Search

JNK, a member of the mitogen activated protein kinases (MAPKs) superfamily, plays a key role in cell death in many neurological disorders, but systemic inhibition of JNK has detrimental side effects. JNK can be regulated by two direct upstream kinases: MAPK kinase 4 (MKK4) and MAPK kinase 7 (MKK7). Here, we investigated the effect of GADD45β-I, a recently designed cell-permeable inhibitor peptide for MKK7, on endoplasmic reticulum (ER) stress-induced cytotoxicity in neuronal HT22 cells. We found that treatment with the ER stress inducer tunicamycin (TM) increased the phosphorylation of JNK and MKK7 in HT22 cells, which was nullified by GADD45β-I. GADD45β-I significantly attenuated TM-induced toxicity via inhibiting apoptotic cell death, as evidenced by decreased number of TUNEL-positive cells and reduced caspase-3 activity. GADD45β-I treatment also decreased expression of ER stress associated pro-apoptotic proteins and prevented morphological changes of the ER after TM exposure. In addition, inhibition of mitochondrial oxidative stress and preservation of intracellular ATP levels were observed in GADD45β-I-treated cells. The experiments using siRNA transfection and Topflash reporter assay revealed a possible involvement of Wnt/β-catenin pathway in GADD45β-I-induced protection in HT22 cells. In summary, our results demonstrated that GADD45β-I exerted protective effects against TM-induced cytotoxicity via regulating JNK-Wnt pathway. Targeting MKK7 could represent a new therapeutic strategy for the treatment of neurological diseases where ER stress associated neuronal injury are involved. [ABSTRACT FROM AUTHOR]

GADD45β is selectively and constitutively expressed in Multiple Myeloma cells, and this expression correlates with an unfavourable clinical outcome. GADD45β physically interacts with the JNK kinase, MKK7, inhibiting its activity to enable the survival of cancer cells. DTP3 is a small peptide inhibitor of the GADD45β/MKK7 complex and is able to restore MKK7/JNK activation, thereby promoting selective cell death of GADD45β-overexpressing cancer cells. Enzymatic MS foot-printing and diazirine-based chemical cross-linking MS (CX-MS) strategies were applied to study the interactions between GADD45β and MKK7 kinase domain (MKK7_KD) and between DTP3 and MKK7_KD. Our data show that the binding between GADD45β and MKK7 largely occurs between GADD45β loop 2 (region 103–117) and the kinase enzymatic pocket. We also show that DTP3 interferes with this GADD45β/MKK7 interaction by contacting the MKK7 peptides, 113–136 and 259–274. Accordingly, an MKK7_KD Δ(101–136) variant lacking Trp135 did not produce a fluorescence quenching effect upon the binding of DTP3. The assessment of the interaction between GADD45β and MKK7 and the elucidation of the recognition surfaces between DTP3 and MKK7 significantly advance the understanding of the mechanism underlying the inhibition of the GADD45β/MKK7 interaction by DTP3 and pave the way to the design of small-molecule DTP3 analogues. [ABSTRACT FROM AUTHOR]

Objectives: Krüppel-like factor 5 (KLF5) modulates multiple cell processes in different cancers. It is frequently deleted and inactivated in prostate cancer and may exert a tumor suppressor function. However, how KLF5 inhibits the progression of prostate cancer is still not clear. In the present study, we identified how KLF5 and tumor necrosis factor α (TNFα) pathway, which can induce apoptosis in cancer, regulate each other in LNCaP prostate cancer cells.Material and Methods: The expression of messenger RNA and protein was detected by real-time polymerase chain reaction assay and western blot analysis, respectively. To identify whether KLF5 regulates the activity of TNFα downstream pathway, we constructed a stable KLF5 knockdown or KLF5 overexpressing cell line with lentivirus-containing short hairpin RNA targeting KLF5 or full-length KLF5 in LNCaP cells. Cell apoptosis was determined through flow cytometry assay. In addition, the regulation of KLF5 on target gene transcription was detected by reporter luciferase activity assay, and the binding of KLF5 on target promoter was detected through oligonucleotides pull-down analysis.Results: We found that TNFα induced the expression of KLF5 at both messenger RNA and protein levels; moreover, TNFα up-regulated KLF5 through TNF receptor 1 but not through TNF receptor 2 in LNCaP cells. Knockdown of KLF5 decreased apoptosis induced by TNFα, whereas cell apoptosis was increased by KLF5 overexpression. Consistently, expression of cleaved PARP and caspase-3 induced by TNFα was decreased by KLF5 knockdown, whereas it was increased by overexpressed KLF5. JNK activity is essential for the apoptosis induced by TNFα. We found that knockdown of KLF5 not only decreased the phosphorylation of JNK induced by TNFα, but also down-regulated the transcription of mitogen-activated protein kinase kinase 7 (MKK7), an upstream kinase of JNK, by binding to the MKK7 promoter.Conclusions: Our results indicate that KLF5 is an essential transcription regulator of MKK7 kinase and promotes the apoptosis induced by TNFα in LNCaP cells. Loss of KLF5 in prostate cancer may decrease cell response to TNFα-inducing apoptosis and facilitate cancer initiation and progression; moreover, KLF5 could be a potential molecular marker for predicting the effect of high-dose TNFα on tumor growth inhibition in prostate cancer. [ABSTRACT FROM AUTHOR]

In this study, we investigated the molecular mechanisms underlying the anti-inflammatory effects of α-chaconine in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages and in LPS-induced septic mice. α-Chaconine inhibited the expressions of cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) at the transcriptional level, and attenuated the transcriptional activity of activator protein-1 (AP-1) by reducing the translocation and phosphorylation of c-Jun. α-Chaconine also suppressed the phosphorylation of TGF-β-activated kinase-1 (TAK1), which lies upstream of mitogen-activated protein kinase kinase 7 (MKK7)/Jun N-terminal kinase (JNK) signaling. JNK knockdown using siRNA prevented the α-chaconine-mediated inhibition of pro-inflammatory mediators. In a sepsis model, pretreatment with α-chaconine reduced the LPS-induced lethality and the mRNA and production levels of pro-inflammatory mediators by inhibiting c-Jun activation. These results suggest that the anti-inflammatory effects of α-chaconine are associated with the suppression of AP-1, and support its possible therapeutic role for the treatment of sepsis. [ABSTRACT FROM AUTHOR]

Abstract: c-Jun N-terminal kinases (JNKs) are the exclusive downstream substrates of mitogen-activated protein kinase kinase 7 (MKK7). Recently, we have shown that a single MKK7 splice variant, MKK7γ1, substantially changes the functions of JNKs in naïve PC12 cells. Here we provide evidence that MKK7γ1 blocks NGF-mediated differentiation and sustains proliferation by interfering with the NGF-triggered differentiation programme at several levels: (i) down-regulation of the NGF receptors TrkA and p75; (ii) attenuation of the differentiation-promoting pathways ERK1/2 and AKT; (iii) increase of JNK1 and JNK2, especially the JNK2 54kDa splice variants; (iv) repression of the cyclin-dependent kinase inhibitor p21WAF1/CIP1, which normally supports NGF-mediated cell cycle arrest; (v) strong induction of the cell cycle promoter CyclinD1, and (vi) profound changes of p53 functions. Moreover, MKK7γ1 substantially changes the responsiveness to stress. Whereas NGF differentiation protects PC12 cells against taxol-induced apoptosis, MKK7γ1 triggers an escape from cell cycle arrest and renders transfected cells sensitive to taxol-induced death. This stress response completely differs from naïve PC12 cells, where MKK7γ1 protects against taxol-induced cell death. These novel aspects on the regulation of JNK signalling emphasise the importance of MKK7γ1 in its ability to reverse basic cellular programmes by simply using JNKs as effectors. Furthermore, our results highlight the necessity for the cells to balance the expression of JNK activators to ensure precise intracellular processes. [Copyright &y& Elsevier]

Abstract: The functions of mitogen-activated protein kinases (MKKs) 4 and 7 are typically associated with the c-Jun N-terminal kinase (JNK) signaling pathway. Both MKKs synergistically phosphorylate different JNK isoforms and are therefore involved in numerous physiological (e.g. differentiation and proliferation) and pathological (e.g. apoptosis and tumorigenesis) processes. MKK4 and MKK7 share similar molecular characteristics as well as several upstream activators and scaffold proteins. However, their functions are non-redundant and determined by different stimuli, biochemical interactions and differential tissue distribution. The central question is how two MKKs regulate or affect the multiple actions of their JNK substrates. Similar to JNKs, MKK4 and MKK7 can simultaneously exert divergent functions in different cellular compartments and signalosomes. It is also important to realize that the MKK effects are splice variant-specific. The present review not only summarizes the various modes of MKK4 and MKK7 activation and activity, but also their functions. We also extensively describe their impact on JNK signaling, their molecular interactions resulting in the formation of context-specific signalosomes and the functional consequences of JNK deficiency. [Copyright &y& Elsevier]

Abstract: The c-Jun N-terminal kinases (JNKs) mediate a diversity of physiological and pathophysiological effects. Apart from isoform-specific JNK activation, upstream kinases are supposed to be the relevant regulators, which are involved in the context- and signalosome-depending functions. In the present study we report the cloning and characterization of the novel rat MKK7γ1, a splice variant of MKK7 with an additional exon in the N-terminal region, in the neuronal pheochromocytoma cell line PC12. Transfected MKK7γ1 increased basal JNK activity, in particular phosphorylation of JNK2. Consequently, JNK signalling was changed in mRNA-, protein- and activation-levels of JNK targets, such as transcription factors (c-Jun, p53, c-Myc), cell cycle regulators (p21, CyclinD1) and apoptotic proteins (Fas, Bim, Bcl-2, Bcl-xl). These alterations promote the sensitivity of MKK7γ1-transfected cells towards cell death and repress cell proliferation under normal cell growth conditions. Complexes of JIP-1, MKK7 and JNK2 were the major JNK signalosomes under basal conditions. After stimulation with taxol (5μM) and tunicamycin (1.4μg/ml), MKK7γ1- but not MKK7β1-transfection, reduced cell death and even increased cell proliferation. Cellular stress also led to an increased phosphorylation of JNK1 and the almost complete abrogation of complexes of JIP-1, MKK7 and JNK2 in MKK7γ1-transfected PC12 cells. Summarizing, MKK7γ1 affects the function and activity of individual JNK isoforms and the formation of their signalosomes. This study demonstrates for the first time that one splice-variant of MKK7 tightly controls JNK signalling and effectively adapts JNK functions to the cellular context. [ABSTRACT FROM AUTHOR]

Abstract: The c-Jun N-terminal kinase (JNK) is a mitogen-activated protein kinase (MAPK) activated by stress-signals and involved in many different diseases. Previous results proved the powerful effect of the cell permeable peptide inhibitor d-JNKI1 (d-retro-inverso form of c-Jun N-terminal kinase-inhibitor) against neuronal death in CNS diseases, but the precise features of this neuroprotection remain unclear. We here performed cell-free and in vitro experiments for a deeper characterization of d-JNKI1 features in physiological conditions. This peptide works by preventing JNK interaction with its c-Jun N-terminal kinase–binding domain (JBD) dependent targets. We here focused on the two JNK upstream MAPKKs, mitogen-activated protein kinase kinase 4 (MKK4) and mitogen-activated protein kinase kinase 7 (MKK7), because they contain a JBD homology domain. We proved that d-JNKI1 prevents MKK4 and MKK7 activity in cell-free and in vitro experiments: these MAPKK could be considered not only activators but also substrates of JNK. This means that d-JNKI1 can interrupt downstream but also upstream events along the JNK cascade, highlighting a new remarkable feature of this peptide. We also showed the lack of any direct effect of the peptide on p38, MEK1, and extracellular signal-regulated kinase (ERK) in cell free, while in rat primary cortical neurons JNK inhibition activates the MEK1–ERK–Ets1/c-Fos cascade. JNK inhibition induces a compensatory effect and leads to ERK activation via MEK1, resulting in an activation of the survival pathway—(MEK1/ERK) as a consequence of the death pathway—(JNK) inhibition. This study should hold as an important step to clarify the strong neuroprotective effect of d-JNKI1. [Copyright &y& Elsevier]

Abstract: The c-Jun N-terminal kinases (JNKs) are activated in response to stress, DNA damage, and cytokines by MKK4 and MKK7. We recently demonstrated that PKC can augment the degree of JNK activation by phosphorylating JNK, which requires the adaptor protein RACK1. Here we report on the conditions required for PKC-dependent JNK activation. In vitro kinase assays reveal that PKC phosphorylation of JNK is not sufficient for its activation but rather augments JNK activation by canonical JNK upstream kinases MKK4 or MKK7 alone or in combination. Further, to enhance JNK activity, PKC phosphorylation of JNK should precede its phosphorylation by MKK4/7. Inhibition of PKC phosphorylation of JNK affects both early and late phases of JNK activation following UV-irradiation and reduces the apoptotic response mediated by JNK. These data provide important insight into the requirements for PKC activation of JNK signaling. [Copyright &y& Elsevier]

Abstract: c-Jun NH2-terminal protein kinase (JNK) is a mitogen-activated protein kinase (MAPK) involved in the regulation of numerous physiological processes during development and in response to stress. Its activity is increased upon phosphorylation by the MAPK kinases, MKK4 and MKK7. Similar to the early embryonic death of mice caused by the targeted deletion of the jnk genes, mice lacking mkk4 or mkk7 die before birth. The inability of MKK4 and MKK7 to compensate for each other''s functions in vivo is consistent with their synergistic effect in mediating JNK activation. However, the phenotypic analysis of the mutant mouse embryos indicates that MKK4 and MKK7 have specific roles that may be due to their selective regulation by extracellular stimuli and their distinct tissue distribution. MKK4 and MKK7 also have different biochemical properties. For example, whereas MKK4 can activate p38 MAPK, MKK7 functions as a specific activator of JNK. Here we summarize the studies that have shed light on the mechanism of activation of MKK4 and MKK7 and on their physiological functions. [Copyright &y& Elsevier]

Abstract: It has been well documented that the activation of c-Jun N-terminal protein kinase (JNK) pathway and caspase-3 signal are involved in the delayed neuronal cell death in cerebral ischemia. In this study, we first detected the activation pattern of JNK signaling including mixed lineage kinase (MLK)3, mitogen-activated protein kinase kinase (MKK)7 and JNK3 in hippocampal CA1 and CA3/DG regions at various time points after 15 min of ischemia. These results indicated that cerebral ischemia induced the continuous activation of MLK3/MKK7/JNK3 cascade, which all had two active waves only in the CA1 region. We also detected the phosphorylation of JNK substrates c-Jun and Bcl-2, and the activation of a key protease of caspase-3 in CA1 region, which only had one active peak, respectively. Because K252a has recently been shown to be a potent inhibitor of MLK3 activity both in vivo and in vitro, we further examined the possible effects and mechanism of this interesting drug in cerebral ischemia. In our present paper, we found that administration of K252a 20 min prior to ischemia inhibited MLK3/MKK7/JNK3 signaling, Bcl-2 phosphorylation, the activation of c-Jun and caspase-3, but had no significant effects on these protein expressions. Additionally, pretreatment of K252a significantly increased the number of the surviving CA1 pyramidal cells at 5 days of reperfusion. Our results suggest that K252a play a neuroprotective role in ischemic injury via inhibition of the JNK pathway, involving the death effector of caspase-3. Thus, JNK signaling may eventually emerge as a prime target for novel therapeutic approaches to treatment of ischemic stroke, and K252a may serve as a potential and important neuroprotectant in therapeutic aspect in ischemic stroke. [Copyright &y& Elsevier]

c-Jun NH2-terminal kinase (JNK), also known as stress-activated protein kinase (SAPK), is activated primarily by inflammatory cytokines and environmental stresses including toxic metal exposure. To reveal the upstream kinase responsible for JNK activation by toxic metals, the phosphorylation status and the activity of JNK were examined in mouse embryonic stem (ES) cells lacking MKK4 or MKK7 following exposure to CdCl2 or HgCl2. Treatment with CdCl2 or HgCl2 induced the phosphorylation of JNK in a dose- and time-dependent manner in wild-type ES cells. In both mkk4-/- and mkk7-/- ES cells, CdCl2- or HgCl2-induced phosphorylation and activation of JNK were suppressed significantly. However, in mkk7-/- ES cells treated with CdCl2 and HgCl2, JNK activation was not abolished (suppressed by 56% and 78%, respectively). These findings suggest that the full activation of JNK by toxic metal exposure requires both MKK4 and MKK7, and these upstream kinases might contribute differentially in JNK activation between mouse ES cells exposed to CdCl2 and HgCl2. [Copyright &y& Elsevier]

• Phosphatidic acid (PA) can bind to MKK7 and MKK9. • MKK7 and MKK9 positively regulate plant salt tolerance with functional redundancy. • PA signal increases MKK7/MKK9-catalyzed phosphorylation of MPK6 under salt stress. • PA induces the translocation of MKK7 and MKK9 to plasma membrane under salt stress. Phosphatidic acid (PA) is a lipid secondary messenger involved in intracellular signaling in eukaryotes. It has been confirmed that PA mediates salt stress signaling by promoting activation of Mitogen-activated Protein Kinase 6 (MPK6) which phosphorylates Na+/H+ antiporter SOS1. However, the MPK6-upstream kinases and their relationship to PA remain unclear. Here, we found that, among the six tested Arabidopsis Mitogen-activated Protein Kinase Kinases (MKKs), PA specifically bound to MKK7 and MKK9 which phosphorylate MPK6, and promoted the activation of MKK7/MKK9. Based on phenotypic and physiological analyses, we found that MKK7 and MKK9 positively regulate Arabidopsis salt tolerance and are functionally redundant. NaCl treatment can induce significant increase in MKK7/MKK9 activities, and this depends, in part, on the Phospholipase Dα1 (PLDα1). MKK7 and MKK9 also mediate the NaCl-induced activation of MPK6. Furthermore, PA or NaCl treatment could induce translocation of MKK7/MKK9 to the plasma membrane, whereas this translocation disappeared in pldα1. These results indicate that PA binds to MKK7 and MKK9, increases their kinase activity and plasma membrane localization during Arabidopsis response to salt stress. Together with the PA-MPK6-SOS1 pathway identified previously, this mechanism may maximize the signal transduction efficiency, providing novel insights into the link between lipid signaling and MAPK cascade. [ABSTRACT FROM AUTHOR]