Extracellular signal regulated kinases

Inactivation of the retinoblastoma protein pathway is the most common abnormality in malignant tumors. We therefore tried to detect agents that induce the cyclin-dependent kinase inhibitor p15INK4b and found that ZD1839 (gefitinib, Iressa) could up-regulate p15INK4b expression. ZD1839 has been shown to inhibit cell cycle progression through inhibition of signaling pathways such as phosphatidylinositol 3′-kinase-Akt and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) cascades. However, the mechanism responsible for the differential sensitivity of the signaling pathways to ZD1839 remains unclear. We here showed that ZD1839 up-regulated p15INK4b, resulting in retinoblastoma hypophosphorylation and G1 arrest in human immortalized keratinocyte HaCaT cells. p15INK4b induction was caused by MAPK/ERK kinase inhibitor (PD98059), but not by Akt inhibitor (SH-6, Akt-III). Moreover, mouse embryo fibroblasts lacking p15INK4b were resistant to the growth inhibi...

Background Type 1 and 2 diabetes confer an increased risk of pancreatic cancer (PaC) of similar magnitude, suggesting a common mechanism. The recent finding that PaC incidence increases linearly with increasing fasting glucose levels supports a central role for hyperglycaemia, which is known to cause carbonyl stress and advanced glycation end-product (AGE) accumulation through increased glycolytic activity and non-enzymatic reactions. This study investigated the impact of hyperglycaemia on invasive tumour development and the underlying mechanisms involved. Methods Pdx1-Cre;LSL-KrasG12D/+ mice were interbred with mitosis luciferase reporter mice, rendered diabetic with streptozotocin and treated or not with carnosinol (FL-926-16), a selective scavenger of reactive carbonyl species (RCS) and, as such, an inhibitor of AGE formation. Mice were monitored for tumour development by in vivo bioluminescence imaging. At the end of the study, pancreatic tissue was collected for histology/immun...

The cells of multicellular organisms receive extracellular signals using surface receptors. The extracellular domains (ECDs) of cell surface receptors function as interaction platforms, and as regulatory modules of receptor activation. Understanding how interactions between ECDs produce signal-competent receptor complexes is challenging because of their low biochemical tractability. In plants, the discovery of ECD interactions is complicated by the massive expansion of receptor families, which creates tremendous potential for changeover in receptor interactions. The largest of these families in Arabidopsis thaliana consists of 225 evolutionarily related leucine-rich repeat receptor kinases (LRR-RKs), which function in the sensing of microorganisms, cell expansion, stomata development and stem-cell maintenance. Although the principles that govern LRR-RK signalling activation are emerging, the systems-level organization of this family of proteins is unknown. Here, to address this, we ...

Naloxone benzoylhydrazone (NalBzoH) is often used as a nociceptin opioid peptide (NOP) receptor antagonist. However, NalBzoH is also a mixed µ antagonist/κ and δ opioid agonist and its actions at the NOP receptor range from an antagonist to partial agonist, depending on the effector response/system studied. We now report that NalBzoH activates endogenously expressed κ 3 opioid, but not NOP, receptors to stimulate extracellular signal-regulated protein kinases 1/2 and, subsequently, to mediate changes in the expression of tyrosine hydroxylase and G protein-coupled receptor kinase 2 in SH-SY5Y human neuroblastoma cells. Furthermore, pretreatment with NalBzoH produces homologous desensitization of the κ 3 opioid receptor as well as heterologous desensitization of the NOP receptor. In contrast, pretreatment with the NOP agonist, orphanin FQ/nociceptin (OFQ/N), desensitizes only NOP but not the NalBzoH response, suggesting the involvement of a receptor, distinct from NOP, in mediating the actions of NalBzoH in SH-SY5Y cells.

The Roman high-(RHA) and low-(RLA) avoidance rats are selectively bred for, respectively, rapid vs. poor acquisition of active avoidance in a shuttle box and differ markedly in emotional reactivity and coping style as well as in their behavioural and neurochemical responses to cocaine. Thus, acute cocaine induces a more robust increase in locomotion in RHA than RLA rats. Moreover, in RHA rats, acute cocaine causes a larger increment in dopamine (DA) output in the shell compartment of the nucleus accumbens (AcbSh:~+119%) as compared with the core (AcbC: +58%), whereas no such accumbal compartment-related difference is observed in RLA rats (AcbSh:~+63%; AcbC:~+52%) [1]. Furthermore, RHA rats learn to self-administer cocaine faster and consume larger amounts of the drug compared with their RLA counterparts [2]. Cocaine administration induces plastic adaptations of cellular mechanisms in limbic brain areas involved in learning and memory, including signaling by extracellular signalregulated kinase (ERK). The present study was therefore designed to compare the effects of acute cocaine on ERK phosphorylation in limbic areas of RHA and RLA rats, such as prefrontal cortex (PFCx), Acb, central amygdala (CeA) and bed nucleus of stria terminalis (BNST). Method: Rats were treated with cocaine (5 mg/kg, i.p.) or vehicle and transcardially perfused 20 min later. Brains were removed and coronal sections were cut with a vibratome. After immunohistochemical staining, the slices were examined with a light microscope to count the pERK-positive neurons in the regions of interest, as previously described [3]. Results: Acute cocaine induced a significant increase in the number of pERK positive neurons in the AcbSh (+51%) and in the infralimbic portion of the PFCx (PFCx-IL, +101%) of RHA rats but not of their RLA counterparts (AcbSh: +12%; PFCx-IL: +36%) [AcbSh: F line (1,16) = 10.97, p < 0.005, F treatment (1,16) = 4.92, p < 0.05, F interaction (1,16) = 2.80, NS; PFCx-IL: F line (1,16) = 15.06, p < 0.01, F treatment (1,16) = 5.36, p < 0.05, F interaction (1,16) = 0.24, NS; two-way ANOVA]. In contrast, acute cocaine failed to modify pERK immunoreactivity in the AcbC and in the prelimbic portion of the PFCx (PFCx-PrL) of either line. Moreover, no line-or cocaine-related changes were observed in the CeA and BNST. Conclusions: In keeping with our previous finding that RHA rats learn to self-administer cocaine faster than RLA rats [2], acute cocaine increased ERK phosphorylation in the AcbSh and the PFCx-IL only in RHA rats (i.e., two areas that are part of the limbic subcircuit that controls motivated behaviors and plays a crucial role in the initial response to the drug). Conversely, acute cocaine failed to modify pERK immunoreactivity in the AcbC and the PFCx-PrL of either line, two areas that are part of a motor subcircuit involved in habit acquisition upon chronic exposure to cocaine. These results provide additional support to the view that the Roman lines may represent a valid model to investigate the neurochemical underpinnings of the individual vulnerability to drug addiction.

In the present work, we described the expression and activity of extracellular signal-related kinases 1-2 (ERK1-2) in mouse primordial germ cells (PGCs) from 8.5-14.5 days post coitum (dpc) and investigated whether these kinases play a role in regulating the various processes of PGC development. Using immunofluorescence and immunoblotting to detect the active phosphorylated form of ERK1-2 (p-ERK1-2), we found that the kinases were present in most proliferating 8.5-10.5 dpc PGCs, low in 11.5 dpc PGCs, and progressively increasing between 12.5-14.5 dpc both in female and male PGCs. In vitro culture experiments showed that inhibiting activation of ERK1-2 with the MEK-specific inhibitor U0126 significantly reduced the growth of 8.5 dpc PGCs in culture but had little effect on 11.5-12.5 dpc PGCs. Moreover, we found that the inhibitor did not affect the adhesion of 11.5 dpc PGCs, but it significantly reduced their motility features onto a cell monolayer. Further, while the ability of female PGCs to begin meiosis was not significantly affected by U0126, their progression through meiotic prophase I was slowed down. Notably, the activity of ERK1-2 was necessary for maintaining the correct expression of oocytespecific genes crucial for germ cells survival and the formation of primordial follicles.

Ligation of the ␣ 6 ␤ 4 integrin induces tyrosine phosphorylation of the ␤ 4 cytoplasmic domain, followed by recruitment of the adaptor protein Shc and activation of mitogen-activated protein kinase cascades. We have used Far Western analysis and phosphopeptide competition assays to map the sites in the cytoplasmic domain of ␤ 4 that are required for interaction with Shc. Our results indicate that, upon phosphorylation, Tyr 1440 , or secondarily Tyr 1422 , interacts with the SH2 domain of Shc, whereas Tyr 1526 , or secondarily Tyr 1642 , interacts with its phosphotyrosine binding (PTB) domain. An inactivating mutation in the PTB domain of Shc, but not one in its SH2 domain, suppresses the activation of Shc by ␣ 6 ␤ 4. In addition, mutation of ␤ 4 Tyr 1526 , which binds to the PTB domain of Shc, but not of Tyr 1422 and Tyr 1440 , which interact with its SH2 domain, abolishes the activation of ERK by ␣ 6 ␤ 4. Phenylalanine substitution of the ␤ 4 tyrosines able to interact with the SH2 or PTB domain of Shc does not affect incorporation of ␣ 6 ␤ 4 in the hemidesmosomes of 804G cells. Exposure to the tyrosine phosphatase inhibitor orthovanadate increases tyrosine phosphorylation of ␤4 and disrupts the hemidesmosomes of 804G cells expressing recombinant wild type ␤ 4. This treatment, however, exerts a decreasing degree of inhibition on the hemidesmosomes of cells expressing versions of ␤ 4 containing phenylalanine substitutions at Tyr 1422 and Tyr 1440 , at Tyr 1526 and Tyr 1642 , or at all four tyrosine phosphorylation sites. These results suggest that ␤ 4 Tyr 1526 interacts in a phosphorylation-dependent manner with the PTB domain of Shc. This event is required for subsequent tyrosine phosphorylation of Shc and signaling to ERK but not formation of hemidesmosomes.

Malignant mesothelioma (MM), lung cancers, and asbestosis are hyperproliferative diseases associated with exposures to asbestos. All have a poor prognosis; thus, the need to develop novel and effective therapies is urgent. Vandetanib (Van) (ZD6474, ZACTIMA) is a tyrosine kinase inhibitor that has shown equivocal results in clinical trials for advanced non-small cell lung cancer. However, tyrosine kinase inhibitors alone have shown no significant clinical activity in phase II trials of patients with unresectable MM. Using epithelioid (HMESO) and sarcomatoid (H2373) human MM lines, the efficacy of tumor cell killing and signaling pathways modulated by Van with and without doxorubicin (Dox) was examined. Van alone reduced total cell numbers in HMESO MM and synergistically increased the toxicity of Dox in HMESO and H2373 cells. Most importantly, we identified two novel cell survival/resistance pathways, ERK5 and cyclic AMP response element binding protein (CREB), that were inhibited by Van and Dox. After silencing of either ERK5 or CREB, significant decreases in cell numbers in the Dox-resistant sarcomatoid H2373 line were observed. Results suggest that a plethora of cell signaling pathways associated with cell survival are induced by Dox but inhibited by the addition of Van in MM. Data from our study support the combined efficacy of Van and Dox as a novel approach in the treatment of MM that is further enhanced by blocking ERK5 or CREB signaling cascades.

Supplementary Figure 1. Analyses of co-localization of MEK1 and tubulin in different regions at various stages of the cell cycle. A, Average values of Pearson's correlation (Rp) and Manders' co-localization coefficients (M) around the centrosomes and midbody in MCH603MEK1act cells at various stages of mitosis and cytokinesis.

Tau neuronal protein has a central role in neurodegeneration and is implicated in Alzheimer disease development. Abnormal phosphorylation of Tau impairs its interaction with other proteins and is associated with its dysregulation in pathological conditions. Molecular mechanisms leading to hyperphosphorylation of Tau in pathological conditions are unknown. Here, we characterize phosphorylation of Tau by extracellular-regulated kinase (ERK2), a mitogen-activated kinase (MAPK) that responds to extracellular signals. Analysis of in vitro phosphorylated Tau by activated recombinant ERK2 with nuclear magnetic resonance spectroscopy (NMR) reveals phosphorylation of 15 Ser/Thr sites. In vitro phosphorylation of Tau using rat brain extract and subsequent NMR analysis identifies the same sites. Phosphorylation with rat brain extract is known to transform Tau into an Alzheimer disease-like state. Our results indicate that phosphorylation of Tau by ERK2 alone is sufficient to produce the same characteristics. We further investigate the mechanism of ERK2 phosphorylation of Tau. Kinases are known to recognize their protein substrates not only by their specificity for a targeted Ser or Thr phosphorylation site but also by binding to linear-peptide motifs called docking sites. We identify two main ERK2 docking sites in Tau sequence using NMR. Our results suggest that ERK2 dysregulation in Alzheimer disease could lead to abnormal phosphorylation of Tau resulting in the pathology of the disease.

Glycemic spikes may negatively affect the long-term prognosis of patients with diabetes. Extracellular signal-regulated kinases (ERKs) are intracellular mediators of cell proliferation, and they can be activated in response to high glucose levels. However, the modifications of their activity in response to hyperglycemia have been poorly investigated, in vivo, in humans. Thus, we sought to determine in circulating monocytes: 1) the role of hyperglycemia in ERKs activity and phosphorylation, and 2) whether hyperglycemia affects mitogen-activated protein kinase kinase (MEK) activity and mitogen-activated protein phosphatase-1 (MKP-1) expression. These goals were performed in five normal subjects. Baseline monocyte ERKs activity was 60 Ϯ 5 pmol/min⅐mg protein; when exogenous hyperglycemia was induced, both monocyte ERKs activity (81 Ϯ 11 pmol/min⅐mg protein; P Ͻ 0.05) and phosphorylation significantly increased (P Ͻ 0.01). MEK activity was significantly increased by hyperglycemia (1251 Ϯ 136 vs. 2000 Ϯ 42 cpm; P ϭ 0.0017), whereas no changes were observed in MKP-1 expression. We conclude that hyperglycemia acutely stimulates ERKs activity and phosphorylation in human monocytes by the MEK pathway in vivo. These findings may be relevant in understanding the negative role of acute hyperglycemia on monocyte pathophysiology.

Background: The role of PPARD was examined in the testes because this receptor can regulate cell differentiation and proliferation. Results: PPARD temporally represses ERK-dependent cell cycle signaling and increases tight junction proteins in Sertoli cells. Conclusion: PPARD is essential for maturation of Sertoli cells to prevent testicular degeneration. Significance: PPARD modulates Sertoli cell function and spermatogenesis. Ppard ؊/؊ mice exhibit smaller litter size compared with Ppard ؉/؉ mice. To determine whether peroxisome proliferator-activated receptor-D (PPARD) could possibly influence this phenotype, the role of PPARD in testicular biology was examined. Atrophic testes and testicular degeneration were observed in Ppard ؊/؊ mice compared with Ppard ؉/؉ mice, indicating that PPARD modulates spermatogenesis. Higher expression of p27 and decreased expression of proliferating cellular nuclear antigen in Sertoli cells were observed in Ppard ؉/؉ mice as compared with Ppard ؊/؊ mice, and these were associated with decreased Sertoli cell number in Ppard ؉/؉ mice. Cyclin D1 and cyclin D2 expression was lower in Ppard ؉/؉ as compared with Ppard ؊/؊ mice. Ligand activation of PPARD inhibited proliferation of a mouse Sertoli cell line, TM4, and an inverse agonist of PPARD (DG172) rescued this effect. Temporal inhibition of extracellular signal-regulated kinase (ERK) activation by PPARD in the testis was observed in Ppard ؉/؉ mice and was associated with decreased serum follicle-stimulating hormone and higher claudin-11 expression along the blood-testis barrier. PPARD-dependent ERK activation also altered expression of claudin-11, p27, cyclin D1, and cyclin D2 in TM4 cells, causing inhibition of cell proliferation, maturation, and formation of tight junctions in Sertoli cells, thus confirming a requirement for PPARD in accurate Sertoli cell function. Combined, these results reveal for the first time that PPARD regulates spermatogenesis by modulating the function of Sertoli cells during early testis development.

The ETS domain transcription factor ELK1 is in a repressive association with growth genes and is transiently activated through phosphorylation by ERK1/2. In prostate cancer (PCa) cells the androgen receptor (AR) is recruited by ELK1, via its amino-terminal domain (A/B), as a transcriptional co-activator, without ELK1 hyper-phosphorylation. Here we elucidate the structural basis of the interaction of AR with ELK1. The ELK1 polypeptide motifs required for co-activation by AR vs. those required for activation of ELK1 by ERK were systematically mapped using a mammalian two-hybrid system and confirmed using a co-immunoprecipitation assay. The mapping precisely identified the two ERK docking sites in ELK1, the D-box and the DEF (Docking site for ERK, FXFP) motif as the essential motifs for its cooperation with AR(A/B) or wtAR. In contrast, the transactivation domain in ELK1 was only required for activation by ERK. ELK1-mediated transcriptional activity of AR(A/B) was optimal in the absenc...

Resistance to RAF inhibitors is generally accompanied by reactivation of extracellular signal-regulated kinase (ERK) signaling. SCH772984, a selective, ATP-competitive inhibitor of ERK1 and ERK2, is effective in BRAF-mutant models in which resistance is the result of ERK reactivation. SCH772984 may also have a role in the treatment of tumors in which ERK is dysregulated by mutant RAS, NF1, or activated receptor tyrosine kinases, settings in which current RAF inhibitors are ineffective.

Resistance to RAF inhibitors is generally accompanied by reactivation of extracellular signal-regulated kinase (ERK) signaling. SCH772984, a selective, ATP-competitive inhibitor of ERK1 and ERK2, is effective in BRAF-mutant models in which resistance is the result of ERK reactivation. SCH772984 may also have a role in the treatment of tumors in which ERK is dysregulated by mutant RAS, NF1, or activated receptor tyrosine kinases, settings in which current RAF inhibitors are ineffective.

Tau neuronal protein has a central role in neurodegeneration and is implicated in Alzheimer disease development. Abnormal phosphorylation of Tau impairs its interaction with other proteins and is associated with its dysregulation in pathological conditions. Molecular mechanisms leading to hyperphosphorylation of Tau in pathological conditions are unknown. Here, we characterize phosphorylation of Tau by extracellular-regulated kinase (ERK2), a mitogen-activated kinase (MAPK) that responds to extracellular signals. Analysis of in vitro phosphorylated Tau by activated recombinant ERK2 with nuclear magnetic resonance spectroscopy (NMR) reveals phosphorylation of 15 Ser/Thr sites. In vitro phosphorylation of Tau using rat brain extract and subsequent NMR analysis identifies the same sites. Phosphorylation with rat brain extract is known to transform Tau into an Alzheimer disease-like state. Our results indicate that phosphorylation of Tau by ERK2 alone is sufficient to produce the same characteristics. We further investigate the mechanism of ERK2 phosphorylation of Tau. Kinases are known to recognize their protein substrates not only by their specificity for a targeted Ser or Thr phosphorylation site but also by binding to linear-peptide motifs called docking sites. We identify two main ERK2 docking sites in Tau sequence using NMR. Our results suggest that ERK2 dysregulation in Alzheimer disease could lead to abnormal phosphorylation of Tau resulting in the pathology of the disease.

The protein kinase CK2 is a pleiotropic and constitutively active kinase that plays crucial roles in cellular proliferation and survival. Overexpression of CK2, particularly the alpha catalytic subunit (CK2α, CSNK2A1), has been implicated in a wide variety of cancers and is associated with poorer survival and with resistance to both conventional and targeted anticancer therapies. Here, we found that CK2α protein is elevated in melanoma cell lines compared to normal human melanocytes. We then tested the involvement of CK2α in drug resistance to FDA-approved single-agent targeted therapies for melanoma. In BRAF-mutant melanoma cells, ectopic CK2α decreased sensitivity to vemurafenib (BRAFi), dabrafenib (BRAFi), and trametinib (MEKi), by a mechanism distinct from that of mutant NRAS. Conversely, knockdown of CK2α sensitized cells to inhibitor treatment. CK2α-mediated RAF-MEK kinase inhibitor resistance was tightly linked to its maintenance of ERK phosphorylation. We found that CK2α pos...

Background Type 1 and 2 diabetes confer an increased risk of pancreatic cancer (PaC) of similar magnitude, suggesting a common mechanism. The recent finding that PaC incidence increases linearly with increasing fasting glucose levels supports a central role for hyperglycaemia, which is known to cause carbonyl stress and advanced glycation end-product (AGE) accumulation through increased glycolytic activity and non-enzymatic reactions. This study investigated the impact of hyperglycaemia on invasive tumour development and the underlying mechanisms involved. Methods Pdx1-Cre;LSL-KrasG12D/+ mice were interbred with mitosis luciferase reporter mice, rendered diabetic with streptozotocin and treated or not with carnosinol (FL-926-16), a selective scavenger of reactive carbonyl species (RCS) and, as such, an inhibitor of AGE formation. Mice were monitored for tumour development by in vivo bioluminescence imaging. At the end of the study, pancreatic tissue was collected for histology/immun...

Background Type 1 and 2 diabetes confer an increased risk of pancreatic cancer (PaC) of similar magnitude, suggesting a common mechanism. The recent finding that PaC incidence increases linearly with increasing fasting glucose levels supports a central role for hyperglycaemia, which is known to cause carbonyl stress and advanced glycation end-product (AGE) accumulation through increased glycolytic activity and non-enzymatic reactions. This study investigated the impact of hyperglycaemia on invasive tumour development and the underlying mechanisms involved. Methods Pdx1-Cre;LSL-KrasG12D/+ mice were interbred with mitosis luciferase reporter mice, rendered diabetic with streptozotocin and treated or not with carnosinol (FL-926-16), a selective scavenger of reactive carbonyl species (RCS) and, as such, an inhibitor of AGE formation. Mice were monitored for tumour development by in vivo bioluminescence imaging. At the end of the study, pancreatic tissue was collected for histology/immun...

Background Type 1 and 2 diabetes confer an increased risk of pancreatic cancer (PaC) of similar magnitude, suggesting a common mechanism. The recent finding that PaC incidence increases linearly with increasing fasting glucose levels supports a central role for hyperglycaemia, which is known to cause carbonyl stress and advanced glycation end-product (AGE) accumulation through increased glycolytic activity and non-enzymatic reactions. This study investigated the impact of hyperglycaemia on invasive tumour development and the underlying mechanisms involved. Methods Pdx1-Cre;LSL-KrasG12D/+ mice were interbred with mitosis luciferase reporter mice, rendered diabetic with streptozotocin and treated or not with carnosinol (FL-926-16), a selective scavenger of reactive carbonyl species (RCS) and, as such, an inhibitor of AGE formation. Mice were monitored for tumour development by in vivo bioluminescence imaging. At the end of the study, pancreatic tissue was collected for histology/immun...

Cyclosporin A (CsA) is known to have an immunosuppressive action. However, it is also attracting attention due to its effects on the nervous system, such as inhibiting the development and expression of morphine-induced tolerance and dependence through unknown mechanisms. It has been shown that CsA modulates the nitric oxide (NO) synthesis and extracellular signal-regulated kinases (ERK) activation, which are potentially involved in signaling pathways in morphine-induced tolerance in cellular models. Therefore, the current study was designed to evaluate the modulatory role of CsA on the MOR tolerance, by targeting the downstream signaling pathway of NO and ERK using an in vitro model. For this purpose, T98G cells were pretreated with CsA, calcineurin autoinhibitory peptide (CAIP), and NG-nitro-l-arginine methyl ester (L-NAME) 30 min before 18 h exposure to MOR. Then, we analyzed the intracellular cyclic adenosine monophosphate (cAMP) levels and also the expression of phosphorylated E...

Matrix metalloproteinases (MMPs) play key roles in vascular remodeling. We characterized the role of inflammatory mediators and extracellular signal-regulated kinases (ERKs) in the control of arterialized vein graft expression of MMP-9, MMP-2, and membrane-type 1-MMP (MT1-MMP) and of the tissue inhibitor of metalloproteinases-2 (TIMP-2). For this purpose we used a canine model of jugular vein to carotid artery interposition graft and analyzed the vein grafts at various postoperative times (30 min to 28 days) using the contralateral vein as a control. To study the role of ERK-1/2, veins were incubated with the mitogen-activated protein kinase kinase (MEK-1/2) inhibitor UO126 for 30 min before being grafted. Vein graft extracts were analyzed for MMPs, TIMP-2, tumor necrosis factor-α (TNF-α), polymorphonuclear neutrophil (PMN) infiltration, myeloperoxidase (MPO), and thrombin activity, and for ERK-1/2 activation. Vein graft arterialization resulted in rapid and sustained (8 h to 28 day...

Recent studies have demonstrated that administration of an electroconvulsive shock produces a rapid and transient increase in tyrosyl phosphorylation of a-40-kDa protein in rat brain. Initial characterization of this protein's chromatographic properties indicated that it might be a member of a recently identified family of kinases, referred to as mitogen-activated protein (MAP) kinases, that are activated by tyrosyl phosphorylation. In the present study, we have used MAP kinase antisera to assess the identity of this protein. We have found that the-40-kDa phosphotyrosine-containing protein comigrates with p42 MAP kinase (~42"'~"~) and not with two other 44-kDa MAP kinase family members detected by these antisera. Western blots of proteins immunoprecipitated with MAP kinase antibodies confirm that ~4 2~"~~ displays increased tyrosyl phosphorylation after an electroconvulsive stimulus. Chromatographic separation of hippocampal extracts indicates that MAP kinase activity elutes in parallel with ~4 2~~"~. Accordingly, these studies identify as a tyrosyl kinase substrate that is activated by this stimulus and suggest that this form of MAP kinase may be selectively regulated by neuronal stimulation.

Alterations of the EGFR/ERK and Hippo/YAP pathway have been found in non-small cell lung cancer (NSCLC). Herein, we show that ERK1 and ERK2 have an effect on the Hippo/YAP pathway in human NSCLC cells. Firstly, inhibition of ERK1/2 by siRNA or small-molecular inhibitors decreased the YAP protein level, the reporter activity of the Hippo pathway, and the mRNA levels of the Hippo downstream genes, CTGF, Gli2, and BIRC5. Secondly, degradation of YAP protein was accelerated after ERK1/2 depletion in NSCLC cell lines, in which YAP mRNA level was not decreased. Thirdly, forced over-expression of the ERK2 gene rescued the YAP protein level and Hippo reporter activity after siRNA knockdown targeting 3&#39;UTR of the ERK2 gene in NSCLC cells. Fourthly, depletion of ERK1/2 reduced the migration and invasion of NSCLC cells. Combined depletion of ERK1/2 had a greater effect on cell migration than depletion of either one separately. Finally, the MEK1/2 inhibitor Trametinib decreased YAP protein ...

Activation of V1 vasopressin (VP) receptors prevents serum deprivation-induced apoptosis in neuronal H32 cells, partially through mitogen activated protein (MAP) kinase-mediated Bad phosphorylation. Here we investigated the role of protein kinases C (PKC) and B (PKB) mediating VP induced antiapoptosis in H32 cells. Serum deprivation increased PKCδ but not PKCα or PKCβ activity, while VP increased PKCα and PKCβ without affecting PKCδ activity. Inhibition of PKCδ prevented caspase-3 activation, indicating that PKCδ mediates the proapoptotic actions of serum deprivation. Simultaneous inhibition of PKC α&β and MAP kinase abolished VP-induced Bad phosphorylation, but it only partially prevented caspase-3 inhibition. Complete abolition of the protective effect of VP on serum deprivation-induced caspase 3 activity required additional blockade of PI3K/protein kinase B (Akt). The data demonstrate that VP exerts antiapoptosis through multiple pathways; while PKCα&β together with ERK/MAP kinase activation mediates Bad phosphorylation (inactivation), the full protective action of VP requires additional activation of PKB (PI3K/Akt) pathway.

Background: The role of dopamine D 1 receptors and Extracellular signal Regulated Kinase (ERK) in the motivational properties of drugs can be studied by place-conditioning. Recent advances have shown that the motivational properties of ethanol, determined by place-conditioning, are mediated by its metabolic conversion into acetaldehyde. To date, the role of D 1 receptors and ERK activation in acetaldehyde-elicited place preference has not been determined. The aim of this study was to assess the role of D 1 receptors blockade and MEK inhibition in the acquisition of acetaldehyde-elicited conditioned place preference. Methods: Male Sprague-Dawley rats were subjected to repeated pairings with 1 compartment of the conditioning apparatus immediately following acetaldehyde (20 mg ⁄ kg i.g.) or ethanol (1 g ⁄ kg i.g.) administration. The D 1 receptor antagonist, SCH 39166 (50 lg ⁄ kg s.c.), was administered 10 minutes before acetaldehyde or ethanol administration. In order to study the role of activated ERK in the acetaldehyde-elicited place preference, rats were administered the MEK inhibitor, PD98059 (1, 30, and 90 lg i.c.v.), 10 or 30 minutes before acetaldehyde. To verify the specificity of these effects, we also studied whether PD98059 pretreatment could affect morphine (1 mg/kg s.c.)-elicited place preference. Results: Both acetaldehyde and ethanol elicited significant place preferences and these were prevented by pretreatment with SCH 39166. In addition, pretreatment with PD98059, dose-(30 and 90 but not 1 lg i.c.v.) and time-(10 but not 30 minutes before) dependently, prevented the acquisition of acetaldehyde-and significantly reduced the acquisition of morphine-elicited conditioned place preference. Conclusions: These results confirm that acetaldehyde and ethanol elicit conditioned place preference and demonstrate that D 1 receptors are critically involved in these effects. Furthermore, the finding that PD98059 prevents the acquisition of acetaldehyde-elicited conditioned place preference highlights the importance of the D 1 receptors-ERK pathway in its motivational effects.

In this study, the homozygous tubby (tub/tub) mutant mouse, with an early progressive hearing loss and photoreceptor degeneration, was used as a model system to examine the effects of systemic administration of a naturally occurring isothiocyanate, sulforaphane (SF), on photoreceptor degeneration. Several novel observations have been made: (i) the mRNA and protein expression of thioredoxin (Trx), thioredoxin reductase (TrxR) and NF-E2-related factor-2 (Nrf2) were significantly reduced even prior to photoreceptor cell degeneration in the retinas of tub/tub mice, suggesting that retinal expression of the Trx system is impaired and that Trx regulation is involved in the pathogenesis of retinal degeneration in this model, (ii) intraperitoneal injection with SF significantly up-regulated retinal levels of Trx, TrxR, and Nrf2, and effectively protected photoreceptor cells in tub/tub mice as evaluated functionally by electroretinography and morphologically by quantitative histology, and (iii) treatment with PD98059, an inhibitor of extracellular signal-regulated kinases (ERKs), blocked SF-mediated ERKs activation and up-regulation of Trx/TrxR/Nrf2 in the retinas of tub/tub mice. This suggests that ERKs and Nrf2 are involved in the mechanism of SF-mediated up-regulation of the Trx system to protect photoreceptor cells in this model. These novel findings are significant and could provide important information for the development of a unique strategy to prevent sensorineural deafness/retinal dystrophic syndromes and also other forms of inherited neurological disorders.

Background: The role of dopamine D 1 receptors and Extracellular signal Regulated Kinase (ERK) in the motivational properties of drugs can be studied by place-conditioning. Recent advances have shown that the motivational properties of ethanol, determined by place-conditioning, are mediated by its metabolic conversion into acetaldehyde. To date, the role of D 1 receptors and ERK activation in acetaldehyde-elicited place preference has not been determined. The aim of this study was to assess the role of D 1 receptors blockade and MEK inhibition in the acquisition of acetaldehyde-elicited conditioned place preference. Methods: Male Sprague-Dawley rats were subjected to repeated pairings with 1 compartment of the conditioning apparatus immediately following acetaldehyde (20 mg ⁄ kg i.g.) or ethanol (1 g ⁄ kg i.g.) administration. The D 1 receptor antagonist, SCH 39166 (50 lg ⁄ kg s.c.), was administered 10 minutes before acetaldehyde or ethanol administration. In order to study the role of activated ERK in the acetaldehyde-elicited place preference, rats were administered the MEK inhibitor, PD98059 (1, 30, and 90 lg i.c.v.), 10 or 30 minutes before acetaldehyde. To verify the specificity of these effects, we also studied whether PD98059 pretreatment could affect morphine (1 mg/kg s.c.)-elicited place preference. Results: Both acetaldehyde and ethanol elicited significant place preferences and these were prevented by pretreatment with SCH 39166. In addition, pretreatment with PD98059, dose-(30 and 90 but not 1 lg i.c.v.) and time-(10 but not 30 minutes before) dependently, prevented the acquisition of acetaldehyde-and significantly reduced the acquisition of morphine-elicited conditioned place preference. Conclusions: These results confirm that acetaldehyde and ethanol elicit conditioned place preference and demonstrate that D 1 receptors are critically involved in these effects. Furthermore, the finding that PD98059 prevents the acquisition of acetaldehyde-elicited conditioned place preference highlights the importance of the D 1 receptors-ERK pathway in its motivational effects.

Leptin resistance is induced by the feedback inhibitors tyrosine phosphatase-1B (PTP1B) and decreased Src homology 2 domain-containing tyrosine phosphatase-2 (SHP-2) signaling. To investigate the participation of PTP1B and SHP-2 in LPS-induced leptin resistance, we injected repeated (6-LPS) intraperitoneal LPS doses (100 μg/kg ip) for comparison with a single (1-LPS) treatment and evaluated the expression of SHP-2, PTP1B, p-ERK1/2, and p-STAT3 in the hypothalamus of male Wistar rats. The single LPS treatment increased the expression of p-STAT3 and PTP1B but not SHP-2. The repeated LPS treatment reduced SHP-2, increased PTP1B, and did not change p-STAT3. We observed that the PTP1B expression induced by the endotoxin was highly colocalized with leptin receptor cells in the hypothalamus of LepRb-IRES-Cre-tdTomato reporter mice. The single, but not the repeated, LPS treatment decreased the food intake and body weight. Leptin had no stimulatory effect on the hypophagia, body weight loss,...

This study examined the anti-obesity effect and mechanism of action of red ginseng extract (RG) in 3T3-L1 cells. To measure the levels of lipid accumulation, we were observed of expression of genes and proteins, which is associated with adipocyte differentiation in 3T3-L1 cells. Eight days following induction for differentiation with MDI-medium and simultaneously with the tested RG, our study found significantly reduced (p < 0.05) intracellular triacylglycerol accumulations of 3T3-L1 cells by does dependent manner. RG decrease adipogenic markers, C/EBPα, C/EBPβ and PPARγ, which is a key regulator of adipocyte differentiation. In this study showed that C/EBPβ is regulated RG decreased the phosphorylations of p38 mitogen-activated protein kinase (MAPK) and extracellular signal–regulated kinases (ERK). These results suggest that the anti-adipogenic effect of RG in 3T3-L1 pro-adipocyte could be associated with the inhibition of adipogenic transcription factors by regulations with p38 MAPK and ERK phosphorylation.