scholarly journals Negative-feedback regulation of FGF signalling by DUSP6/MKP-3 is driven by ERK1/2 and mediated by Ets factor binding to a conserved site within the DUSP6/MKP-3 gene promoter

2008 ◽  
Vol 412 (2) ◽  
pp. 287-298 ◽  
Author(s):  
Maria Ekerot ◽  
Marios P. Stavridis ◽  
Laurent Delavaine ◽  
Michael P. Mitchell ◽  
Christopher Staples ◽  
...  
Keyword(s):  
Binding Site ◽  
Negative Feedback ◽  
Fluorescent Protein ◽  
Gene Promoter ◽  
Negative Regulator ◽  
Mitogen Activated Protein Kinase ◽  
Negative Feedback Regulation ◽  
Fgf Signalling

DUSP6 (dual-specificity phosphatase 6), also known as MKP-3 [MAPK (mitogen-activated protein kinase) phosphatase-3] specifically inactivates ERK1/2 (extracellular-signal-regulated kinase 1/2) in vitro and in vivo. DUSP6/MKP-3 is inducible by FGF (fibroblast growth factor) signalling and acts as a negative regulator of ERK activity in key and discrete signalling centres that direct outgrowth and patterning in early vertebrate embryos. However, the molecular mechanism by which FGFs induce DUSP6/MKP-3 expression and hence help to set ERK1/2 signalling levels is unknown. In the present study, we demonstrate, using pharmacological inhibitors and analysis of the murine DUSP6/MKP-3 gene promoter, that the ERK pathway is critical for FGF-induced DUSP6/MKP-3 transcription. Furthermore, we show that this response is mediated by a conserved binding site for the Ets (E twenty-six) family of transcriptional regulators and that the Ets2 protein, a known target of ERK signalling, binds to the endogenous DUSP6/MKP-3 promoter. Finally, the murine DUSP6/MKP-3 promoter coupled to EGFP (enhanced green fluorescent protein) recapitulates the specific pattern of endogenous DUSP6/MKP-3 mRNA expression in the chicken neural plate, where its activity depends on FGFR (FGF receptor) and MAPK signalling and an intact Ets-binding site. These findings identify a conserved Ets-factor-dependent mechanism by which ERK signalling activates DUSP6/MKP-3 transcription to deliver ERK1/2-specific negative-feedback control of FGF signalling.

scholarly journals Genome-wide CRISPR-Cas9 screen identified KLF11 as a druggable suppressor for sarcoma cancer stem cells

2021 ◽  
Vol 7 (5) ◽  
pp. eabe3445
Author(s):  
Yicun Wang ◽  
Jinhui Wu ◽  
Hui Chen ◽  
Yang Yang ◽  
Chengwu Xiao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cancer Stem Cells ◽  
Negative Feedback ◽  
Direct Interaction ◽  
Therapy Resistance ◽  
Negative Regulator ◽  
Chemotherapy Response ◽  
Genome Wide

Cancer stem cells (CSCs) are involved in tumorigenesis, recurrence, and therapy resistance. To identify critical regulators of sarcoma CSCs, we performed a reporter-based genome-wide CRISPR-Cas9 screen and uncovered Kruppel-like factor 11 (KLF11) as top candidate. In vitro and in vivo functional annotation defined a negative role of KLF11 in CSCs. Mechanistically, KLF11 and YAP/TEAD bound to adjacent DNA sites along with direct interaction. KLF11 recruited SIN3A/HDAC to suppress the transcriptional output of YAP/TEAD, which, in turn, promoted KLF11 transcription, forming a negative feedback loop. However, in CSCs, this negative feedback was lost because of epigenetic silence of KLF11, causing sustained YAP activation. Low KLF11 was associated with poor prognosis and chemotherapy response in patients with sarcoma. Pharmacological activation of KLF11 by thiazolidinedione effectively restored chemotherapy response. Collectively, our study identifies KLF11 as a negative regulator in sarcoma CSCs and potential therapeutic target.

scholarly journals Inhibition of Ubiquitination and Stabilization of Human Ubiquitin E3 Ligase PIRH2 by Measles Virus Phosphoprotein

2005 ◽  
Vol 79 (18) ◽  
pp. 11824-11836 ◽  
Author(s):  
Mingzhou Chen ◽  
Jean-Claude Cortay ◽  
Ian R. Logan ◽  
Vasileia Sapountzi ◽  
Craig N. Robson ◽  
...  
Keyword(s):  
Binding Site ◽  
Measles Virus ◽  
Fluorescent Protein ◽  
E3 Ligase ◽  
Yeast Two Hybrid ◽  
Ubiquitin E3 Ligase ◽  
P Protein ◽  
Two Hybrid

ABSTRACT Using a C-terminal domain (PCT) of the measles virus (MV) phosphoprotein (P protein) as bait in a yeast two-hybrid screen, a cDNA identical to the recently described human p53-induced-RING-H2 (hPIRH2) cDNA was isolated. A glutathione S-transferase-hPIRH2 fusion protein expressed in bacteria was able to pull down P protein when mixed with an extract from P-expressing HeLa cells in vitro, and myc-tagged hPIRH2 could be reciprocally coimmunoprecipitated with MV P protein from human cells. Additionally, immunoprecipitation experiments demonstrated that hPIRH2-myc, MV P, and nucleocapsid (N) proteins form a ternary complex. The hPIRH2 binding site was mapped to the C-terminal X domain region of the P protein by using a yeast two-hybrid assay. The PCT binding site was mapped on hPIRH2 by using a novel yeast two-hybrid tagged PCR approach and by coimmunoprecipitation of hPIRH2 cysteine mutants and mouse/human PIRH2 chimeras. The hPIRH2 C terminus could mediate the interaction with MV P which was favored by the RING-H2 motif. When coexpressed with an enhanced green fluorescent protein-tagged hPIRH2 protein, MV P alone or in a complex with MV N was able to redistribute hPIRH2 to outside the nucleus, within intracellular aggregates. Finally, MV P efficiently stabilized hPIRH2-myc expression and prevented its ubiquitination in vivo but had no effect on the stability or ubiquitination of an alternative ubiquitin E3 ligase, Mdm2. Thus, MV P protein is the first protein from a pathogen that is able to specifically interact with and stabilize the ubiquitin E3 ligase hPIRH2 by preventing its ubiquitination.

scholarly journals Extracellular Signal-Regulated Kinases Phosphorylate Mitogen-Activated Protein Kinase Phosphatase 3/DUSP6 at Serines 159 and 197, Two Sites Critical for Its Proteasomal Degradation

2005 ◽  
Vol 25 (2) ◽  
pp. 854-864 ◽  
Author(s):  
Sandrine Marchetti ◽  
Clotilde Gimond ◽  
Jean-Claude Chambard ◽  
Thomas Touboul ◽  
Danièle Roux ◽  
...  
Keyword(s):  
Map Kinases ◽  
Fluorescent Protein ◽  
Proteasomal Degradation ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Double Mutation ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

ABSTRACT Mitogen-activated protein (MAP) kinase phosphatases (MKPs) are dual-specificity phosphatases that dephosphorylate phosphothreonine and phosphotyrosine residues within MAP kinases. Here, we describe a novel posttranslational mechanism for regulating MKP-3/Pyst1/DUSP6, a member of the MKP family that is highly specific for extracellular signal-regulated kinase 1 and 2 (ERK1/2) inactivation. Using a fibroblast model in which the expression of either MKP-3 or a more stable MKP-3-green fluorescent protein (GFP) chimera was induced by tetracycline, we found that serum induces the phosphorylation of MKP-3 and its subsequent degradation by the proteasome in a MEK1 and MEK2 (MEK1/2)-ERK1/2-dependent manner. In vitro phosphorylation assays using glutathione S-transferase (GST)-MKP-3 fusion proteins indicated that ERK2 could phosphorylate MKP-3 on serines 159 and 197. Tetracycline-inducible cell clones expressing either single or double serine mutants of MKP-3 or MKP-3-GFP confirmed that these two sites are targeted by the MEK1/2-ERK1/2 module in vivo. Double serine mutants of MKP-3 or MKP-3-GFP were more efficiently protected from degradation than single mutants or wild-type MKP-3, indicating that phosphorylation of either serine by ERK1/2 enhances proteasomal degradation of MKP-3. Hence, double mutation caused a threefold increase in the half-life of MKP-3. Finally, we show that the phosphorylation of MKP-3 has no effect on its catalytic activity. Thus, ERK1/2 exert a positive feedback loop on their own activity by promoting the degradation of MKP-3, one of their major inactivators in the cytosol, a situation opposite to that described for the nuclear phosphatase MKP-1.

Microtubule-entrained kinase activities associated with the cortical cytoskeleton during cytokinesis

1997 ◽  
Vol 110 (12) ◽  
pp. 1373-1386 ◽  
Author(s):  
G.R. Walker ◽  
C.B. Shuster ◽  
D.R. Burgess
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Protein Phosphorylation ◽  
Immunoblot Analysis ◽  
Negative Regulator ◽  
Mitogen Activated Protein Kinase ◽  
Mitotic Apparatus ◽  
Cortical Cytoskeleton

Research over the past few years has demonstrated the central role of protein phosphorylation in regulating mitosis and the cell cycle. However, little is known about how the mechanisms regulating the entry into mitosis contribute to the positional and temporal regulation of the actomyosin-based contractile ring formed during cytokinesis. Recent studies implicate p34cdc2 as a negative regulator of myosin II activity, suggesting a link between the mitotic cycle and cytokinesis. In an effort to study the relationship between protein phosphorylation and cytokinesis, we examined the in vivo and in vitro phosphorylation of actin-associated cortical cytoskeletal (CSK) proteins in an isolated model of the sea urchin egg cortex. Examination of cortices derived from eggs or zygotes labeled with 32P-orthophosphate reveals a number of cortex-associated phosphorylated proteins, including polypeptides of 20, 43 and 66 kDa. These three major phosphoproteins are also detected when isolated cortices are incubated with [32P]ATP in vitro, suggesting that the kinases that phosphorylate these substrates are also specifically associated with the cortex. The kinase activities in vivo and in vitro are stimulated by fertilization and display cell cycle-dependent activities. Gel autophosphorylation assays, kinase assays and immunoblot analysis reveal the presence of p34cdc2 as well as members of the mitogen-activated protein kinase family, whose activities in the CSK peak at cell division. Nocodazole, which inhibits microtubule formation and thus blocks cytokinesis, significantly delays the time of peak cortical protein phosphorylation as well as the peak in whole-cell histone H1 kinase activity. These results suggest that a key element regulating cortical contraction during cytokinesis is the timing of protein kinase activities associated with the cortical cytoskeleton that is in turn regulated by the mitotic apparatus.

scholarly journals Treatment of inflammatory arthritis via targeting of tristetraprolin, a master regulator of pro-inflammatory gene expression

2016 ◽  
Vol 76 (3) ◽  
pp. 612-619 ◽  
Author(s):  
E A Ross ◽  
A J Naylor ◽  
J D O'Neil ◽  
T Crowley ◽  
M L Ridley ◽  
...  
Keyword(s):  
Inflammatory Arthritis ◽  
Inflammatory Responses ◽  
Vascular Endothelial Cells ◽  
Bone Erosion ◽  
Negative Regulator ◽  
Mitogen Activated Protein Kinase ◽  
Critical Determinant ◽  
Anti Inflammatory

ObjectivesTristetraprolin (TTP), a negative regulator of many pro-inflammatory genes, is strongly expressed in rheumatoid synovial cells. The mitogen-activated protein kinase (MAPK) p38 pathway mediates the inactivation of TTP via phosphorylation of two serine residues. We wished to test the hypothesis that these phosphorylations contribute to the development of inflammatory arthritis, and that, conversely, joint inflammation may be inhibited by promoting the dephosphorylation and activation of TTP.MethodsThe expression of TTP and its relationship with MAPK p38 activity were examined in non-inflamed and rheumatoid arthritis (RA) synovial tissue. Experimental arthritis was induced in a genetically modified mouse strain, in which endogenous TTP cannot be phosphorylated and inactivated. In vitro and in vivo experiments were performed to test anti-inflammatory effects of compounds that activate the protein phosphatase 2A (PP2A) and promote dephosphorylation of TTP.ResultsTTP expression was significantly higher in RA than non-inflamed synovium, detected in macrophages, vascular endothelial cells and some fibroblasts and co-localised with MAPK p38 activation. Substitution of TTP phosphorylation sites conferred dramatic protection against inflammatory arthritis in mice. Two distinct PP2A agonists also reduced inflammation and prevented bone erosion. In vitro anti-inflammatory effects of PP2A agonism were mediated by TTP activation.ConclusionsThe phosphorylation state of TTP is a critical determinant of inflammatory responses, and a tractable target for novel anti-inflammatory treatments.

scholarly journals High doses of TGF-β potently suppress type I collagen via the transcription factor CUX1

2011 ◽  
Vol 22 (11) ◽  
pp. 1836-1844 ◽  
Author(s):  
Maria Fragiadaki ◽  
Tetsurou Ikeda ◽  
Abigail Witherden ◽  
Roger M Mason ◽  
David Abraham ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Transforming Growth Factor ◽  
Interstitial Fibrosis ◽  
Transforming Growth Factor Β ◽  
Negative Regulator ◽  
Mitogen Activated Protein Kinase ◽  
Type I ◽  
Aberrant Expression

Transforming growth factor-β (TGF-β) is an inducer of type I collagen, and uncontrolled collagen production leads to tissue scarring and organ failure. Here we hypothesize that uncovering a molecular mechanism that enables us to switch off type I collagen may prove beneficial in treating fibrosis. For the first time, to our knowledge, we provide evidence that CUX1 acts as a negative regulator of TGF-β and potent inhibitor of type I collagen transcription. We show that CUX1, a CCAAT displacement protein, is associated with reduced expression of type I collagen both in vivo and in vitro. We show that enhancing the expression of CUX1 results in effective suppression of type I collagen. We demonstrate that the mechanism by which CUX1 suppresses type I collagen is through interfering with gene transcription. In addition, using an in vivo murine model of aristolochic acid (AA)-induced interstitial fibrosis and human AA nephropathy, we observe that CUX1 expression was significantly reduced in fibrotic tissue when compared to control samples. Moreover, silencing of CUX1 in fibroblasts from kidneys of patients with renal fibrosis resulted in increased type I collagen expression. Furthermore, the abnormal CUX1 expression was restored by addition of TGF-β via the p38 mitogen-activated protein kinase pathway. Collectively, our study demonstrates that modifications of CUX1 expression lead to aberrant expression of type I collagen, which may provide a molecular basis for fibrogenesis.

scholarly journals A Direct Binding Site for Grb2 Contributes to Transformation and Leukemogenesis by the Tel-Abl (ETV6-Abl) Tyrosine Kinase

2004 ◽  
Vol 24 (11) ◽  
pp. 4685-4695 ◽  
Author(s):  
Ryan P. Million ◽  
Nari Harakawa ◽  
Sergei Roumiantsev ◽  
Lyuba Varticovski ◽  
Richard A. Van Etten
Keyword(s):  
Small Bowel ◽  
Binding Site ◽  
Mitogen Activated Protein Kinase ◽  
Scaffolding Protein ◽  
Common Mechanism ◽  
Human Leukemia ◽  
Abl Tyrosine Kinase ◽  
Direct Binding

ABSTRACT A direct binding site for the Grb2 adapter protein is required for the induction of fatal chronic myeloid leukemia (CML)-like disease in mice by Bcr-Abl. Here, we demonstrate direct binding of Grb2 to the Tel-Abl (ETV6-Abl) fusion protein, the product of complex (9;12) chromosomal translocations in human leukemia, via tyrosine 314 encoded by TEL exon 5. A Tel-Abl point mutant (Y314F) and a splice variant without TEL exon 5 sequences (Δe5) lacked Grb2 interaction and exhibited decreased binding and phosphorylation of the scaffolding protein Gab2 and impaired activation of phosphatidylinositol 3-kinase, Akt, and extracellular signal-regulated kinase/mitogen-activated protein kinase in hematopoietic cells. Tel-Abl Y314F and Δe5 were unable to transform fibroblasts to anchorage-independent growth and were defective for B-lymphoid transformation in vitro and lymphoid leukemogenesis in vivo. Previously, we demonstrated that full-length Tel-Abl induced two distinct myeloproliferative diseases in mice: CML-like leukemia similar to that induced by Bcr-Abl and a novel syndrome of small-bowel myeloid infiltration endotoxemia and hepatic and renal failure. Lack of the Grb2 binding site had no effect on development of small bowel syndrome but significantly attenuated the induction of CML-like disease by Tel-Abl. These results suggest that direct binding of Grb2 is a common mechanism contributing to leukemogenesis by oncogenic Abl fusion proteins.

LIF transduces contradictory signals on capillary outgrowth through induction of stat3 and (P41/43)MAP kinase

2000 ◽  
Vol 113 (23) ◽  
pp. 4331-4339 ◽  
Author(s):  
H. Paradis ◽  
R.L. Gendron
Keyword(s):  
Signaling Pathways ◽  
Tyrosine Kinases ◽  
Mapk Pathway ◽  
Present Report ◽  
Binding Activity ◽  
Negative Regulator ◽  
Mapk Cascade ◽  
Mitogen Activated Protein Kinase

The signaling pathways regulating blood vessel growth and development are not well understood. In the present report, an in vitro model was used to identify signaling pathways regulating capillary formation in embryonic endothelial cells. Basic fibroblast growth factor (bFGF) plus leukemia inhibitory factor (LIF) optimally stimulate the formation of capillary-like structures of the embryonic endothelial cell line IEM. LIF stimulation of IEM cells leads to activation of the Stat3 as well as the (P41/43)mitogen-activated protein kinase ((P41/43)MAPK) cascade, while bFGF does not activate Stat3 but does induce the (P41/43)MAPK cascade. Inhibition of Stat3 DNA-binding activity by expression of a dominant inhibitory Stat3 mutant increases the capillary outgrowth of the IEM cells induced by LIF. Increased Stat3 activity by overexpression of the wild-type Stat3 greatly reduced capillary outgrowth. In contrast, inhibition of the (P41/43)MAPK cascade using a MEK-1 inhibitor dramatically inhibits the LIF-induced capillary outgrowth. Moreover, the increased formation of capillary-like structures of the IEM cells mediated by Stat3 inhibition does not overcome the requirement for activation of the (P41/43)MAPK pathway for capillary outgrowth. Stat3 activity correlates with the LIF-induced expression of the negative feedback regulators of the Janus (JAK) family of tyrosine kinases, SOCS-1 and SOCS-3. These results provide evidence that Stat3 acts as a negative regulator of capillary outgrowth, possibly by increasing SOCS-1 or SOCS-3 expression. The contradictory signals stimulated by LIF could be necessary to control the intensity of the response leading to capillary outgrowth in vivo.

scholarly journals Flumequine-Mediated Upregulation of p38 MAPK and JNK Results in Melanogenesis in B16F10 Cells and Zebrafish Larvae

Biomolecules ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 596 ◽  
Author(s):  
Wisurumuni Arachchilage Hasitha Maduranga Karunarathne ◽  
Ilandarage Menu Neelaka Molagoda ◽  
Myung Sook Kim ◽  
Yung Hyun Choi ◽  
Matan Oren ◽  
...  
Keyword(s):  
P38 Mapk ◽  
Negative Regulator ◽  
Mitogen Activated Protein Kinase ◽  
Melanin Content ◽  
Melanin Pigmentation ◽  
Zebrafish Larvae ◽  
B16f10 Cells ◽  
Mitogen Activated Protein

Flumequine is a well-known second generation quinolone antibiotic that induces phototoxicity. However, the effect of flumequine on skin melanogenesis is unclear. Therefore, we, for the first time, investigated whether flumequine regulates melanogenesis. The present study showed that flumequine slightly inhibited in vitro mushroom tyrosinase activity but significantly increased extracellular and intracellular melanin content in B16F10 cells and promoted the expression of microphthalmia-associated transcription factor (MITF) and tyrosinase. Additionally, flumequine remarkably increased melanin pigmentation in zebrafish larvae without any toxicity. We also found that flumequine stimulated p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) phosphorylation; inhibition of p38 MAPK and JNK resulted in significant downregulation of extracellular and intracellular melanin content in B16F10 cells and pigmentation of zebrafish larvae accompanied with suppression of MITF and tyrosinase expression, indicating that flumequine-mediated p38 and JNK promote melanogenesis in vitro and in vivo. According to the molecular docking prediction, flumequine targeted dual-specificity MAPK phosphatase 16 (DUSP16), which is a major negative regulator of p38 MAPK and JNK. Our findings demonstrate that flumequine induces an increase in melanin content in B16F10 cells and zebrafish larvae by activating p38 MAPK and JNK. These data show the potential of flumequine for use as an anti-vitiligo agent.

Mitogen-activated protein kinase signaling is necessary for the maintenance of skeletal muscle mass

2009 ◽  
Vol 296 (5) ◽  
pp. C1040-C1048 ◽  
Author(s):  
Hao Shi ◽  
Jason M. Scheffler ◽  
Caiyun Zeng ◽  
Jonathan M. Pleitner ◽  
Kevin M. Hannon ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Mass ◽  
Skeletal Muscle Mass ◽  
Fluorescent Protein ◽  
Mapk Signaling ◽  
26S Proteasome ◽  
Mitogen Activated Protein Kinase ◽  
Reporter Activity

The signal transduction cascades that maintain muscle mass remain to be fully defined. Herein, we report that inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling in vitro decreases myotube size and protein content after 3-day treatment with a MEK inhibitor. Neither p38 nor JNK inhibitors had any effect on myotube size or morphology. ERK1/2 inhibition also upregulated gene transcription of atrogin-1 and muscle-specific RING finger protein 1 and downregulated the phosphorylation of Akt and its downstream kinases. Forced expression of enhanced green fluorescent protein-tagged MAPK phosphatase 1 (MKP-1) in soleus and gastrocnemius muscles decreased both fiber size and reporter activity. This atrophic effect of MKP-1 was time dependent. Analysis of the reporter activity in vivo revealed that the activities of nuclear factor-κB and 26S proteasome were differentially activated in slow and fast muscles, suggesting muscle type-specific mechanisms may be utilized. Together, these findings suggest that MAPK signaling is necessary for the maintenance of skeletal muscle mass because inhibition of these signaling cascades elicits muscle atrophy in vitro and in vivo.

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