Mechanism of Activation and Role of Extracellular Signal Regulated Kinase in ADP-Induced Thromboxane A2 Generation in Platelets.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3567-3567
Author(s):  
Analia Garcia ◽  
Haripriya Shankar ◽  
Satya P. Kunapuli
Keyword(s):  
Thromboxane A2 ◽  
P2y Receptors ◽  
Extracellular Calcium ◽  
Negative Regulation ◽  
Erk Activation ◽  
Erk Phosphorylation ◽  
Extracellular Signal ◽  
Upstream Kinase ◽  
Erk2 Activation ◽  
Induced Aggregation

Abstract We have previously shown that ADP-induced thromboxane A2 generation in platelets requires co-ordinated signaling events from the Gq-coupled P2Y1 receptor and the Gi-coupled P2Y12 receptor in addition to outside-in signaling. It is also known that ADP-induced thromboxane A2 generation is completely abolished in the presence of extracellular calcium, but the mechanism of this negative regulation is not known. In this study we sought to identify the important signaling molecules in ADP-induced thromboxane A2 generation in platelets and characterize the regulation of these molecules by extracellular calcium. Erk2 activation occurred when outside-in signaling was blocked, indicating that it is a downstream event from the P2Y receptors. However, blockade of either P2Y1 or the P2Y12 receptors with corresponding antagonists completely abolished Erk phosphorylation, indicating that both P2Y receptors are required for ADP-induced Erk activation. However, blockade of Erk upstream kinase MEK had not effect on ADP-induced aggregation in aspirin-treated platelets, but dramatically inhibited aggregation as well as secretion in non-aspirinated platelets, suggesting that Erk might be important for thromboxane A2 generation. Finally, PP1 and PP2, inhibitors of Src family kinases, but not PP3, an inactive analog, abolished ADP-induced Erk phosphorylation and thromboxane A2 generation. Interestingly, ADP-induced Erk phosphorylation was completely inhibited in the presence of extracellular calcium, indicating that Erk is a key signaling molecule regulated by extracellular calcium in the negative regulation of thromboxane A2 generation. We conclude that Erk2 is activated downstream of P2Y receptors through a complex mechanism involving Src kinases and plays an important role in ADP-induced thromboxane A2 generation. We also conclude that extracellular calcium blocks ADP-induced thromboxane A2 generation through the inhibition of Erk activation.

scholarly journals Regulation and functional consequences of ADP receptor-mediated ERK2 activation in platelets

2007 ◽  
Vol 404 (2) ◽  
pp. 299-308 ◽  
Author(s):  
Analia Garcia ◽  
Haripriya Shankar ◽  
Swaminathan Murugappan ◽  
Soochong Kim ◽  
Satya P. Kunapuli
Keyword(s):  
Thromboxane A2 ◽  
Underlying Mechanism ◽  
P2y Receptors ◽  
Extracellular Calcium ◽  
Mitogen Activated Protein Kinase ◽  
Erk Activation ◽  
Erk Phosphorylation ◽  
Mitogen Activated Protein ◽  
Upstream Kinase ◽  
Adp Receptor

We have previously shown that ADP-induced thromboxane generation in platelets requires signalling events from the Gq-coupled P2Y1 receptor (platelet ADP receptor coupled to stimulation of phospholipase C) and the Gi-coupled P2Y12 receptor (platelet ADP receptor coupled to inhibition of adenylate cyclase) in addition to outside-in signalling. While it is also known that extracellular calcium negatively regulates ADP-induced thromboxane A2 generation, the underlying mechanism remains unclear. In the present study we sought to elucidate the signalling mechanisms and regulation by extracellular calcium of ADP-induced thromboxane A2 generation in platelets. ERK (extracllular-signal-regulated kinase) 2 activation occurred when outside-in signalling was blocked, indicating that it is a downstream event from the P2Y receptors. However, blockade of either P2Y1 or the P2Y12 receptors with corresponding antagonists completely abolished ERK phosphorylation, indicating that both P2Y receptors are required for ADP-induced ERK activation. Inhibitors of Src family kinases or the ERK upstream kinase MEK [MAPK (mitogen-activated protein kinase)/ERK kinase] abrogated ADP-induced ERK phosphorylation and thromboxane A2 generation. Finally ADP- or Gi+Gz-induced ERK phosphorylation was blocked in the presence of extracellular calcium. The present studies show that ERK2 is activated downstream of P2Y receptors through a complex mechanism involving Src kinases and this plays an important role in ADP-induced thromboxane A2 generation. We also conclude that extracellular calcium blocks ADP-induced thromboxane A2 generation through the inhibition of ERK activation.

Urea signaling to ERK phosphorylation in renal medullary cells requires extracellular calcium but not calcium entry

2001 ◽  
Vol 280 (1) ◽  
pp. F162-F171 ◽  
Author(s):  
Xiao-Yan Yang ◽  
Hongyu Zhao ◽  
Zheng Zhang ◽  
Karin D. Rodland ◽  
Jean-Baptiste Roullet ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Calcium Release ◽  
Calcium Entry ◽  
Extracellular Calcium ◽  
Urea Treatment ◽  
Erk Activation ◽  
Calcium Dependent ◽  
Erk Phosphorylation ◽  
Extracellular Signal ◽  
A Cell

The renal cell line mIMCD3 exhibits markedly upregulated phosphorylation of the extracellular signal-regulated kinase (ERK) 1 and 2 in response to urea treatment (200 mM for 5 min). Previous data have suggested the involvement of a classical protein kinase C (cPKC)-dependent pathway in downstream events related to urea signaling. We now show that urea-inducible ERK activation requires extracellular calcium; unexpectedly, it occurs independently of activation of cPKC isoforms. Pharmacological inhibitors of known intracellular calcium release pathways and extracellular calcium entry pathways fail to inhibit ERK activation by urea. Fura 2 ratiometry was used to assess the effect of urea treatment on intracellular calcium mobilization. In single-cell analyses using subconfluent monolayers and in population-wide analyses using both confluent monolayers and cells in suspension, urea failed to increase intracellular calcium concentration. Taken together, these data indicate that urea-inducible ERK activation requires calcium action but not calcium entry. Although direct evidence is lacking, one possible explanation could include involvement of a calcium-dependent extracellular moiety of a cell surface-associated protein.

scholarly journals Oxytocin antagonism prevents pregnancy-associated aortic dissection in a mouse model of Marfan syndrome

2019 ◽  
Vol 11 (490) ◽  
pp. eaat4822 ◽  
Author(s):  
Jennifer Pardo Habashi ◽  
Elena Gallo MacFarlane ◽  
Rustam Bagirzadeh ◽  
Caitlin Bowen ◽  
Nicholas Huso ◽  
...  
Keyword(s):  
Aortic Dissection ◽  
Mouse Model ◽  
Marfan Syndrome ◽  
Uterine Contraction ◽  
Aortic Wall ◽  
Erk Activation ◽  
Extracellular Signal Regulated Kinase ◽  
Erk Phosphorylation ◽  
Extracellular Signal ◽  
Erk Kinase

Women with Marfan syndrome (MFS) are at high risk for pregnancy-associated aortic dissection. Pathogenic models that singularly invoke hemodynamic stress are difficult to reconcile with predominant postnatal occurrence of aortic tear, often occurring weeks to months after delivery. In consideration of events that peak at term, are sustained after delivery, and might synergize with previously defined signaling pathways implicated in aneurysm progression, we examined the hormone oxytocin, which initiates uterine contraction and milk letdown for the duration of lactation through phosphorylation of extracellular signal–regulated kinase (ERK). In a mouse model of MFS that shows highly penetrant postnatal aortic dissection, risk was strongly attenuated by preventing lactation or use of an oxytocin receptor antagonist. Survival correlated inversely with the extent of ERK activation in the aortic wall, and strong protection was observed upon attenuation of ERK phosphorylation using an inhibitor of ERK kinase (MEK) or the U.S. Food and Drug Administration–approved medication hydralazine, offering potential therapeutic strategies for pregnancy-associated vascular catastrophe in the setting of MFS.

scholarly journals Regulation of ERK phosphorylation in differentiated arterial muscle of rabbits

2001 ◽  
Vol 281 (1) ◽  
pp. H114-H123 ◽  
Author(s):  
Paul H. Ratz
Keyword(s):  
Adrenergic Receptor ◽  
Map Kinases ◽  
Mek Inhibitor ◽  
Erk Activation ◽  
Differentiated Cells ◽  
Erk Phosphorylation ◽  
Extracellular Signal ◽  
Protein Map ◽  
Mitogen Activated Protein ◽  
Isolated Arteries

Extracellular signal-regulated kinases (ERK) and mitogen-activated protein (MAP) kinases participate in cell signaling, regulating cell growth. In differentiated cells, the role ERK plays is less well known. This study quantified the degree of basal and stimulated ERK phosphorylation and contraction in freshly isolated arteries. The level of basal ERK phosphorylation was identical in preloaded and slack arteries, was greater in media than in the whole artery, and was reduced by the MAP or ERK kinase (MEK) inhibitor PD-98059. Chemical denudation using 1 H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one did not elevate basal ERK phosphorylation. PD-98059 reduced maximum phenylephrine (PE)-stimulated ERK phosphorylation but not force. Pervanadate elevated ERK phosphorylation without causing contraction. Contractions produced by PE and relaxations produced by PE washout preceded the ERK phosphorylation. K+ depolarization, muscle stretch, and angiotensin II elevated ERK phosphorylation transiently, whereas PE maintained ERK phosphorylation for 30 min. The α1A-adrenergic receptor antagonist WB-4101 reduced PE-stimulated force by 70% and abolished PE-induced ERK phosphorylation. Afterloaded and zero-load contractions produced by K+ depolarization displayed identical increases in ERK phosphorylation. These data indicate that ERK was active basally in the differentiated artery but regulated by the endothelium and that ERK phosphorylation was not load dependent. A strong correlation between PE-induced force and ERK phosphorylation supports the hypothesis that ERK activation may reflect a signal “notifying” the cell of the degree of α1-adrenergic receptor-induced contraction.

scholarly journals Influence of ERK activation on decreased chemotaxis of mature human cord blood monocyte–derived dendritic cells to CCL19 and CXCL12

Blood ◽  
2006 ◽  
Vol 109 (8) ◽  
pp. 3173-3176 ◽  
Author(s):  
Geling Li ◽  
Sunanda Basu ◽  
Myung-Kwan Han ◽  
Young-June Kim ◽  
Hal E. Broxmeyer
Keyword(s):  
Dendritic Cells ◽  
Cord Blood ◽  
Human Cord Blood ◽  
Erk Activation ◽  
Extracellular Signal Regulated Kinase ◽  
Graft Versus Host ◽  
Erk Phosphorylation ◽  
Extracellular Signal ◽  
Insight Into ◽  
Dc Maturation

Abstract Dendritic cells (DCs) are important regulators in graft-versus-host disease (GVHD). To gain insight into cord blood (CB) DC immunology, we compared chemotactic responses of mature monocyte-derived DCs and maturation agent lipopolysaccharide (LPS)–induced signaling between CB and adult blood (AB). Mature CB DCs expressed reduced CCR7, but increased CXCR4. This was associated with reduced migratory efficiency toward both CCR7 ligand CCL19 and CXCR4 ligand CXCL12. LPS induced higher extracellular signal-regulated kinase (ERK) phosphorylation in CB than in AB DCs. Specific inhibition of ERK during CB DC maturation enhanced LPS-induced up-regulation of CCR7 and CXCR4 on CB DCs and their chemotaxis toward CCL19 and CXCL12, to a level similar to that of mature AB DCs. Overall, monocyte-derived CB DCs responded to LPS with stronger and sustained ERK activation, which negatively correlated with LPS-induced up-regulation of CCR7 and CXCR4 on CB DCs and their migratory responses. These findings may have potential relevance to better understanding DC function in CB transplantation.

scholarly journals ShcA Mediates the Dominant Pathway to Extracellular Signal-Regulated Kinase Activation during Early Thymic Development

2006 ◽  
Vol 26 (23) ◽  
pp. 9035-9044 ◽  
Author(s):  
Paul Trampont ◽  
Li Zhang ◽  
Kodi S. Ravichandran
Keyword(s):  
Ex Vivo ◽  
Dominant Role ◽  
Cell Receptor ◽  
Thymocyte Development ◽  
Erk Activation ◽  
Thymic Development ◽  
Extracellular Signal Regulated Kinase ◽  
Erk Phosphorylation ◽  
Extracellular Signal

ABSTRACT During thymic development, the β selection checkpoint is regulated by pre-T-cell receptor-initiated signals. Progression through this checkpoint is influenced by phosphorylation and activation of the serine/threonine kinases extracellular signal-regulated kinase 1 (ERK1) and ERK2, but the in vivo relevance of specific upstream players leading to ERK activation is not known. Here, using mice with a conditional loss of the shc1 gene or expressing mutants of ShcA, we demonstrate that the adapter protein ShcA is responsible for up to 70% of ERK activation in double-negative (DN) thymocytes in vivo and ex vivo. We also identify two specific tyrosines on ShcA that promote ERK phosphorylation in vivo, and mice expressing ShcA with mutations of these tyrosines show impaired DN thymocyte development. This work provides the first in vivo demonstration of the relative requirement of upstream adapters in controlling ERK activation during β selection and suggests a dominant role for ShcA.

Stimulation of fibroblast proliferation by the plant cysteine protease CMS2MS2 is independent of its proteolytic activity and requires ERK activation

2009 ◽  
Vol 390 (12) ◽  
Author(s):  
Marco Túlio R. Gomes ◽  
Andréia P. Turchetti ◽  
Miriam T.P. Lopes ◽  
Carlos E. Salas
Keyword(s):  
Proteolytic Activity ◽  
Cysteine Protease ◽  
Fibroblast Proliferation ◽  
Mitogenic Effect ◽  
Erk Activation ◽  
Proliferative Effect ◽  
Erk Phosphorylation ◽  
Extracellular Signal ◽  
L929 Fibroblast ◽  
Stimulation Of

Abstract The cysteine protease CMS2MS2 from Carica candamarcensis latex has been shown to enhance proliferation of L929 fibroblast and to activate the extracellular signal-regulated protein kinase (ERK). In experiments with CMS2MS2 irreversibly inhibited by E-64, the proliferative effect on fibroblasts remains unaffected. ERK phosphorylation mediated by CMS2MS2 was abolished in the presence of PD 98059 or U0126, both MAPK cascade inhibitors. In addition, these inhibitors suppress the mitogenic activity of intact CMS2MS2 or CMS2MS2-E-64. Furthermore, ERK phosphorylation and the mitogenic effect are partially suppressed by a phospholipase C (PLC) inhibitor. These data suggest that the mitogenic effect of CMS2MS2 on fibroblasts is independent of its proteolytic activity, requires ERK phosphorylation, and involves activation of PLC.

Exercise effects on muscle β-adrenergic signaling for MAPK-dependent NKCC activity are rapid and persistent

2002 ◽  
Vol 93 (4) ◽  
pp. 1457-1465 ◽  
Author(s):  
Aidar R. Gosmanov ◽  
Nicholas C. Nordtvedt ◽  
Richard Brown ◽  
Donald B. Thomason
Keyword(s):  
Fold Increase ◽  
Signal Pathways ◽  
Plantaris Muscle ◽  
Acute Bout ◽  
Erk Activation ◽  
Akt Activation ◽  
Exercise Adaptation ◽  
Erk Phosphorylation ◽  
Extracellular Signal ◽  
Exercise Induced

This study investigated exercise adaptation of signaling mechanisms that control Na+-K+-2Cl− cotransporter (NKCC) activity in rat skeletal muscle. An acute bout of exercise increased total and NKCC-mediated 86Rb influx. Inhibition of extracellular signal-regulated kinase (ERK) activation abolished the exercise-induced NKCC upregulation. Treadmill training (20 m/min, 20% grade, 30 min/day, 5 days/wk) stimulated total 86Rb influx and increased NKCC activity in the soleus muscle after 2 wk and in the plantaris muscle after 4 wk. Exercise-induced NKCC activity was associated with a 1.4- to 2-fold increase in ERK phosphorylation. Isoproterenol, which activates ERK and NKCC in sedentary muscle, caused a remarkable inhibition of the exercise-induced NKCC activity. Furthermore, isoproterenol inhibition of exercise-induced NKCC activity was accompanied with decreased ERK phosphorylation in the plantaris muscle. Akt (protein kinase B) phosphorylation on both Thr308 and Ser473, which activates Akt and inhibits NKCC activity in sedentary muscle, was stimulated by acute and chronic exercise. This Akt activation was unaffected by isoproterenol. These results indicate an immediate and persistent exercise adaptation of the signal pathways that participate in the control of potassium transport.

MEK1 is required for PDGF-induced ERK activation and DNA synthesis in tracheal myocytes

1997 ◽  
Vol 272 (3) ◽  
pp. L558-L565 ◽  
Author(s):  
A. Y. Karpova ◽  
M. K. Abe ◽  
J. Li ◽  
P. T. Liu ◽  
J. M. Rhee ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Active Form ◽  
Dominant Negative ◽  
Airway Smooth Muscle Cells ◽  
Dependent Manner ◽  
Erk Activation ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Erk2 Activation ◽  
Constitutively Active

We tested whether activation of mitogen-activated protein kinase/ extracellular signal-regulated kinase kinase-1 (MEK1) is required and sufficient for extracellular signal-regulated kinase (ERK) activation in airway smooth muscle cells. First, we transiently cotransfected bovine tracheal myocytes with an epitope-tagged ERK2 and a dominant-negative or a constitutively active form of the gene encoding MEK1 and assessed ERK2 activation by in vitro phosphorylation assay. Expression of the dominant-negative MEK1 inhibited platelet-derived growth factor (PDGF)-induced ERK2 activation, whereas expression of the constitutively active MEK1 induced ERK2 activation, suggesting that MEK1 is required and sufficient for ERK activation in these cells. Next, we assessed the effect of PD-98059, a synthetic MEK inhibitor, on PDGF-induced MEK1 and ERK activation. PD-98059 (10 microM) inhibited MEK1 and ERK activation, confirming that MEK1 is required for ERK activation in bovine tracheal myocytes. PD-98059 had no effect on Src or Raf-1 activity, evidence that PD-98059 is a specific inhibitor of MEK in this system. Finally, PD-98059 reduced PDGF-induced [(3)H]thymidine incorporation in a concentration-dependent manner, suggesting that catalytic activation of MEK1 and ERKs is required for DNA synthesis. We conclude that MEK1 is required for PDGF-induced ERK activation in bovine tracheal myocytes and that MEK1 and ERKs are required for PDGF-induced DNA synthesis in these cells.

scholarly journals The Arf GAP AGAP2 interacts with β-arrestin2 and regulates β2-adrenergic receptor recycling and ERK activation

2013 ◽  
Vol 452 (3) ◽  
pp. 411-421 ◽  
Author(s):  
Yuanjun Wu ◽  
Yu Zhao ◽  
Xiaojie Ma ◽  
Yunjuan Zhu ◽  
Jaimin Patel ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Adrenergic Receptors ◽  
Adrenergic Receptor ◽  
Receptor Activation ◽  
Recycling Endosomes ◽  
Erk Activation ◽  
Erk Phosphorylation ◽  
Extracellular Signal ◽  
Β2 Adrenergic Receptor ◽  
Adp Ribosylation Factor

AGAP2 [Arf (ADP-ribosylation factor) GAP (GTPase-activating protein) with GTP-binding-protein-like, ankyrin repeat and PH (pleckstrin homology) domains] is a multidomain Arf GAP that was shown to promote the fast recycling of transferrin receptors. In the present study we tested the hypothesis that AGAP2 regulates the trafficking of β2-adrenergic receptors. We found that AGAP2 formed a complex with β-arrestin1 and β-arrestin2, proteins that are known to regulate β2-adrenergic receptor signalling and trafficking. AGAP2 co-localized with β-arrestin2 on the plasma membrane, and knockdown of AGAP2 expression reduced plasma membrane association of β-arrestin2 upon β2-adrenergic receptor activation. AGAP2 also co-localized with internalized β2-adrenergic receptors on endosomes, and overexpression of AGAP2 slowed accumulation of β2-adrenergic receptor in the perinuclear recycling endosomes. In contrast, knockdown of AGAP2 expression prevented the recycling of the β2-adrenergic receptor back to the plasma membrane. In addition, AGAP2 formed a complex with endogenous ERK (extracellular-signal-regulated kinase) and overexpression of AGAP2 potentiated ERK phosphorylation induced by β2-adrenergic receptors. Taken together, these results support the hypothesis that AGAP2 plays a role in the signalling and recycling of β2-adrenergic receptors.

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