scholarly journals SHOC2 complex-driven RAF dimerization selectively contributes to ERK pathway dynamics

2019 ◽  
Vol 116 (27) ◽  
pp. 13330-13339 ◽  
Author(s):  
Isabel Boned del Río ◽  
Lucy C. Young ◽  
Sibel Sari ◽  
Greg G. Jones ◽  
Benjamin Ringham-Terry ◽  
...  
Keyword(s):  
Erk Pathway ◽  
Ras Proteins ◽  
Oncogenic Signaling ◽  
Erk Activation ◽  
Pathway Activation ◽  
Complex Functions ◽  
Inhibitory Site ◽  
Pathway Inhibition ◽  
Mutant Cells

Despite the crucial role of RAF kinases in cell signaling and disease, we still lack a complete understanding of their regulation. Heterodimerization of RAF kinases as well as dephosphorylation of a conserved “S259” inhibitory site are important steps for RAF activation but the precise mechanisms and dynamics remain unclear. A ternary complex comprised of SHOC2, MRAS, and PP1 (SHOC2 complex) functions as a RAF S259 holophosphatase and gain-of-function mutations in SHOC2, MRAS, and PP1 that promote complex formation are found in Noonan syndrome. Here we show that SHOC2 complex-mediated S259 RAF dephosphorylation is critically required for growth factor-induced RAF heterodimerization as well as for MEK dissociation from BRAF. We also uncover SHOC2-independent mechanisms of RAF and ERK pathway activation that rely on N-region phosphorylation of CRAF. In DLD-1 cells stimulated with EGF, SHOC2 function is essential for a rapid transient phase of ERK activation, but is not required for a slow, sustained phase that is instead driven by palmitoylated H/N-RAS proteins and CRAF. Whereas redundant SHOC2-dependent and -independent mechanisms of RAF and ERK activation make SHOC2 dispensable for proliferation in 2D, KRAS mutant cells preferentially rely on SHOC2 for ERK signaling under anchorage-independent conditions. Our study highlights a context-dependent contribution of SHOC2 to ERK pathway dynamics that is preferentially engaged by KRAS oncogenic signaling and provides a biochemical framework for selective ERK pathway inhibition by targeting the SHOC2 holophosphatase.

scholarly journals FcγR-induced production of superoxide and inflammatory cytokines is differentially regulated by SHIP through its influence on PI3K and/or Ras/Erk pathways

Blood ◽  
2006 ◽  
Vol 108 (2) ◽  
pp. 718-725 ◽  
Author(s):  
Latha P. Ganesan ◽  
Trupti Joshi ◽  
Huiqing Fang ◽  
Vijay Kumar Kutala ◽  
Julie Roda ◽  
...  
Keyword(s):  
Inflammatory Cytokines ◽  
Clustering Analysis ◽  
Molecular Mechanisms ◽  
Erk Pathway ◽  
Erk Activation ◽  
Coated Particles ◽  
Inositol Phosphatase ◽  
Enhanced Production ◽  
Gtp Binding

Phagocytosis of IgG-coated particles via FcγR is accompanied by the generation of superoxide and inflammatory cytokines, which can cause collateral tissue damage in the absence of regulation. Molecular mechanisms regulating these phagocytosis-associated events are not known. SHIP is an inositol phosphatase that downregulates PI3K-mediated activation events. Here, we have examined the role of SHIP in FcγR-induced production of superoxide and inflammatory cytokines. We report that primary SHIP-deficient bone marrow macrophages produce elevated levels of superoxide upon FcγR clustering. Analysis of the molecular mechanism revealed that SHIP regulates upstream Rac-GTP binding, an obligatory event for superoxide production. Likewise, SHIP-deficient macrophages displayed enhanced IL-1β and IL-6 production in response to FcγR clustering. Interestingly, whereas IL-6 production required activation of both PI3K and Ras/Erk pathways, IL-1β production was dependent only on Ras/Erk activation, suggesting that SHIP may also regulate the Ras/Erk pathway in macrophages. Consistently, SHIP-deficient macrophages displayed enhanced activation of Erk upon FcγR clustering. Inhibition of Ras/Erk or PI3K suppressed the enhanced production of IL-6 in SHIP-deficient macrophages. In contrast, inhibition of Ras/Erk, but not PI3K, suppressed IL-1β production in these cells. Together, these data demonstrate that SHIP regulates phagocytosis-associated events through the inhibition of PI3K and Ras/Erk pathways.

scholarly journals Adenovirus Type 7 Induces Interleukin-8 Production via Activation of Extracellular Regulated Kinase 1/2

2001 ◽  
Vol 75 (14) ◽  
pp. 6450-6459 ◽  
Author(s):  
M. J. Alcorn ◽  
J. L. Booth ◽  
K. M. Coggeshall ◽  
J. P. Metcalf
Keyword(s):  
Epithelial Cells ◽  
Neutrophil Infiltration ◽  
Adenovirus Type ◽  
Mek Inhibitor ◽  
Interleukin 8 ◽  
Erk Pathway ◽  
Erk Activation ◽  
Extracellular Regulated Kinase ◽  
Rapid Activation

ABSTRACT Infection with adenovirus serotype 7 (Ad7) frequently causes lower respiratory pneumonia and is associated with severe lung inflammation and neutrophil infiltration. Earlier studies indicated release of proinflammatory cytokines, specifically interleukin-8 (IL-8), by pulmonary epithelial cells following infection by Ad7. However, the mechanism of IL-8 induction by Ad7 is unclear. We have explored the role of the Ras/Raf/MEK/Erk pathway in the Ad7-associated induction of IL-8 using a model system of A549 epithelial cells. We found that Ad7 infection induced a rapid activation of epithelial cell-derived Erk. The MEK-specific inhibitors PD98059 and U0126 blocked Erk activation and release of IL-8 following infection with Ad7. Treatment with PD98059 is cytostatic and not cytotoxic, as treated cells regain the ability to phosphorylate Erk and secrete IL-8 after removal of the drug. The expression of a mutated form of Ras in A549 epithelial cells blocked the induction of IL-8 promoter activity, and MEK inhibitor blocked induction of IL-8 mRNA. These results suggest that the Ras/Raf/MEK/Erk pathway is necessary for the Ad7 induction of IL-8 and that induction occurs at the level of transcription. Further, the kinetics of Erk activation and IL-8 induction suggest that an early viral event, such as receptor binding, may be responsible for the observed inflammatory response.

scholarly journals Role of ERK activation in H. pylori-induced disruption of cell-cell tight junctions

2020 ◽  
Author(s):  
Amita Sekar ◽  
Bow Ho
Keyword(s):  
Tight Junction ◽  
Barrier Function ◽  
Cell Junction ◽  
Erk Pathway ◽  
Erk Activation ◽  
Infected Cells ◽  
H Pylori ◽  
Tight Junction Disruption ◽  
Cell Cell

AbstractBackgroundTight junctions, a network of claudins and other proteins, play an important role in maintaining barrier function and para-cellular permeability. H. pylori, the major etiological agent of various gastroduodenal diseases, is known to cause tight junction disruption. However, the molecular events that triggered cell-cell tight junction disruption in H. pylori-infected cells, remain largely elusive.Materials and MethodsTrans-epithelial electrical resistance (TEER) and FITC-Dextran permeability measurement were performed to determine the barrier function in H. pylori 88-3887-infected polarized MKN28 cells. For visualization of tight junction protein localization, immunofluorescence and immunoblotting techniques were used. To examine the role of ERK activation in tight junction disruption, U0126, a MEK inhibitor, was employed. To further support the study, computational analyses of H. pylori-infected primary gastric cells were carried out to decipher the transcriptomic changes.ResultsThe epithelial barrier of polarized MKN28 cells when infected with H. pylori displayed disruption of cell-cell junctions as shown by TEER & FITC-dextran permeability tests. Claudin-4 was shown to delocalize from host cytoplasm to nucleus in H. pylori-infected cells. In contrast, delocalization of claudin-4 was minimized when ERK activation was inhibited. Interestingly, transcriptomic analyses revealed the upregulation of genes associated with cell-junction assembly and ERK pathway forming a dense interacting network of proteins.ConclusionTaken together, evidence from this study indicates that H. pylori regulates ERK pathway triggering cell-cell junction disruption, contributing to host pathogenesis. It indicates the vital role of ERK in regulating key events associated with the development of H. pylori-induced gastroduodenal diseases.

Atypical Protein Kinase Cs Promote CSF-1-Dependent Erk Activation and Proliferation in Myeloid Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4227-4227 ◽  
Author(s):  
Angel W. Lee
Keyword(s):  
Drug Targets ◽  
Phorbol Esters ◽  
Myeloid Cells ◽  
Dominant Negative ◽  
Erk Pathway ◽  
Erk Activation ◽  
Cell Biol ◽  
Pkc Inhibitors

Abstract Macrophages are integral components of the innate immune system and essential players in inflammation. Enhanced macrophage numbers underlie these pathological states. Colony stimulating factor-1 (CSF-1) is the major physiological regulator of proliferation and survival of cells of the monocyte/macrophage lineage. CSF-1 binds to a receptor tyrosine kinase, the CSF-1 receptor (CSF-1R). CSF-1 and CSF-1R have emerged as drug targets in several diseases where inflammation is a critical component, e.g. breast cancer and rheumatoid arthritis. Multiple pathways are activated downstream of the CSF-1R; however, it is not clear which of these pathways regulate proliferation and survival. Atypical PKCs (aPKCs) are implicated in cell proliferation and survival. They include the isoforms PKCζ and PKCλ/ι. Unlike the classical and novel PKCs, aPKCs are insensitive to Ca2+ and phorbol esters. In this study, we investigated the role of aPKCs in CSF-1-mediated proliferation in myeloid cells. CSF-1 is a proliferation and survival factor for 32D.R cells, a myeloid progenitor cell line transfected with the CSF-1R. Western blotting shows that PKCα, PKCδ, PKCε and PKCζ/λ/I are expressed in 32D.R. Based on studies with PKC inhibitors that have different specificities towards aPKCs (GF109203X, Ro-31-8220, Go6983 and a Myr-PKCζ peptide), maximal CSF-1-dependent proliferation in 32D.R cells appears to depend on the activity of either aPKCs or PKCε. Using phospho-specific antibodies that detect the activation state of PKCζ as well as in vitro kinase assays, we showed that CSF-1 activates aPKCs in 32D.R and bone marrow derived macrophages. In contrast, CSF-1-induced activation of PKCε was not observed. We next asked how aPKC affects CSF-1 signaling. PKCζ promotes activation of the MEK-Erk pathway in different cell types (Corbit, K.C. et al. Mol. Cell. Biol. 20, 5392). In 32D.R cells, treatment with the MEK inhibitor, U0126, reduced CSF-1-provoked proliferation by 60–70%, consistent with the inhibition observed with PKC inhibitors. Previous work from our lab showed that CSF-1 activates the Erk pathway through A-Raf and not Raf-1 (Lee and States, Mol. Cell. Biol. 18, 6779). We found that aPKC inhibitors do not affect CSF-1 induced Ras and A-Raf activity but markedly reduce MEK and Erk activity, implying that aPKC inputs into the CSF-1 Erk pathway at the level of MEK. Transient transfections with dominant-negative and constitutively active (CA) PKCζ confirmed that aPKC promotes CSF-1-induced Erk activation. aPKC inhibition does not affect CSF-1-stimulated Akt activation. To investigate the role of PKCζ in CSF-1-dependent proliferation, we established stable 32D.R mass populations overexpressing wildtype (WT) or CA PKCζ at levels 2-fold above endogenous. Comparing cells expressing CA-PKCζ to WT-PKCζ, the EC50 for CSF-1-dependent proliferation and the cell doubling time at maximal CSF-1 concentration were both reduced, consistent with a role for PKCζ in CSF-1 dependent proliferation. We will use our stable cell lines to elucidate the pathways modulated by PKCζ. Altogether, our results identify atypical PKCs as new targets of CSF-1 signaling.

Cadmium induced apoptosis in mouse primary hepatocytes: the role of oxidative stress-mediated ERK pathway activation and the involvement of histone H3 phosphorylation

RSC Advances ◽  
2015 ◽  
Vol 5 (40) ◽  
pp. 31798-31806 ◽  
Author(s):  
Jing Wang ◽  
Minglu Hao ◽  
Chunguang Liu ◽  
Rutao Liu
Keyword(s):  
Oxidative Stress ◽  
Histone H3 ◽  
Erk Signaling ◽  
Erk Pathway ◽  
Primary Hepatocytes ◽  
H3 Phosphorylation ◽  
Pathway Activation ◽  
Induced Apoptosis ◽  
Histone H3 Phosphorylation

Time-delayed apoptosis induced by cadmium in primary hepatocytes through DNA damage, histone modification and ERK signaling cascade, which are all mediated by oxidative stress.

scholarly journals RSL3 Drives Ferroptosis through NF-κB Pathway Activation and GPX4 Depletion in Glioblastoma

2021 ◽  
Vol 2021 ◽  
pp. 1-10
Author(s):  
Shengbiao Li ◽  
Yuping He ◽  
Kexin Chen ◽  
Jiaojiao Sun ◽  
Lulu Zhang ◽  
...  
Keyword(s):  
Tumor Model ◽  
Underlying Mechanism ◽  
Glioblastoma Cells ◽  
Recurrence Rates ◽  
Antitumor Effects ◽  
Pathway Activation ◽  
Pathway Inhibition ◽  
Related Proteins

Glioblastoma, the most aggressive form of malignant glioma, is very difficult to treat because of its aggressively invasive nature and high recurrence rates. RAS-selective lethal 3 (RSL3), a well-known inhibitor of glutathione peroxidase 4 (GPX4), could effectively induce oxidative cell death in glioblastoma cells through ferroptosis, and several signaling pathways are involved in this process. However, the role of the nuclear factor kappa-B (NF-κB) pathway in glioblastoma cell ferroptosis has not yet been investigated. Therefore, we aimed to clarify the underlying mechanism of the NF-κB pathway in RSL3-induced ferroptosis in glioblastoma cells. We found that RSL3 led to an increase in lipid ROS concentration and downregulation of ferroptosis-related proteins such as GPX4, ATF4, and SLC7A11 (xCT) in glioblastoma cells. Additionally, the NF-κB pathway was activated by RSL3, and its inhibition by BAY 11-7082 could alleviate ferroptosis. The murine xenograft tumor model indicated that NF-κB pathway inhibition could mitigate the antitumor effects of RSL3 in vivo. Furthermore, we found that GPX4 knockdown could not effectively induce ferroptosis. However, NF-κB pathway activation coupled with GPX4 silencing induced ferroptosis. Additionally, ATF4 and xCT expression might be regulated by the NF-κB pathway. Collectively, our results revealed that the NF-κB pathway plays a novel role in RSL3-induced ferroptosis in glioblastoma cells and provides a new therapeutic strategy for glioblastoma treatment.

scholarly journals Mitotic Phosphorylation of Golgi Reassembly Stacking Protein 55 by Mitogen-activated Protein Kinase ERK2

2001 ◽  
Vol 12 (6) ◽  
pp. 1811-1817 ◽  
Author(s):  
Stephen A. Jesch ◽  
Timothy S. Lewis ◽  
Natalie G. Ahn ◽  
Adam D. Linstedt
Keyword(s):  
Protein Kinase ◽  
Specific Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Erk Pathway ◽  
Erk Activation ◽  
Mitogen Activated Protein ◽  
Mitotic Phosphorylation

The role of the mitogen-activated protein kinase kinase (MKK)/extracellular-activated protein kinase (ERK) pathway in mitotic Golgi disassembly is controversial, in part because Golgi-localized targets have not been identified. We observed that Golgi reassembly stacking protein 55 (GRASP55) was phosphorylated in mitotic cells and extracts, generating a mitosis-specific phospho-epitope recognized by the MPM2 mAb. This phosphorylation was prevented by mutation of ERK consensus sites in GRASP55. GRASP55 mitotic phosphorylation was significantly reduced, both in vitro and in vivo, by treatment with U0126, a potent and specific inhibitor of MKK and thus ERK activation. Furthermore, ERK2 directly phosphorylated GRASP55 on the same residues that generated the MPM2 phospho-epitope. These results are the first demonstration of GRASP55 mitotic phosphorylation and indicate that the MKK/ERK pathway directly phosphorylates the Golgi during mitosis.

scholarly journals Role of the MAPK/ERK pathway in valvular interstitial cell calcification

2009 ◽  
Vol 296 (6) ◽  
pp. H1748-H1757 ◽  
Author(s):  
Xiaoxiao Gu ◽  
Kristyn S. Masters
Keyword(s):  
Mapk Pathway ◽  
Cell Types ◽  
Heart Valve Disease ◽  
Pcr Analysis ◽  
Nodule Formation ◽  
Erk Pathway ◽  
Dramatic Decrease ◽  
Pathway Inhibition ◽  
Erk Inhibition

Much remains to be discovered about the etiology of heart valve disease and the molecular level mechanisms that drive it. The MAPK/ERK pathway influences calcification in many cell types and has been linked to the expression of a contractile phenotype in valvular interstitial cells (VICs). However, a direct correlation between MAPK/ERK pathway activity and VIC calcification has not been previously described. Thus the role of the MAPK pathway in the calcification of VIC cultures was investigated by measuring ERK activation in both calcifying and noncalcifying VIC environments and then, conversely, analyzing the effects of ERK pathway inhibition on VIC calcification and phenotype. Prolonged elevation of phosphorylated ERK-1/2 was found in calcifying VIC cultures, whereas directly blocking phosphorylation of ERK-1/2 resulted in a dramatic decrease in nodule number, nodule size, and total calcified area. Application of the ERK pathway inhibitor was also associated with a dramatic decrease in apoptosis, which may have contributed to the decreased nodule formation obtained via ERK inhibition. Real-time PCR analysis revealed that calcified samples exhibited significantly elevated expression of several myofibroblastic and osteoblastic markers, while ERK inhibition substantially reduced the expression of these markers, often to levels comparable to the noncalcifying control. These data suggest that the MAPK pathway plays an important role in regulating the phenotype and calcification of VICs, wherein sustained pathway activation is associated with increased VIC calcification. These findings may be used to further elucidate the mechanisms of valvular disease and identify potential treatment targets.

Retinoic acid activation of the ERK pathway is required for embryonic stem cell commitment into the adipocyte lineage

2002 ◽  
Vol 361 (3) ◽  
pp. 621-627 ◽  
Author(s):  
Frédéric BOST ◽  
Leslie CARON ◽  
Irène MARCHETTI ◽  
Christian DANI ◽  
Yannick LE MARCHAND-BRUSTEL ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Es Cells ◽  
Embryonic Stem ◽  
Lipid Binding ◽  
Embryoid Bodies ◽  
Acid Activation ◽  
Erk Pathway ◽  
Erk Activation ◽  
Cell Commitment

Mouse embryonic stem (ES) cells are pluripotent cells that differentiate into multiple cell lineages. The commitment of ES cells into the adipocyte lineage is dependent on an early 3-day treatment with all-trans retinoic acid (RA). To characterize the molecular mechanisms underlying this process, we examined the contribution of the extracellular-signal-regulated kinase (ERK) pathway. Treatment of ES cell-derived embryoid bodies with RA resulted in a prolonged activation of the ERK pathway, but not the c-Jun N-terminal kinase, p38 mitogen-activated protein kinase or phosphoinositide 3-kinase pathways. To investigate the role of ERK activation, co-treatment of RA with PD98059, a specific inhibitor of the ERK signalling pathway, prevented both adipocyte formation and expression of the adipogenic markers, adipocyte lipid-binding protein and peroxisome-proliferator-activated receptor γ. Furthermore, we show that ERK activation is required only during RA treatment. PD98059 does not interfere with the commitment of ES cells into other lineages, such as neurogenesis, myogenesis and cardiomyogenesis. As opposed to the controversial role of the ERK pathway in terminal differentiation, our results clearly demonstrate that this pathway is specifically required at an early stage of adipogenesis, corresponding to the RA-dependent commitment of ES cells.

scholarly journals The Activation of MEK1 by Enhanced Homodimerization Drives Tumorigenesis

2017 ◽  
Author(s):  
Jimin Yuan ◽  
Wan Hwa Ng ◽  
Zizi Tian ◽  
Jiajun Yap ◽  
Manuela Baccarini ◽  
...  
Keyword(s):  
Cancer Biology ◽  
Differential Resistance ◽  
Erk Signaling ◽  
Activation Loop ◽  
Erk Activation ◽  
Pathway Activation ◽  
Key Steps

SummaryHyperactive RAS/RAF/MEK/ERK signaling has a well-defined role in cancer biology. Aberrant pathway activation occurs mostly upstream of MEK; however, MEK mutations are prevalent in some cancer subsets. Here we show that cancer-related MEK mutants can be classified as those activated by relieving the inhibitory role of helix A, and those with in-frame deletions of β3-αC loop, which exhibit differential resistance to MEK inhibitors in vitro and in vivo. The β3-αC loop deletions activate MEK1 through enhancing homodimerization that can drive intradimer cross-phosphorylation of activation loop. Further, we demonstrate that MEK1 dimerization is required both for its activation by RAF and for its catalytic activity towards ERK. Our study identifies a novel group of MEK mutants, illustrates some key steps in RAF/MEK/ERK activation, and has important implications for the design of therapies targeting hyperactive RAS/RAF/MEK/ERK signaling in cancers.

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