Phosphorylation of Argonaute 2 at serine-387 facilitates its localization to processing bodies

2008 ◽  
Vol 413 (3) ◽  
pp. 429-436 ◽  
Author(s):  
Yan Zeng ◽  
Heidi Sankala ◽  
Xiaoxiao Zhang ◽  
Paul R. Graves
Keyword(s):  
Protein Kinase ◽  
P38 Mapk ◽  
Kinase Inhibitor ◽  
Mapk Pathway ◽  
Phosphorylation Site ◽  
Mitogen Activated Protein Kinase ◽  
Processing Bodies ◽  
Mitogen Activated Protein

Ago (Argonaute) proteins are essential effectors of RNA-mediated gene silencing. To explore potential regulatory mechanisms for Ago proteins, we examined the phosphorylation of human Ago2. We identified serine-387 as the major Ago2 phosphorylation site in vivo. Phosphorylation of Ago2 at serine-387 was significantly induced by treatment with sodium arsenite or anisomycin, and arsenite-induced phosphorylation was inhibited by a p38 MAPK (mitogen-activated protein kinase) inhibitor, but not by inhibitors of JNK (c-Jun N-terminal kinase) or MEK [MAPK/ERK (extracellular-signal-regulated kinase) kinase]. MAPKAPK2 (MAPK-activated protein kinase-2) phosphorylated bacterially expressed full-length human Ago2 at serine-387 in vitro, but not the S387A mutant. Finally, mutation of serine-387 to an alanine residue or treatment of cells with a p38 MAPK inhibitor reduced the localization of Ago2 to processing bodies. These results suggest a potential regulatory mechanism for RNA silencing acting through Ago2 serine-387 phosphorylation mediated by the p38 MAPK pathway.

scholarly journals Mitogen-activated protein kinase kinase 1 (MKK1) is negatively regulated by threonine phosphorylation

1994 ◽  
Vol 14 (3) ◽  
pp. 1594-1602
Author(s):  
A J Rossomando ◽  
P Dent ◽  
T W Sturgill ◽  
D R Marshak
Keyword(s):  
Protein Kinase ◽  
Phosphorylation Site ◽  
Mitogen Activated Protein Kinase ◽  
Dual Specificity ◽  
Mitogen Activated Protein ◽  
Kinase Cascade ◽  
Protein Kinase Cascade ◽  
Protein Kinase Kinase

Mitogen-activated protein kinase kinase 1 (MKK1), a dual-specificity tyrosine/threonine protein kinase, has been shown to be phosphorylated and activated by the raf oncogene product as part of the mitogen-activated protein kinase cascade. Here we report the phosphorylation and inactivation of MKK1 by phosphorylation on threonine 286 and threonine 292. MKK1 contains a consensus phosphorylation site for p34cdc2, a serine/threonine protein kinase that regulates the cell division cycle, at Thr-286 and a related site at Thr-292. p34cdc2 catalyzes the in vitro phosphorylation of MKK1 on both of these threonine residues and inactivates MKK1 enzymatic activity. Both sites are phosphorylated in vivo as well. The data presented in this report provide evidence that MKK1 is negatively regulated by threonine phosphorylation.

Mechanisms of Resistance to Mitogen-Activated Protein Kinase Pathway Inhibition in BRAF-Mutant Melanoma

2012 ◽  
pp. 680-684 ◽  
Author(s):  
Eva M. Goetz ◽  
Levi A. Garraway
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Mapk Pathway ◽  
Drug Binding ◽  
Mitogen Activated Protein Kinase ◽  
Braf V600e ◽  
Mechanisms Of Resistance ◽  
Mitogen Activated Protein

Overview: Anticancer drug resistance remains a crucial impediment to the care of many patients with cancer. Although the exact mechanisms of resistance may differ for each therapy, common mechanisms of resistance predominate, including drug inactivation or modification, mutation of the target protein, reduced drug accumulation, or bypass of target inhibition. With the discovery and use of targeted therapies (such as small-molecule kinase inhibitors), resistance has received renewed attention—especially in light of the dramatic responses that may emerge from such therapeutics in particular genetic or molecular contexts. Recently, the mitogen-activated protein kinase (MAPK) pathway has become exemplary in this regard, since it is activated in many different cancers. Drugs targeting RAF and MAPK kinase (MEK) are currently in clinical trials for the treatment of several types of cancer. Vemurafenib, a selective RAF kinase inhibitor recently approved for the treatment of BRAF(V600E) melanoma, shows strong efficacy initially; however, the development of resistance is nearly ubiquitous. In vitro testing and analysis of patient samples have uncovered several mechanisms of resistance to RAF inhibition. Surprisingly, mutations in the drug-binding pocket have not thus far been observed; however, other alterations at the level of RAF, as well as downstream activation of MEK and bypass of MEK/extracellular signal-regulated kinase (ERK) signaling altogether, confer resistance to vemurafenib. Looking forward, combined RAF and MEK inhibitor treatments may improve efficacy—yet we must anticipate mechanisms of resistance to this combination as well. Therefore, understanding and/or determining the mechanism of resistance are paramount to effective cancer treatment.

scholarly journals PDK1 Homologs Activate the Pkc1–Mitogen-Activated Protein Kinase Pathway in Yeast

1999 ◽  
Vol 19 (12) ◽  
pp. 8344-8352 ◽  
Author(s):  
Maiko Inagaki ◽  
Tobias Schmelzle ◽  
Kyoko Yamaguchi ◽  
Kenji Irie ◽  
Michael N. Hall ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mapk Pathway ◽  
Phosphorylation Site ◽  
Genetic Selection ◽  
Mitogen Activated Protein Kinase ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Mutant Form ◽  
Mitogen Activated Protein

ABSTRACT PDK1 (phosphoinositide-dependent kinase 1) is a mammalian growth factor-regulated serine/threonine kinase. Using a genetic selection based on a mutant form of the yeast MAP kinase kinase Ste7, we isolated a gene, PKH2, encoding a structurally and functionally conserved yeast homolog of PDK1. Yeast cells lacking bothPKH2 and PKH1, encoding another PDK1 homolog, were nonviable, indicating that Pkh1 and Pkh2 share an essential function. A temperature-sensitive mutant, pkh1D398Gpkh2, was phenotypically similar to mutants defective in the Pkc1–mitogen-activated protein kinase (MAPK) pathway. Genetic epistasis analyses, the phosphorylation of Pkc1 by Pkh2 in vitro, and reduced Pkc1 activity in the pkh1D398G pkh2mutant indicate that Pkh functions upstream of Pkc1. The Pkh2 phosphorylation site in Pkc1 (Thr-983) is part of a conserved PDK1 target motif and essential for Pkc1 function. Thus, the yeast PDK1 homologs activate Pkc1 and the Pkc1-effector MAPK pathway.

scholarly journals Combined VEGFR and MAPK pathway inhibition in angiosarcoma

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Michael J. Wagner ◽  
Yasmin A. Lyons ◽  
Jean H. Siedel ◽  
Robert Dood ◽  
Archana S. Nagaraja ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mapk Pathway ◽  
Combined Treatment ◽  
Mek Inhibitor ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
In Vivo Model ◽  
Mitogen Activated Protein

AbstractAngiosarcoma is an aggressive malignancy of endothelial cells that carries a high mortality rate. Cytotoxic chemotherapy can elicit clinical responses, but the duration of response is limited. Sequencing reveals multiple mutations in angiogenesis pathways in angiosarcomas, particularly in vascular endothelial growth factor (VEGFR) and mitogen-activated protein kinase (MAPK) signaling. We aimed to determine the biological relevance of these pathways in angiosarcoma. Tissue microarray consisting of clinical formalin-fixed paraffin embedded tissue archival samples were stained for phospho- extracellular signal-regulated kinase (p-ERK) with immunohistochemistry. Angiosarcoma cell lines were treated with the mitogen-activated protein kinase kinase (MEK) inhibitor trametinib, pan-VEGFR inhibitor cediranib, or combined trametinib and cediranib and viability was assessed. Reverse phase protein array (RPPA) was performed to assess multiple oncogenic protein pathways. SVR angiosarcoma cells were grown in vivo and gene expression effects of treatment were assessed with whole exome RNA sequencing. MAPK signaling was found active in over half of clinical angiosarcoma samples. Inhibition of MAPK signaling with the MEK inhibitor trametinib decreased the viability of angiosarcoma cells. Combined inhibition of the VEGF and MAPK pathways with cediranib and trametinib had an additive effect in in vitro models, and a combinatorial effect in an in vivo model. Combined treatment led to smaller tumors than treatment with either agent alone. RNA-seq demonstrated distinct expression signatures between the trametinib treated tumors and those treated with both trametinib and cediranib. These results indicate a clinical study of combined VEGFR and MEK inhibition in angiosarcoma is warranted.

An Inhibitor of p38 Mitogen-Activated Protein Kinase Abrogates Procoagulant and Proinflammatory Effects of Antiphospholipid Antibodies In Vivo.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 136-136
Author(s):  
Silvia S. Pierangeli ◽  
Mariano E. Vega-Ostertag ◽  
Xiaowei Liu
Keyword(s):  
Protein Kinase ◽  
Carotid Artery ◽  
P38 Mapk ◽  
Specific Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Peritoneal Cells ◽  
Mitogen Activated Protein ◽  
Pre Treatment

Abstract Background: Activation of p38 mitogen-activated protein kinase (p38 MAPK) has been shown to play a fundamental role in antiphospholipid-induced up regulation of tissue factor (TF) expression and function in monocytes and in endothelial cells (ECs) and increased expression of intercellular adhesion molecule -1 (ICAM-1) in vitro. Those effects correlate with the thrombogenic and pro-inflammatory effects of aPL in vivo. However, It is not clear whether aPL-induceTF in vivo. Methods: To examine this question, we treated CD1 male mice, in groups of 4, with IgG from 3 patients with Antiphospholipid Syndrome (IgG-APS) or with control IgG from healthy controls (IgG-NHS), twice. Seventy-two hours after the first injection, the adhesion of leukocytes per capillary venule (#WBC) to EC in cremaster muscle (as an indication of EC activation in vivo), as well the size of an induced thrombus in the femoral vein of the mice were examined. Some mice were infused i.p. with 25 mg/kg of SB203580 (a p38 MAPK-specific inhibitor) 30 minutes prior to the each IgG-APS injection. TF activity was determined using a chromogenic assay that measures the conversion of factor X into Factor Xa, in homogenates of carotid artery, and in peritoneal cells of mice treated with IgG-APS or with IgG-NHS. Expression of TF and ICAM-1 was determined by cyto-ELISA on cultured HUVECs after treatment of the cells with IgG-APS or with IgG-NHS. Results: At the time of the surgical procedures, the mean aCL titer in the sera of the mice injected with IgG-APS was 73 ± 34 GPL. In vivo, IgG-APS increased significantly the #WBC adhering to EC, when compared to control mice (5.25 ± 0.96 vs 1.85 ± 0.72) and these effects were significantly reduced (2.1 ± 0.74), when mice were pre-treated with SB203580. IgG-APS increased significantly the thrombus size when compared to IgG-NHS-treated mice (3189 ± 558 μm2 vs 1468 ± 401 μm2) and SB203580 inhibited this effect by 65%. Treatment of the mice with IgG-APS also induced significantly increased TF function in peritoneal cells and in homogenates of carotid artery when compared to IgG-NHS-treated mice (17.5 ±11.1 pM vs. 0.8 ±0.2 pM and 8.31 ± 1.59 vs 0.69 ± 0.03, respectively). Pre-treatment of the mice with SB203580 abrogated completely those effects (0.61 ± 0.06 pM in peritoneal cells and 0.75 ± 0.28 pM in carotid artery preparations of mice treated with IgG-APS). Significant expression of TF and ICAM-1 was observed in vitro when HUVECs were treated with any of the three IgG-APS. TF upregulation and ICAM-1 expression were significantly reduced by pre-treatment of the cells with SB203580 (49–97% for TF and 25–69% for ICAM-1). Conclusions: The data show that IgG-APS up regulates TF function in vivo, and this correlates with an in vivo pro-inflammatory and pro-thrombotic effect. Importantly, those effects were abrogated in vivo by a p38 MAPK specific inhibitor. These findings may be important in designing new modalities of targeted therapies to treat thrombosis in patients with APS.

scholarly journals Ptc1, a Type 2C Ser/Thr Phosphatase, Inactivates the HOG Pathway by Dephosphorylating the Mitogen-Activated Protein Kinase Hog1

2001 ◽  
Vol 21 (1) ◽  
pp. 51-60 ◽  
Author(s):  
Janel Warmka ◽  
Jennifer Hanneman ◽  
Ji Lee ◽  
Dipesh Amin ◽  
Irene Ota
Keyword(s):  
Protein Kinase ◽  
Osmotic Stress ◽  
Metal Ion ◽  
Mapk Pathway ◽  
Negative Regulator ◽  
Mitogen Activated Protein Kinase ◽  
Hog Pathway ◽  
Mitogen Activated Protein

ABSTRACT The HOG (high-osmolarity glycerol) mitogen-activated protein kinase (MAPK) pathway regulates the osmotic stress response in the yeast Saccharomyces cerevisiae. Three type 2C Ser/Thr phosphatases (PTCs), Ptc1, Ptc2, and Ptc3, have been isolated as negative regulators of this pathway. Previously, multicopy expression of PTC1 and PTC3 was shown to suppress lethality of the sln1Δ strain due to hyperactivation of the HOG pathway. In this work, we show thatPTC2 also suppresses sln1Δ lethality. Furthermore, the phosphatase activity of these PTCs was needed for suppression, as mutation of a conserved Asp residue, likely to coordinate a metal ion, inactivated PTCs. Further analysis of Ptc1 function in vivo showed that it inactivates the MAPK, Hog1, but not the MEK, Pbs2. In the wild type, Hog1 kinase activity increased transiently, ∼12-fold in response to osmotic stress, while overexpression of PTC1 limited activation to ∼3-fold. In contrast, overexpression of PTC1 did not inhibit phosphorylation of Hog1 Tyr in the phosphorylation lip, suggesting that Ptc1 does not act on Pbs2. Deletion of PTC1 also strongly affected Hog1, leading to high basal Hog1 activity and sustained Hog1 activity in response to osmotic stress, the latter being consistent with a role for Ptc1 in adaptation. In vitro, Ptc1 but not the metal binding site mutant, Ptc1D58N, inactivated Hog1 by dephosphorylating the phosphothreonine but not the phosphotyrosine residue in the phosphorylation lip. Consistent with its role as a negative regulator of Hog1, which accumulates in the nucleus upon activation, Ptc1 was found in both the nucleus and the cytoplasm. Thus, one function of Ptc1 is to inactivate Hog1.

scholarly journals Mitogen-activated protein kinase kinase 1 (MKK1) is negatively regulated by threonine phosphorylation.

1994 ◽  
Vol 14 (3) ◽  
pp. 1594-1602 ◽  
Author(s):  
A J Rossomando ◽  
P Dent ◽  
T W Sturgill ◽  
D R Marshak
Keyword(s):  
Protein Kinase ◽  
Phosphorylation Site ◽  
Mitogen Activated Protein Kinase ◽  
Dual Specificity ◽  
Mitogen Activated Protein ◽  
Kinase Cascade ◽  
Protein Kinase Cascade ◽  
Protein Kinase Kinase

Mitogen-activated protein kinase kinase 1 (MKK1), a dual-specificity tyrosine/threonine protein kinase, has been shown to be phosphorylated and activated by the raf oncogene product as part of the mitogen-activated protein kinase cascade. Here we report the phosphorylation and inactivation of MKK1 by phosphorylation on threonine 286 and threonine 292. MKK1 contains a consensus phosphorylation site for p34cdc2, a serine/threonine protein kinase that regulates the cell division cycle, at Thr-286 and a related site at Thr-292. p34cdc2 catalyzes the in vitro phosphorylation of MKK1 on both of these threonine residues and inactivates MKK1 enzymatic activity. Both sites are phosphorylated in vivo as well. The data presented in this report provide evidence that MKK1 is negatively regulated by threonine phosphorylation.

scholarly journals Elimination of Protein Kinase MK5/PRAK Activity by Targeted Homologous Recombination

2003 ◽  
Vol 23 (21) ◽  
pp. 7732-7741 ◽  
Author(s):  
Yu Shi ◽  
Alexey Kotlyarov ◽  
Kathrin Laaß ◽  
Achim D. Gruber ◽  
Elke Butt ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Homologous Recombination ◽  
P38 Mapk ◽  
Knockout Mice ◽  
Endotoxic Shock ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein ◽  
Deficient Mice

ABSTRACT MK5 (mitogen-activated protein kinase [MAPK]-activated protein kinase 5), also designated PRAK (p38-regulated and -activated kinase), was deleted from mice by homologous recombination. Although no MK5 full-length protein and kinase activity was detected in the MK5 knockout mice, the animals were viable and fertile and did not display abnormalities in tissue morphology or behavior. In addition, these mice did not show increased resistance to endotoxic shock or decreased lipopolysaccharide-induced cytokine production. Hence, MK5 deletion resulted in a phenotype very different from the complex inflammation-impaired phenotype of mice deficient in MK2, although MK2 and MK5 exhibit evolutional, structural, and apparent extensive functional similarities. To explain this discrepancy, we used wild-type cells and embryonic fibroblasts from both MK2 and MK5 knockout mice as controls to reexamine the mechanism of activation, the interaction with endogenous p38 MAPK, and the substrate specificity of both enzymes. In contrast to MK2, which shows interaction with and chaperoning properties for p38 MAPK and which is activated by extracellular stresses such as arsenite or sorbitol treatment, endogenous MK5 did not show these properties. Furthermore, endogenous MK5 is not able to phosphorylate Hsp27 in vitro and in vivo. We conclude that the differences between the phenotypes of MK5- and MK2-deficient mice result from clearly different functional properties of both enzymes.

A novel vasculo-angiogenic effect of cilostazol mediated by cross-talk between multiple signalling pathways including the ERK/p38 MAPK signalling transduction cascade

2012 ◽  
Vol 123 (3) ◽  
pp. 147-159 ◽  
Author(s):  
Ting-Hsing Chao ◽  
Shih-Ya Tseng ◽  
Yi-Heng Li ◽  
Ping-Yen Liu ◽  
Chung-Lung Cho ◽  
...  
Keyword(s):  
Protein Kinase ◽  
P38 Mapk ◽  
Umbilical Vein ◽  
Tube Formation ◽  
Mitogen Activated Protein Kinase ◽  
Signalling Pathways ◽  
No Production ◽  
Angiogenic Effect

Cilostazol is an anti-platelet agent with vasodilatory activity that acts by increasing intracellular concentrations of cAMP. Recent reports have suggested that cilostazol may promote angiogenesis. In the present study, we have investigated the effect of cilostazol in promoting angiogenesis and vasculogenesis in a hindlimb ischaemia model and have also examined its potential mechanism of action in vitro and in vivo. We found that cilostazol treatment significantly increased colony formation by human early EPCs (endothelial progenitor cells) through a mechanism involving the activation of cAMP/PKA (protein kinase A), PI3K (phosphoinositide 3-kinase)/Akt/eNOS (endothelial NO synthase) and ERK (extracellular-signal-regulated kinase)/p38 MAPK (mitogen-activated protein kinase) signalling pathways. Cilostazol also enhanced proliferation, chemotaxis, NO production and vascular tube formation in HUVECs (human umbilical vein endothelial cells) through activation of multiple signalling pathways downstream of PI3K/Akt/eNOS. Cilostazol up-regulated VEGF (vascular endothelial growth factor)-A165 expression and secretion of VEGF-A in HUVECs through activation of the PI3K/Akt/eNOS pathway. In a mouse hindlimb ischaemia model, recovery of blood flow ratio (ipsilateral/contralateral) 14 days after surgery was significantly improved in cilostazol-treated mice (10 mg/kg of body weight) compared with vehicle-treated controls (0.63±0.07 and 0.43±0.05 respectively, P<0.05). Circulating CD34+ cells were also increased in cilostazol-treated mice (3614±670 compared with 2151±608 cells/ml, P<0.05). Expression of VEGF and phosphorylation of PI3K/Akt/eNOS and ERK/p38 MAPK in ischaemic muscles were significantly enhanced by cilostazol. Our data suggest that cilostazol produces a vasculo-angiogenic effect by up-regulating a broad signalling network that includes the ERK/p38 MAPK, VEGF-A165, PI3K/Akt/eNOS and cAMP/PKA pathways.

scholarly journals Cross-talk between IRAK-4 and the NADPH oxidase1

2007 ◽  
Vol 403 (3) ◽  
pp. 451-461 ◽  
Author(s):  
Sandrine Pacquelet ◽  
Jennifer L. Johnson ◽  
Beverly A. Ellis ◽  
Agnieszka A. Brzezinska ◽  
William S. Lane ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nadph Oxidase ◽  
P38 Mapk ◽  
Interleukin 1 ◽  
Mitogen Activated Protein Kinase ◽  
Free System ◽  
Mitogen Activated Protein ◽  
A Cell ◽  
Tlr4 Activation

Exposure of neutrophils to LPS (lipopolysaccharide) triggers their oxidative response. However, the relationship between the signalling downstream of TLR4 (Toll-like receptor 4) after LPS stimulation and the activation of the oxidase remains elusive. Phosphorylation of the cytosolic factor p47phox is essential for activation of the NADPH oxidase. In the present study, we examined the hypothesis that IRAK-4 (interleukin-1 receptor-associated kinase-4), the main regulatory kinase downstream of TLR4 activation, regulates the NADPH oxidase through phosphorylation of p47phox. We show that p47phox is a substrate for IRAK-4. Unlike PKC (protein kinase C), IRAK-4 phosphorylates p47phox not only at serine residues, but also at threonine residues. Target residues were identified by tandem MS, revealing a novel threonine-rich regulatory domain. We also show that p47phox is phosphorylated in granulocytes in response to LPS stimulation. LPS-dependent phosphorylation of p47phox was enhanced by the inhibition of p38 MAPK (mitogen-activated protein kinase), confirming that the kinase operates upstream of p38 MAPK. IRAK-4-phosphorylated p47phox activated the NADPH oxidase in a cell-free system, and IRAK-4 overexpression increased NADPH oxidase activity in response to LPS. We have shown that endogenous IRAK-4 interacts with p47phox and they co-localize at the plasma membrane after LPS stimulation, using immunoprecipitation assays and immunofluorescence microscopy respectively. IRAK-4 was activated in neutrophils in response to LPS stimulation. We found that Thr133, Ser288 and Thr356, targets for IRAK-4 phosphorylation in vitro, are also phosphorylated in endogenous p47phox after LPS stimulation. We conclude that IRAK-4 phosphorylates p47phox and regulates NADPH oxidase activation after LPS stimulation.

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