Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.24 (mitogen-activated protein kinase)
95,810 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Adenosine exerts a mitogenic effect on human endothelial cells via stimulation of the A2A-adenosine receptor. This effect can also be elicited by the beta2-adrenergic receptor but is not mimicked by elevation of intracellular cAMP levels. In the present work, we report that stimulation of the A2A-adenosine receptor and of the beta2-adrenergic receptor activates mitogen-activated protein kinase (MAP kinase) in human endothelial cells based on the following criteria: adenosine analogues and beta-adrenergic agonists cause an (i) increase in tyrosine phosphorylation of the p42 isoform and to a lesser extent of the p44 isoform of MAP kinase and (ii) stimulate the phosphorylation of myelin basic protein by MAP kinase; (iii) this is accompanied by a redistribution of the enzyme to the perinuclear region. Pretreatment of the cells with cholera toxin (to down-regulate Gsalpha) abolishes activation of MAP kinase by isoproterenol but not that induced by adenosine analogues. In addition, MAP kinase stimulation via the A2A-adenosine receptor is neither impaired following pretreatment of the cells with pertussis toxin (to block Gi-dependent pathways) nor affected by GF109203X (1 microM; to inhibit typical protein kinase C isoforms) nor by a monoclonal antibody, which blocks epidermal growth factor-dependent signaling. In contrast, MAP kinase activation is blocked by PD 098059, an inhibitor of MAP kinase kinase 1 (MEK1) activation, which also blunts the A2A-adenosine receptor-mediated increase in [3H]thymidine incorporation. Activation of the A2A-adenosine receptor is associated with increased levels of GTP-bound p21(ras). Thus, our experiments define stimulation of MAP kinase as the candidate cellular target mediating the mitogenic action of the A2A-adenosine receptor on primary human endothelial cells; the signaling pathway operates via p21(ras) and MEK1 but is independent of Gi, Gs, and the typical protein kinase C isoforms. This implies an additional G protein which links this prototypical Gs-coupled receptor to the MAP kinase cascade.
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PMID:Stimulation of the mitogen-activated protein kinase via the A2A-adenosine receptor in primary human endothelial cells. 903 93

PC12 cell differentiation was induced by one week of nerve growth factor (NGF) treatment and adenosine A2A receptor expression and activity were analysed. Undifferentiated PC12 cells expressed very high levels of adenosine A2A receptors (approximately equal to 2 pmol/mg) and exhibited strong cyclic AMP (cAMP) responses when stimulated with the selective adenosine A2A receptor agonist 2-[p-(2-carbonylethyl) phenylethylamino-5'-N-ethylcarboxamidoadenosine]. NGF-induced differentiation was accompanied by a down-regulation of adenosine A2A receptors: receptor binding decreased to 500 fmol/mg, immunoreactive A2A receptor protein was decreased by about half and cAMP production was reduced by 60%. In situ hybridization experiments demonstrated a heterogenous distribution of A2A receptor mRNA and a decreased number of strongly labelled cells after NGF treatment. Stimulation of the cells with the non-selective adenosine receptor agonist N-ethylcarboxamidoadenosine (NECA) inhibited NGF-induced mitogen-activated protein kinase activation. These results thus show that NGF-induced differentiation of PC12 cells is accompanied by a decrease in A2A receptor-mediated cAMP accumulation. This might be a way for PC12 cells to counteract an inhibitory effect of A2A receptor activation on some aspects of neurotrophin signalling.
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PMID:Down-regulation of adenosine A2A receptors upon NGF-induced differentiation of PC12 cells. 936 87

Promiscuous coupling between G protein-coupled receptors and multiple species of heterotrimeric G proteins provides a potential mechanism for expanding the diversity of G protein-coupled receptor signaling. We have examined the mechanism and functional consequences of dual Gs/Gi protein coupling of the beta3-adrenergic receptor (beta3AR) in 3T3-F442A adipocytes. The beta3AR selective agonist disodium (R, R)-5-[2[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1, 3-benzodioxole-2,2-dicarboxylate (CL316,243) stimulated a dose-dependent increase in cAMP production in adipocyte plasma membrane preparations, and pretreatment of cells with pertussis toxin resulted in a further 2-fold increase in cAMP production by CL316,243. CL316,243 (5 microM) stimulated the incorporation of 8-azido-[32P]GTP into Galphas (1.57 +/- 0.12; n = 3) and Galphai (1. 68 +/- 0.13; n = 4) in adipocyte plasma membranes, directly demonstrating that beta3AR stimulation results in Gi-GTP exchange. The beta3AR-stimulated increase in 8-azido-[32P]GTP labeling of Galphai was equivalent to that obtained with the A1-adenosine receptor agonist N6-cyclopentyladenosine (1.56 +/- 0.07; n = 4), whereas inclusion of unlabeled GTP (100 microM) eliminated all binding. Stimulation of the beta3AR in 3T3-F442A adipocytes led to a 2-3-fold activation of mitogen-activated protein (MAP) kinase, as measured by extracellular signal-regulated kinase-1 and -2 (ERK1/2) phosphorylation. Pretreatment of cells with pertussis toxin (PTX) eliminated MAP kinase activation by beta3AR, demonstrating that this response required receptor coupling to Gi. Expression of the human beta3AR in HEK-293 cells reconstituted the PTX-sensitive stimulation of MAP kinase, demonstrating that this phenomenon is not exclusive to adipocytes or to the rodent beta3AR. ERK1/2 activation by the beta3AR was insensitive to the cAMP-dependent protein kinase inhibitor H-89 but was abolished by genistein and AG1478. These data indicate that constitutive beta3AR coupling to Gi proteins serves both to restrain Gs-mediated activation of adenylyl cyclase and to initiate additional signal transduction pathways, including the ERK1/2 MAP kinase cascade.
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PMID:The beta3-adrenergic receptor activates mitogen-activated protein kinase in adipocytes through a Gi-dependent mechanism. 1020 24

The A(2A)-adenosine receptor, a prototypical G(s)-coupled receptor, activates mitogen-activated protein (MAP) kinase in a manner independent of cAMP in primary human endothelial cells. In order to delineate signaling pathways that link the receptor to the regulation of MAP kinase, the human A(2A) receptor was heterologously expressed in Chinese hamster ovary (CHO) and HEK293 cells. In both cell lines, A(2A) agonist-mediated cAMP accumulation was accompanied by activation of the small G protein rap1. However, rap1 mediates A(2A) receptor-dependent activation of MAP kinase only in CHO cells, the signaling cascade being composed of G(s), adenylyl cyclase, rap1, and the p68 isoform of B-raf. This isoform was absent in HEK293 cells. Contrary to CHO cells, in HEK293 cells activation of MAP kinase by A(2A) agonists was not mimicked by 8-bromo-cAMP, was independent of Galpha(s), and was associated with activation of p21(ras). Accordingly, overexpression of the inactive S17N mutant of p21(ras) and of a dominant negative version of mSos (the exchange factor of p21(ras)) blocked MAP kinase stimulation by the A(2A) receptor in HEK 293 but not in CHO cells. In spite of the close homology between p21(ras) and rap1, the S17N mutant of rap1 was not dominant negative because (i) overexpression of rap1(S17N) failed to inhibit A(2A) receptor-dependent MAP kinase activation, (ii) rap1(S17N) was recovered in the active form with a GST fusion protein comprising the rap1-binding domain of ralGDS after A(2A) receptor activation, and (iii) A(2A) agonists promoted the association of rap1(S17N) with the 68-kDa isoform of B-raf in CHO cells. We conclude that the A(2A) receptor has the capacity two activate MAP kinase via at least two signaling pathways, which depend on two distinct small G proteins, namely p21(ras) and rap1. Our observations also show that the S17N version of rap1 cannot be assumed a priori to act as a dominant negative interfering mutant.
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PMID:Activation of mitogen-activated protein kinase by the A(2A)-adenosine receptor via a rap1-dependent and via a p21(ras)-dependent pathway. 1046 24

Nerve growth factor (NGF) induces differentiation of the rat pheochromocytoma clone (PC12) by activating the high affinity receptor, p140(trkA), linked to mitogen-activated protein kinase. While the physiological role of the low affinity NGF receptor (p75) has not been clearly defined, this receptor promotes activation of nuclear factor (NF) kappaB in Schwann cells. PC12 cells express the A(2A) adenosine receptor (AR), whose expression is significantly decreased by NGF treatment. In this study, we determined whether TrkA or p75 is involved in NGF-mediated regulation of A(2A)AR expression. NGF treatment decreased A(2A)AR in a time-dependent manner, with maximal effects observed by 1 day, and continued down-regulation of the receptor for up to 3 days in the presence of NGF. The decrease in A(2A)AR was associated with a more delayed decrease in the steady-state levels of the A(2A)AR mRNA. Down-regulation of the A(2A)AR at 1 day was mimicked by activators of NFkappaB, such as H(2)O(2), and ceramide, and was attenuated by the inhibitor pyrrolidine dithiocarbamate or following transient transfection of PC12 cells with a dominant negative IkappaBalpha mutant. Moreover, NGF stimulated nuclear accumulation of p65 subunits of NFkappaB (but not p50 subunits) in PC12 cells, as determined by electrophoretic mobility shift assays and by Western blotting. In contrast, inhibition of TrkA by AG879 or of TrkA-dependent mitogen-activated protein kinase mitogen-activated protein kinase kinase with PD98059 blocked PC12 cell differentiation without affecting A(2A)AR down-regulation, suggesting dissociation between these two phenomena. Taken together, these data provide strong support for the involvement of the p75/NFkappaB pathway in NGF-mediated down-regulation of A(2A)AR in PC12 cells.
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PMID:A role of p75 in NGF-mediated down-regulation of the A(2A) adenosine receptors in PC12 cells. 1053 99

Fusion proteins between the human A(1) adenosine receptor and the pertussis toxin resistant (Cys351Gly) mutant of the G-protein alpha subunit G(i1)alpha (A1/Gi), and between the human A(1) adenosine receptor, the Aequorea victoria green fluorescent protein (GFP) and Cys351Gly G(i1)alpha (A1/GFP/Gi), were expressed in CHO cells. The agonist NECA caused a stimulation of [(35)S]GTPgammaS binding at both fusion proteins with similar concentration dependence as at the native receptor. However in the presence of pertussis toxin NECA stimulation of [(35)S]GTPgammaS binding was only seen at the A1/GFP/Gi fusion protein. The regulation of the adenylyl cyclase and MAP kinase effector systems by both fusion proteins was attenuated following pertussis toxin treatment. These studies demonstrate for the first time the characterisation of a fusion protein between a G-protein coupled receptor, GFP and a G-protein alpha subunit.
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PMID:Functional analysis of a human A(1) adenosine receptor/green fluorescent protein/G(i1)alpha fusion protein following stable expression in CHO cells. 1058 92

Adenosine inhibits growth of vascular smooth muscle cells. The goals of this study were to determine which adenosine receptor subtype mediates the antimitogenic effects of adenosine and to investigate the signal transduction mechanisms involved. In rat aortic vascular smooth muscle cells, platelet-derived growth factor-BB (PDGF-BB) (25 ng/mL) stimulated DNA synthesis ([(3)H]thymidine incorporation), cellular proliferation (cell number), collagen synthesis ([(3)H]proline incorporation), total protein synthesis ([(3)H]leucine incorporation), and mitogen-activated protein (MAP) kinase activity. The adenosine receptor agonists 2-chloroadenosine and 5'-N-methylcarboxamidoadenosine, but not N(6)-cyclopentyladenosine or CGS21680, inhibited the growth effects of PDGF-BB, an agonist profile consistent with an A(2B) receptor-mediated effect. The adenosine receptor antagonists KF17837 and 1,3-dipropyl-8-p-sulfophenylxanthine, but not 8-cyclopentyl-1, 3-dipropylxanthine, blocked the growth-inhibitory effects of 2-chloroadenosine and 5'-N-methylcarboxamidoadenosine, an antagonist profile consistent with an A(2) receptor-mediated effect. Antisense, but not sense or scrambled, oligonucleotides to the A(2B) receptor stimulated basal and PDGF-induced DNA synthesis, cell proliferation, and MAP kinase activity. Moreover, the growth-inhibitory effects of 2-chloroadenosine, 5'-N-methylcarboxamidoadenosine, and erythro-9-(2-hydroxy-3-nonyl) adenine plus iodotubericidin (inhibitors of adenosine deaminase and adenosine kinase, respectively) were abolished by antisense, but not scrambled or sense, oligonucleotides to the A(2B) receptor. Our findings strongly support the hypothesis that adenosine causes inhibition of vascular smooth muscle cell growth by activating A(2B) receptors coupled to inhibition of MAP kinase activity. Pharmacological or molecular biological activation of A(2B) receptors may prevent vascular remodeling associated with hypertension, atherosclerosis, and restenosis following balloon angioplasty.
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PMID:A(2B) receptors mediate antimitogenesis in vascular smooth muscle cells. 1064 9

Recent studies suggest that p38 mitogen-activated protein kinase (MAPK) may be involved in ischemic preconditioning (PC). To further test this possibility, the regulation of MAPK-activated protein kinase 2 (MAPKAPK2), a kinase immediately downstream from p38 MAPK, and the activity of c-Jun NH(2)-terminal kinase (JNK), a second MAPK, were examined in preconditioned hearts. Isolated, perfused rabbit hearts were subjected to 20 to 30 minutes of global ischemia. Ventricular biopsies before treatment and after 20 minutes of ischemia were homogenized, and the activities of MAPKAPK2 and JNK were evaluated. For the MAPKAPK2 experiments, 7 groups were studied, as follows: control hearts; preconditioned hearts; hearts treated with 500 nmol/L R(-) N(6)-(2-phenylisopropyl) adenosine (PIA), an A(1)-adenosine receptor agonist; preconditioned hearts pretreated with 100 micromol/L 8-(p-sulfophenyl) theophylline (SPT), an adenosine receptor antagonist; preconditioned hearts also treated with SB 203580, a potent inhibitor of p38 MAPK activation; hearts treated with 50 ng/mL anisomycin (a p38 MAPK/JNK activator); and hearts treated with both anisomycin (50 ng/mL) and the tyrosine kinase inhibitor genistein (50 micromol/L). MAPKAPK2 activity was not altered in control hearts after 20 minutes of global ischemia. By contrast, there was a 3.8-fold increase in activity during ischemia in preconditioned hearts. Activation of MAPKAPK2 in preconditioned hearts was blocked by both SPT and SB 203580. MAPKAPK2 activity during ischemia increased 3.5-fold and 3.3-fold in hearts pretreated with PIA or anisomycin, respectively. MAPKAPK2 activation during ischemia in hearts pretreated with anisomycin was blocked by genistein. In separate hearts, anisomycin mimicked the anti-infarct effect of PC, and that protection was abolished by genistein. JNK activity was measured in control and preconditioned hearts. There was a comparable, modest decline in activity during 30 minutes of global ischemia in both groups. As a positive control, a third group of hearts was treated with anisomycin before global ischemia, and in these, JNK activity increased by 290% above baseline. These results confirm that the p38 MAPK/MAPKAPK2 pathway is activated during ischemia only if the heart is in a preconditioned state. These data further support p38 MAPK as an important signaling component in ischemic PC.
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PMID:Ischemic preconditioning activates MAPKAPK2 in the isolated rabbit heart: evidence for involvement of p38 MAPK. 1066 9

In this study the effect of insulin and A(1)-adenosine receptor stimulation on protein kinase B (PKB) activation has been investigated in the hamster vas deferens smooth muscle cell line DDT(1)MF-2. Increases in PKB phosphorylation were determined by Western blotting using an antibody that detects PKB phosphorylation at Ser(473). Insulin, a recognized activator of PKB, stimulated a concentration-dependent increase in PKB phosphorylation in DDT(1)MF-2 cells (EC(50) 5+/-1 pM). The selective A(1)-adenosine receptor agonist N(6)-cyclopentyladenosine (CPA) stimulated time and concentration-dependent increases in PKB phosphorylation in DDT(1)MF-2 cells (EC(50) 1.3+/-0.5 nM). CPA-mediated increases in PKB phosphorylation were antagonized by the A(1)-adenosine receptor selective antagonist 1,3-dipropylcyclopentylxanthine (DPCPX) yielding an apparent K(D) value of 2.3 nM. Pre-treatment of DDT(1)MF-2 cells with pertussis toxin (PTX, 100 ng ml(-1) for 16 h), to block G(i)/G(o)-dependent pathways, abolished CPA (1 microM) induced phosphorylation of PKB. In contrast, responses to insulin (100 nM) were resistant to PTX pre-treatment. The phosphatidylinositol 3-kinase (PI-3K) inhibitors wortmannin (IC(50) 10.3+/-0.6 nM) and LY 294002 (IC(50) 10.3+/-1.2 microM) attenuated the phosphorylation of PKB elicited by CPA (1 microM) in a concentration-dependent manner. Wortmannin (30 nM) and LY 294002 (30 microM) also blocked responses to insulin (100 nM). Removal of extracellular Ca(2+) and chelation of intracellular Ca(2+) with BAPTA had no significant effect on CPA-induced PKB phosphorylation. Similarly, pretreatment (30 min) with inhibitors of protein kinase C (Ro 31-8220; 10 microM), tyrosine kinase (genistein; 100 microM), mitogen-activated protein (MAP) kinase kinase (PD 98059; 50 microM) and p38 MAPK (SB 203580; 20 microM) had no significant effect on CPA-induced PKB phosphorylation. In conclusion, these data demonstrate that A(1)-adenosine receptor stimulation in DDT(1)MF-2 cells increases PKB phosphorylation through a PTX and PI-3K-sensitive pathway.
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PMID:Activation of protein kinase B by the A(1)-adenosine receptor in DDT(1)MF-2 cells. 1086 94

Ischemic preconditioning improves liver resistance to hypoxia and reduces reperfusion injury following transplantation. However, the intracellular signals that mediate the development of liver hypoxic preconditioning are largely unknown. We have investigated the signal pathway leading to preconditioning in freshly isolated rat hepatocytes. Hepatocytes were preconditioned by 10-minute incubation under hypoxic conditions followed by 10 minutes of reoxygenation and subsequently exposed to 90 minutes of hypoxia. Preconditioning reduced hepatocyte killing by hypoxia by about 35%. A similar protection was also obtained by preincubation with chloro-adenosine or with A(2A)-adenosine receptor agonist CGS21680, whereas A(1)-adenosine receptor agonist N-phenyl-isopropyladenosine (R-PIA) was inactive. Conversely, the development of preconditioning was blocked by A(2)-receptor antagonist 3,7-dimethyl-1-propargylxanthine (DMPX), but not by A(1)-receptor antagonist 8-cyclopenthyl-1, 3-dipropylxanthine (DPCPX). In either preconditioned or CGS21680-treated hepatocytes a selective activation of delta and epsilon protein kinase C (PKC) isoforms was also evident. Inhibition of heterotrimeric G(i) protein or of phospholypase C by, respectively, pertussis toxin or U73122, prevented PKC activation as well as the development of preconditioning. MEK inhibitor PD98509 did not interfere with preconditioning that was instead blocked by p38 MAP kinase inhibitor SB203580. The direct activation of p38 MAPK by anisomycin A mimicked the protection against hypoxic injury given by preconditioning. Consistently, an increased phosphorylation of p38 MAPK was observed in preconditioned or CGS21680-treated hepatocytes, and this effect was abolished by PKC-blocker, chelerythrine. We propose that a signal pathway involving A(2A)-adenosine receptors, G(i)-proteins, phospholypase C, delta- and epsilon-PKCs, and p38 MAPK, is responsible for the development of liver ischemic preconditioning.
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PMID:Signal pathway involved in the development of hypoxic preconditioning in rat hepatocytes. 1112 29


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