scholarly journals Reciprocal Role of ERK and Nf-κb Pathways in Survival and Activation of Osteoclasts

2000 ◽  
Vol 148 (2) ◽  
pp. 333-342 ◽  
Author(s):  
Tsuyoshi Miyazaki ◽  
Hideki Katagiri ◽  
Yumi Kanegae ◽  
Hiroshi Takayanagi ◽  
Yasuhiro Sawada ◽  
...  
Keyword(s):  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Erk Activation ◽  
Iκb Kinase ◽  
Mitogen Activated Protein ◽  
Osteoclast Activation ◽  
Osteoclast Survival ◽  
Constitutively Active

To examine the role of mitogen-activated protein kinase and nuclear factor kappa B (NF-κB) pathways on osteoclast survival and activation, we constructed adenovirus vectors carrying various mutants of signaling molecules: dominant negative Ras (RasDN), constitutively active MEK1 (MEKCA), dominant negative IκB kinase 2 (IKKDN), and constitutively active IKK2 (IKKCA). Inhibiting ERK activity by RasDN overexpression rapidly induced the apoptosis of osteoclast-like cells (OCLs) formed in vitro, whereas ERK activation after the introduction of MEKCA remarkably lengthened their survival by preventing spontaneous apoptosis. Neither inhibition nor activation of ERK affected the bone-resorbing activity of OCLs. Inhibition of NF-κB pathway with IKKDN virus suppressed the pit-forming activity of OCLs and NF-κB activation by IKKCA expression upregulated it without affecting their survival. Interleukin 1α (IL-1α) strongly induced ERK activation as well as NF-κB activation. RasDN virus partially inhibited ERK activation, and OCL survival promoted by IL-1α. Inhibiting NF-κB activation by IKKDN virus significantly suppressed the pit-forming activity enhanced by IL-1α. These results indicate that ERK and NF-κB regulate different aspects of osteoclast activation: ERK is responsible for osteoclast survival, whereas NF-κB regulates osteoclast activation for bone resorption.

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.

Synergistic Activation of Mitogen-Activated Protein Kinase by Cyclic AMP and Myeloid Growth Factors Opposes Cyclic AMP’s Growth-Inhibitory Effects

Blood ◽  
1999 ◽  
Vol 93 (2) ◽  
pp. 537-553 ◽  
Author(s):  
Angel Wai-mun Lee
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Cell Line ◽  
Mapk Pathway ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Erk Activation ◽  
Mitogen Activated Protein ◽  
Erk Activity ◽  
Myeloid Progenitors

Abstract Colony-stimulating factors (CSFs) promote the proliferation, differentiation, commitment, and survival of myeloid progenitors, whereas cyclic AMP (cAMP)-mediated signals frequently induce their growth arrest and apoptosis. The ERK/mitogen-activated protein kinase (MAPK) pathway is a target for both CSFs and cAMP. We investigated how costimulation by cAMP and colony-stimulating factor-1 (CSF-1) or interleukin-3 (IL-3) modulates MAPK in the myeloid progenitor cell line, 32D. cAMP dramatically increased ERK activity in the presence of CSF-1 or IL-3. IL-3 also synergized with cAMP to activate ERK in another myeloid cell line, FDC-P1. The increase in ERK activity was transmitted to a downstream target, p90rsk. cAMP treatment of 32D cells transfected with oncogenic Ras was found to recapitulate the superactivation of ERK seen with cAMP and CSF-1 or IL-3. ERK activation in the presence of cAMP did not appear to involve any of the Raf isoforms and was blocked by expression of dominant-negative MEK1 or treatment with a MEK inhibitor, PD98059. Although cAMP had an overall inhibitory effect on CSF-1–mediated proliferation and survival, the inhibition was markedly increased if ERK activation was blocked by PD98059. These findings suggest that upregulation of the ERK pathway is one mechanism induced by CSF-1 and IL-3 to protect myeloid progenitors from the growth-suppressive and apoptosis-inducing effects of cAMP elevations.

Epithelial injury induces Egr-1 and Fos expression by a pathway involving protein kinase C and ERK

1999 ◽  
Vol 276 (2) ◽  
pp. G322-G330 ◽  
Author(s):  
Brian K. Dieckgraefe ◽  
Danielle M. Weems
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Dominant Negative Mutant ◽  
Mrna Levels ◽  
Mobility Shift ◽  
Erk Activation ◽  
Kinase C

The signaling pathways activated in response to gastrointestinal injury remain poorly understood. Previous work has implicated the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase as a mediator of wound-signal transduction and a possible regulator of epithelial restitution. Monolayer injury resulted in rapid activation of p42 and p44 ERK. Injury-induced ERK activation was blocked by protein kinase C inhibition or by disruption of the cell cytoskeleton. Significant increases in Fos and early growth response (Egr)-1 mRNA levels were stimulated by injury, peaking by 20 min. ERK activation and the induction of Egr-1 mRNA were inhibited in a dose-dependent fashion with PD-98059. Fos mRNA expression was partially blocked by PD-98059. Western blot analysis demonstrated strong expression and nuclear localization of Fos and Egr after wounding. Electrophoretic mobility shift assays demonstrated that nuclear extracts contained a protein that specifically bound double-stranded oligonucleotides containing the Egr consensus binding element. Gel supershift assays demonstrated that the protein-DNA complexes were recognized by anti-Egr antibody. Inhibition of injury-induced ERK activation by PD-98059 or direct interference with Egr by expression of a dominant negative mutant led to significantly reduced in vitro monolayer restitution.

scholarly journals MEKK1 activates both IκB kinase α and IκB kinase β

1998 ◽  
Vol 95 (16) ◽  
pp. 9319-9324 ◽  
Author(s):  
Frank S. Lee ◽  
Robert T. Peters ◽  
Luan C. Dang ◽  
Tom Maniatis
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Mitogen Activated Protein Kinase ◽  
Site Specific ◽  
Iκb Kinase ◽  
Mitogen Activated Protein ◽  
Bona Fide ◽  
Factor Α

A critical step in the signal-induced activation of the transcription factor NF-κB is the site-specific phosphorylation of its inhibitor, IκB, that targets the latter for degradation by the ubiquitin–proteasome pathway. We have previously shown that mitogen-activated protein kinase/ERK kinase kinase 1 (MEKK1) can induce both this site-specific phosphorylation of IκBα at Ser-32 and Ser-36 in vivo and the activity of a high molecular weight IκB kinase complex in vitro. Subsequently, others have identified two proteins, IκB kinase α (IKK-α) and IκB kinase β (IKK-β), that are present in a tumor necrosis factor α-inducible, high molecular weight IκB kinase complex. These kinases are believed to directly phosphorylate IκB based on the examination of the kinase activities of IKK immunoprecipitates, but more rigorous proof of this has yet to be demonstrated. We show herein that recombinant IKK-α and IKK-β can, in fact, directly phosphorylate IκBα at Ser-32 and Ser-36, as well as homologous residues in IκBβ in vitro, and thus are bona fide IκB kinases. We also show that MEKK1 can induce the activation of both IKK-α and IKK-β in vivo. Finally, we show that IKK-α is present in the MEKK1-inducible, high molecular weight IκB kinase complex and treatment of this complex with MEKK1 induces phosphorylation of IKK-α in vitro. We conclude that IKK-α and IKK-β can mediate the NF-κB-inducing activity of MEKK1.

scholarly journals Regulation of Cyclooxygenase 2 mRNA Stability by the Mitogen-Activated Protein Kinase p38 Signaling Cascade

2000 ◽  
Vol 20 (12) ◽  
pp. 4265-4274 ◽  
Author(s):  
Marina Lasa ◽  
Kamal R. Mahtani ◽  
Andrew Finch ◽  
Gary Brewer ◽  
Jeremy Saklatvala ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mrna Stability ◽  
Active Form ◽  
Cyclooxygenase 2 ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Signaling Cascade ◽  
Mitogen Activated Protein ◽  
Cox 2 ◽  
Constitutively Active

ABSTRACT A tetracycline-regulated reporter system was used to investigate the regulation of cyclooxygenase 2 (Cox-2) mRNA stability by the mitogen-activated protein kinase (MAPK) p38 signaling cascade. The stable β-globin mRNA was rendered unstable by insertion of the 2,500-nucleotide Cox-2 3′ untranslated region (3′ UTR). The chimeric transcript was stabilized by a constitutively active form of MAPK kinase 6, an activator of p38. This stabilization was blocked by SB203580, an inhibitor of p38, and by two different dominant negative forms of MAPK-activated protein kinase 2 (MAPKAPK-2), a kinase lying downstream of p38. Constitutively active MAPKAPK-2 was also able to stabilize chimeric β-globin–Cox-2 transcripts. The MAPKAPK-2 substrate hsp27 may be involved in stabilization, as β-globin–Cox-2 transcripts were partially stabilized by phosphomimetic mutant forms of hsp27. A short (123-nucleotide) fragment of the Cox-2 3′ UTR was necessary and sufficient for the regulation of mRNA stability by the p38 cascade and interacted with a HeLa protein immunologically related to AU-rich element/poly(U) binding factor 1.

scholarly journals Hepatitis C Virus (HCV) Constitutively Activates STAT-3 via Oxidative Stress: Role of STAT-3 in HCV Replication

2005 ◽  
Vol 79 (3) ◽  
pp. 1569-1580 ◽  
Author(s):  
Gulam Waris ◽  
James Turkson ◽  
Tarek Hassanein ◽  
Aleem Siddiqui
Keyword(s):  
Oxidative Stress ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Hcv Rna ◽  
Pyrrolidine Dithiocarbamate ◽  
Constitutive Activation ◽  
Mitogen Activated Protein

ABSTRACT The hepatitis C virus (HCV) causes chronic hepatitis, which often results in liver cirrhosis and hepatocellular carcinoma. We have previously shown that HCV nonstructural proteins induce activation of STAT-3 via oxidative stress and Ca2+ signaling (G. Gong, G. Waris, R. Tanveer, and A. Siddiqui, Proc. Natl. Acad. Sci. USA 98:9599-9604, 2001). In this study, we focus on the signaling pathway leading to STAT-3 activation in response to oxidative stress induced by HCV translation and replication activities. Here, we demonstrate the constitutive activation of STAT-3 in HCV replicon-expressing cells. The HCV-induced STAT-3 activation was inhibited in the presence of antioxidant (pyrrolidine dithiocarbamate) and Ca2+ chelators (BAPTA-AM and TMB-8). Previous studies have shown that maximum STAT-3 transactivation requires Ser727 phosphorylation in addition to tyrosine phosphorylation. Using a series of inhibitors and dominant negative mutants, we show that HCV-induced activation of STAT-3 is mediated by oxidative stress and influenced by the activation of cellular kinases, including p38 mitogen-activated protein kinase, JNK, JAK-2, and Src. Our results also suggest a potential role of STAT-3 in HCV RNA replication. We also observed the constitutive activation of STAT-3 in the liver biopsy of an HCV-infected patient. These studies provide an insight into the mechanisms by which HCV induces intracellular events relevant to liver pathogenesis associated with the viral infection.

scholarly journals Positive and negative selection invoke distinct signaling pathways.

1996 ◽  
Vol 184 (1) ◽  
pp. 9-18 ◽  
Author(s):  
J Alberola-Ila ◽  
K A Hogquist ◽  
K A Swan ◽  
M J Bevan ◽  
R M Perlmutter
Keyword(s):  
T Cell ◽  
Positive Selection ◽  
Negative Selection ◽  
Mapk Pathway ◽  
Cell Receptor ◽  
Dominant Negative ◽  
Mapk Cascade ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein

During T cell development, interaction of the T cell receptor (TCR) with cognate ligands in the thymus may result in either maturation (positive selection) or death (negative selection). The intracellular pathways that control these opposed outcomes are not well characterized. We have generated mice expressing dominant-negative Ras (dnRas) and Mek-1 (dMek) transgenes simultaneously, either in otherwise normal animals, or in animals expressing a transgenic TCR, thereby permitting a comprehensive analysis of peptide-specific selection. In this system, thymocyte maturation beyond the CD4+8+ stage is blocked almost completely, whereas negative selection, assessed using an in vitro deletion protocol, is quantitatively intact. This suggests that activation of the mitogen-activated protein kinase (MAPK) cascade is necessary for positive selection, but irrelevant for negative selection. Generation of gamma/delta and of CD4-8- alpha/beta T cells proceeds normally despite blockade of the MAPK cascade. Hence, only cells that mature via conventional, TCR-mediated repertoire selection require activation of the MAPK pathway to complete their maturation.

scholarly journals Akt-Dependent Phosphorylation Specifically Regulates Cot Induction of NF-κB-Dependent Transcription

2002 ◽  
Vol 22 (16) ◽  
pp. 5962-5974 ◽  
Author(s):  
Lawrence P. Kane ◽  
Marianne N. Mollenauer ◽  
Zheng Xu ◽  
Christoph W. Turck ◽  
Arthur Weiss
Keyword(s):  
Protein Kinase ◽  
Signaling Pathways ◽  
Cellular Transformation ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Iκb Kinase ◽  
Ikk Complex ◽  
Mitogen Activated Protein ◽  
Tumor Necrosis Factor Induction ◽  
Necrosis Factor

ABSTRACT The Akt (or protein kinase B) and Cot (or Tpl-2) serine/threonine kinases are associated with cellular transformation. These kinases have also been implicated in the induction of NF-κB-dependent transcription. As a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, Cot can also activate MAP kinase signaling pathways that target AP-1 and NFAT family transcription factors. Here we show that Akt and Cot physically associate and functionally cooperate. Akt appears to function upstream of Cot, as Akt can enhance Cot induction of NF-κB-dependent transcription, and dominant-negative Cot blocks the activation of this element by Akt. Furthermore, deletion analysis shows that binding to Akt is critical for Cot function. The regulation of NF-κB-dependent transcription by Cot requires Akt-dependent phosphorylation of serine 400 (S400), near the carboxy terminus of Cot. However, phosphorylation at this site is not required for Cot kinase activity or AP-1 induction, suggesting it specifically regulates Cot effector function at the level of the NF-κB pathway. Mutation of S400 in Cot does indeed abolish its ability to activate IκB-kinase (IKK) complexes, but paradoxically it allows for increased Cot association with the IKK complex. This mutated form of Cot also acts as a dominant negative for T-cell antigen receptor/CD28- or Akt/phorbol myristate acetate-induced NF-κB induction, while having relatively little effect on tumor necrosis factor induction of NF-κB. These findings suggest that the activation of different signaling pathways by MAP3Ks may be regulated separately and may provide evidence for how such discrimination by one member of this kinase family occurs.

scholarly journals Differential inhibition of signaling pathways by dominant-negative SH2/SH3 adapter proteins.

1995 ◽  
Vol 15 (12) ◽  
pp. 6829-6837 ◽  
Author(s):  
M Tanaka ◽  
R Gupta ◽  
B J Mayer
Keyword(s):  
Protein Kinase ◽  
Signaling Pathways ◽  
Tyrosine Kinases ◽  
Egf Receptor ◽  
Sh3 Domain ◽  
Sh2 Domain ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Erk Activation ◽  
Mitogen Activated Protein

SH2/SH3 adapters are thought to function in signal transduction pathways by coupling inputs from tyrosine kinases to downstream effectors such as Ras. Members of the mitogen-activated protein kinase family are known to be activated by a variety of mitogenic stimuli, including tyrosine kinases such as Abl and the epidermal growth factor (EGF) receptor. We have used activation of the mitogen-activated protein kinase Erk-1 as a model system with which to examine whether various dominant-negative SH2/SH3 adapters (Grb2, Crk, and Nck) could block signaling pathways leading to Erk activation. Activation of Erk-1 by oncogenic Abl was effectively inhibited by Grb2 with mutations in either its SH2 or SH3 domain or by Crk-1 with an SH3 domain mutation. The Crk-1 SH2 mutant was less effective, while Nck SH2 and SH3 mutants had little or no effect on Erk activation. These results suggest that both Crk and Grb2 may contribute to the activation of Erk by oncogenic Abl, whereas Nck is unlikely to participate in this pathway. Next we examined whether combinations of these dominant-negative adapters could inhibit Erk activation more effectively than each mutant alone. When combinations of Crk-1 and Grb2 mutants were analyzed, the combination of the Crk-1 SH3 mutant plus the Grb2 SH3 mutant gave a striking synergistic effect. This finding suggests that in Abl-transformed cells, more than one class of tyrosine-phosphorylated sites (those that bind the Grb2 SH2 domain and those that bind the Crk SH2 domain) can lead to Ras activation. In contrast to results with Abl, Erk activation by EGF was strongly inhibited only by Grb2 mutants; Crk and Nck mutants had little or no effect. This finding suggests that Grb2 is the only adapter involved in the activation of Erk by EGF. Dominant-negative adaptors provide a novel means to identify binding interactions important in vivo for signaling in response to a variety of stimuli.

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