Substituting c-Jun N-terminal kinase-3 (JNK3) ATP-binding site amino acid residues with their p38 counterparts affects binding of JNK- and p38-selective inhibitors

c-Jun N-terminal kinase (JNK) activation is linked to the aberrant cell death in several neurodegenerative disorders, including Parkinson's and Alzheimer's disease. The sequence similarity among the JNK isoforms and fellow MAP kinase family member p38 has rendered the challenge of producing JNK3-specific inhibitors difficult. Using the crystal structure of JNK3 complexed with JNK inhibitors, potential compound-interacting amino acid residues were mutated to the corresponding residues in p38. The effects of these mutations on the kinetic parameters with three compounds were examined: a JNK3- (vs. p38-) selective inhibitor (SP 600125); a p38-selective inhibitor (Merck Z); and a potent combined JNK3 and p38 inhibitor (Merck Y). The data confirm the role of the JNK3 residues Ile-70 and Val-196 in both inhibitor and ATP-binding. Remarkably, the Ile-70-Val and Val-196-Ala mutations caused an increase and decrease, respectively, in the binding affinity of the p38-specific compound, Merck Z, of 10-fold. The Ile-70-Val effect may be due to the increased capacity of the active site to accommodate Merck Z, whereas the Val-196-Ala mutant may induce an unfavourable conformational change. Conservative mutations of the Asn-152 and Gln-155 residues inactivated the JNK3 enzyme, possibly interfering with protein folding in a critical hinge region of the protein.     

Adenosine A 2B receptors modulate cAMP levels and induce CREB but not ERK1/2 and p38 phosphorylation in rat skeletal muscle cells

The present study examined the existence of the adenosine A(1),A(2A), and A(2B) receptors and the effect of receptor activation on cAMP accumulation and protein phosphorylation in primary rat skeletal muscle cells. Presence of mRNA and protein for all three receptors was demonstrated in both cultured and adult rat skeletal muscle. NECA (10(-9)-10(-4)M) increased the cAMP concentration in cultured muscle cells with an EC(50) of (95% confidence interval)=15 (5.9-25.1) micro M, whereas CGS 21680 (10(-9)-10(-4)M) had no effect on cAMP accumulation. Concentrations of [R]-PIA below 10(-6)M had no effect on cAMP accumulation induced by either isoproterenol or forskolin. NECA resulted in phosphorylation of CREB with an EC(50) of (95% confidence interval)=1.7 (0.40-7.02) micro M, whereas ERK1/2 and p38 phosphorylation was unchanged. The results show that, although the A(1),A(2A), and A(2B) receptors are all present in skeletal muscle cells, the effect of adenosine on adenylyl cyclase activation and phosphorylation of CREB is mainly mediated via the adenosine A(2B) receptor.     

Detection of phosphorylated mitogen-activated protein kinase in the developing spinal cord of the mouse embryo

Global understanding of the proteome is a major research topic. The comprehensive visualization of the distribution of proteins in vivo or the construction of in situ protein atlases may be a valuable strategy for proteomic researchers. Information about the distribution of various proteins under physiological and pathological conditions should be extremely valuable for the basic and clinical sciences.The mitogen-activated protein kinase (MAPK) cascade plays an essential role in intracellular signaling in organisms. This cascade also regulates biological processes involving development, differentiation, and proliferation. Phosphorylation and dephosphorylation are integral reactions in regulating the activity of MAPKs. Changes in the phosphorylation state of MAPKs are rapid and reversible; therefore, the localizations of physiologically phosphorylated MAPKs in vivo are difficult to accurately detect. Furthermore, phosphorylated MAPKs are likely to change phosphorylated states through commonly used experimental manipulations.In the present study, as a step toward the construction of in situ phosphoprotein atlases, we attempted to detect physiologically phosphorylated MAPKs in vivo in developing spinal cords of mice. We previously reported an improved immunohistochemical method for detecting unstable phosphorylated MAPKs. The distribution patterns of phosphorylated MAPKs in the spinal cords of embryonic mice from embryonic day 13 (E13) to E17 were observed with an improved immunohistochemical method. Phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) and phosphorylated c-Jun N-terminal kinase 1/2 (p-JNK1/2) were strongly observed in the marginal layer and the dorsal horn from E13 to E17. Our results suggest that p-ERK1/2 and p-JNK1/2 play critical roles in the developing spinal cord. Constructing phosphoprotein atlases will be possible in the future if this work is systematically developed on a larger scale than we presented here.     

Ca2+ stimulates COX-2 expression through calcium-sensing receptor in fibroblasts

Fibroblasts isolated from jaw cysts expressed calcium-sensing receptor (CasR). In the fibroblasts elevated extracellular Ca(2+) ([Ca(2+)](o)) increased fluo-3 fluorescence intensity, and the production of inositol(1,4,5)trisphosphate and active protein kinase C. Phospholipase C inhibitor U-73122 attenuated the Ca(2+)-induced increase in fluo-3 fluorescence intensity. Elevated [Ca(2+)](o) enhanced the expression of cyclooxygenase-2 (COX-2) mRNA and protein, and the secretion of prostaglandin E(2) in the fibroblasts. CasR activator neomycin also increased the expression of COX-2 mRNA, and U-73122 attenuated the Ca(2+)-induced expression of COX-2 mRNA. Elevated [Ca(2+)](o)-induced phosphorylation of extracellular signal-regulated protein kinase-1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK), and U-73122 inhibited the Ca(2+)-induced phosphorylation. The inhibitors for each kinase, PD98059, SB203580, and SP600125, attenuated the Ca(2+)-induced expression of COX-2 mRNA. These results suggest that in jaw cyst fibroblasts elevated extracellular Ca(2+) may enhance COX-2 expression via the activation of ERK1/2, p38 MAPK, and JNK through CasR.     

Role of Extracellular Signal-Regulated Protein Kinases 1 and 2 in Oligodendroglial Process Extension

Abstract: The relationship between extracellular signal-regulated protein kinase (ERK) activation and process extension in cultured bovine oligodendrocytes (OLGs) was investigated. Process extension was induced through the exposure of cultured OLGs to phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), for various intervals. During the isolation of these OLGs from bovine brain, the original processes were lost. Therefore, any reinitiation of process extension via PMA stimulation was easily discernible through morphological monitoring. It was found that exposure of OLGs to PMA for 10 min was enough to induce OLG process extension 24–72 h later. Furthermore, this extension was still evident at least 1 week after the initial PMA stimulation, indicating that OLGs do not need continuous PKC activation to sustain process extension. Control and PMA-stimulated OLGs were also subjected to immunocytochemistry using an anti-ERK antibody selective for the mitogen-activated protein kinases p42 Erk2 (ERK2) and p44 Erk1 (ERK1) isoforms. ERK immunoreactivity in the nucleus was evident after PMA stimulation of OLGs but not in control OLGs. In parallel experiments, the control and PMA-stimulated OLGs were purified by Mono Q fractionation and subjected to ERK phosphotransferase assays using [γ-³²P]ATP and either myelin basic protein (MBP) or a synthetic peptide substrate based on the Thr⁹⁷ phosphorylation site in MBP. These assays indicated that in PMA-treated OLGs, ERK activation was at least 12-fold higher than in control OLGs. Anti-ERK and anti-phosphotyrosine western blots of the assay fractions verified an enhanced phosphorylation of ERK1 and ERK2 in PMA-treated fractions relative to control fractions. When OLGs were pretreated for 15 min with the ERK kinase (MEK) inhibitor PD 098059 before PMA stimulation, they exhibited a 67% decrease in ERK activation as compared with cells treated with PMA alone. Furthermore, these MEK inhibitor-pretreated cells were still viable but showed no process extensions up to 1 week later. Therefore, we propose that a threshold level of ERK activity is required for the initiation of OLG process extension.     

Specific inhibitor of MEK-mediated cross-talk between ERK and p38 MAPK during differentiation of human osteosarcoma cells

Osteosarcoma is the most common primary malignant bone tumor, accounting for approximately 20% of all primary sarcomas in bone. Although treatment modalities have been improved over the past decades, it is still a tumor with a high mortality rate in children and young adults. Based on histological considerations, osteosarcoma arises from impaired differentiation of these immature cells into more mature types and that correction of this impairment may reduce malignancy and increase the efficiency of chemotherapy. The purpose of this study was to determine the effect of specific inhibitors of MAPK extracellular signaling-regulated kinase (ERK) kinase (MEK) and p38 on the differentiation of human osteosarcoma cell line SaOS-2 cells. We found that PD98059, a specific inhibitor of MEK, inhibited the serum-stimulated proliferation of SaOS-2 cells; whereas SB203580, a specific inhibitor of p38 MAPK, had little effect on it. SB203580 suppressed ALPase activity, gene expression of type I collagen, and expression of ALP and BMP-2 mRNAs; whereas PD98059 upregulated them dose dependently. In addition, immunoblot and immunostaining analysis revealed that phosphorylation of ERK was increased by treatment with SB203580; whereas PD98059 increased the phosphorylation of p38, which implies a seesaw-like balance between ERK and p38 phosphorylation. We suggest that osteosarcoma cell differentiation is regulated by the balance between the activities of the ERK and p38 pathways and that the MEK/ERK pathway negatively regulates osteosarcoma cell differentiation, whereas the p38 pathway does so positively. MEK inhibitor may thus be a good candidate for altering the expression of the osteosarcoma malignant phenotype.     

Comparison Between the Timing of JNK activation, c-Jun Phosphorylation, and Onset of Death Commitment in Sympathetic Neurones

Abstract: We have investigated the relationship between c-Jun N-terminal kinase (JNK) activity, apoptosis, and the potential of survival factors to rescue primary rat sympathetic neurones deprived of trophic support. Incubation of sympathetic neurones in the absence of nerve growth factor (NGF) caused a time-dependent increase in JNK activity, which became apparent by 3 h and attained maximal levels that were three- to fourfold higher than activity measured in neurones maintained for the same periods with NGF. Continuous culture in the presence of either NGF or the cyclic AMP analogue 4-(8-chlorophenylthio) cyclic AMP (CPTcAMP) not only prevented JNK activation from occurring, but also suppressed JNK activity that had been elevated by prior culture of the neurones in the absence of trophic support. When either NGF or CPTcAMP was added to cultures that had been initially deprived of neurotrophic support for up to 10 h, this resulted in complete suppression of total JNK activity, arrest of apoptosis, and rescue of >90% of the neurones that did not display apoptotic morphology by this time. However, when either agent was added after more protracted periods of initial neurotrophin deprivation (≥ 14 h), although this also resulted in near-complete suppression of total JNK activity and short-term arrest of apoptosis, not all of the neurones that appeared to be nonapoptotic at the time of agent addition were rescued. The lack of death commitment after 10 h of maintained JNK activity was not due to a late induction of c-Jun expression, because the majority of newly isolated sympathetic neurones had already been expressing high levels of c-Jun in their nuclei for several hours, yet were capable of being rescued by NGF. Elevation of JNK activity as a result of neurotrophic-factor deprivation was also associated with enhanced phosphorylation of c-Jun, assessed by immunoblot analysis and immunocytochemistry, and addition of NGF to cultures previously deprived of neurotrophic support resulted in a reversion of the state of phospho-c-Jun to that observed in cultures that had been maintained in the continuous presence of trophic support. We conclude that activation of JNK and c-Jun phosphorylation are not necessarily rate-limiting for apoptosis induction. In some neurones undergoing prolonged NGF deprivation, suppression of JNK activity and c-Jun dephosphorylation by NGF may be insufficient to effect their rescue. Thus, if c-Jun mediates death by increasing the expression of “death” genes, these must become effective very close to the death commitment point.     

Regulation of inflammatory arthritis by the upstream kinase mitogen activated protein kinase kinase 7 in the c-Jun N-Terminal kinase pathway

Introduction

The c-Jun N-terminal kinase (JNK) is a key regulator of matrix metalloproteinase (MMP) and cytokine production in rheumatoid arthritis (RA) and JNK deficiency markedly protects mice in animal models of arthritis. Cytokine-induced JNK activation is strictly dependent on the mitogen-activated protein kinase kinase 7 (MKK7) in fibroblast-like synoviocytes (FLS). Therefore, we evaluated whether targeting MKK7 using anti-sense oligonucleotides (ASO) would decrease JNK activation and severity in K/BxN serum transfer arthritis.

Methods

Three 2''-O-methoxyethyl chimeric ASOs for MKK7 and control ASO were injected intravenously in normal C57BL/6 mice. PBS, control ASO or MKK7 ASO was injected from Day -8 to Day 10 in the passive K/BxN model. Ankle histology was evaluated using a semi-quantitative scoring system. Expression of MKK7 and JNK pathways was evaluated by quantitative PCR and Western blot analysis.

Results

MKK7 ASO decreased MKK7 mRNA and protein levels in ankles by about 40% in normal mice within three days. There was no effect of control ASO on MKK7 expression and MKK7 ASO did not affect MKK3, MKK4 or MKK6. Mice injected with MKK7 ASO had significantly less severe arthritis compared with control ASO (P < 0.01). Histologic evidence of synovial inflammation, bone erosion and cartilage damage was reduced in MKK7 ASO-treated mice (P < 0.01). MKK7 deficiency decreased phospho-JNK and phospho-c-Jun in ankle extracts (P < 0.05), but not phospho-MKK4. Interleukin-1beta (IL-1β), MMP3 and MMP13 gene expression in ankle joints were decreased by MKK7 ASO (P < 0.01).

Conclusions

MKK7 plays a critical regulatory role in the JNK pathway in a murine model of arthritis. Targeting MKK7 rather than JNK could provide site and event specificity when treating synovitis.     

Inhibition of aberrant and constitutive phosphorylation of the high-molecular-mass neurofilament subunit by CEP-1347 (KT7515), an inhibitor of the stress-activated protein kinase signaling pathway

Previous studies have implicated stress-activated protein kinases (SAPKs) in aberrant phosphorylation of the high-molecular-mass neurofilament subunit (NFH). We now present direct evidence for this involvement using CEP-1347, a specific inhibitor of SAPK activation. Inhibition by this drug of stress-induced phosphorylation of NFH and the middle-molecular-mass neurofilament subunit in the perikaryon of dorsal root ganglion (DRG) neurons paralleled the decrease in levels of activated SAPKs and was essentially complete at 1 microM: CEP-1347. In addition, a role for SAPKs in the constitutive phosphorylation of NFH was demonstrated. Longterm treatment of unstressed DRG neurons with CEP-1347 lowered the steady-state phosphorylation level of NFH in neurites. No such effect was seen in neurons treated with PD 098059, which blocks activation of extracellular signal-regulated kinase 1/2. DRG neurites were shown to contain high basal levels of activated SAPKs. These included a 55-kDa SAPK whose activation was completely abolished at 0.05 microM: CEP-1347 and a 45-kDa SAPK that was not affected by the drug. These results indicate that SAPKs are involved in both stress-induced and constitutive phosphorylation of NFH. The differing responses of SAPKs in neurites and cell bodies to CEP-1347 inhibition further suggest the presence of different signaling pathways in the two neuronal compartments.     

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.     

Regulation of adenylyl cyclase, ERK1/2, and CREB by Gz following acute and chronic activation of the delta-opioid receptor

Opioid tolerance and physical dependence in mammals can be rapidly induced by chronic exposure to opioid agonists. Recently, opioid receptors have been shown to interact with the pertussis toxin (PTX)-insensitive Gz (a member of the Gi subfamily), which inhibits adenylyl cyclase and stimulates mitogen-activated protein kinases (MAPKs). Here, we established stable human embryonic kidney 293 cell lines expressing delta-opioid receptors with or without Gz to examine the role of Gz in opioid receptor-regulated signaling systems. Each cell line was acutely or chronically treated with [D-Pen2,D-Pen5]enkephalin (DPDPE), a delta-selective agonist, in the absence or presence of PTX. Subsequently, the activities of adenylyl cyclase, cyclic AMP (cAMP)-dependent response element-binding proteins (CREBs), and MAPKs were measured by determining cAMP accumulation and phosphorylation of CREBs and the extracellular signal-regulated protein kinases (ERKs) 1 and 2. In cells coexpressing Gz, DPDPE inhibited forskolin-stimulated cAMP accumulation in a PTX-insensitive manner, but Gz could not replace Gi to mediate adenylyl cyclase supersensitization upon chronic opioid treatment. DPDPE-induced adenylyl cyclase supersensitization was not associated with an increase in the phosphorylation of CREBs. Both Gi and Gz mediated DPDPE-induced activation of ERK1/2, but these responses were abolished by chronic opioid treatment. Collectively, our results show that although Gz mediated opioid-induced inhibition of adenylyl cyclase and activation of ERK1/2, Gz alone was insufficient to mediate opioid-induced adenylyl cyclase supersensitization.     

Physiological roles of mitogen-activated-protein-kinase-activated p38-regulated/activated protein kinase

Mitogen-activated protein kinases (MAPKs) are a family of proteins that constitute signaling pathways involved in processes that control gene expression, cell division, cell survival, apoptosis, metabolism, differentiation and motility. The MAPK pathways can be divided into conventional and atypical MAPK pathways. The first group converts a signal into a cellular response through a relay of three consecutive phosphorylation events exerted by MAPK kinase kinases, MAPK kinase, and MAPK. Atypical MAPK pathways are not organized into this three-tiered cascade. MAPK that belongs to both conventional and atypical MAPK pathways can phosphorylate both non-protein kinase substrates and other protein kinases. The latter are referred to as MAPK-activated protein kinases. This review focuses on one such MAPK-activated protein kinase, MAPK-activated protein kinase 5 (MK5) or p38-regulated/activated protein kinase (PRAK). This protein is highly conserved throughout the animal kingdom and seems to be the target of both conventional and atypical MAPK pathways. Recent findings on the regulation of the activity and subcellular localization, bona fide interaction partners and physiological roles of MK5/PRAK are discussed.     

Activation of extracellular signal-regulated kinase (ERK) and Akt by human serotonin 5-HT(1B) receptors in transfected BE(2)-C neuroblastoma cells is inhibited by RGS4

Regulator of G protein signaling (RGS) proteins are GTPase-activating proteins for heterotrimeric G proteins. One of the best-studied RGS proteins, RGS4, accelerates the rate of GTP hydrolysis by all G(i) and G(q) alpha subunits yet has been shown to exhibit receptor selectivity. Although RGS4 is expressed primarily in brain, its effect on modulating the activity of serotonergic receptors has not yet been reported. In the present study, transfected BE(2)-C human neuroblastoma cells expressing human 5-HT(1B) receptors were used to demonstrate that RGS4 can inhibit the coupling of 5-HT(1B) receptors to cellular signals. Serotonin and sumatriptan were found to stimulate activation of extracellular signal-regulated kinase. This activation was attenuated, but not completely inhibited, by RGS4. Similar inhibition by RGS4 of the protein kinase Akt was also observed. As RGS4 is expressed at high levels in brain, these results suggest that it may play a role in regulating serotonergic pathways.     

Progression of dystrophic features and activation of mitogen-activated protein kinases and calcineurin by physical exercise,in hearts of mdx mice

We have previously demonstrated that calcineurin and p38 mitogen-activated protein kinase (MAPK) are up-regulated in the hearts of mdx mice. However, the degree of up-regulation observed was variable, which may reflect variable levels of daily physical activities among the mice. To investigate whether or not exercise affects dystrophic features and activates intracellular signaling molecules in mdx hearts, we subjected mdx and C57BL/10 mice to treadmill exercise and examined intracellular signaling molecules in cardiac muscles, at the protein level. The heart to body weight ratio was significantly increased in exercised mdx mice. Histopathology in exercised mdx hearts showed extensive infiltration of inflammatory cells, together with increases in interstitial fibrosis and adipose tissues, all of which were not observed either in exercised C57BL/10 or non-exercised mdx hearts. Phosphorylated p38 MAPK, phosphorylated extracellular signal-regulated kinase 1/2 and calcineurin, but not phosphorylated c-Jun N-terminal kinase 1, were up-regulated in exercised mdx hearts compared to exercised C57BL/10 or non-exercised mdx hearts. These data suggest that physical exercise accelerates the dystrophic process through activation of intracellular signaling molecules in dystrophin-deficient hearts.     

Identification of mitogen-activated protein kinase homologues from Leishmania mexicana

Mitogen-activated protein kinases are key-regulatory elements in the differentiation, proliferation, apoptosis and stress response of eukaryotic cells. Our recent identification of a mitogen-activated protein kinase homologue in Leishmania mexicana which is essential for the proliferation of the amastigote stage of the parasite living in the parasitophorous vacuole of the infected macrophage prompted us to screen the genome of L. mexicana for additional mitogen-activated protein kinase homologues using degenerate oligonucleotide primers in a polymerase chain reaction amplification approach. We cloned and sequenced the genes for eight new mitogen-activated protein kinase homologues which were subsequently shown to be present in one copy per haploid genome. The mRNA levels of the kinases varied significantly in pro- and amastigote life stages of the parasite. We used the structural information of the p38 stress-activated protein kinase, which belongs to the family of mitogen-activated protein kinases, for the alignment of the deduced proteins and the verification of the predicted secondary structure elements. All new mitogen-activated protein kinases reveal the typical 12 subdomain primary structure, the conserved residues characterising serine/threonine protein kinases and the characteristic TXY motif in the phosphorylation lip. Typical features of some of the molecules are amino acid insertions between the subdomains and long carboxy-terminal amino acid extensions carrying putative src-homology 3-binding motifs.