basket/JNK
The p21-activated protein kinases (PAKs) are activated through direct interaction with the GTPases Rac and Cdc42Hs, which are implicated in the control of the mitogen-activated protein kinase (MAP kinase) c-Jun N-terminal kinase (JNK) and the reorganization of the actin cytoskeleton. The exact role of the PAK proteins in these signaling pathways is not entirely clear. To elucidate the biological function of Pak2 and to identify its molecular targets, a novel two-hybrid system, the Ras recruitment system (RRS), was used. RRS aims to detect protein-protein interactions at the inner surface of the plasma membrane. The Pak2 regulatory domain (PakR) was fused at the carboxyl terminus of a RasL61 mutant protein and screened against a myristoylated rat pituitary cDNA library. Four clones were identified that interact specifically with PakR and three were subsequently shown to encode a previously unknown homolog of the GTPase Cdc42Hs. This approximately 36 kDa protein, designated Chp, exhibits an overall sequence identity to Cdc42Hs of approximately 52%. Chp contains two additional sequences at the amino and carboxyl termini that are not found in any known GTPase. The amino terminus contains a polyproline sequence, typically found in Src homology 3 (SH3)-binding domains; the carboxyl terminus appears to be important for Pak2 binding. Results from the microinjection of Chp into cells implicates Chp in the induction of lamellipodia and shows that Chp activates the JNK MAP kinase cascade (Aronheim, 1998).
The Rho, Rac and Cdc42 GTPases coordinately regulate the organization of the actin cytoskeleton and the JNK MAP kinase pathway. Mutational analysis of Rac has shown that these two activities are mediated by distinct cellular targets, though their identities are not known. Two Rac targets, p65(PAK) and MLK, are ser/thr kinases that have been reported to be capable of activating the JNK pathway. Evidence is presented that neither is the Rac target mediating JNK activation in Cos-1 cells. Yeast two-hybrid selection identified POSH as a new target of Rac. This protein consists of four SH3 domains and ectopic expression leads to the activation of the JNK pathway and to nuclear translocation of NF-kappaB. When overexpressed in fibroblasts, POSH is a strong inducer of apoptosis. It is proposed that POSH acts as a scaffold protein and contributes to Rac-induced signal transduction pathways leading to diverse gene transcriptional changes (Tapon, 1998).
The multidomain protein POSH (plenty of SH3s) acts as a scaffold for the JNK pathway of neuronal death. This pathway consists of a sequential cascade involving activated Rac1/Cdc42, mixed-lineage kinases (MLKs), MAP kinase kinases (MKKs) 4 and 7, c-Jun N-terminal kinases (JNKs) and c-Jun, and is required for neuronal death induced by various means including nerve growth factor (NGF) deprivation. In addition to binding GTP-Rac1, POSH binds MLKs both in vivo and in vitro, and complexes with MKKs 4 and 7 and with JNKs. POSH overexpression promotes apoptotic neuronal death and this is suppressed by dominant-negative forms of MLKs, MKK4/7 and c-Jun, and by an MLK inhibitor. Moreover, a POSH antisense oligonucleotide and a POSH small interfering RNA (siRNA) suppress c-Jun phosphorylation and neuronal apoptosis induced by NGF withdrawal. Thus, POSH appears to function as a scaffold in a multiprotein complex that links activated Rac1 and downstream elements of the JNK apoptotic cascade (Xu, 2003).
During neuromuscular junction formation, agrin secreted from motor neurons causes muscle cell surface acetylcholine receptors (AChRs) to cluster at synaptic sites by mechanisms that are insufficiently understood. The Rho family of small guanosine triphosphatases (GTPases), including Rac and Cdc42, can mediate focal reorganization of the cell periphery in response to extracellular signals. The role of Rac and Cdc42 in coupling agrin signaling to AChR clustering was investigated. Agrin causes marked muscle-specific activation of Rac and Cdc42 in differentiated myotubes, as detected by biochemical measurements. Moreover, this activation is crucial for AChR clustering, since the expression of dominant interfering mutants of either Rac or Cdc42 in myotubes blocks agrin-induced AChR clustering. In contrast, constitutively active Rac and Cdc42 mutants cause AChR to aggregate in the absence of agrin. Activation of AChR clustering was detected using three separate measurements and was demonstrated both with endogenous Rac in nontransfected muscle cultures and with ectopically expressed Rac and Cdc42 in transfected myotubes. The most direct demonstration involves agrin-induced increases in the selective binding of activated (GTP-bound) Rac and Cdc42 to a Rac/Cdc42-binding domain derived from PAK, a downstream effector molecule that is thought to link Rac activation to actin polymerization. Additional evidence for agrin stimulation of Rac/Cdc42 was obtained by recording the activation of a second downstream component, JNK, and the conclusion that JNK activation by agrin is indeed mediated by Rac and Cdc42 was confirmed by showing that this activation is blocked by overexpression of dominant negative mutants of either G protein. By indicating that agrin-dependent activation of Rac and Cdc42 constitutes a critical step in the signaling pathway leading to AChR clustering, these findings suggest a novel role for these Rho-GTPases: the coupling of neuronal signaling to a key step in neuromuscular synaptogenesis. How might Rac and Cdc42 mediate the agrin-initiated clustering of AChR and other constituents of the neuromuscular junction? The ability of activated Rac/Cdc42 to induce reorganization of cortical actin by modulating the dynamics of actin polymerization is well documented. Moreover, surface AChR is thought to be attached to the actin cytoskeleton via a complex in which rapsyn links AChR to the actin-binding protein utrophin, and recent findings indicate that agrin-induced AChR aggregation involves the clustering of these diffusely distributed complexes at sites of MuSK activation. Thus it is thought that agrin-induced activation of Rac/Cdc42 produces highly localized reorganization of cortical actin cytoskeleton, resulting in redistribution of actin-anchored AChR-containing complexes into clusters (Weston, 2000).
A genetic mosaic system has been used to conduct an in vivo analysis of the effects of Rac1 activation on the developing intestinal epithelium. Expression of a constitutively active human Rac1 (Rac1Leu61) in the 129/Sv-derived small intestinal epithelium of C57Bl/6-ROSA26 -> 129/Sv chimeric mice leads to precocious differentiation of some lineages with accompanying alterations in their apical actin. The underlying mechanisms have been explored in this study. Rac1Leu61 leads to accumulation of the 46 kDa form of phosphorylated Jun N-terminal kinase (p-Jnk) in the apical cytoplasm, but not in the nucleus of E18.5 proliferating and differentiating intestinal epithelial cells. The effect is cell-autonomous, selective for this mitogen-activated protein kinase family member, and accompanied by apical cytoplasmic accumulation of p21-activated kinase. c-Jun, a downstream nuclear target of p-Jnk, does not show evidence of enhanced phosphorylation, providing functional evidence for cytoplasmic sequestration of p-Jnk in Rac1Leu61-expressing epithelium. In adult chimeras, Rac1 activation augments cell proliferation in crypts of Lieberkühn, without a compensatory change in basal apoptosis and produces a dramatic, very unusual widening of villi. These results reveal a novel in vivo paradigm for Rac1 activation involving p-Jnk-mediated signaling at a distinctive extra-nuclear site, with associated alterations in the actin cytoskeleton. They also provide a new perspective about the determinants of small intestinal villus morphogenesis (Stappenbeck, 2001).
CrkII, a cellular homolog of v-crk, belongs to a family of adaptor proteins that play a central role in signal transduction cascades. CrkII interacts directly with c-Jun N-terminal kinase 1 (JNK1). A proline-rich sequence of JNK1 is critical for the interaction of the kinase with the N-terminal Src homology 3 (SH3) domain of CrkII. JNK1 is localized with CrkII in membrane ruffles of Crk-overexpressing cells in a Rac1-dependent manner. A JNK1 mutant (K340A) that fails to interact with CrkII is defective in Rac/epidermal growth factor-induced activation, but remains responsive to UVC irradiation. Furthermore, CrkII recruits JNK1 to a p130Cas multiprotein complex (see CAS/CSE1 segregation protein) where it may be activated through a hematopoietic progenitor kinase 1- and mitogen-activated protein kinase kinase 4-dependent pathway. Together, the results presented here argue for a new mechanism of regulation of the JNK pathway through the CrkII-p130Cas adaptor complex (Girardin, 2001).
The data presented here strongly support the hypothesis that the CrkII-JNK1 interaction is functionally linked to JNK1 activation by CrkII. (1) By using CrkDelta(N)SH3, a CrkII mutant protein harboring single mutations which alter the structure of the N-terminal SH3 domain, it has been shown that JNK1 interacts with CrkII through CrkII's N-terminal SH3 domain. While wild-type CrkII activates JNK1, CrkDelta(N)SH3 overexpression fails to do so. (2) Since mutation in the CrkII N-terminal SH3 domain could also affect other signaling pathways, the role of CrkII-JNK1 interaction in JNK1 activation was directly assessed by mutating a lysine residue in a proline-rich cluster of JNK1 that shares high homology with the CrkII N-terminal SH3 binding domain motif. Indeed, this lysine to alanine substitution strongly decreases both the CrkII-JNK1 interaction and CrkII-induced JNK activation. Since the activation of JNK1 by CrkII is concomitant with the appearance of N-terminally phosphorylated c-Jun in the nucleus, it is very likely that CrkII regulates the transcription of genes dependent upon the activity of c-Jun. However, the possibility that JNK1 also phosphorylates cytoplasmic proteins cannot be excluded (Girardin, 2001).
In conclusion, these results argue for a role of the p130Cas-CrkII interface as a scaffolding complex regulated by Rac1, which is involved in a specific signaling pathway leading to JNK1 activation. These findings suggest that different routes leading to JNK activation may be physically separated by different scaffolding proteins, such as JIP-1 or the p130Cas-CrkII complex. The sequestration of kinase cascades could, in turn, allow cells to modulate their responses to various stimuli (Girardin, 2001).
FAK and its associated signaling pathways mediate cell cycle progression by integrins. The potential role and mechanism of Pyk2, a tyrosine kinase closely related to FAK, in cell cycle regulation was investigated by using tetracycline-regulated expression system as well as chimeric molecules. Induction of Pyk2 inhibits G(1) to S phase transition whereas comparable induction of FAK expression accelerates it. Furthermore, expression of a chimeric protein containing Pyk2 N-terminal and kinase domain and FAK C-terminal domain (PFhy1) increases cell cycle progression as does FAK. Conversely, the complementary chimeric molecule containing FAK N-terminal and kinase domain and Pyk2 C-terminal domain (FPhy2) inhibit cell cycle progression to an even greater extent than Pyk2. Biochemical analyses indicate that Pyk2 and FPhy2 stimulated JNK activation whereas FAK or PFhy1 have little effect on it, suggesting that differential activation of JNK by Pyk2 may contribute to its inhibition of cell cycle progression. In addition, Pyk2 and FPhy2 to a greater extent also inhibit Erk activation in cell adhesion whereas FAK and PFhy1 stimulate it, suggesting a role for Erk activation in mediating differential regulation of cell cycle by Pyk2 and FAK. A role for Erk and JNK pathways in mediating the cell cycle regulation by FAK and Pyk2 was also confirmed by using chemical inhibitors for these pathways. While FAK and PFhy1 are present in focal contacts, Pyk2 and FPhy2 were localized in the cytoplasm. Interestingly, both Pyk2 and FPhy2 (to a greater extent) are tyrosine phosphorylated and associated with Src and Fyn. This suggests that they may inhibit Erk activation in an analogous manner as the mislocalized FAK mutant DeltaC14 by competing with endogenous FAK for binding signaling molecules such as Src and Fyn. This model is further supported by an inhibition of endogenous FAK association with active Src by Pyk2 and FPhy2 and a partial rescue by FAK of Pyk2-mediated cell cycle inhibition (Zhao, 2000).
Axin negatively regulates the Wnt pathway during axis formation and plays a central role in cell growth control and tumorigenesis. Axin also serves as a scaffold protein for mitogen-activated protein kinase activation and the structural requirement for this activation have been determined. Overexpression of Axin in 293T cells leads to differential activation of mitogen-activated protein kinases, with robust induction for c-Jun NH(2)-terminal kinase (JNK)/stress-activated protein kinase, moderate induction for p38, and negligible induction for extracellular signal-regulated kinase. Axin forms a complex with MEKK1 through a novel domain that is termed MEKK1-interacting domain. MKK4 and MKK7, which act downstream of MEKK1, are also involved in Axin-mediated JNK activation. Domains essential in Wnt signaling, that is, binding sites for adenomatous polyposis coli, glycogen synthase kinase-3beta, and beta-catenin, are not required for JNK activation, suggesting distinct domain utilization between the Wnt pathway and JNK signal transduction. Dimerization/oligomerization of Axin through its C terminus is required for JNK activation, although MEKK1 is capable of binding C terminus-deleted monomeric Axin. Furthermore, Axin without the MEKK1-interacting domain has a dominant-negative effect on JNK activation by wild-type Axin. The results suggest that Axin, in addition to its function in the Wnt pathway, may play a dual role in cells through its activation of JNK/stress-activated protein kinase signaling cascade (Zhang, 1999).
Axin is a scaffold protein that controls multiple important pathways, including the canonical Wnt pathway and JNK signaling. An Axin-interacting protein, Aida, blocks Axin-mediated JNK activation by disrupting Axin homodimerization. During investigation of in vivo functions of Axin/JNK signaling and aida in development, it was found that Axin, besides ventralizing activity by facilitating β-catenin degradation, possesses a dorsalizing activity that is mediated by Axin-induced JNK activation. This dorsalizing activity is repressed when aida is overexpressed in zebrafish embryos. Whereas Aida-MO injection leads to dorsalized embryos, JNK-MO and MKK4-MO can ventralize embryos. The anti-dorsalization activity of aida is conferred by its ability to block Axin-mediated JNK activity. It is further demonstrated that dorsoventral patterning regulated by Axin/JNK signaling is independent of maternal or zygotic Wnt signaling. Thus a dorsalization pathway has been identified that is exerted by Axin/JNK signaling and its inhibitor Aida during vertebrate embryogenesis (Rui, 2007).
Stimulation of a variety of cell surface receptors enhances the enzymatic activity of MAPKs. MAPKs have been classified in three subfamilies: extracellular signal-regulated kinases (ERKs), stress-activated protein kinases or c-Jun NH2-terminal kinases (SAPKs/JNKs), and p38 kinase. Whereas the pathway linking cell surface receptors to ERKs has been partially elucidated, the activation mechanism of JNKs is still poorly understood. Stimulation of G protein-coupled receptors can effectively induce JNK in NIH 3T3 cells. Stimulation of m1 and m2 muscarinic receptors (mAChRs) leads to JNK activation; however, this effect is not mimicked by expression of activated forms of G protein alpha subunits. In contrast, overexpression of Gbetagamma subunits potently induces JNK activity. Signaling from m1 and m2 mAChRs to JNK involves betagamma subunits of heterotrimeric G proteins, acting on a Ras and Rac1-dependent pathway (Coso, 1996).
Retinoic acid induces the differentiation of P19 mouse embryonal carcinoma cells into endoderm, and increases expression of the heterotrimeric G-protein subunits Galpha12 and Galpha13. Retinoic acid was found to induce differentiation and sustain activation of c-Jun amino-terminal kinase, but not for ERK1,2 or of p38 mitogen-activated protein kinases. Much like retinoic acid, expression of constitutively active forms of Galpha12 and Galpha13 induce differentiation and constitutive activation of c-Jun amino-terminal kinase. Expression of the dominant negative form of c-Jun amino-terminal kinase 1 blocks both the activation of c-Jun amino-terminal kinase and the induction of endodermal differentiation in the presence of retinoic acid. These data implicate c-Jun amino-terminal kinase as downstream of Galpha12 or Galpha13 and as obligate for retinoic acid-induced differentiation (Jho, 1997).
Integrins induce the formation of large complexes of cytoskeletal and signaling proteins, which regulate many intracellular processes. The activation and assembly of signaling complexes involving focal adhesion kinase (FAK: Drosophila homolog Focal adhesion kinase-like) occurs late in integrin signaling, downstream from actin polymerization. Integrin-mediated activation of the non-receptor tyrosine kinase Syk in hematopoietic cells is independent of FAK and actin polymerization, and suggests the existence of a distinct signaling pathway regulated by Syk. Multiple proteins are activated by Syk, downstream of engagement of the platelet/megakaryocyte-specific integrin alphaIIbbeta3. The guanine nucleotide exchange factor Vav1 is inducibly phosphorylated in a Syk-dependent manner in cells following their attachment to fibrinogen. Together, Syk and Vav1 trigger lamellipodia formation in fibrinogen-adherent cells; both Syk and Vav1 colocalize with alphaIIbbeta3 in lamellipodia but not in focal adhesions. Additionally, Syk and Vav1 cooperatively induce activation of Jun N-terminal kinase (JNK), extracellular-signal-regulated kinase 2 (ERK2) and the kinase Akt, and phosphorylation of the oncoprotein Cbl in fibrinogen-adherent cells. Activation of all of these proteins by Syk and Vav1 is not dependent on actin polymerization. It is concluded that Syk and Vav1 regulate a unique integrin signaling pathway that differs from the FAK pathway in its proximity to the integrin itself, its localization to lamellipodia, and its activation, which is independent of actin polymerization. This pathway may regulate multiple downstream events in hematopoietic cells, including Rac-induced lamellipodia formation, tyrosine phosphorylation of Cbl, and activation of JNK, ERK2 and the phosphatidylinositol 3'-kinase-regulated kinase Akt (Miranti, 1998).
The extracellular matrix exerts a stringent control on the proliferation of normal cells, suggesting the existence of a mitogenic signaling pathway activated by integrins, but not significantly by growth factor receptors. Evidence has been found that integrins cause a significant and protracted activation of Jun NH2-terminal kinase (JNK), while several growth factors cause more modest or no activation of this enzyme. Integrin-mediated stimulation of JNK requires the association of focal adhesion kinase (FAK) with a Src kinase and p130CAS, the phosphorylation of p130CAS, and subsequently, the recruitment of Crk. Ras and PI-3K are not required. FAK-JNK signaling is necessary for proper progression through the G1 phase of the cell cycle. These findings establish a role for FAK in both the activation of JNK and the control of the cell cycle, and identify a physiological stimulus for JNK signaling that is consistent with the role of Jun in both proliferation and transformation (Oktay, 1999).
What is the mechanism by which FAK activates JNK? Upon activation, FAK undergoes autophosphorylation at tyrosine 397 and combines with the SH2 domain of Src or Fyn. The most prominent substrates of the FAK/Src complex are the docking adaptor proteins p130CAS and paxillin. Both contain tyrosine phosphorylation sites conforming to the consensus for binding to the adaptor protein Crk. However, while paxillin has only two such sites and does not appear to associate efficiently with Crk in response to integrin ligation, p130CAS contains nine Crk-binding motifs and associates well with Crk in cells adhering to fibronectin. The expression of dominant-negative versions of FAK, Src, p130CAS, and Crk suppress the activation of JNK by integrins. These findings provide evidence that integrin-mediated activation of JNK requires the association of FAK with Src (or Fyn) and p130CAS, and the recruitment of Crk. Thus, it appears that the beta1 and alphav integrins activate JNK and ERK via two separate pathways. By contrast, the alpha6beta4 integrin, which is presumably unable to activate FAK because it does not contain the sequences required for its recruitment, is coupled to JNK signaling via the Ras-PI-3K-Rac pathway. The identity of genes regulated by JNK is largely unknown, but they must include genes important for cell proliferation. The evidence for this is several fold: (1) deregulated expression of c-Jun or its mutated viral version v-Jun is sufficient to cause neoplastic transformation of primary avian and mammalian fibroblasts; (2) primary fibroblasts derived from c-Jun minus mice display a severe proliferation defect; and (3) several oncoproteins, including v-Src, activated Ras, v-Crk, Bcr-Abl, and Met, potently activate JNK and there is evidence to suggest that this activation is required to cause neoplastic transformation. Despite the clear requirement for c-Jun transcriptional activity in cell proliferation, it has been difficult to identify a physiological, nonstress stimulus for JNK consistent with its role in the regulation of AP-1 transcription. With the notable exception of EGF, mitogenic neuropeptides, and muscarinic receptor ligands, which indeed activate FAK or the related kinase PYK-2, most growth factors cause a relatively modest activation of JNK. The results indicating that integrin ligation causes a significant activation of JNK and TRE-dependent transcription provide a physiological stimulus for JNK signaling that is consistent with its role in the control of cell proliferation (Oktay, 1999 and references).
Palytoxin is a novel skin tumor promoter that does not activate protein kinase C. Palytoxin stimulates a sodium-dependent signaling pathway that activates the c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK) in Swiss 3T3 fibroblasts. A JNK kinase known as the stress-activated protein kinase/extracellular signal-regulated kinase-1 (SEK1) plays an important role in the regulation of JNK by palytoxin. Palytoxin stimulates the sustained activation of both JNK and SEK1 in COS7 and HeLa cells. Transiently expressed SEK1 isolated from palytoxin-treated cells can phosphorylate and activate JNK, which, in turn, can phosphorylate c-Jun. Furthermore, expression of a dominant negative mutant of SEK1 blocks activation of JNK by palytoxin. Sodium appears to play an important role in the regulation of JNK and SEK1 by palytoxin. Activation of JNK and SEK1 by palytoxin, but not anisomycin, requires extracellular sodium. The sodium ionophore gramicidin can mimic palytoxin by regulating JNK and SEK1 through a sodium-dependent mechanism (Kuroki, 1997).
In mammalian cells TGF-ß initiates a signaling cascade leading to Stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) activation. Expression of dominant-interfering forms of various components of the SAPK/JNK signaling pathways (including Rho-like GTPases, mitogen-activated protein kinase (MAPK) kinase kinase 1 (MEKK1), MAPK kinase 4 (MKK4), SAPK/JNK, and cJun) abolishes TGF-ß-mediated signaling (Atfi, 1997a). A distinct murine member of the MAPKKK family, TAK1 (Drosophila homolog: TGF-ß activated kinase 1), is a potential component of both TGF-ß and BMP4, in that the kinase activity of TAK1 is stimulated in response to both ligands. TAK1 activates SAPK/JNK, possibly by direct modification of TAK1 by type I and type II TGF-ß receptors, or indirectly through another cascade (Yamaguchi, 1995).
Transforming growth factor-beta (TGF-beta) exerts its effects on cell proliferation, differentiation and migration in part through its modulation of extracellular matrix components, such as fibronectin and plasminogen activator inhibitor-1 (PAI-1). Although the SMAD family of proteins recently has been shown to be a key participant in TGF-beta signaling, other signaling pathways have also been shown to be activated by TGF-beta. c-Jun N-terminal kinase (JNK), a member of the MAP kinase family, is activated in response to TGF-beta in a human fibrosarcoma line. Stable expression of dominant-negative forms of JNK1 and MKK4, an upstream activator of JNK, results in loss of TGF-beta-stimulated fibronectin mRNA and protein induction, while having little effect on TGF-beta-induced levels of PAI-1. The human fibronectin promoter contains three CRE elements, one of which has been shown to bind a c-Jun-ATF-2 heterodimer. There is a decrease in transactivating potential of GAL4-c-Jun and GAL4-ATF-2 in dominant-negative JNK1- and MKK4-expressing cells. TGF-beta-induced fibronectin synthesis is independent of Smad4. These results demonstrate that TGF-beta-mediated fibronectin induction requires activation of JNK, which in turn modulates the activity of c-Jun and ATF-2 in a Smad4 independent manner (Hocevar, 1999).
In mammalian cells, a specific stress-activated protein kinase (SAPK/JNK) pathway is activated in response to inflammatory cytokines, injury from heat, chemotherapeutic drugs and UV or ionizing radiation. The mechanisms that link these stimuli to activation of the SAPK/JNK pathway in different tissues remain to be identified. Hematopoietic progenitor kinase 1 (hpk1) encodes a serine/threonine kinase sharing similarity with the kinase domain of Ste20. HPK1 specifically activates the SAPK/JNK pathway after transfection into COS1 cells, but does not stimulate the p38/RK or mitogen-activated ERK signaling pathways. Activation of SAPK requires a functional HPK1 kinase domain and HPK1 signals via the SH3-containing mixed lineage kinase MLK-3 and the known SAPK activator SEK1. HPK1 therefore provides an example of a cell type-specific input into the SAPK/JNK pathway. The developmental specificity of its expression suggests a potential role in hematopoietic lineage decisions and growth regulation (Kiefer, 1996).
Mixed lineage kinase-3 (MLK-3) is a 97 kDa serine/threonine kinase of unknown function with multiple interaction domains, including a Cdc42 binding motif. Cdc42 and the related small GTP binding protein Rac1 (See Drosophila Rac1) can activate the SAPK/JNK and p38/RK stress-responsive kinase cascades, suggesting that MLK-3 may have a role in upstream regulation of these pathways. MLK-3 can specifically activate the SAPK/JNK and p38/RK pathways, but has no effect on the activation of ERKs. Immunoprecipitated MLK-3 catalyzes the phosphorylation of SEK1 in vitro, and co-transfect MLK-3 induced phosphorylation of SEK1 and MKK3 at sites required for activation, suggesting direct regulation of these protein kinases. Interactions between MLK-3 and SEK and MLK-3 and MKK6 have been observed in co-precipitation experiments. Kinase-dead mutants of MLK-3 block activation of the SAPK pathway by a newly identified mammalian analog of Ste20, germinal center kinase, but not by MEKK, suggesting that MLK-3 functions to activate the SAPK/JNK and p38/RK cascades in response to stimuli transduced by Ste20-like kinases (Tibbles, 1996).
The c-Jun N-terminal kinase (JNK), or stress-activated protein kinase, plays a crucial role in cellular responses stimulated by environmental stress and proinflammatory cytokines. However, the mechanisms that lead to the activation of the JNK pathway have not been elucidated. A cDNA encoding a novel protein kinase was identified that has significant sequence similarity to human germinal center kinase (GCK) and human hematopoietic progenitor kinase 1. The novel GCK-like kinase (GLK) has a nucleotide sequence that encodes an ORF of 885 amino acids with 11 kinase subdomains. Endogenous GLK could be activated by UV radiation and proinflammatory cytokine tumor necrosis factor alpha. When transiently expressed in 293 cells, GLK specifically activates the JNK, but not the p42/44(MAPK)/extracellular signal-regulated kinase or p38 kinase signaling pathways. Interestingly, deletion of amino acids 353-835 in the putative C-terminal regulatory region, or mutation of Lys-35 in the putative ATP-binding domain, markedly reduces the ability of GLK to activate JNK. This result indicates that both kinase activity and the C-terminal region of GLK are required for maximal activation of JNK. Furthermore, GLK-induced JNK activation can be inhibited by a dominant-negative mutant of mitogen-activated protein kinase kinase kinase 1 (MEKK1) or mitogen-activated protein kinase kinase 4/SAPK/ERK kinase 1 (SEK1), suggesting that GLK may function upstream of MEKK1 in the JNK signaling pathway (Diener, 1997).
In rat liver epithelial cells (GN4), angiotensin II (Ang II) and thapsigargin stimulate a novel calcium-dependent tyrosine kinase (CADTK) also known as PYK2, CAKbeta, or RAFTK. Activation of CADTK by a thapsigargin-dependent increase in intracellular calcium fails to stimulate the extracellular signal-regulated protein kinase pathway but is well correlated with a 30-50-fold activation of c-Jun N-terminal kinase (JNK). In contrast, Ang II, which increases both protein kinase C (PKC) activity and intracellular calcium, stimulates extracellular signal-regulated protein kinase but produced a smaller, less sustained, JNK activation than thapsigargin. These findings suggest either that CADTK is not involved in JNK activation or that PKC activation inhibits the CADTK to JNK pathway. A 1-min phorbol pretreatment of GN4 cells inhibits thapsigargin-dependent JNK activation by 80-90%. The consequence of PKC-dependent JNK inhibition is reflected in c-Jun and c-Fos mRNA induction following treatment with thapsigargin and Ang II. Thapsigargin, which only minimally induces c-Fos, produces a much greater and more prolonged c-Jun response than Ang II. In summary, two pathways stimulate JNK in cells expressing CADTK, a calcium-dependent pathway modifiable by PKC and cAMP-dependent protein kinase and a stress-activated pathway independent of CADTK and PKC; the inhibition by PKC can ultimately alter gene expression initiated by a calcium signal (Li, 1997).
Staurosporine, a protein kinase inhibitor, is known to mimic the effect of nerve growth factor (NGF) in promoting neurite outgrowth. Staurosporine induces the activation of a kinase with an apparent molecular mass of 57 kDa. The dose of staurosporine required to activate this kinase is consistent with that required to induce neurite outgrowth. Interestingly, the staurosporine-activated kinase is immunoprecipitated by anti-c-Jun NH2-terminal kinase (JNK) isoforms antibody, but not by anti-JNK1-specific antibody or anti-ERK1 antibody, raising the possibility that this kinase is a novel JNK isoform. The substrate specificity of the kinase is distinct from those of osmotic shock-activated JNKs and NGF-activated ERK1. The kinase phosphorylates transcription factors, including c-Jun, Elk-1, and ATF2, as well as myelin basic protein; this suggests that it plays a role in gene induction. Staurosporine induces immediate-early genes, including Nur77 and fos, but not jun. The activation of the staurosporine-activated kinase, as well as the induction of neurite outgrowth, does not require Ras function; however, Ras is required for the activation of ERKs and neurite outgrowth induced by NGF. Taken together, these results indicate staurosporine specifically activates a JNK isoform, which may contribute to biological activities, including neurite outgrowth (Yao, 1997).
Lysophosphatidylcholine (lyso-PC), a natural lipid generated through the action of phospholipase A2 on membrane phosphatidylcholine, has been implicated in atherogenesis and the inflammatory process. In vitro studies have established a role for lyso-PC in modulation of gene expression and other cellular responses including differentiation and proliferation. There is also evidence that lyso-PC may act as an intracellular second messenger transducing signals elicited from membrane-associated receptors. The mechanisms underlying the diverse activities of lyso-PC are poorly understood. Treatment of cultured cells with exogenous lyso-PC, at nontoxic concentrations, potently induces activator protein-1 (AP-1) DNA binding and transcriptional activity independent of well known AP-1 activators, either protein kinase C or mitogen-activated protein kinases ERK1 and ERK2. Lyso-PC also activates the c-Jun N-terminal kinase (JNK/SAPK). The stimulated JNK and AP-1 activities probably mediate or contribute to some bioactive effects of lyso-PC 9 (Fang, 1997).
Vav is a hematopoietic cell-specific guanine nucleotide exchange factor (GEF) whose activation is mediated by receptor engagement. The relationship of Vav localization to its function is presently unclear. Vav redistributes to the plasma membrane in response to Fcin receptor I (FcinRI) engagement. The redistribution of Vav is mediated by its Src homology 2 (SH2) domain and requires Syk activity. The FcinRI and Vav colocalize and are recruited to glycosphingolipid-enriched microdomains (GEMs). The scaffold protein, linker for activation of T cells (LAT), and Rac1 (a target of Vav activity) were constitutively present in GEMs. Expression of an SH2 domain-containing COOH-terminal fragment of Vav inhibits Vav phosphorylation and movement to the GEMs but had no effect on the tyrosine phosphorylation of the adaptor protein, SLP-76 (SH2 domain-containing leukocyte protein of 76 kD), and LAT. However, assembly of the multiprotein complex containing these proteins was inhibited. In addition, FcinRI-dependent activation of c-Jun NH(2)-terminal kinase 1 (JNK1) is also inhibited. Thus, Vav localization to the plasma membrane is mediated by its SH2 domain and may serve to regulate downstream effectors like JNK1 (Arudchandran, 2000).
c-Jun N-terminal kinases (JNKs) are potently activated by a number of cellular stimuli. Small GTPases, in particular Rac, are responsible for initiating the activation of the JNK pathways. So far, the signals leading from extracellular stimuli to the activation of Rac have remained elusive. The Src homology 2 (SH2)- and Src homology 3 (SH3)-containing adaptor protein Crk is capable of activating JNK when ectopically expressed. Transient expression of Crk induces JNK activation, and this activation is dependent on both the SH2- and SH3-domains of Crk. Expression of p130(Cas), a major binding protein for the Crk SH2-domain, also induces JNK activation, which is blocked by the SH2-mutant of Crk. JNK activation by Cas and Crk is effectively blocked by a dominant-negative form of Rac, suggesting a linear pathway from the Cas-Crk-complex to the Rac-JNK activation. Many of the stimuli that activate the Rac-JNK pathway enhance engagement of the Crk SH2-domain. JNK activation by these stimuli, such as epidermal growth factor, integrin ligand binding and v-Src, is efficiently blocked by dominant-negative mutants of Crk. In turn, a dominant-negative form of Cas blocks integrin-mediated JNK activation, but not that of epidermal growth factor nor v-Src. Together, these results demonstrate an important role for Crk in connecting multiple cellular stimuli to the Rac-JNK pathway, and a role for the Cas-Crk complex in integrin-mediated JNK activation (Dolfi, 1998).
Malfolded proteins in the endoplasmic reticulum (ER) induce cellular stress and activate c-Jun amino-terminal kinases (JNKs or SAPKs). Mammalian homologs of yeast IRE1 (the product of the inositol auxotrophy gene), which activate chaperone genes in response to ER stress, also activate JNK; IRE1alpha-/- fibroblasts are impaired in JNK activation by ER stress. The cytoplasmic part of IRE1 binds TRAF2, an adaptor protein that couples plasma membrane receptors to JNK activation. Dominant-negative TRAF2 inhibits activation of JNK by IRE1. Activation of JNK by endogenous signals initiated in the ER proceeds by a pathway similar to that initiated by cell surface receptors in response to extracellular signals (Urano, 2000).
Retinoic acid (RA), a potent teratogen, produces a characteristic set of embryonic cardiovascular malformations similar to those observed in neural crest ablated avians. A subset of cranial neural crest, the cardiac neural crest, has been implicated in cardiovascular development. This population extends from the midotic placode to the third or fourth somite and appears to be highly sensitive to retinoids. The aortic arches and the outflow tract malformations described in human retinoic acid embryopathy have long been observed in animals exposed to high doses of RA. While the effects of RA on neural crest are well described, the molecular mechanism(s) of RA action on these cells is less clear. The present study examines the relationship between RA and mitogen-activated protein kinase signaling in neural crest cells and demonstrates that c-Jun N-terminal kinase (JNK) activation is severely repressed by RA. RA suppresses migration and proliferation of primary cultures of mouse neural crest cells treated in vitro as well as from animals treated in vivo. On Western blots, JNK activation/phosphorylation in neural crest cultures is reduced, while neither extracellular signal-regulated kinase (ERK) nor p38 pathways are affected. Both the dose-dependent stimulation of neural crest outgrowth and JNK phosphorylation by platelet-derived growth factor AA, which promotes outgrowth but not proliferation of neural crest cultures, are completely abrogated by RA. To establish the relevance of the JNK signaling pathway to cardiac neural crest migration, dominant negative adenoviral constructs were used to inhibit upstream activation of JNK or c-Jun downstream responses. Both adenoviral constructs markedly reduce neural crest cell outgrowth, while a dominant negative inhibitor of the p38 pathway has no effect. These data demonstrate that the JNK signaling pathway and c-Jun activation are critical for cardiac neural crest outgrowth and are potential targets for the action of RA (Li, 2001).
Presenilin 1 (PS1) plays a pivotal role in Notch signaling and the intracellular metabolism of the amyloid ß-protein. To understand intracellular signaling events downstream of PS1, the action of PS1 on mitogen-activated protein kinase pathways has been investigated. Overexpressed PS1 suppresses the stress-induced stimulation of stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase (JNK) in human embryonic kidney 293 cells. Interestingly, two functionally inactive PS1 mutants, PS1(D257A) and PS1(D385A), fail to inhibit UV-stimulated SAPK/JNK. Furthermore, H2O2- or UV-stimulated SAPK activity is higher in mouse embryonic fibroblast (MEF) cells from PS1-null mice than in MEF cells from PS+/+ mice. MEFPS1(-/-) cells were more sensitive to the H2O2-induced apoptosis than MEFPS1(+/+) cells. Ectopic expression of PS1 in MEFPS1(-/-) cells suppressesH2O2-stimulated SAPK/JNK activity and apoptotic cell death. Together, these data suggest that PS1 inhibits the stress-activated signaling by suppressing the SAPK/JNK pathway (Kim, 2001).
Thus, PS1 inhibits the SAPK/JNK pathway. Ectopically expressed PS1 blocks the stress-induced stimulation of SAPK/JNK and its upstream kinases, including SEK1 and MEKK1. FAD-linked PS1 mutants, M146V, C410Y, and L286V, are also able to inhibit the SAPK stimulation. Interestingly, biologically inactive PS1 mutants D257A and D385A, both of which have been shown to lack gamma-secretase activation and PS1 endoproteolysis, fail to inhibit SAPK stimulation. Furthermore, PS1DeltaEx9, which lacks the endoproteolysis site but is competent for activation of gamma-secretase activity, retains the inhibitory effect on the SAPK/JNK pathway. These data suggest that the gamma-secretase activation, rather than the PS1 endoproteolysis, is required for the PS1-induced inhibition of the SAPK/JNK pathway. gamma-Secretase has two major substrates, APP and Notch. The cleavage of APP or Notch by gamma-secretase produces Aß or the intracellular domain of Notch, Notch-IC, respectively. Aß1-42 does not inhibit the SAPK/JNK activity. In comparison, preliminary data showed that overexpression of the Notch intracellular domain, which is the active form of intracellular Notch, results in suppression of SAPK/JNK activation. These findings imply that PS1-mediated cleavage of Notch might be involved in the mechanism of PS1-induced suppression of the SAPK pathway. In this regard, Notch has been previously proposed to play a role in the regulation of the SAPK/JNK pathway (Kim, 2001 and references therein).
Several lines of evidence suggest that presenilins are involved in apoptosis. Overexpression of PS2 has been shown to potentiate apoptosis of PC12 cells induced by NGF withdrawal or neurotoxic Aß1-42. ALG3, a truncated form of murine PS2, reduces T cell receptor-induced or Fas-induced apoptosis in a mouse T cell hybridoma. Studies using PS1-null mice have demonstrated that PS1 is involved in neuronal survival. Ectopic PS1 suppresses the H2O2-induced apoptosis in B103 neuroblastoma cells. Moreover, deficiency of PS1 causes an elevation in the H2O2-induced apoptosis in MEF cells from PS1-null mice, as compared with MEF cells from PS1+/+ wild-type mice. The H2O2-induced apoptosis is blocked by overexpression of SEK1(K129R), suggesting that the SAPK/JNK pathway is involved in the mechanism of the H2O2-induced apoptosis. These findings suggest that PS1, by inhibiting the SAPK pathway, can protect cells from stress-induced apoptotic cell death. However, further study is needed to determine the precise mechanism by which PS1 inhibits the SAPK/JNK pathway (Kim, 2001).
Genetic studies have identified Drosophila Naked Cuticle (Nkd) as an antagonist of the canonical Wnt/ß-catenin signaling pathway, but its mechanism of action remains obscure. A mammalian homolog of Naked cuticle, mNkd, has been cloned. mNkd interacts directly with Dishevelled. Dishevelled is an intracellular mediator of both the canonical Wnt pathway and planar cell polarity (PCP) pathway. Activation of the c-Jun-N-terminal kinase has been implicated in the PCP pathway. mNkd has been shown to acts in a cell-autonomous manner not only to inhibit the canonical Wnt pathway but also to stimulate c-Jun-N-terminal kinase activity. Expression of mNkd disrupts convergent extension in Xenopus, consistent with a role for mNkd in the PCP pathway. These data suggest that mNkd may act as a switch to direct Dishevelled activity toward the PCP pathway, and away from the canonical Wnt pathway (Yan, 2001).
Activation of JNK seems to be an important step in the PCP pathway, and a vertebrate cognate of the Drosophila PCP pathway controls convergent extension movements during vertebrate development. In both Xenopus and Drosophila, hyperactivation of this pathway disrupts PCP signaling without affecting the canonical Wnt pathway. Consistent with its ability to activate JNK in vitro, mNkd overexpression inhibits the normal elongation of Xenopus embryos. The normal formation of anterior structures in these embryos indicates that the phenotype is not the result of ventralization, suggesting that mNkd inhibits convergent extension. To assess more directly the effects of mNkd on convergent extension, open-face Keller explants of the dorsal mesoderm were examined. Such explants made from control embryos elongate and change shape significantly, whereas explants made from embryos expressing mNkd fail to elongate. These effects are similar to those elicited by overexpression of other wild-type components of the planar cell polarity cascade, such as Xdsh and Xfz-8, indicating a role for mNkd in controlling the PCP pathway (Yan, 2001).
Genetic and biochemical studies have shown that Dishevelled controls cell polarity by acting as an upstream activator of the JNK pathway both in vivo and in vitro. Because mNkd is directly associated with Dishevelled, whether mNkd participates in the JNK pathway was tested. NIH 3T3 cells were transfected with expression constructs of mNkd and c-Jun, in which c-Jun served to monitor JNK activities. In this assay, expression of mNkd or mDvl alone induces a strong phosphorylation of c-Jun that was detected by blotting with an antibody specific for phosphoserine-63. These data show that mNkd has an effect similar to Dishevelled in activating the JNK pathway in mammalian cell culture assays (Yan, 2001).
The corepressors N-CoR and SMRT partner with histone deacetylases (HDACs) in diverse repression pathways. GPS2, a protein involved in intracellular signaling, is an integral subunit of the N-CoR-HDAC3 complex. Structural motifs that direct the formation of a highly stable and active deacetylase complex have been determined. GPS2 and TBL1, another component of the N-CoR-HDAC3 complex, interact cooperatively with repression domain 1 of N-CoR to form a heterotrimeric structure and are indirectly linked to HDAC3 via an extended N-CoR SANT domain that also activates latent HDAC3 activity. More importantly, the N-CoR-HDAC3 complex inhibits JNK activation through the associated GPS2 subunit and thus could potentially provide an alternative mechanism for hormone-mediated antagonism of AP-1 function (Zhang, 2002).
The MLK family of mitogen activated protein kinase kinase kinases (MAPKKK) has been shown to activate Jun N-terminal kinase/stress-activated protein kinase 1 (JNK/SAPK1). However, little is known of the in vivo functions of the MLKs. A Xenopus laevis MLK has been identified that shows highest homology with mammalian MLK2 (62%) and, like MLK2, interacts preferentially with the Rho-family GTPase Rac. xMLK2 was expressed zygotically from late gastrula/early neurula. Surprisingly, this expression is restricted to the cement gland, the brain, and the pronephros. In the differentiating cement gland, xMLK2 expression correlates with cell elongation and the onset of a previously unobserved apoptotic phase, while in the pronephros, expression corresponds with the differentiation and opening of the nephric tubules. Overexpression of xMLK2 in COS7 cells leads to a SEK1/MKK4 (MAPKK)-dependent hyperactivation of JNK in response to UV irradiation. xMLK2 has been shown to be required for normal cement gland development and pronephric tubule formation using antisense inactivation and a dominant negative xMLK2. The data suggest a novel role for the MLKs as tissue-restricted mediators of signal transduction. They also suggest that tissue-specific responses to common extracellular signals may in part result from the programmed expression of MAPKKKs with differing specificities (Poitras, 2003).
MEKK1-deficient mice show an eye open at birth phenotype caused by impairment in embryonic eyelid closure. MEK kinase 1 (MEKK1) is highly expressed in the growing tip of the eyelid epithelium, which displays loose cell-cell contacts and prominent F-actin fibers in wild-type mice, but compact cell contacts, lack of polymerized actin and a concomitant impairment in c-Jun N-terminal phosphorylation in MEKK1-deficient mice. In cultured keratinocytes, MEKK1 is essential for JNK activation by TGF-ß and activin, but not by TGF-alpha. MEKK1-driven JNK activation is required for actin stress fiber formation, c-Jun phosphorylation and cell migration. However, MEKK1 ablation does not impair other TGF-ß/activin functions, such as nuclear translocation of Smad4. These results establish a specific role for the MEKK1-JNK cascade in transmission of TGF-ß and activin signals that control epithelial cell movement, providing the mechanistic basis for the regulation of eyelid closure by MEKK1. This study also suggests that the signaling mechanisms that control eyelid closure in mammals and dorsal closure in Drosophila are evolutionarily conserved (Zhang, 2003).
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. The molecular cloning is described 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 has demonstrated that JIP3 is present in the cytoplasm and accumulates 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 (Kelkar, 2000).
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: (1) artificially induced dimerization of dual leucine zipper-bearing kinase (DLK) confirmed that DLK dimerization is sufficient to induce DLK activation; (2) under basal conditions, DLK associated with JIP is held in a monomeric, unphosphorylated and catalytically inactive state; (3) JNK recruitment to JIP coincides with significantly decreased affinity of JIP and DLK. JNK promotes the dimerization, phosphorylation and activation of JIP-associated DLK. Similarly, treatment of cells with okadaic acid inhibits DLK association with JIP and results 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 (Nihalani, 2001).
Using a yeast two-hybrid method, amyloid precursor protein (APP)-interacting molecules were sought by screening mouse and human brain libraries. In addition to known interacting proteins containing a phosphotyrosine-interaction-domain (PID), the following were identified as novel APP-interacting molecules: Fe65, Fe65L, Fe65L2, X11, and mDab1, a PID-containing isoform of mouse JNK-interacting protein-1 (JIP-1b) and its human homolog IB1 (the established scaffold proteins for JNK). The APP amino acids Tyr682, Asn684, and Tyr687 in the G681YENPTY687 region are all essential for APP/JIP-1b interaction, but neither Tyr653 nor Thr668 are necessary. APP-interacting ability is specific for this additional isoform containing PID and is shared by both human and mouse homologs. JIP-1b expressed by mammalian cells is efficiently precipitated by the cytoplasmic domain of APP in the extreme Gly681-Asn695 domain-dependent manner. Reciprocally, both full-length wild-type and familial Alzheimer's disease mutant APPs are precipitated by PID-containing JIP constructs. Antibodies raised against the N and C termini of JIP-1b coprecipitate JIP-1b and wild-type or mutant APP in non-neuronal and neuronal cells. Moreover, human JNK1ß1 forms a complex with APP in a JIP-1b-dependent manner. Confocal microscopic examination demonstrates that APP and JIP-1b share similar subcellular localization in transfected cells. These data indicate that JIP-1b/IB1 scaffolds APP with JNK, providing a novel insight into the role of the JNK scaffold protein as an interface of APP with intracellular functional molecules (Matsuda, 2001).
JIP-1 was initially characterized as a cytoplasmic inhibitor of JNK family kinases and subsequently found to interact with MKK7, MLK, DLK, and HPK-1 in addition to JNK. Coexpression of JIP-1 and JNK with MKK7 or MLK3 increases JNK activation. These findings have established that JIP-1 scaffolds the kinase components of the JNK signaling pathway. An additional isoform of JIP-1 has been reported in mouse (JIP-1b), rat [islet-brain-1 (IB1)], and human (IB1). This isoform contains a 47-residue insertion that completes the PID region at the C terminus, which was originally identified in Shc interaction with NPXY in the cytoplasmic domain of the epidermal growth factor receptor. Neither the physiological nor the pathological role of the JIP-1 proteins has become totally clear, whereas expression of JIP-1 has been reported to transcriptionally activate the GLUT2 promoter and is implicated in the pathogenesis of a form of type 2 familial diabetes mellitus and in the cytoprotection of insulin-secreting cells. The present study thus provides the first line of evidence that the JNK scaffold protein, abundant in the brain and in islet ß-cells, could be relevant to Alzheimer's disease. Interestingly, it has been reported that in vivo, neurotoxicity by hippocampal administration of Ab1-42 occurs only in diabetic rats (Matsuda, 2001 and references therein).
Analysis of subcellular localization using transfected cells indicates that JIP-1b and APP colocalize in the cytoplasm but both are not detected in the nuclei. Similar cytoplasmic localization of JIP-1b has been reported although other studies have reported that IB1 is a nuclear protein. Nuclear localization of JIP-1 proteins have been demonstrated in cerebellar granule cells, and JIP-1 proteins localize in the cytoplasm in unpolarized NIE115 and PC12 cells but are concentrated at neurites when the cells are polarized. These differences in JIP-1 localization thus may reflect different functions of JIP-1 proteins assigned in different cell environments. Although the present study provides evidence that JIP-1b interacts with APP inside the transfected cells, it would be necessary to investigate whether endogenous APP and JIP-1b interact in nontransfected cells. Yet the notion that JIP-1b/IB1 colocalizes with APP is consistent with earlier studies indicating that the subcellular and brain regional localizations of JIP proteins considerably overlap with those of APP. Because the putative alpha-secretase ADAM10 and the putative ß-secretase BACE are expressed in the same neurons that express APP in the mouse brain, APP cleavage by these putative secretases would lose the interaction of APP with JIP-1b/IB1, causing, in turn, a loss in the ability of JIP-1b/IB1 to specifically colocalize signaling molecules with APP. Although so far coimmunoprecipitate APP with IB1 from rat brain homogenates has not been demonstrated, it remains unclear whether this failure is caused by inappropriate experimental conditions for specific immunoprecipitation of the APP/IB1 complex from solubilized brain homogenates or whether it implies that, with the APP/IB1 complex being a minor fraction, the majority of APP and IB1 in the brain does not complex with each other or form complexes with different partners. The latter notion is consistent, at least in part, with the observed relatively lower maximal binding of JIP-1b to the cytoplasmic domain of APP, as compared with those of the other PID-containing proteins tested (Matsuda, 2001).
By constructing deletion and point mutants, it has been shown that the domains necessary for the APP/JIP-1b interaction are the cytoplasmic G681YENPTY687 region in APP and the PID region in JIP-1b, completed by the insertion specific for this isoform. This accounts for the PID-nonbearing isoform JIP-1 not interacting with APP. As noted above, X11, Fe65, Fe65L, and mDab1 have been shown to interact with the C terminus of APP. The present study indicates that the APP/JIP-1b interaction requires Tyr682, Asn684, and Tyr687 contained in the G681YENPTY687 region. This is different from the mode of APP interaction with Fe65 and X11 and similar to that with mDab1. Interestingly, the JIP-1b isoform, which is interactive with APP, is the major transcript in the brain, and the noninteractive JIP-1 transcript is hardly detected, pointing to certain specific roles of the JIP-1b isoform in neuronal functions (Matsuda, 2001).
The mechanism underlying the observed JIP-1b/IB1 interaction with APP is thus consistent with the established NPXY motif interaction of PID in Shc and IRS-1. Yet in the present GST pull-down experiments, the cytoplasmic domains of APP, APLP1, and APLP2, all of which contain the same NPXY structure GYENPTY, show largely different binding intensities for JIP-1b/IB1, with APP being the strongest among them. These different binding characteristics might reflect the difference in the primary to ternary structures surrounding the NPXY motif, suggesting the presence of an additional structural requirement allowing NPXY to interact efficiently with PID. In support of this idea, the most recent literature, in which PID of JIP-1b is shown to interact with p190 rhoGEF, indicates that the binding region of p190 does not contain the classical NPXY motif (Matsuda, 2001).
Because JIP-1b shows binding similar to full-length APP regardless of the presence of four different FAD mutations, JIP-1b is most likely involved in the basic function of APP. Although the binding of Fe65 or X11 to APP has been shown to affect Aß secretion from APP, so far remarkable changes in Aß42 secretion from NL-APP have not been demonstrated by cotransfection with JIP-1b. The JIP-1 proteins have been shown to serve as scaffold proteins for the organization of active JNK signaling complexes. In fact, APP associates with JNK via JIP-1b. It has also been established that APP interacts with the GTP-binding protein Go through the middle portion in the APP cytoplasmic domain adjacent to the NPXY-containing C terminus. It is likely, therefore, that APP may serve as a membrane-anchoring protein that further scaffolds the JIP-scaffolding complex with other signaling molecules. Taking into account the recently cloned members of the JIP family, JIP2 and JIP3 (PID is contained in JIP2 but not in JIP3), it may be worthwhile to investigate whether APP might regulate the JNK signaling pathway through the binding of these various JIP proteins to the cytoplasmic domain of APP (Matsuda, 2001).
Overactivation of ionotropic glutamate receptors can induce neuronal death, a phenomenon known as excitotoxicity. Cell survival during this response is determined by a balance among signaling cascades, including those that recruit the Akt and JNK pathways. A novel interaction is described between Akt1 and JNK interacting protein 1 (JIP1), a JNK pathway scaffold. Direct association between Akt1 and JIP1 is observed in primary neurons. Neuronal exposure to an excitotoxic stimulus decreases the Akt1-JIP1 interaction and concomitantly increases association between JIP1 and JNK. Akt1 interaction with JIP1 inhibits 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 excitotoxicity 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 (Yano, 2002).
In several systems, the JNK pathway plays a positive role in apoptosis. Glutamate or kainate exposure activates the JNK pathway in primary neurons; this activation is responsible for subsequent apoptotic death. How is an excitotoxicity-specific JNK response generated? The JIP family of JNK scaffolds (also islet-brain [IB] or JNK/stress-activated kinase-associated protein [JSAP]) has been suggested to play a critical role in assembling specific JNK signaling pathway components. Each member of this scaffold family can bind JNK, MKK7, and a mixed-lineage kinase (MLK, a MAPKKK family) on different regions of JIP. Experiments using transiently transfected cell lines have suggested that JIP1 can amplify MLK-induced JNK activation. Recent evidence from mice genetically deficient in JIP1 has demonstrated that this scaffold is a stimulus-specific, positive regulator of JNK activity in vivo. Significantly, JIP1 gene deletion confers higher resistance to kainate-induced neuronal death in mice and neuronal culture, indicating that JIP1 plays an positive role in AMPA/kainate receptor-mediated apoptotic signaling (Yano, 2002).
JNK activity can be antagonized by Akt kinase activity in numerous cell systems, and this crosstalk may underlie many of the prosurvival effects of Akt. The Akt family of Ser/Thr-directed protein kinases (Akt1-3 or protein kinase Balpha-gamma) are important mediators of cell survival in response to growth factors and stimuli that elicit calcium influx. Akt kinases have been suggested to phosphorylate a number of proapoptotic proteins directly, thereby leading to suppression of death signals. This study describes a novel mechanism of Akt-JNK crosstalk in neurons undergoing excitotoxic apoptosis. Evidence that Akt1 binding to JIP1 decreases JIP1's ability to enhance JNK activity by interfering with JIP1-mediated assembly of an active JNK signaling complex. Excitotoxic kainate exposure decreases the neuronal interaction between Akt1 and JIP1 and increases formation of JNK-JIP1 complexes, suggesting that Akt1 interaction with JIP1 acts as a negative switch for JNK activity. Consistent with this model, Akt1 gene deletion renders neurons more susceptible to kainate-induced neuronal death, and ectopic expression of Akt1 binding mutants decreases kainate toxicity (Yano, 2002).
The radial migration of differentiating neurons provides an essential step in the generation of laminated neocortex, although the molecular mechanism of this migration is not fully understood. The protein levels of a JNK activator kinase, MUK/DLK/ZPK, and JNK activity increase potently and temporally in newly generated neurons in developing mouse telencephalon during radial migration. MUK/DLK/ZPK, as well as other mixed lineage kinases have been shown to associate with scaffold proteins, JIPs (which also interact directly with MKK7, a MAPKK class protein kinase), and with JNK. JIPs have been identified as cargo of the kinesin motor functioning in vesicle transport. In addition, JIPs carrying MUK/DLK/ZPK also bind to a Reelin receptor, ApoER2. The ectopic expression of MUK/DLK/ZPK in neural precursor cells in utero impairs radial migration, whereas it allows these cells to leave the ventricular zone and differentiate into neural cells. The MUK/DLK/ZPK protein is associated with dotted structures that are frequently located along microtubules and with Golgi apparatus in cultured embryonic cortical cells. In COS-1 cells, MUK/DLK/ZPK overexpression impairs the radial organization of microtubules without massive depolymerization. These results suggest that MUK/DLK/ZPK and JNK regulate radial cell migration via microtubule-based events (Hirai, 2002).
The effect of exogenous MUK/DLK/ZPK expression is cell-autonomous, and the radial glial scaffold and radial migration of other neurons are apparently not affected. Therefore, MUK/DLK/ZPK expression may affect the interaction with radial glia and/or cell migration itself. It should also be noted that the MUK/DLK/ZPK protein associates with dotted structures that are frequently located along microtubules and that the overexpression of MUK/DLK/ZPK in COS-1 cells induces microtubule reorganization, as characterized by the disappearance of radial microtubule organization without massive depolymerization of the microtubules and disruption of centrosomes. Since a well-organized microtubule network is essential for the directed migration of cells, these observations may explain, at least in part, the neuronal migration disorder observed with the constitutive expression of MUK/DLK/ZPK. This unique type of microtubule disorganization has not been commonly reported except in the case of Lis1 overexpression in COS-7 cells. The LIS1 gene is responsible for a neural cell migration disorder and type I lissencephaly in humans: the Lis1 protein associates with the dynein-dynactin complex, a microtubule motor. Therefore, the similar effects of MUK/DLK/ZPK and Lis1 overexpression on microtubule organization support the idea that the MUK/DLK/ZPK-JNK pathway regulates neural cell migration via a microtubule-based event (Hirai, 2002 and references therein).
Genetic arguments about the function of JIPs, which are scaffold proteins for the MUK/DLK/ZPK-JNK pathway, also support this model of regulation. Sunday driver, a Drosophila homolog of JIP3/JSAP1, has recently been identified as a receptor for kinesin motors; it functions in vesicle transport. Moreover, Caenorhabditis elegans JNK and JNK kinases, as well as UNC-16, a nematode homolog of JIP3, have been shown to regulate vesicle transport in neurons. Since vesicle transport supports cell locomotion by driving the endocytic cycle, JIP and JNK may be essential not only for axonal transport but also for neural cell migration. The MUK-associated dotted structures observed in the primary culture of cortical cells may correspond to vesicular cargo in secretory or endocytic pathways. Notably, mammalian JIPs bind to a Reelin receptor, ApoER2, as well as to the kinesin light chain. Therefore, they may mediate the transport of vesicular cargoes containing the Reelin receptor, which is essential for the radial migration of neural cells in the cortical plate. Even though this possibility has not been tested, the results suggest the intriguing possibility that the MUK/DLK/ZPK-JNK pathway supported by JIPs transfers a signal from Reelin to microtubules for the regulation of neural cell migration (Hirai, 2002).
In combination with studies that show that MUK/DLK/ZPK is a MAPKKK for the JNK pathway, the coincidence of MUK/DLK/ZPK expression and JNK activation in the developing cortex indicates that the JNK activity is regulated by the expression level of MUK/DLK/ZPK. The preferential activation of JNK among three MAPK-related protein kinases in the E16 telencephalon is consistent with observations in COS cells overexpressing MUK/DLK/ZPK. The ratio of active JNK to total JNK in the intermediate zone of the E16 cortex is roughly comparable to that in NIH3T3 cells irradiated with UV, a potent activator of JNK, indicating the potency of JNK activity in differentiating neurons. Although the relationship between the activation of the MUK/DLK/ZPK-JNK pathway and the migration rate is not clear at present, the observations suggest that MUK/DLK/ZPK-JNK modulates microtubule organization and temporally disrupts the unipolar cell shape, and probably also the radial migration of immature neurons just after they leave the ventricular zone. This step may be required for the newly generated neurons to prepare for maturation and provide them with a chance for tangential dispersion, which is also essential for the formation of a functional neocortex. With JNK activation, radial migration is retarded or pauses; this may provide a chance for tangential migration. The MUK protein level and JNK activity are reduced upon the progression of neural differentiation or by specific factors present on the pial side of the intermediate zone. Then, a definite leading edge is formed, and radial migration is accelerated (Hirai, 2002).
Lipopolysaccharide (LPS) is recognized by Toll-like receptor (TLR) 4 and activates NF-kappaB and a set of MAP kinases. Proteins associated with the cytoplasmic domain of mouse TLR4 have been investigated by yeast two-hybrid screening: JNK-interacting protein 3 (JIP3), a scaffold protein for JNK, was identified as a TLR4-associated protein. The homolog of JIP3 has been reported in Drosophila and Caenorhabiditis elegans (Bowman, 2000; Byrd, 2001), indicating JIP3 is evolutionally well conserved. In mammalian cells, JIP3, through its N-terminal region, constitutively associates with TLR4. The association is specific to JIP3, as evidenced by the observation that the two other JIPs, JIP1 and JIP2, fail to bind TLR4. In HEK 293 cells exogenously expressing TLR4, MD2 and CD14, co-expression of JIP3 significantly increases the complex formation of TLR4-JNK and LPS-mediated JNK activation. In contrast, expression of C-terminally truncated forms of JIP3 impairs LPS-induced JNK activation in a mouse macrophage cell line, RAW264.7. Moreover, RNA interference of JIP3 inhibits 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, these data indicate the involvement of JIP3 in JNK activation in downstream signals of some TLRs (Byrd, 2001).
The c-Jun NH2-terminal kinase (JNK) is activated during obesity. One consequence of obesity is that JNK phosphorylates the adapter protein insulin receptor substrate 1 (IRS-1) on Ser 307 and inhibits signaling by the insulin receptor. JNK can therefore cause peripheral insulin resistance during obesity and may contribute to the development of type 2 diabetes. The JNK-interacting protein 1 (JIP1) scaffold protein, which binds components of the JNK signaling module, is essential for JNK activation in the adipose tissue of obese mice. These data identify JIP1 as a novel molecular target for therapeutic intervention in the development of obesity (Jaeschke, 2004).
Previous studies have demonstrated that the JIP1 scaffold protein is required for sustained JNK activation in neurons caused by ischemia but is not required for JNK activation caused by other stimuli. This study demonstrates that JIP1 is also essential for obesity-induced JNK activation in fat and muscle. This finding provides a new example of a MAP kinase signaling module that is regulated by a scaffold protein in vivo. JIP1-dependent JNK activation in fat contributes to the regulation of insulin sensitivity and adipose tissue mass. Drugs that inhibit JNK may be useful for the treatment of obesity and diabetes, but this approach may be limited by potential toxicity. The identification of an essential role for the JIP1 scaffold protein is therefore important. Inhibition of JIP1-dependent JNK activation is likely to provide greater therapeutic specificity than a general inhibitor of JNK activity (Jaeschke, 2004).
The development of neuronal polarity is essential for the determination of neuron connectivity and for correct brain function. The c-Jun N-terminal kinase (JNK)-interacting protein-1 (JIP1) is highly expressed in neurons and has previously been characterized as a regulator of JNK signaling. JIP1 has been shown to localize to neurites in various neuronal models, but the functional significance of this localization is not fully understood. JIP1 is also a cargo of the motor protein kinesin-1, which is important for axonal transport. This study demonstrated that before primary cortical neurons become polarized, JIP1 specifically localizes to a single neurite and that after axonal specification, it accumulates in the emerging axon. JIP1 is necessary for normal axonal development and promotes axonal growth dependent upon its binding to kinesin-1 and via a newly described interaction with the c-Abl tyrosine kinase. JIP1 associates with and is phosphorylated by c-Abl, and the mutation of the c-Abl phosphorylation site on JIP1 abrogates its ability to promote axonal growth. The kinesin-1-dependent localization of JIP1 to developing axonal growth cones and its colocalization with dynamic microtubules and exploratory processes emanating from the growth cone suggest that it may be important for the regulation of cytoskeletal structures. c-Abl is a well-established regulator of cytoskeletal dynamics, and JIP1 may be an important downstream effector of c-Abl in cytoskeletal reorganization. The JIP1 scaffold protein, therefore, has the potential to act as a crucial link between extracellular signals and the regulation of cytoskeletal dynamics that lead to axonal development (Dajas-Bailador, 2008).
JNK proteins are ubiquitously expressed, evolutionarily conserved MAP kinases that are involved in stress responses. The JNK cascade in Xenopus oocytes exhibits sustained, all-or-none responses to graded, transient stimuli. The character of the JNK cascade's response has been examined in mammalian cells. The steady-state responses of JNK to sorbitol and anisomycin are highly ultrasensitive in HeLa cells, HEK 293 cells, and Jurkat T cells. The JNK responses are also reversible, not sustained, as is the case in oocytes. Jurkat cells activate their JNK in response to phorbol myristate acetate (PMA), and the response of the entire population of Jurkat cells was graded. However, analysis of subpopulations of the PMA-treated Jurkat cells reveals that the steady-state responses of both JNK and CD69, a T cell surface activation marker, are essentially all-or-none in character. These studies show that the JNK cascade commonly exhibits switch-like responses to a variety of stimuli (Bagowski, 2003).
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