MEKK1-/- cells, confirming that MEKK4 is indeed another mitogen-activated protein kinase kinase kinase that is specifically involved in Axin-mediated JNK activation independently of MEKK1. We have also identified an additional domain between MEKK1- and MEKK4-binding sites as being required for JNK activation by Axin. MEKK1 and MEKK4 compete for Axin binding even though they bind to sites far apart, suggesting that Axin may selectively bind to MEKK1 or MEKK4 depending on distinct signals or cellular context. Our findings will provide new insights into how scaffold proteins mediate ultimate activation of different mitogen-activated protein kinase kinase kinases.     

Interaction of a mitogen-activated protein kinase signaling module with the neuronal protein JIP3

The c-Jun NH(2)-terminal kinase (JNK) group of mitogen-activated protein kinases (MAPKs) is activated in response to the treatment of cells with inflammatory cytokines and by exposure to environmental stress. JNK activation is mediated by a protein kinase cascade composed of a MAPK kinase and a MAPK kinase kinase. Here we describe the molecular cloning of a putative molecular scaffold protein, JIP3, that binds the protein kinase components of a JNK signaling module and facilitates JNK activation in cultured cells. JIP3 is expressed in the brain and at lower levels in the heart and other tissues. Immunofluorescence analysis demonstrated that JIP3 was present in the cytoplasm and accumulated in the growth cones of developing neurites. JIP3 is a member of a novel class of putative MAPK scaffold proteins that may regulate signal transduction by the JNK pathway.     

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.     

Activated mitogen-activated protein kinase kinase 7 redistributes to the cytosol and binds to Jun N-terminal kinase-interacting protein 1 involving oxidative stress during early reperfusion in rat hippocampal CA1 region

Mitogen-activated protein kinase kinase (MKK) 7, a specific upstream activator of Jun N-terminal kinases (JNKs) in the stress-activated protein kinase (SAPK)/JNK signaling pathway, plays an important role in response to global cerebral ischemia. We investigated the subcellular localization of activated (phosphorylated) MKK (p-MKK) 7 using western blotting, immunoprecipitation and immunohistochemistry analysis in rat hippocampus. Transient forebrain ischemia was induced by the four-vessel occlusion method on Sprague-Dawley rats. Our results showed that both protein expression and activation of MKK7 were increased rapidly with peaks at 10 min of reperfusion in the nucleus of the hippocampal CA1 region. Simultaneously, in the cytosol activated MKK7 enhanced gradually and peaked at 30 min of reperfusion. In addition, we also detected JNK-interacting protein (JIP) 1, which accumulated in the perinuclear region of neurons at 30 min of reperfusion. Interestingly, at the same time-point the binding of JIP-1 to p-MKK7 reached a maximum. Consequently, we concluded that MKK7 was rapidly activated and then translocated from the nucleus to the cytosol depending on its activation in the hippocampal CA1 region. To further elucidate the possible mechanism of MKK7 activation and translocation, the antioxidant N-acetylcysteine was injected into the rats 20 min before ischemia. The result showed that the levels of MKK7 activation, translocation and binding of p-MKK7 to JIP-1 were obviously limited by N-acetylcysteine in the cytosol at 30 min after reperfusion. The findings suggested that MKK7 activation, translocation and binding to JIP-1 were closely associated with reactive oxygen species and might play a pivotal role in the activation of the JNK signaling pathway in brain ischemic injury.