The AP-1 repressor, JDP2, is a bona fide substrate for the c-Jun N-terminal kinase

Phenylalanine hydroxylase (PAH) is activated by its substrate phenylalanine and inhibited by its cofactor tetrahydrobiopterin (BH(4)). The crystal structure of PAH revealed that the N-terminal sequence of the enzyme (residues 19-29) partially covered the enzyme active site, and suggested its involvement in regulation. We show that the protein lacking this N-terminal sequence does not require activation by phenylalanine, shows an altered structural response to phenylalanine, and is not inhibited by BH(4). Our data support the model where the N-terminal sequence of PAH acts as an intrasteric autoregulatory sequence, responsible for transmitting the effect of phenylalanine activation to the active site.     

Phosphorylation of microtubule-associated protein tau by isoforms of c-Jun N-terminal kinase (JNK)

Microtubule-associated protein tau in a hyperphosphorylated state is the major component of the filamentous lesions that define a number of neurodegenerative diseases commonly referred to as tauopathies. Hyperphosphorylation of tau at most sites appears to precede filament assembly. Many of the hyperphosphorylated sites are serine/threonine-proline sequences. Here we show that c-Jun N-terminal kinases JNK1, JNK2 and JNK3 phosphorylate tau at many serine/threonine-prolines, as assessed by the generation of the epitopes of phosphorylation-dependent anti-tau antibodies. Of the three protein kinases, JNK2 phosphorylated the most sites in tau, followed by JNK3 and JNK1. Phosphorylation by JNK isoforms resulted in a greatly reduced ability of tau to promote microtubule assembly. These findings extend the number of candidate protein kinases for the hyperphosphorylation of tau in Alzheimer's disease and other neurodegenerative disorders.     

Cell-type-specific activation of c-Jun N-terminal kinase by salicylates

Salicylates inhibit signaling by tumor necrosis factor (TNF), including TNF-induced activation of mitogen-activated protein kinases (MAPKs). On the other hand, we recently showed that in normal human diploid fibroblasts sodium salicylate (NaSal) elicits activation of p38 MAPK but not activation of c-Jun N-terminal kinase (JNK). Here we show that NaSal treatment of COS-1 or HT-29 cells produced a sustained c-Jun N-terminal kinase (JNK) activation. Activation of JNK or p38 MAPK by NaSal (or aspirin) was not due to a nonspecific hyperosmotic effect because much higher molar concentrations of sorbitol or NaCl were required to produce a similar activation. Three structurally unrelated nonsteroidal antiinflammatory drugs (ibuprofen, acetaminophen, and indomethacin) failed to induce significant activation of JNK or p38 MAPK, suggesting that cyclooxygenase inhibition is not the underlying mechanism whereby salicylates induce p38 MAPK and JNK activation. Activation of JNK and p38 MAPKs may be relevant for some antiinflammatory actions of salicylates.     

Type 2 cGMP-dependent protein kinase regulates homeostasis by blocking c-Jun N-terminal kinase in the colon epithelium

Analysis of knockout animals indicates that 3′,5′cyclic guanosine monophosphate (cGMP) has an important role in gut homeostasis but the signaling mechanism is not known. The goals of this study were to test whether increasing cGMP could affect colon homeostasis and determine the mechanism. We increased cGMP in the gut of Prkg2^+/+ and Prkg2^−/− mice by treating with the PDE5 inhibitor Vardenafil (IP). Proliferation, differentiation and apoptosis in the colon mucosa were then quantitated. Vardenafil (Vard) treatment increased cGMP in colon mucosa of all mice, but reduced proliferation and apoptosis, and increased differentiation only in Prkg2^+/+ mice. Vard and cGMP treatment also increased dual specificity protein phosphatase 10 (DUSP10) expression and reduced phospho-c-Jun N-terminal kinase (JNK) levels in the colon mucosa of Prkg2^+/+ but not Prkg2^−/− mice. Treatment of Prkg2^−/− mice with the JNK inhibitor SP600125 reversed the defective homeostasis observed in these animals. Activation of protein kinase G2 (PKG2) in goblet-like LS174T cells increased DUSP10 expression and reduced JNK activity. PKG2 also increased goblet cell-specific MUC2 expression in LS174T cells, and this process was blocked by DUSP10-specific siRNA. The ability of cGMP signaling to inhibit JNK-induced apoptosis in vivo was demonstrated using dextran sodium sulfate (DSS) to stress the colon epithelium. Vard was a potent inhibitor of DSS-induced epithelial apoptosis, and significantly blocked pathological endpoints in this model of experimental colitis. In conclusion, Vard treatment activates cGMP signaling in the colon epithelium. Increased PKG2 activity alters homeostasis by suppressing proliferation and apoptosis while promoting differentiation. The PKG2-dependent mechanism was shown to involve increased DUSP10 and subsequent inhibition of JNK activity.     

Regulation of c-Jun N-terminal kinase by MEKK-2 and mitogen-activated protein kinase kinase kinases in rheumatoid arthritis

The mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK) is a critical regulator of collagenase-1 production in rheumatoid arthritis (RA). The MAPKs are regulated by upstream kinases, including MAPK kinases (MAPKKs) and MAPK kinase kinases (MAP3Ks). The present study was designed to evaluate the expression and regulation of the JNK pathway by MAP3K in arthritis. RT-PCR studies of MAP3K gene expression in RA and osteoarthritis synovial tissue demonstrated mitogen-activated protein kinase/ERK kinase kinase (MEKK) 1, MEKK2, apoptosis-signal regulating kinase-1, TGF-beta activated kinase 1 (TAK1) gene expression while only trace amounts of MEKK3, MEKK4, and MLK3 mRNA were detected. Western blot analysis demonstrated immunoreactive MEKK2, TAK1, and trace amounts of MEKK3 but not MEKK1 or apoptosis-signal regulating kinase-1. Analysis of MAP3K mRNA in cultured fibroblast-like synoviocytes (FLS) showed that all of the MAP3Ks examined were expressed. Western blot analysis of FLS demonstrated that MEKK1, MEKK2, and TAK1 were readily detectable and were subsequently the focus of functional studies. In vitro kinase assays using MEKK2 immunoprecipitates demonstrated that IL-1 increased MEKK2-mediated phosphorylation of the key MAPKKs that activate JNK (MAPK kinase (MKK)4 and MKK7). Furthermore, MEKK2 immunoprecipitates activated c-Jun in an IL-1 dependent manner and this activity was inhibited by the selective JNK inhibitor SP600125. Of interest, MEKK1 immunoprecipitates from IL-1-stimulated FLS appeared to activate c-Jun through the JNK pathway and TAK1 activation of c-Jun was dependent on JNK, ERK, and p38. These data indicate that MEKK2 is a potent activator of the JNK pathway in FLS and that signal complexes including MEKK2, MKK4, MKK7, and/or JNK are potential therapeutic targets in RA.     

Phosphorylation of proteins and apoptosis induced by c-Jun N-terminal kinase1 activation in rat cardiomyocytes by H(2)O(2) stimulation

Cytokines and various cellular stresses are known to activate c-Jun N-terminal kinase-1 (JNK1), which is involved in physiological function. Here, we investigate the activation of JNK1 by oxidative stress in H9c2 cells derived from rat cardiomyocytes. H(2)O(2) (100 microM) significantly induces the tyrosine phosphorylation of JNK1 with a peak 25 min after the stimulation. The amount of JNK1 protein remains almost constant during stimulation. Immunocytochemical observation shows that JNK1 staining in the nucleus is enhanced after H(2)O(2) stimulation. To clarify the physiological role of JNK1 activation under these conditions, we transfected antisense JNK1 DNA into H9c2 cells. The antisense DNA (2 microM) inhibits JNK1 expression by 80% as compared with expression in the presence of the sense DNA, and significantly blocks H(2)O(2)-induced cell death. Consistent with the decrease in cell number, we detected condensation of the nuclei, a hallmark of apoptosis, 3 h after H(2)O(2) stimulation in the presence of the sense DNA for JNK1. The antisense DNA of JNK1 inhibits the condensation of nuclei by H(2)O(2). Under these conditions, the H(2)O(2)-induced phosphorylation of proteins with molecular masses of 55, 72, and 78 kDa is blocked by treatment with the antisense DNA for JNK1 as compared with the sense DNA for JNK1. These findings suggest that JNK1 induces apoptotic cell death in response to H(2)O(2), and that the cell death may be involved in the phosphorylations of 55, 72, and 78 kDa proteins induced by JNK1 activation.     

Bcl-2 enhances neurite extension via activation of c-Jun N-terminal kinase

Recent studies suggest that Bcl-2 may play an active role in neuronal differentiation. Here, we showed a marked neurite extension in MN9D dopaminergic neuronal cells overexpressing Bcl-2 (MN9D/Bcl-2) or Bcl-X(L) (MN9D/Bcl-X(L)). We found a specific increase in phosphorylation of c-Jun N-terminal kinase (JNK) accompanied by neurite extension in MN9D/Bcl-2 but not in MN9D/Bcl-X(L) cells. Consequently, neurite extension in MN9D/Bcl-2 but not in MN9D/Bcl-X(L) cells was suppressed by treatment with SP600125, a specific inhibitor of JNK. Inhibition of other mitogen-activated protein kinases-including p38 and extracellular signal-regulated kinase-did not affect Bcl-2-mediated neurite extension in MN9D cells. While the expression levels of such protein markers of maturation as SNAP-25, phosphorylated NF-H, and neuron-specific enolase were increased in MN9D/Bcl-2 cells, only upregulation of SNAP-25 was inhibited after treatment with SP600125. Thus, the JNK signal activated by Bcl-2 seems to play an important role during morphological and certain biochemical differentiation in cultured dopaminergic neurons.     

Regulation of myostatin signaling by c-Jun N-terminal kinase in C2C12 cells

Myostatin, a member of the transforming growth factor beta (TGF-beta) superfamily, is a negative regulator of skeletal muscle growth. We found that myostatin could activate c-Jun N-terminal kinase (JNK) signaling pathway in both proliferating and differentiating C2C12 cells. Using small interfering RNA (siRNA) mediated activin receptor type IIB (ActRIIB) knockdown, the myostatin-induced JNK activation was significantly reduced, indicating that ActRIIB was required for JNK activation by myostatin. Transfection of C2C12 cells with TAK1-specific siRNA reduced myostatin-induced JNK activation. In addition, JNK could not be activated by myostatin when the expression of MKK4 was suppressed with MKK4-specific siRNA, suggesting that TAK1-MKK4 cascade was involved in myostatin-induced JNK activation. We also found that blocking JNK signaling pathway by pretreatment with JNK specific inhibitor SP600125, attenuated myostatin-induced upregulation of p21 and downregulation of the differentiation marker gene expression. Furthermore, it was also observed that the presence of SP600125 almost annulled the growth inhibitory role of myostatin. Our findings provide the first evidence to reveal the involvement of JNK signaling pathway in myostatin's function as a negative regulator of muscle growth.     

Endothelin promotes neurite elongation by a mechanism dependent on c-Jun N-terminal kinase

Endothelin (ET), which is known as a vasoconstrictive peptide, is associated with a lot of biological functions. Although endothelin receptors are expressed in the central nervous system (CNS), little is known about the effects of endothelin on neuronal function. In this study, we reported that endothelins elongate cortical neurites via the endothelin A receptor. All the endothelin isoforms tested, endothelin-1, endothelin-2, and endothelin-3, promoted neurite elongation. ET-1-induced neurite elongation was specifically inhibited by treatment with BQ123, an antagonist for the endothelin A receptor. In addition, inhibition of ET-1-induced c-Jun N-terminal kinase (JNK) activation by treatment with SP600125, a JNK inhibitor, also prevented the ET-1-mediated promotion of neurite elongation. Thus, endothelin induces cortical neurite elongation through the endothelin A receptor by a mechanism dependent on JNK.     

Regression of abdominal aortic aneurysm by inhibition of c-Jun N-terminal kinase

Abdominal aortic aneurysm (AAA) is a common disease among elderly people that, when surgical treatment is inapplicable, results in progressive expansion and rupture of the aorta with high mortality. Although nonsurgical treatment for AAA is much awaited, few options are available because its molecular pathogenesis remains elusive. Here, we identify JNK as a proximal signaling molecule in the pathogenesis of AAA. Human AAA tissue showed a high level of phosphorylated JNK. We show that JNK programs a gene expression pattern in different cell types that cooperatively enhances the degradation of the extracellular matrix while suppressing biosynthetic enzymes of the extracellular matrix. Selective inhibition of JNK in vivo not only prevented the development of AAA but also caused regression of established AAA in two mouse models. Thus, JNK promotes abnormal extracellular matrix metabolism in the tissue of AAA and may represent a therapeutic target.     

Phosphorylation of Bcl-2 and activation of caspase-3 via the c-Jun N-terminal kinase pathway in ursolic acid-induced DU145 cells apoptosis

There is currently no successful therapy for androgen-independent prostate cancer. Ursolic acid (UA), a pentacyclic triterpenoid compound, has been shown to have an anti-proliferative effect on various tumors. We investigated the effect of UA on cell viability in the human hormone-refractory prostate cancer cell line DU145, as well as the molecular mechanisms underlying its growth inhibiting effect. We demonstrated that UA induces apoptosis and the activation of caspase-3 in DU145 cells. UA also causes the activation of c-Jun N-terminal kinase (JNK), but has no effect on extracellular signal-regulated protein kinases (ERK1/2) and p38 MAP kinases (p38). UA-induced JNK activation could result in Bcl-2 phosphorylation (Ser70) and degradation in DU145 cells, which may be one of the molecular mechanisms by which it induces apoptosis. Although further evaluation, such as in vivo testing, is clearly needed, the present results suggest the potential utility of UA as a novel therapeutic agent in advanced prostate cancer.     

Mitogen-activated protein kinase kinase kinase kinase 4 as a putative effector of Rap2 to activate the c-Jun N-terminal kinase

Little is known about the specific signaling roles of Rap2, a Ras family small GTP-binding protein. In a search for novel Rap2-interacting proteins by the yeast two-hybrid system, we isolated isoform 3 of the human mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), a previously described but uncharacterized isoform. Other isoforms of MAP4K4 in humans and mice are known as hematopoietic progenitor kinase (HPK)/germinal center kinase (GCK)-like kinase and Nck-interacting kinase, respectively. MAP4K4 belongs to the STE20 group of protein kinases and regulates c-Jun N-terminal kinase (JNK). MAP4K4 interacted with Rap2 through its C-terminal citron homology domain but did not interact with Rap1 or Ras. Interaction with Rap2 required the intact effector region of Rap2. MAP4K4 interacted preferentially with GTP-bound Rap2 over GDP-bound Rap2 in vitro. In cultured cells, MAP4K4 colocalized with Rap2, while a mutant MAP4K4 lacking the citron homology domain failed to do so. Furthermore, Rap2 enhanced MAP4K4-induced activation of JNK. These results suggest that MAP4K4 is a putative effector of Rap2 mediating the activation of JNK by Rap2.     

Phosphorylation by the protein kinase CK2 promotes calpain-mediated degradation of IkappaBalpha.

Rapid IkappaBalpha turnover has been implicated in the high basal NF-kappaB activity in WEHI 231 B immature IgM(+) B cells. Here we show that treatment of WEHI 231 cells with apigenin, a selective inhibitor of the protein kinase CK2, decreased the rate of IkappaBalpha turnover and nuclear levels of NF-kappaB. Turnover of IkappaBalpha in these cells is mediated in part by the protease calpain. Since both CK2 and calpain target the proline-glutamic acid-serine-threonine (PEST) domain, we investigated the role of CK2 in the degradation of IkappaBalpha by calpain using an in vitro phosphorylation/degradation assay. CK2 phosphorylation enhanced mu-calpain-mediated degradation of wild-type IkappaBalpha, but not of mutant 3CIkappaBalpha, with S283A, T291A, and T299A mutations in phosphorylation sites within the PEST domain. Roles for CK2 and calpain in IkappaBalpha turnover were similarly shown in CH31 immature and CH12 mature IgM(+) B cells, but not in A20 and M12 IgG(+) B cells. These findings demonstrate for the first time that CK2 phosphorylation of serine/threonine residues in the PEST domain promotes calpain-mediated degradation of IkappaBalpha and thereby increases basal NF-kappaB levels in IgM(+) B cells.     

Synthesis and SAR of 2-phenoxypyridines as novel c-Jun N-terminal kinase inhibitors

The design and synthesis of a novel series of c-jun N-terminal kinase (JNK3) inhibitors is described. The development and optimization of the 2-phenoxypyridine series was carried out from an earlier pyrimidine series of JNK1 inhibitors. Through the optimization of the scaffold 2, several potent compounds with good in vivo profiles were discovered.     

Cyclin-dependent kinase 5 prevents neuronal apoptosis by negative regulation of c-Jun N-terminal kinase 3

Cyclin-dependent kinase 5 (cdk5) is a serine/threonine kinase activated by associating with its neuron-specific activators p35 and p39. Analysis of cdk5(-/-) and p35(-/-) mice has demonstrated that both cdk5 and p35 are essential for neuronal migration, axon pathfinding and the laminar configuration of the cerebral cortex, suggesting that the cdk5-p35 complex may play a role in neuron survival. However, the targets of cdk5 that regulate neuron survival are unknown. Here, we show that cdk5 directly phosphorylates c-Jun N-terminal kinase 3 (JNK3) on Thr131 and inhibits its kinase activity, leading to reduced c-Jun phosphorylation. Expression of cdk5 and p35 in HEK293T cells inhibits c-Jun phosphorylation induced by UV irradiation. These effects can be restored by expression of a catalytically inactive mutant form of cdk5. Moreover, cdk5-deficient cultured cortical neurons exhibit increased sensitivity to apoptotic stimuli, as well as elevated JNK3 activity and c-Jun phosphorylation. Taken together, these findings show that cdk5 may exert its role as a key element by negatively regulating the c-Jun N-terminal kinase/stress-activated protein kinase signaling pathway during neuronal apoptosis.     

The T4 RI antiholin has an N-terminal signal anchor release domain that targets it for degradation by DegP

Lysis inhibition (LIN) of T4-infected cells was one of the foundational experimental systems for modern molecular genetics. In LIN, secondary infection of T4-infected cells results in a dramatically protracted infection cycle in which intracellular phage and endolysin accumulation can continue for hours. At the molecular level, this is due to the inhibition of the holin, T, by the antiholin, RI. RI is only 97 residues and contains an N-terminal hydrophobic domain and a C-terminal hydrophilic domain; expression of the latter domain fused to a secretory signal sequence is sufficient to impose LIN, due to its specific interaction with the periplasmic domain of the T holin. Here we show that the N-terminal sequence comprises a signal anchor release (SAR) domain, which causes the secretion of RI in a membrane-tethered form and then its subsequent release into the periplasm, without proteolytic processing. Moreover, the SAR domain confers both functional lability and DegP-mediated proteolytic instability on the released form of RI, although LIN is not affected in a degP host. These results are discussed in terms of a model for the activation of RI in the establishment of the LIN state.     

Phosphorylation of Bcl-associated death protein (Bad) by erythropoietin-activated c-Jun N-terminal protein kinase 1 contributes to survival of erythropoietin-dependent cells

The glycoprotein erythropoietin (Epo) is a hematopoietic cytokine necessary for the survival of erythrocytes from immature erythroid cells. The mitogen-activated c-Jun N-terminal kinase 1 (JNK1) plays an important role in the proliferation and survival of erythroid cells in response to Epo. However, the precise mechanism of JNK1 activation promoting erythroid cell survival is incompletely understood. Here, we reported that JNK1 is required for Epo-mediated cell survival through phosphorylation and inactivation of the pro-apoptotic, Bcl-2 homology domain 3 (BH3)-only Bcl-associated death protein (Bad). Upon Epo withdrawal, HCD57 cells, a murine Epo-dependent cell line, displayed increased apoptotic cell death that was associated with decreased JNK1 activity. Epo withdrawal-induced apoptosis was promoted by inhibition of JNK1 activity but suppressed by expression of a constitutively active JNK1. Furthermore, Epo-activated JNK1 phosphorylated Bad at threonine 201, thereby inhibiting the association of Bad with the anti-apoptotic molecule B-cell lymphoma-extra large (Bcl-X(L)). Replacement of threonine 201 by alanine in Bad promoted Epo withdrawal-induced apoptosis. Thus, our results provide a molecular mechanism by which JNK1 contributes to the survival of erythroid cells.     

Regulation of apoptotic c-Jun N-terminal kinase signaling by a stabilization-based feed-forward loop

A sequential kinase cascade culminating in activation of c-Jun N-terminal kinases (JNKs) plays a fundamental role in promoting apoptotic death in many cellular contexts. The mechanisms by which this pathway is engaged in response to apoptotic stimuli and suppressed in viable cells are largely unknown. Here, we show that apoptotic stimuli increase endogenous cellular levels of pathway components, including POSH, mixed lineage kinases (MLKs), and JNK interacting protein 1, and that this effect occurs through protein stabilization and requires the presence of POSH as well as activation of MLKs and JNKs. Our findings suggest a self-amplifying, feed-forward loop mechanism by which apoptotic stimuli promote the stabilization of JNK pathway components, thereby contributing to cell death.