Inhibition Of c-Jun Phosphorylation Research Articles

Development of a pentylenetetrazole-induced seizure model to evaluate kinase inhibitor efficacy in the central nervous system

c-Jun N-terminal kinases (JNKs) are implicated in cell death in neurodegenerative disorders. Therefore, JNK inhibitors could act as neuroprotective agents. To evaluate potential candidates, reproducible and quantitative CNS in vivo models are required. To that end, a pentylenetetrazole-induced seizure model was explored. c-Jun phosphorylation was detected in hippocampal extracts by blotting c-Jun immunoprecipitates with phosphorylation-specific antibodies. Pentylenetetrazole administration induced rapid and reproducible increases in c-Jun phosphorylation. However, special attention had to be paid to the composition of the extraction buffer to ensure stabilization of protein phosphorylation, as demonstrated using internal standards of phosphorylated recombinant c-Jun. As JNK and its upstream activator MKK4 are activated by phosphorylation, these events were also evaluated. In principle, kinase inhibitors could act at the level of JNK or upstream kinases to inhibit c-Jun phosphorylation. MKK4 phosphorylation was dramatically increased in response to pentylenetetrazole but, again, only when appropriate phosphatase inhibitors were in the extraction buffer. In contrast, JNK was found to be constitutively phosphorylated and unaltered upon pentylenetetrazole treatment. The JNK inhibitor SP600125 was shown to inhibit c-Jun phosphorylation without affecting MKK4 phosphorylation. Our procedures enable analysis of JNK pathway signalling in a CNS model and, also, should be applicable to that of other protein phosphorylation events in vivo.

The neuroprotective action of JNK3 inhibitors based on the 6,7-dihydro-5 H-pyrrolo[1,2- a]imidazole scaffold

Imidazole-based structures of p38 inhibitors served as a starting point for the design of JNK3 inhibitors. Construction of a 6,7-dihydro-5 H-pyrrolo[1,2- a]imidazole scaffold led to the synthesis of the ( S)-enantiomers, which exhibited p38/JNK3 IC 50 ratio of up to 10 and were up to 20 times more potent inhibitors of JNK3 than the relevant ( R)-enantiomers. The JNK3 inhibitory potency correlated well with inhibition of c-Jun phosphorylation and neuroprotective properties of the compounds in low K +-induced cell death of rat cerebellar granule neurones.

The tumor suppressor p16INK4a prevents cell transformation through inhibition of c-Jun phosphorylation and AP-1 activity

Inactivation of the p16(INK4a) tumor suppressor protein is critical for the development of human cancers, including human melanoma. However, the molecular basis of the protein's inhibitory effect on cancer development is not clear. Here we investigated a possible mechanism for p16(INK4a) inhibition of neoplastic transformation and UV-induced skin cancer. We show that p16(INK4a) suppresses the activity of c-Jun N-terminal kinases (JNKs) and that it binds to the glycine-rich loop of the N-terminal domain of JNK3. Although p16(INK4a) does not affect the phosphorylation of JNKs, its interaction with JNK inhibits c-Jun phosphorylation induced by UV exposure. This, in turn, interferes with cell transformation promoted by the H-Ras-JNK-c-Jun-AP-1 signaling axis.

Regulation of vitamin D receptor expression via estrogen-induced activation of the ERK 1/2 signaling pathway in colon and breast cancer cells

We previously demonstrated that 17beta-estradiol (E2) regulates the transcription and expression of the vitamin D receptor (VDR) in rat colonocytes and duodenocytes in vivo. The aim of the present study was to assess whether the extracellular signal-regulated kinase (ERK) induced by E2 is involved in regulating VDR expression. We compared E2-associated signaling activity in HT29 colon cancer cells, a non-classical E2-target, with that in MCF-7 breast cancer cells, the natural targets of the hormone. E2 did not affect proliferation of HT29 cells, but enhanced proliferation of MCF-7 cells. Vitamin D inhibited proliferation of both cell lines and the combined treatment induced potentiation of vitamin D activity. E2 upregulated VDR transcription and protein expression concomitantly with ERK 1/2 phosphorylation in both cell lines. PD98059, a specific mitogen-activated protein kinase (MAPK) inhibitor, prevented E2-mediated activation of ERK 1/2, with concomitant inhibition of VDR expression. ICI182780 inhibited VDR expression in HT29 and MCF-7 cell lines. A conjugate of E2 and bovine serum albumin upregulated phosphorylation of ERK 1/2 and concomitantly enhanced VDR expression in a similar fashion as the nonconjugated hormone. Expression of ERalpha and ERbeta was detected in MCF-7 and HT29 cell lines respectively, which localized to the nuclei, cytosol and caveolar membrane rather than non-caveolar membrane. Disruption of lipid rafts/caveolae by depleting cellular cholesterol with the cholesterol-binding reagent beta-methylcyclodextrin blocked ERK 1/2 phosphorylation concomitantly with VDR upregulation. The tyrosine phosphorylation inhibitor suramin and src kinase inhibitor PP2 inhibited both ERK 1/2 phosphorylation and VDR expression. E2 induced phosphorylation of Raf and Jun in a time-dependent manner. The Ras/Raf dependent inhibitor of transactivation sulindac sulfide also blocked E2 effects. The specific c-Jun phosphorylation inhibitor SP600125 dose dependently inhibited c-Jun phosphorylation and VDR expression. The MAPK/ERK kinase inhibitor PD 98059 downregulated both c-Jun phosphorylation and VDR expression indicating that upstream and downstream events in the signaling cascade are all related to the control of VDR expression. Taken together, our data suggest that E2 binds to receptors compartmentalized to membranal caveolar domains in HT29 and MCF-7 cells, inducing ERK 1/2 activation and transcriptional activity, which finally results in upregulation of expression of the VDR gene.

Ceramide Promotes Apoptosis in Lung Cancer-Derived A549 Cells by a Mechanism Involving c-Jun NH2-Terminal Kinase

Ceramide regulates diverse signaling pathways involving cell senescence, the cell cycle, and apoptosis. Ceramide is known to potently activate a number of stress-regulated enzymes, including the c-Jun NH(2)-terminal kinase (JNK). Although ceramide promotes apoptosis in human lung cancer-derived A549 cells, a role for JNK in this process is unknown. Here, we report that ceramide promotes apoptosis in A549 cells by a mechanism involving JNK. The JNK inhibitor SP600125 proved effective at protecting cells from the lethal effects of ceramide. To understand which JNK-mediated pathway may be involved, a number of JNK target proteins were examined, including the transcription factor, c-Jun, and the apoptotic regulatory proteins Bcl-X(L) and Bim. A549 cells exhibited basal levels of phosphorylated c-Jun in nuclear fractions, revealing that active c-Jun is present in these cells. Ceramide was found to inhibit c-Jun phosphorylation, suggesting that JNK-mediated phosphorylation of c-Jun is not likely involved in ceramide-induced apoptosis. Ceramide did not promote Bcl-X(L) phosphorylation. On the other hand, ceramide promoted phosphorylation of Bim and induced translocation of active JNK from the nucleus to the cytoplasm and mitochondrial fraction. Ceramide-mediated changes in localization of JNK were consistent with the observed changes in phosphorylation status of c-Jun and Bim. Furthermore, ceramide promoted Bim translocation to the mitochondria. Mitochondrial localization of Bim has been shown recently to promote apoptosis. These results suggest that JNK may participate in ceramide-induced apoptosis in A549 cells by a mechanism involving Bim.

Transformation suppressor protein Pdcd4 interferes with JNK-mediated phosphorylation of c-Jun and recruitment of the coactivator p300 by c-Jun.

The transformation suppressor gene Pdcd4 (programmed cell death gene 4) inhibits the tumor-promoter mediated transformation of mouse keratinocytes and has recently been implicated as a potential tumor suppressor gene in the development of human lung cancer. Biochemical analysis has suggested that the Pdcd4 protein is involved in protein translation as well as in nuclear events. Recent work has shown that Pdcd4 suppresses the transactivation of AP-1 responsive promoters by c-Jun, suggesting that the transformation-suppressor activity of Pdcd4 might be due, at least in part, to the inhibition of c-Jun activity. Here, we have addressed how Pdcd4 inhibits c-Jun. We show that Pdcd4 interferes with the phosphorylation of c-Jun by Jun N-terminal kinase (JNK). The inhibition of c-Jun phosphorylation by Pdcd4 appears not to be due to a general suppression of JNK activity, our data rather suggest that Pdcd4 interacts with c-Jun and thereby blocks phosphorylation of c-Jun. In addition to affecting c-Jun phosphorylation, Pdcd4 blocks the recruitment of the coactivator p300 by c-Jun. Taken together, our results strongly suggest that Pdcd4 is directly involved in the regulation of c-Jun activity.

279 Inhibition of C-JUN phosphorylation sensitizes to CD95 mediated apoptosis independent of P53 and P21

279 Inhibition of C-JUN phosphorylation sensitizes to CD95 mediated apoptosis independent of P53 and P21

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.

Analysis of the NF-κB and PI 3-Kinase/Akt Survival Pathways in Nerve Growth Factor-Dependent Neurons

Nerve growth factor (NGF) readdition to NGF-deprived neurons can halt Jun N-terminal kinase (JNK) activation, cytochrome c release, and cell death through mechanisms that may involve phosphatidylinositol (PI) 3-kinase, Akt, and nuclear factor kappa B (NF-κB). We found that expression of the NF-κB protein c-Rel in NGF-deprived neurons blocks cytochrome c release but does not inhibit c-Jun phosphorylation. Conversely, inhibition of NF-κB in NGF-maintained neurons promotes cytochrome c release and cell death. In contrast to c-Rel, activated PI 3-kinase and Akt inhibit c-Jun phosphorylation but have only a small effect on cytochrome c release. Finally, although c-Rel can protect neurons from death caused by inhibitors of PI 3-kinase or Akt, NF-κB function is not critical for Akt-promoted survival. These results suggest that the PI 3-kinase/Akt and NF-κB survival pathways target distinct cell death events in neurons.

Inhibition of JNK by Overexpression of the JNK Binding Domain of JIP-1 Prevents Apoptosis in Sympathetic Neurons

Studies in non-neuronal cells show that c-Jun N-terminal kinases (JNK) play a key role in apoptotic cell death. In some neurons JNK is also thought to initiate cell death by the activation of c-Jun. JNK inhibition has been achieved pharmacologically by inhibiting upstream kinases, but there has been no direct demonstration that inhibition of JNK can prevent neuronal death. We have therefore examined whether the JNK binding domain (JBD) of JNK-interacting protein-1 (JIP-1, a scaffold protein and specific inhibitor of JNK) can inhibit c-Jun phosphorylation and support the survival of sympathetic neurons deprived of NGF. We show that expression of the JBD in >80% of neurons was sufficient to prevent the phosphorylation of c-Jun and its nuclear accumulation as well as abrogate neuronal cell death induced by NGF deprivation. JBD expression also preserved the capacity of mitochondria to reduce MTT. Interestingly, although the PTB domain of JIP was reported to interact with rhoGEF, expression of the JBD domain was sufficient to localize the protein to the membrane cortex and growth cones. Hence, JNK activation is a key event in apoptotic death induced by NGF withdrawal, where its point of action lies upstream of mitochondrial dysfunction.

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Effects of the steroidal anti-inflammatory drug dexamethasone on the antigeninduced activation of c-Jun N-terminal kinase (JNK) was examined. Antigen stimulation of IgE-sensitized rat basophilic leukemia RBL-2 H3 cells induced activation of JNK within a few minutes with maximum activity attained 40 min later. The increase in JNK activity was accompanied with an increase in phosphorylation of c-Jun in the cells. The antigen-induced JNK activation was inhibited by the phosphatidylinositol 3-kinase inhibitors wortmannin and LY 294002 but not by the protein kinase C inhibitors calphostin C and Ro 31-8425. Pretreatment with dexamethasone (10 and 100 nM) for 18 h prominently inhibited the antigen-induced increase in JNK activity in a concentration-dependent manner, while protein levels of JNK were slightly decreased. At least 6-h preincubation with dexamethasone was necessary to inhibit the antigen-induced JNK activation. The phosphorylation of c-Jun induced by the antigen stimulation was reduced by pretreatment with dexamethasone without reduction of the content of c-Jun protein. The antigen-induced activation of the JNK kinase kinase MEKK-1 was also inhibited by pretreatment with dexamethasone. These findings indicate that dexamethasone inhibits the antigen-induced activation of JNK. In conclusion, the inhibition by dexamethasone of JNK activation resulting in the inhibition of c-Jun phosphorylation might be a new mechanism by which dexamethasone down-regulates AP-1 activity and inhibits cytokine production.

Inhibition by Dexamethasone of Antigen-Induced c-Jun N-Terminal Kinase Activation in Rat Basophilic Leukemia Cells

Antigen stimulation of IgE-sensitized rat basophilic leukemia RBL-2H3 cells induced activation of c-Jun N-terminal kinase (JNK) within a few minutes with maximum activity attained 40 min later. The increase in JNK activity was accompanied with an increase in phosphorylation of c-Jun in the cells. The Ag-induced JNK activation was inhibited by the phosphatidylinositol 3-kinase inhibitors wortmannin (10-100 nM) and LY 294002 (100 microM) but not by the protein kinase C inhibitors calphostin C (1 and 3 microM) and Ro 31-8425 (1 and 3 microM). Pretreatment with dexamethasone (10 and 100 nM) for 18 h inhibited the Ag-induced increase in JNK activity in a concentration-dependent manner. At least 6 h of preincubation with dexamethasone was necessary to inhibit the Ag-induced JNK activation. The phosphorylation of c-Jun induced by the Ag stimulation was reduced by pretreatment with dexamethasone without reduction of the content of c-Jun protein. The Ag-induced activation of the JNK kinase kinase mitogen-activated protein kinase-extracellular signal-regulated kinase kinase-1 was also inhibited by pretreatment with dexamethasone at 10 and 100 nM. These findings indicate that dexamethasone reduces JNK protein level and inhibits the Ag-induced activation of JNK resulting in the inhibition of c-Jun phosphorylation.

A peptide encoding the c-Jun delta domain inhibits the activity of a c-jun amino-terminal protein kinase

Evidence suggests that the c-Jun protooncogene delta (delta) domain (amino acids 31-60) helps regulate the transcriptional activating capacity of c-Jun by modulating the amino-terminal phosphorylation of this protein. By using a peptide encoding the delta domain and purified amino-terminal c-Jun protein kinase, we demonstrate that the delta domain peptide inhibits phosphorylation of the amino terminus of both c-Jun and the related protein JunD. The delta domain peptide inhibited the activation of the c-Jun amino-terminal protein kinase by phorbol esters in permeabilized U937 leukemic cells. Mutation of c-Jun followed by transfection into U937 leukemic cells demonstrated that partial deletions of the delta domain are sufficient to block phosphorylation of the amino terminus of c-Jun. In vitro deletion of the amino-terminal (amino acids 31-44) half of the delta domain inhibited the phosphorylation of c-Jun. However, deletion of the carboxyl-terminal (amino acids 45-60) half only partially inhibited c-Jun phosphorylation. Therefore, these results indicate that the delta domain sequence is an important regulator of c-Jun amino-terminal phosphorylation.