Crystal structure of human mono-phosphorylated ERK1 at Tyr204

Extracellular signal-regulated kinase (ERK) is a member of the MAP kinase family, and can regulate several cellular responses. The isoforms ERK1 and ERK2 have markedly similar amino acid sequences, but exhibit distinctive physiological functions. As well as ERK2, ERK1 was auto- and mono-phosphorylated at Tyr204 in the activation loop during Escherichia coli production, resulting in basal level activity, approximately 500-fold less compared with fully-active ERK1 dual-phosphorylated at Thr202 and Tyr204. Crystal structure demonstrated that the mono-phosphorylated ERK1 kinase possessed a novel conformation distinguishable from the un-phosphorylated (inactive) and the dual-phosphorylated (full-active) forms. The characteristic structural features in both the C-helix and the activation loop likely contribute to the basal activity of the mono-phosphorylated ERK1. The structural dissection of ERK1 compared to ERK2 suggests that the structural differences in the D-motif binding site and in the backside binding site are putative targets for development of selective ERK1/ERK2 inhibitors.     

脊髓细胞外调节激酶1/2在大鼠后足切割痛中的表达和作用

目的：探讨大鼠后足切割后脊髓ERK的表达情况。方法：以大鼠右后足切割作为急性疼痛模型;用免疫组织化学法测试脊髓磷酸化ERK（pERK）表达情况。ERK抑制剂U0126（1μg）在切割前20min或切割后20min鞘内注射。用von Frey纤维测试大鼠机械性痛敏。结果：大鼠后足切割后1min,在切割侧L4-L5脊髓浅层背侧角（板层Ⅰ和板层Ⅱ）ERK被迅速地激活,并在5min达到峰值,随后恢复到基础值。切割前鞘内给予U0126能显著减轻机械性痛敏,然而,切割后鞘内给予U0126对机械性痛敏的作用并不明显。结论：脊髓ERK在大鼠后足切割痛中产生机械性痛敏发挥了重要的作用。     

Nitric Oxide Participates in the Induction of Brain Ischemic Tolerance via Activating ERK1/2 Signaling Pathways

The present study was undertaken to observe in vivo changes of expression and phosphorylation of ERK1/2 proteins during brain ischemic preconditioning and effects of inhibiting generation of nitric oxide (NO) on the changes to determine the role of ERKs in the involvement of NO participating in the acquired tolerance. Fifty-five Wistar rats were used. Brain ischemic preconditioning was performed with four-vessel occlusion for 3 min. Total ERK1/2 proteins and phospho-ERK1/2 in the CA1 hippocampus were assayed with Western immunoblot. Total ERK1/2 proteins did not change in period from 5 min to 5 days of reperfusion after preconditioning stimulus. While the level of phospho-ERK1/2 increased obviously to 223, 237, 300, 385 and 254% of sham level at times of 5 min, 2 h, 1, 3 and 5 days after preconditioning stimulus, respectively (P < 0.01). Administration of L-NAME, an inhibitor of NO synthase, 30 min prior to preconditioning stimulus failed to induce change in total ERK1/2 proteins (P > 0.05). However, phospho-ERK1/2 increased only to 138 and 176% of sham level at 2 h and 3 days after preconditioning stimulus, respectively, when animals were pretreated with L-NAME. The magnitudes of the increase were obviously low compared with those (237 and 385%) in animals untreated with L-NAME at corresponding time points (P < 0.01), which indicated that phosphorylation of ERK1/2 normally induced by preconditioning stimulus was blocked apparently by administration of L-NAME. The results suggested that phosphorylation of ERK1/2, rather than synthesis of ERK1/2 proteins, was promoted in brain ischemic preconditioning, and that the promotion was partly mediated by NO signal pathway.     

In silico identification and biochemical characterization of novel inhibitors of NQO1

From in silico docking and COMPARE analysis, novel inhibitors of human NAD(P)H quinone oxidoreductase (NQO1) have been identified from the NCI compound database, the most potent of which has an observed IC₅₀ of 0.7 μM. The inhibitors exhibit a diverse range of scaffolds. The ability of docking calculations to predict experimentally determined binding affinities for NQO1 is discussed, considering the influence of target flexibility and scoring function.     

Signaling pathway of MAPK/ERK in cell proliferation,differentiation, migration,senescence and apoptosis

Abstract

The generic mitogen-activated protein kinases (MAPK) signaling pathway is shared by four distinct cascades, including the extracellular signal-related kinases (ERK1/2), Jun amino-terminal kinases (JNK1/2/3), p38-MAPK and ERK5. Mitogen-activated protein kinases/extracellular signal-regulated kinase (MAPK/ERK) pathway is reported to be associated with the cell proliferation, differentiation, migration, senescence and apoptosis. The literatures were searched extensively and this review was performed to review the role of MAPK/ERK signaling pathway in cell proliferation, differentiation, migration, senescence and apoptosis.     

TGF-beta suppresses POEM expression through ERK1/2 and JNK in osteoblasts

POEM, also called nephronectin, is an extracellular matrix protein that is considered to play a critical role as an adhesion molecule in the development and functioning of various tissues, such as kidneys and bones. In the present study, we examined the molecular mechanism of POEM gene expression, and found that transforming growth factor-beta (TGF-beta) strongly inhibited POEM expression in the mouse osteoblastic cell line, MC3T3-E1. TGF-beta-induced decrease of POEM expression occurred in both time- and dose-dependent manners through the activation of TGF-beta receptor I and extracellular signal-regulated kinase/c-Jun N-terminal kinase pathways.     

Phosphatase Resistance of ERK2 Brain Kinase PK40erk2

Abstract: We have previously shown that a brain protein kinase, termed PK40, catalyzes the multiple phosphorylation of the KSP-repeat site of neurofilaments (NFs) and also can transform τ proteins into the paired helical filament-like state as found in Alzheimer's disease (AD) brains. Protein sequence analysis suggests that PK40 is a form of the extracellular signal-regulated kinase ERK2. A subpopulation of ERK2 species in soluble brain fractions can be efficiently phosphorylated and activated in cell-free systems, simply by adding Mg²⁺-ATP. Two phosphoisoforms of PK40^erk2 are formed in this process, which have a reduced gel mobility, very much like the ERK2 form obtained in cell culture by stimulation with growth factors. One of these low-mobility forms cannot be inactivated with protein phosphatase 2A (PP2A) or with tyrosine phosphatases. The second form can be slowly inactivated by PP2A. In this case two Ser/Thr phosphates are removed at different rates during inactivation: One phosphate is very quickly removed to result in the formation of a high-mobility 39-kDa ERK2 species without consequence for activity; the other, slowly removed Ser/Thr phosphate controls the activity but has no effect on the gel mobility of ERK2. These results show that forms of ERK2 exist with properties different from the previously characterized ERK2 (p42^mapk) from stimulated cell cultures. The active ERK2 forms produced in the presence of Mg²⁺-ATP alone could provide an explanation for the existence of constitutive ERK2-like NF phosphorylation in vivo. Excessive formation of an ERK2 species resistant to inactivation by PP2A might be relevant to the persistent pathological τ hyperphosphorylation in AD.     

Epidermal growth factor and transforming growth factor-beta1 enhance HK-2 cell migration through a synergistic increase of matrix metalloproteinase and sustained activation of ERK signaling pathway

Epidermal growth factor (EGF) and transforming growth factor-beta1 (TGF-beta1), upregulated in renal diseases, have a combinational effect on epithelial-mesenchymal transformation (EMT) of renal proximal tubular cells. The aim of this study was to examine the mechanism regarding the combinational effect of EGF and TGF-beta1 on cell migration following EMT. The results demonstrated that EGF (10 ng/ml) and TGF-beta1 (3 ng/ml) synergistically increased cell migration, accompanied by an increase in matrix metalloproteinase-9 (MMP-9) gene expression, production and activity. Inhibition of MMP-9 production and activity by an MMP-2/MMP-9-specific inhibitor blocked the synergistic effect of EGF and TGF-beta1 on cell migration. The kinetic profile of extracellular signal-regulated kinase (ERK) signals demonstrated that ERK1/2 activation was rapidly and strongly induced by EGF but delayed and less marked by TGF-beta1 stimulation. In contrast, co-administration of EGF and TGF-beta1 caused an early pronounced and persistent ERK1/2 activation. Inhibition of the ERK1/2 activity by PD98059 abrogated the synergistic effect of EGF and TGF-beta1 on cell migration, MMP-9 production and activity, indicating that EGF and TGF-beta1 converged at the ERK signaling pathway to mediate cell migration. This study demonstrates that EGF and TGF-beta1 synergistically stimulate proximal tubular cell migration through the increased MMP-9 function and enhanced ERK1/2 activation.     

The COX-2 inhibitor SC-236 exerts anti-inflammatory effects by suppressing phosphorylation of ERK in a murine model

SC-236, (4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1-pyrazol-1-]benzenesulfonamide; C(16)H(11)ClF(3)N(3)O(2)S) is a highly selective cyclooxygenase (COX)-2 inhibitor. Recently, there have been reports that SC-236 protects against cartilage damage in addition to reducing inflammation and pain for those with osteoarthritis. However, the mechanism involved in an inflammatory allergic reaction in a murine model has not been examined. The aim of the present study is to elucidate whether and how SC-236 modulates the inflammatory allergic reaction in a murine model. In this study, the anti-allergic effect was investigated using rat peritoneal mast cells, IgE-induced passive cutaneous anaphylaxis (PCA), and the ear-swelling model in mice. Also, we examined the inhibitory effect of SC-236 on the expression of interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha. SC-236 was found to inhibit the ear-swelling response and histamine release in the murine model. Additionally, SC-236 was revealed to inhibit the PCA response and COX-2 expression. As a final step, the inhibitory mechanism of SC-236 was shown to occur through phosphorylation of extracellular signal-regulated protein kinase (ERK). These in vitro and in vivo results provide new insight into the pharmacological actions of SC-236 as a potential molecule for therapy for inflammatory allergic diseases.     

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.     

Erythropoietin receptor Y479 couples to ERK1/2 activation via recruitment of phospholipase Cgamma

Red blood cell development is primarily controlled by erythropoietin (EPO). Several studies have revealed the importance of EPO-R Y343 and Y479 for erythroid cell growth, differentiation, and survival. In order to isolate critical signaling proteins that bind to EPO-R, we initiated a Cloning of Ligand Target (COLT) screen using a murine embryonic day 16 phage library and a biotinylated EPO-R Y343 phosphopeptide. One of the clones isolated encodes Phospholipase C (PLC)gamma1. PLCgamma1 is rapidly tyrosine phosphorylated upon EPO stimulation and associates with EPO-R in an SH2-domain-dependent manner. Although PLCgamma1 bound EPO-R Y343, Y401, Y429, Y431, and Y479 in the COLT screen, PLCgamma1 required Y479 for association with EPO-R in Ba/F3-EPO-R cells. Studies have identified EPO-R Y479 as important for ERK activation. Since PI3-kinase binds EPO-R Y479, one group has suggested that ERK activation downstream of PI3-kinase accounts for the importance of this residue in EPO signaling. However, we show that inhibition of PI3-kinase does not abolish ERK activation. Furthermore, we demonstrate interaction of PLCgamma1 with Grb2 and SOS2. Hence, we have identified a novel adapter function for PLCgamma1 in EPO signaling in which recruitment of PLCgamma1 to EPO-R may lead to activation of the ERK pathway.     

Lactate regulates autophagy through ROS-mediated activation of ERK1/2/m-TOR/p-70S6K pathway in skeletal muscle

The role of autophagy and lysosomal degradation pathway in the regulation of skeletal muscle metabolism was previously studied. However, underlying molecular mechanisms are poorly understood. L-lactate which is utilized as an energetic substrate by skeletal muscle can also augment genes expression related to metabolism and up-regulate those being responsive to reactive oxygen species (ROS). Since ROS is the most important regulator of autophagy in skeletal muscle, we tested if there is a link between cellular lactate metabolism and autophagy in differentiated C2C12 myotubes and the gastrocnemius muscle of male wistar rats. C2C12 mouse skeletal muscle was exposed to 2, 6, 10, and 20 mM lactate and evaluated for lactate autophagic effects. Lactate dose-dependently increased autophagy and augmented ROS generation in differentiated C2C12 myotubes. The autophagic effect of lactate deterred in N-acetylcysteine presence (NAC, a ROS scavenger) indicated lactate regulates autophagy with ROS participation. Lactate-induced up-regulation of extracellular signal-regulated kinase 1/2 (ERK1/2) through ROS was required to regulate the autophagy by lactate. Further analysis about ERK1/2 up- and downstream indicated that lactate regulates autophagy through ROS-mediated the activation of ERK1/2/mTOR/p70S6K pathway in skeletal muscle. The in vitro effects of lactate on autophagy also occurred in the gastrocnemius muscle of male Wistar rats. In conclusion, we provided the lactate-associated regulation evidence of autophagy in skeletal muscle by activating ROS-mediated ERK1/2/mTOR/p70S6K pathway. Since the increase in cellular lactate concentration is a hallmark of energy deficiency, the results provide insight into a skeletal muscle mechanism to fulfill its enhanced energy requirement.     

Structure-based identification of inhibitors targeting obstruction of the HIVgp41 N-heptad repeat trimer

The viral protein HIVgp41 is an attractive and validated drug target that proceeds through a sequence of conformational changes crucial for membrane fusion, which facilitates viral entry. Prior work has identified inhibitors that interfere with the formation of a required six-helix bundle, composed of trimeric C-heptad (CHR) and N-heptad (NHR) repeat elements, through blocking association of an outer CHR helix or obstructing formation of the inner NHR trimer itself. In this work, we employed similarity-based scoring to identify and experimentally characterize 113 compounds, related to 2 small-molecule inhibitors recently reported by Allen et al. (Bioorg. Med. Chem Lett. 2015, 25 2853–59), proposed to act via the NHR trimer obstruction mechanism. The compounds were first tested in an HIV cell-cell fusion assay with the most promising evaluated in a second, more biologically relevant viral entry assay. Of the candidates, compound #11 emerged as the most promising hit (IC₅₀ = 37.81 µM), as a result of exhibiting activity in both assays with low cytotoxicity, as was similarly seen with the known control peptide inhibitor C34. The compound also showed no inhibition of VSV-G pseudotyped HIV entry compared to a control inhibitor suggesting it was specific for HIVgp41. Molecular dynamics simulations showed the predicted DOCK pose of #11 interacts with HIVgp41 in an energetic fashion (per-residue footprints) similar to the four native NHR residues (IQLT) which candidate inhibitors were intended to mimic.     

Extracellular signal-regulated kinase 2 mRNA expression in the rat brain during aging

Aging is accompanied by the loss of memory and cognitive functions. The extracellular signal-regulated kinase (ERK) pathway has been shown to play an essential role in synaptic plasticity and memory. Although a reduction in basal ERK1/2 activity has been found in the cerebral cortex in aged rats, changes in ERK1/2 mRNA expression during aging have not been described. In this study, we investigated age differences in the mRNA expression of ERK2 in different brain regions of male Fisher 344 rats (three age-groups) using quantitative in situ hybridization. No age-dependent changes in ERK2 mRNA were detected in the cerebellum or cortical areas. However, in the hippocampus, a 20% decline in mRNA levels was observed in the CA3 region in the 12-month-old group as compared to the 3-month-old group. These results suggest that the impairment in ERK1/2 activity observed during aging is probably not regulated at the gene expression level.     

Serotonin acts as a novel regulator of interleukin-6 secretion in osteocytes through the activation of the 5-HT2B receptor and the ERK1/2 signalling pathway

Interleukin-6 (IL-6) is a potent stimulator of osteoclastic bone resorption. Osteocyte secretion of IL-6 plays an important role in bone metabolism. Serotonin (5-HT) has recently been reported to regulate bone metabolism. The aim of this study was to evaluate the effect of serotonin on osteocyte expression of IL-6. The requirement for the 5-HT receptor(s) and the role of the extracellular signal-regulated kinase 1/2 (ERK1/2) in serotonin-induced IL-6 synthesis were examined. In this study, real-time PCR and ELISA were used to analyse IL-6 gene and protein expression in serotonin-stimulated MLO-Y4 cells. ERK1/2 pathway activation was determined by Western blot. We found that serotonin significantly activated the ERK1/2 pathway and induced IL-6 mRNA expression and protein synthesis in cultured MLO-Y4 cells. However, these effects were abolished by pre-treatment of MLO-Y4 cells with a 5-HT_2B receptor antagonist, RS127445 or the ERK1/2 inhibitor, PD98059. Our results indicate that serotonin stimulates osteocyte secretion of IL-6 and that this effect is associated with activation of 5-HT_2B receptor and the ERK1/2 pathway. These findings provide support for a role of serotonin in bone metabolism by indicating serotonin regulates bone remodelling by mediating an inflammatory cytokine.     

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.     

Endothelial cell-pericyte cocultures induce PLA2 protein expression through activation of PKCalpha and the MAPK/ERK cascade

Little is known about the regulatory mechanisms of endothelial cell (EC) proliferation by retinal pericytes and vice versa. In a model of coculture with bovine retinal pericytes lasting for 24 h, rat brain ECs showed an increase in arachidonic acid (AA) release, whereas Western blot and RT-PCR analyses revealed that ECs activated the protein expression of cytosolic phospholipase A(2) (cPLA(2)) and its phosphorylated form and calcium-independent intracellular phospholipase A(2) (iPLA(2)). No activation of the same enzymes was seen in companion pericytes. In ECs, the protein level of phosphorylated extracellular signal-regulated kinase (ERK) 1/2 was also enhanced significantly, a finding not observed in cocultured pericytes. The expression of protein kinase C-alpha (PKCalpha) and its phosphorylated form was also enhanced in ECs. Wortmannin, LY294002, and PD98059, used as inhibitors of upstream kinases (the PI3-kinase/Akt/PDK1 or MEK-1 pathway) in cultures, markedly attenuated AA release and the expression of phosphorylated forms of endothelial cPLA(2), PKCalpha, and ERK1/2. By confocal microscopy, activation of PKCalpha in perinuclear regions of ECs grown in coculture as well as strong activation of cPLA(2) in ECs taken from a model of mixed culture were clearly observed. However, no increased expression of both enzymes was found in cocultured pericytes. Our findings indicate that a sequential activation of PKCalpha contributes to endothelial ERK1/2 and cPLA(2) phosphorylation induced by either soluble factors or direct cell-to-cell contact, and that the PKCalpha-cPLA(2) pathway appears to play a key role in the early phase of EC-pericyte interactions regulating blood retina or blood-brain barrier maturation.     

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.