Mixed lineage kinase-dependent JNK activation is governed by interactions of scaffold protein JIP with MAPK module components.

It has been proposed that JNK-interacting proteins (JIP) facilitate mixed lineage kinase-dependent signal transduction to JNK by aggregating the three components of a JNK module. A new model for the assembly and regulation of these modules is proposed based on several observations. First, artificially induced dimerization of dual leucine zipper-bearing kinase (DLK) confirmed that DLK dimerization is sufficient to induce DLK activation. Secondly, under basal conditions, DLK associated with JIP is held in a monomeric, unphosphorylated and catalytically inactive state. Thirdly, JNK recruitment to JIP coincided with significantly decreased affinity of JIP and DLK. JNK promoted the dimerization, phosphorylation and activation of JIP-associated DLK. Similarly, treatment of cells with okadaic acid inhibited DLK association with JIP and resulted in DLK dimerization in the presence of JIP. In summary, JIP maintains DLK in a monomeric, unphosphorylated, inactive state. Upon stimulation, JNK-JIP binding affinity increases while JIP-DLK interaction affinity is attenuated. Dissociation of DLK from JIP results in subsequent DLK dimerization, autophosphorylation and module activation. Evidence is provided that this model holds for other MLK-dependent JNK modules.     

Src family kinases directly regulate JIP1 module dynamics and activation

JIP1 is a mammalian scaffold protein that assembles and participates in regulating the dynamics and activation of components of the mixed-lineage kinase-dependent JNK module. Mechanisms governing JIP1-JNK module regulation remain unclear. JIP1 is a multiply phosphorylated protein; for this reason, it was hypothesized that signaling by unidentified protein kinases or phosphatases might determine module function. We find that Src family kinases directly bind and tyrosine phosphorylate JIP1 under basal conditions in several naturally occurring systems and, by doing so, appear to provide a regulated signal that increases the affinity of JIP1 for DLK and maintains the JIP-JNK module in a catalytically inactive state.     

Cross-talk between JIP3 and JIP1 during glucose deprivation: SEK1-JNK2 and Akt1 act as mediators

We have previously observed that glucose deprivation activates the ASK1-MEK-MAPK signal transduction pathway. In the present study, we reveal that two scaffolding proteins, JIP1 and JIP3, have a cross-talk that leads to the regulation of the ASK1-SEK1-JNK signal during glucose deprivation. Glucose deprivation rapidly increases the interaction between ASK1 and JIP3, and the consequently activated ASK1 phosphorylates SEK1 on the Thr-261 residue. The activated SEK1 dissociates from JIP3 and phosphorylates JNK2 on the Tyr-185 residue. Phosphorylated JNK2 binds to JIP1, and the phosphorylation of the Thr-183 residue of JNK2 occurs. JNK2 phosphorylates JIP1 on the Thr-103 residue and leads to dissociation of Akt1 from JIP1. Dissociated Akt1 binds to SEK1 and ASK1 and inhibits their enzyme activity by phosphorylating SEK1 on the Ser-80 residue and ASK1 on the Ser-83 residue. Taken together, our data demonstrate that cross-talk between JIP3 and JIP1 is mediated through SEK1-JNK2 and Akt1.     

Dissociation of Akt1 from its negative regulator JIP1 is mediated through the ASK1-MEK-JNK signal transduction pathway during metabolic oxidative stress: a negative feedback loop

We have previously observed that metabolic oxidative stress-induced death domain-associated protein (Daxx) trafficking is mediated by the ASK1-SEK1-JNK1-HIPK1 signal transduction pathway. The relocalized Daxx from the nucleus to the cytoplasm during glucose deprivation participates in a positive regulatory feedback loop by binding to apoptosis signal-regulating kinase (ASK) 1. In this study, we report that Akt1 is involved in a negative regulatory feedback loop during glucose deprivation. Akt1 interacts with c-Jun NH(2)-terminal kinase (JNK)-interacting protein (JIP) 1, and Akt1 catalytic activity is inhibited. The JNK2-mediated phosphorylation of JIP1 results in the dissociation of Akt1 from JIP1 and subsequently restores Akt1 enzyme activity. Concomitantly, Akt1 interacts with stress-activated protein kinase/extracellular signal-regulated kinase (SEK) 1 (also known as MKK4) and inhibits SEK1 activity. Knockdown of SEK1 leads to the inhibition of JNK activation, JIP1-JNK2 binding, and the dissociation of Akt1 from JIP1 during glucose deprivation. Knockdown of JIP1 also leads to the inhibition of JNK activation, whereas the knockdown of Akt1 promotes JNK activation during glucose deprivation. Altogether, our data demonstrate that Akt1 participates in a negative regulatory feedback loop by interacting with the JIP1 scaffold protein.     

Reassembly of JIP1 Scaffold Complex in JNK MAP Kinase Pathway Using Heterologous Protein Interactions

Formation of signaling protein complexes is crucial for proper signal transduction. Scaffold proteins in MAP kinase pathways are thought to facilitate complex assembly, thereby promoting efficient and specific signaling. To elucidate the assembly mechanism of scaffold complexes in mammals, we attempted to rationally rewire JIP1-dependent JNK MAP kinase pathway via alternative assembly of JIP1 complex. When JIP1-JNK docking interaction in the complex was replaced with heterologous protein interaction domains, such as PDZ domains and JNK-binding domains, a functional scaffold complex was reconstituted, and JNK signaling was rescued. Reassembly of JIP1 complex using heterologous protein interactions was sufficient for restoring of JNK MAP kinase pathway to induce signaling responses, including JNK activation and cell death. These results suggest a simple yet modular mechanism for JIP1 scaffold assembly in mammals.     

Disassembly of the JIP1/JNK molecular scaffold by caspase-3-mediated cleavage of JIP1 during apoptosis

We report here the cleavage of the c-Jun N-terminal Kinase (JNK) pathway scaffold protein, JNK Interacting Protein-1 (JIP1), by caspases during both Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) and staurosporine-induced apoptosis in HeLa cells. During the initiation of apoptosis, maximal JNK activation is observed when JIP1 is intact, whereas cleavage of JIP1 correlates with JNK inactivation and progression of apoptosis. JIP1 is cleaved by caspase-3 at two sites, leading to disassembly of the JIP1/JNK complex. Inhibition of JIP1 cleavage by the caspase-3 inhibitor DEVD.fmk inhibits this disassembly, and is accompanied by sustained JNK activation. These data suggest that TRAIL and staurosporine induce JNK activation in a caspase-3-independent manner and that caspase-3-mediated JIP1 cleavage plays a role in JNK inactivation via scaffold disassembly during the execution phase of apoptosis. Caspase-mediated cleavage of JIP scaffold proteins may therefore represent an important mechanism for modulation of JNK signalling during apoptotic cell death.     

The docking properties of SHIP2 influence both JIP1 tyrosine phosphorylation and JNK activity

SHIP2 (SH2-containing inositol polyphosphate 5-phosphatase 2) is an ubiquitously expressed phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P(3)) 5-phosphatase which contains various motifs susceptible to mediate protein-protein interaction. In cell models, evidence has been provided that SHIP2 plays a role in insulin and growth factor signaling, cytoskeletal organization, cell adhesion and migration. Herein we describe the c-Jun NH2-terminal kinase (JNK)-interacting protein 1 (JIP1) as a new protein partner of SHIP2. The interaction between SHIP2 and JIP1 was confirmed in both overexpression systems and native cells. Without modifying the association of JIP1 with the MAPKs in the scaffold complex and with no apparent change of Akt phosphorylation, SHIP2 positively modulated the MLK3/JIP1-mediated JNK1 activation. Moreover, SHIP2 positively regulated the tyrosine phosphorylation of JIP1. This up-regulation was prevented by inhibitors of the Src family and Abl kinases, PP2 and Glivec. The effects of SHIP2 on JNK activity and JIP1 tyrosine phosphorylation were independent of the SHIP2 phosphoinositide 5-phosphatase activity, as similar results were obtained when using a SHIP2 catalytic inactive mutant instead of wild-type SHIP2. Together, these data suggest that by its docking properties, SHIP2 can modulate JIP1-mediated JNK pathway signaling.     

Crosstalk between PSD-95 and JIP1-mediated signaling modules: the mechanism of MLK3 activation in cerebral ischemia

Our previous study indicates that global ischemia facilitates the assembly of the GluR6.PSD-95.MLK3 signaling module, which in turn activated MLK3, leading to exacerbated ischemic neuron death. In addition, JIP1, functioning as a scaffold protein, could couple MLK3-MKK7-JNK to form a specific signaling module and facilitate the activation of the JNK signal pathway. However, the organization, regulation, and function between the two signaling modules and the effects they have on MLK3 activation remain incompletely understood. Here, we show that JIP1 maintains MLK3 in an inactive and monomeric state; once activated, MLK3 binds to PSD-95 and then dimerizes and autophosphorylates. In addition, a GluR6 C-terminus-containing peptide (Tat-GluR6-9c) and antisense oligonucleotides (AS-ODNs) against PSD-95 inhibit the integration of PSD-95 and MLK3 and the dimerization of MLK3, facilitate the interaction of JIP1 and MLK3, and, consequently, perform neuroprotection on neuron death. However, AS-ODNs against JIP1 play a negative role compared to that mentioned above. The findings show that the crosstalk occurs between PSD-95 and the JIP1-mediated signaling module, which may be involved in brain ischemic injury and contribute to the regulation of MLK3 activation. Thus, specific blockade of PSD-95-MLK3 coupling may reduce the extent of ischemia-reperfusion-induced neuronal cell death.     

Regulation of stress-associated scaffold proteins JIP1 and JIP3 on the c-Jun NH2-terminal kinase in ischemia-reperfusion

Ischemia-reperfusion (IR)-induced cell apoptosis involves the activation of c-Jun NH2-terminal kinase (JNK). The activation of JNK requires the presence of scaffold proteins called JNK-interacting proteins (JIP), which bind several members of a signaling cascade for proper signaling specificity. In this study, the expression of scaffold proteins JIP1 and JIP3 and their roles in the regulation of JNK activity were investigated in simulated IR in a cell model (H9c2). JIP1 protein expression was significantly decreased, whereas JIP3 protein expression was increased in IR H9c2 cells. Adenovirus-induced overexpression of JIP1 reduced IR-induced JNK activity and apoptosis. Conversely, overexpression of JIP3 increased JNK activity and apoptosis following IR. Depletion of endogenous JIP1 by siRNA treatment increased the IR-induced JNK activity, whereas siRNA-mediated depletion of endogenous JIP3 inhibited JNK activity. These results suggest that JIP1 and JIP3 play important roles in the activation of JNK during simulated IR challenge in H9c2 cells.     

Structural basis for the selective inhibition of JNK1 by the scaffolding protein JIP1 and SP600125

The c-jun N-terminal kinase (JNK) signaling pathway is regulated by JNK-interacting protein-1 (JIP1), which is a scaffolding protein assembling the components of the JNK cascade. Overexpression of JIP1 deactivates the JNK pathway selectively by cytoplasmic retention of JNK and thereby inhibits gene expression mediated by JNK, which occurs in the nucleus. Here, we report the crystal structure of human JNK1 complexed with pepJIP1, the peptide fragment of JIP1, revealing its selectivity for JNK1 over other MAPKs and the allosteric inhibition mechanism. The van der Waals contacts by the three residues (Pro157, Leu160, and Leu162) of pepJIP1 and the hydrogen bonding between Glu329 of JNK1 and Arg156 of pepJIP1 are critical for the selective binding. Binding of the peptide also induces a hinge motion between the N- and C-terminal domains of JNK1 and distorts the ATP-binding cleft, reducing the affinity of the kinase for ATP. In addition, we also determined the ternary complex structure of pepJIP1-bound JNK1 complexed with SP600125, an ATP-competitive inhibitor of JNK, providing the basis for the JNK specificity of the compound.     

Phosphorylation-dependent scaffolding role of JSAP1/JIP3 in the ASK1-JNK signaling pathway. A new mode of regulation of the MAP kinase cascade

JSAP1 (also termed JIP3) is a scaffold protein that interacts with specific components of the JNK signaling pathway. Apoptosis signal-regulating kinase (ASK) 1 is a MAP kinase kinase kinase that activates the JNK and p38 mitogen-activated protein (MAP) kinase cascades in response to environmental stresses such as reactive oxygen species. Here we show that JSAP1 bound ASK1 and enhanced ASK1- and H(2)O(2)-induced JNK activity. ASK1 phosphorylated JSAP1 in vitro and in vivo, and the phosphorylation facilitated interactions of JSAP1 with SEK1/MKK4, MKK7 and JNK3. Furthermore, ASK1-dependent phosphorylation was required for JSAP1 to recruit and thereby activate JNK in response to H(2)O(2). We thus conclude that JSAP1 functions not only as a simple scaffold, but it dynamically participates in signal transduction by forming a phosphorylation-dependent signaling complex in the ASK1-JNK signaling module.     

Differential activation of the JNK signal pathway by UV irradiation and glucose deprivation

Exposure of mammalian cells to ultraviolet (UV) light or glucose deprivation activates c-Jun NH2-terminal protein kinase (JNK). However, the exact mechanism by which UV induces JNK activation is not yet understood completely. Previously, we have observed that glucose deprivation activates the ASK1-SEK1-JNK signal transduction pathway. In the present study, we reveal that UVC irradiation-induced JNK activation has a different signal transduction pathway from glucose deprivation. UVC irradiation increases the interaction between JIP3 and MEKK1, SEK1, while glucose deprivation increases the interaction between JIP3 and ASK1, SEK1, and JNK. UVC irradiation activates MEKK1 rather than ASK1. We also observed that MEKK1 interacted with Grb2 and Grb2-MEKK1 complex was recruited to epidermal growth factor receptor (EGFR) after UVC irradiation. Taken together, our data demonstrate that UVC-induced JNK activation adopts a different signaling cascade (EGFR-Grb2-MEKK1-SEK1-JNK) from glucose deprivation (ASK1-SEK1-JNK).     

Control of a kinesin-cargo linkage mechanism by JNK pathway kinases

Long-distance organelle transport toward axon terminals, critical for neuron development and function, is driven along microtubules by kinesins [1, 2]. The biophysics of force production by various kinesins is known in detail. However, the mechanisms of in vivo transport processes are poorly understood because little is known about how motor-cargo linkages are controlled. A c-Jun N-terminal kinase (JNK)-interacting protein (JIP1) has been identified previously as a linker between kinesin-1 and certain vesicle membrane proteins, such as Alzheimer's APP protein and a reelin receptor ApoER2 [3, 4]. JIPs are also known to be scaffolding proteins for JNK pathway kinases [5, 6]. Here, we report evidence that a Drosophila ubiquitin-specific hydrolase and a JNK signaling pathway that it modulates can regulate a JIP1-kinesin linkage. The JNK pathway includes a MAPKKK (Wallenda/DLK), a MAPKK (Hemipterous/MKK7), and the Drosophila JNK homolog Basket. Genetic tests indicate that those kinases are required for normal axonal transport. Biochemical tests show that activation of Wallenda (DLK) and Hemipterous (MKK7) disrupts binding between kinesin-1 and APLIP1, which is the Drosophila JIP1 homolog. This suggests a control mechanism in which an activated JNK pathway influences axonal transport by functioning as a kinesin-cargo dissociation factor.     

Akt1 regulates a JNK scaffold during excitotoxic apoptosis

Cell survival is determined by a balance among signaling cascades, including those that recruit the Akt and JNK pathways. Here we describe a novel interaction between Akt1 and JNK interacting protein 1 (JIP1), a JNK pathway scaffold. Direct association between Akt1 and JIP1 was observed in primary neurons. Neuronal exposure to an excitotoxic stimulus decreased the Akt1-JIP1 interaction and concomitantly increased association between JIP1 and JNK. Akt1 interaction with JIP1 inhibited JIP1-mediated potentiation of JNK activity by decreasing JIP1 binding to specific JNK pathway kinases. Consistent with this view, neurons from Akt1-deficient mice exhibited higher susceptibility to kainate than wild-type littermates. Overexpression of Akt1 mutants that bind JIP1 reduced excitotoxic apoptosis. These results suggest that Akt1 binding to JIP1 acts as a regulatory gate preventing JNK activation, which is released under conditions of excitotoxic injury.     

JNK-interacting protein 3 associates with Toll-like receptor 4 and is involved in LPS-mediated JNK activation

Lipopolysaccharide (LPS) is recognized by Toll-like receptor (TLR) 4 and activates NF-kappaB and a set of MAP kinases. Here we have investigated proteins associated with the cytoplasmic domain of mouse TLR4 by yeast two-hybrid screening and identified JNK-interacting protein 3 (JIP3), a scaffold protein for JNK, as a TLR4-associated protein. In mammalian cells, JIP3, through its N-terminal region, constitutively associates with TLR4. The association is specific to JIP3, as the two other JIPs, JIP1 and JIP2, failed to bind TLR4. In HEK 293 cells exogenously expressing TLR4, MD2 and CD14, co-expression of JIP3 significantly increased the complex formation of TLR4-JNK and LPS-mediated JNK activation. In contrast, expression of C-terminally truncated forms of JIP3 impaired LPS-induced JNK activation in a mouse macrophage cell line, RAW264.7. Moreover, RNA interference of JIP3 inhibited LPS-mediated JNK activation. In RAW264.7 cells, JIP3 associates MEKK-1, but not with TAK-1. Finally, JIP3 also associates with TLR2 and TLR9, but not with TLR1 or TLR6. Altogether, our data indicate the involvement of JIP3 in JNK activation in downstream signals of some TLRs.     

Hyperphosphorylation of JNK-interacting protein 1, a protein associated with Alzheimer disease

The c-Jun N-terminal kinase (JNK) group of mitogen-activated protein (MAP) kinases are activated by pleiotropic signals including environmental stresses, growth factors, and hormones. JNK-interacting protein 1 (JIP1) is a scaffold protein that assembles and facilitates the activation of the mixed lineage kinase-dependent JNK module and also establishes an interaction with beta-amyloid precursor protein that has been partially characterized. Here we show that, similarly to other proteins involved in various neurological diseases, JIP1 becomes hyperphosphorylated following activation of stress-activated and MAP kinases. By immobilized metal affinity chromatography and a combined microcapillary LC/MALDI-TOF/ESI-ion trap mass spectrometry approach, we identified 35 sites of mitotic phosphorylation within JIP1, among which eight were present within (Ser/Thr)-Pro sequence. This motif is modified by various kinases in aggregates of the microtubule-associated protein tau, which generates typical intraneuronal lesions occurring in Alzheimer disease. Most of the post-translational modifications found were located within the JNK, MAP kinase kinase, and RAC-alpha Ser/Thr protein kinase binding regions; no modifications occurred in protein Src homology 3 and phosphotyrosine interaction domains, which are essential for binding to kinesin, beta-amyloid precursor protein, and MAP kinase kinase kinase. Protein phosphorylation is known to affect stability and protein-protein interactions. Thus, the findings that JIP1 is extensively phosphorylated after activation of stress-activated and MAP kinases indicate that these signaling pathways might modulate JIP1 signaling by regulating its stability and association with some, but not all, interacting proteins.