scholarly journals Novel Membrane-Targeted ERK1 and ERK2 Chimeras Which Act as Dominant Negative, Isotype-Specific Mitogen-Activated Protein Kinase Inhibitors of Ras-Raf-Mediated Transcriptional Activation of c-fos in NIH 3T3 Cells

1999 ◽  
Vol 19 (12) ◽  
pp. 8052-8065 ◽  
Author(s):  
Franz Hochholdinger ◽  
Gottfried Baier ◽  
Anto Nogalo ◽  
Birgit Bauer ◽  
Hans H. Grunicke ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Transcriptional Activation ◽  
Fusion Proteins ◽  
Nuclear Translocation ◽  
Protein Kinase Inhibitors ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Wild Type ◽  
Mitogen Activated Protein ◽  
Constitutively Active

ABSTRACT Expression of constructs encoding fusion proteins of ERK1 and ERK2 containing a C-terminal farnesylation motif (CAAX) is predominantly localized at the cell membrane and was activated by coexpression of constitutively active Ha-RasL61 and epidermal growth factor. Both fusion proteins significantly inhibit the transcriptional activation of a c-fos–chloramphenicol acetyltransferase reporter induced by RasL61, constitutively active MEK1, or constitutively active RafBXB. The corresponding SAAX chimeras or overexpression of the wild-type ERKs did not interfere with the transcriptional activation of c-fos. The inhibition of the Ras-mediated c-fosinduction by ERK2-CAAX can in part be rescued by coexpression of a wild-type ERK2 but not by wild-type ERK1. We find that ERK1-CAAX acts in the same fashion, indicating that mitogen-activated protein kinase (MAPK)–CAAX chimeras interact in an isotype-specific manner. It is demonstrated that both ERK1-CAAX and ERK2-CAAX associate with the corresponding endogenous ERKs, which explains the isotype-specific inhibitory effects of the ERK-CAAX chimeras. Evidence is presented that expression of ERK-CAAX fusion proteins inhibits the nuclear translocation of the corresponding endogenous ERKs. Disruption of MAPK translocation by membrane targeting provides additional, independent proof that nuclear translocation of ERKs is essential for the transcriptional activation of c-fos.

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 Rac-PAK Signaling Stimulates Extracellular Signal-Regulated Kinase (ERK) Activation by Regulating Formation of MEK1-ERK Complexes

2002 ◽  
Vol 22 (17) ◽  
pp. 6023-6033 ◽  
Author(s):  
Scott T. Eblen ◽  
Jill K. Slack ◽  
Michael J. Weber ◽  
Andrew D. Catling
Keyword(s):  
Protein Kinase ◽  
Signaling Pathway ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Wild Type ◽  
Erk Activation ◽  
Extracellular Signal Regulated Kinase ◽  
Signaling Complex ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

ABSTRACT Utilizing mutants of extracellular signal-regulated kinase 2 (ERK2) that are defective for intrinsic mitogen-activated protein kinase or ERK kinase (MEK) binding, we have identified a convergent signaling pathway that facilitates regulated MEK-ERK association and ERK activation. ERK2-Δ19-25 mutants defective in MEK binding could be phosphorylated in response to mitogens; however, signaling from the Raf-MEK pathway alone was insufficient to stimulate their phosphorylation in COS-1 cells. Phosphorylation of ERK2-Δ19-25 but not of wild-type ERK2 in response to Ras V12 was greatly inhibited by dominant-negative Rac. Activated forms of Rac and Cdc42 could enhance the association of wild-type ERK2 with MEK1 but not with MEK2 in serum-starved adherent cells. This effect was p21-activated kinase (PAK) dependent and required the putative PAK phosphorylation sites T292 and S298 of MEK1. In detached cells placed in suspension, ERK2 was complexed with MEK2 but not with MEK1. However, upon replating of cells onto a fibronectin matrix, there was a substantial induction of MEK1-ERK2 association and ERK activation, both of which could be inhibited by dominant-negative PAK1. These data show that Rac facilitates the assembly of a mitogen-activated protein kinase signaling complex required for ERK activation and that this facilitative signaling pathway is active during adhesion to the extracellular matrix. These findings reveal a novel mechanism by which adhesion and growth factor signals are integrated during ERK activation.

scholarly journals Transcriptional Regulators of theSchizosaccharomyces pombe fbp1Gene Include Two Redundant Tup1p-like Corepressors and the CCAAT Binding Factor Activation Complex

Genetics ◽  
2001 ◽  
Vol 157 (3) ◽  
pp. 1205-1215 ◽  
Author(s):  
Rozmin T K Janoo ◽  
Lori A Neely ◽  
Burkhard R Braun ◽  
Simon K Whitehall ◽  
Charles S Hoffman
Keyword(s):  
Protein Kinase ◽  
Fold Increase ◽  
Transcriptional Regulators ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Lacz Expression ◽  
Protein Subunit ◽  
Plasmid Library ◽  
Binding Factor ◽  
Mitogen Activated Protein

AbstractThe Schizosaccharomyces pombe fbp1 gene, which encodes fructose-1,6-bis-phosphatase, is transcriptionally repressed by glucose through the activation of the cAMP-dependent protein kinase A (PKA) and transcriptionally activated by glucose starvation through the activation of a mitogen-activated protein kinase (MAPK). To identify transcriptional regulators acting downstream from or in parallel to PKA, we screened an adh-driven cDNA plasmid library for genes that increase fbp1 transcription in a strain with elevated PKA activity. Two such clones express amino-terminally truncated forms of the S. pombe tup12 protein that resembles the Saccharomyces cerevisiae Tup1p global corepressor. These clones appear to act as dominant negative alleles. Deletion of both tup12 and the closely related tup11 gene causes a 100-fold increase in fbp1-lacZ expression, indicating that tup11 and tup12 are redundant negative regulators of fbp1 transcription. In strains lacking tup11 and tup12, the atf1-pcr1 transcriptional activator continues to play a central role in fbp1-lacZ expression; however, spc1 MAPK phosphorylation of atf1 is no longer essential for its activation. We discuss possible models for the role of tup11- and tup12-mediated repression with respect to signaling from the MAPK and PKA pathways. A third clone identified in our screen expresses the php5 protein subunit of the CCAAT-binding factor (CBF). Deletion of php5 reduces fbp1 expression under both repressed and derepressed conditions. The CBF appears to act in parallel to atf1-pcr1, although it is unclear whether or not CBF activity is regulated by PKA.

scholarly journals Attenuation of CHOP-mediated Myocardial Apoptosis in Pressure-overloaded Dominant Negative p38α Mitogen-activated Protein Kinase Mice

2011 ◽  
Vol 27 (5) ◽  
pp. 487-496 ◽  
Author(s):  
Flori R. Sari ◽  
Bambang Widyantoro ◽  
Rajarajan A. Thandavarayan ◽  
Meilei Harima ◽  
Arun Prasath Lakshmanan ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Myocardial Apoptosis ◽  
Mitogen Activated Protein

scholarly journals TRAF6-Dependent Mitogen-Activated Protein Kinase Activation Differentially Regulates the Production of Interleukin-12 by Macrophages in Response to Toxoplasma gondii

2004 ◽  
Vol 72 (10) ◽  
pp. 5662-5667 ◽  
Author(s):  
Nicola J. Mason ◽  
Jim Fiore ◽  
Takashi Kobayashi ◽  
Katherine S. Masek ◽  
Yongwon Choi ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Toxoplasma Gondii ◽  
Signaling Pathways ◽  
Intracellular Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Interleukin 12 ◽  
Wild Type ◽  
Kinase Activation ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

ABSTRACT The production of interleukin-12 (IL-12) is critical to the development of innate and adaptive immune responses required for the control of intracellular pathogens. Many microbial products signal through Toll-like receptors (TLR) and activate NF-κB family members that are required for the production of IL-12. Recent studies suggest that components of the TLR pathway are required for the production of IL-12 in response to the parasite Toxoplasma gondii; however, the production of IL-12 in response to this parasite is independent of NF-κB activation. The adaptor molecule TRAF6 is involved in TLR signaling pathways and associates with serine/threonine kinases involved in the activation of both NF-κB and mitogen-activated protein kinase (MAPK). To elucidate the intracellular signaling pathways involved in the production of IL-12 in response to soluble toxoplasma antigen (STAg), wild-type and TRAF6−/− mice were inoculated with STAg, and the production of IL-12(p40) was determined. TRAF6−/− mice failed to produce IL-12(p40) in response to STAg, and TRAF6−/− macrophages stimulated with STAg also failed to produce IL-12(p40). Studies using Western blot analysis of wild-type and TRAF6−/− macrophages revealed that stimulation with STAg resulted in the rapid TRAF6-dependent phosphorylation of p38 and extracellular signal-related kinase, which differentially regulated the production of IL-12(p40). The studies presented here demonstrate for the first time that the production of IL-12(p40) in response to toxoplasma is dependent upon TRAF6 and p38 MAPK.

scholarly journals A single amino acid substitution (N297A) in the conserved NPXXY sequence of the human N-formyl peptide receptor results in inhibition of desensitization and endocytosis, and a dose-dependent shift in p42/44 mitogen-activated protein kinase activation and chemotaxis

2000 ◽  
Vol 352 (2) ◽  
pp. 399-407 ◽  
Author(s):  
Jeannie M. GRIPENTROG ◽  
Algirdas J. JESAITIS ◽  
Heini M. MIETTINEN
Keyword(s):  
Protein Kinase ◽  
Transmembrane Domain ◽  
Mitogen Activated Protein Kinase ◽  
Formyl Peptide Receptor ◽  
Wild Type ◽  
Peptide Receptor ◽  
Formyl Peptide ◽  
Mitogen Activated Protein ◽  
Dose Dependent Increase ◽  
Dose Dependent

The formyl peptide receptor (FPR) is a G-protein-coupled receptor (GPCR) that mediates chemotaxis and stimulates the mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase pathway. We have examined the functional effects of substitutions of a conserved aspartic acid residue in the second transmembrane domain (D71A) and of residues in the conserved NPXXY motif in the seventh transmembrane domain (N297A and Y301A). These mutated receptors, expressed in Chinese hamster ovary (CHO) cells, bind ligand with affinities similar to wild-type FPR, but the D71A mutant is uncoupled from G-protein [Miettinen, Mills, Gripentrog, Dratz, Granger and Jesaitis (1997) J. Immunol 159, 4045–4054]. In the present study, we show that both the D71A and N297A mutations resulted in defective endocytosis. The N297A substitution also prevented desensitization, as determined by intracellular calcium mobilization by sequential stimulation with ligand. In chemotaxis assays, the N297A mutation resulted in cell migration towards gradients of up to 100nM N-formyl-methionyl-leucyl-phenylalanine (fMLF), whereas cells expressing the wild-type FPR and the Y301A mutant were no longer chemotactically responsive at 10–100nM fMLF. Maximal activation of p42/44 MAPK occurred in CHO cells expressing wild-type FPR at 10nM–100nM fMLF, whereas cells expressing the N297A mutant showed a dose-dependent increase in the amount of phosphorylated p42/44 MAPK up to 1–10µM fMLF. Since the MAPK kinase inhibitor PD98059 blocked fMLF-induced chemotaxis, our results suggest that the dose-dependent increase in p42/44 MAPK activation may correlate with the increased chemotactic migration of N297A transfectants at 10nM–100nM fMLF.

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.

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