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)

Phosphorylation of serine and threonine residues in the carboxyl-terminal region of many G-protein-coupled receptors directs the rapid uncoupling from signal transduction pathways. In Chinese hamster ovary cells, we have stably expressed a truncated mutant of the angiotensin II (AT1A) receptor devoid of the carboxyl-terminal 45 amino acids, encompassing 13 serine/threonine residues. One clone, designated TL314 to indicate truncation after leucine 314, expressed a single class of angiotensin II receptors with a dissociation constant of 1.08 nM and a receptor density of 560 fmol/mg of protein (approximately 75,000 receptors/cell). A nonhydrolyzable analog of GTP accelerated the angiotensin II-induced dissociation of [125I]angiotensin II from TL314 plasma membranes 3.6-fold, indicating G-protein coupling. In TL314 cells, angiotensin II stimulated the release of intracellular calcium and the induction of mitogen-activated protein kinase activity, the level of which were comparable with the full-length AT1A receptor. The AII-stimulated calcium response was rapidly desensitized in both full-length and truncated AT1A receptors. Interestingly, angiotensin II-induced endocytosis of the truncated receptor was almost completely inhibited, suggesting that a recognition motif within the carboxyl-terminal 45 amino acids of the AT1A receptor promotes sequestration. Thus, truncation of the AT1A receptor after leucine 314 inhibits agonist-induced internalization without affecting the capacity of the expressed protein to adopt the correct conformation necessary for high affinity binding of angiotensin II, coupling to G-proteins, and activation of signal transduction pathways. The rapid desensitization and refractoriness of the angiotensin II-induced calcium transient in the TL314 cell line, in which putative carboxyl-terminal phosphorylation sites are absent, suggests that the mechanism of AT1A receptor desensitization differs from that of other prototypical G-protein-coupled receptors.
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PMID:Stable expression of a truncated AT1A receptor in CHO-K1 cells. The carboxyl-terminal region directs agonist-induced internalization but not receptor signaling or desensitization. 781 75

G-protein coupled Angiotensin II receptors (AT1A), mediate cellular responses through multiple signal transduction pathways. In AT1A receptor-transfected CHO-K1 cells (T3CHO/AT1A), angiotensin II (AII) stimulated a dose-dependent EC50 = 3.3 nM) increase in cAMP accumulation, which was inhibited by the selective AT1, nonpeptide receptor antagonist EXP3174. Activation of protein kinase C, or increasing intracellular Ca2+ with ATP, the calcium ionophore A23187 or ionomycin failed to stimulate cAMP accumulation. Thus, AII-induced cAMP accumulation was not secondary to activation of a protein kinase C- or ca2+/calmodulin-dependent pathway. Since cAMP has an established role in cellular growth responses, we investigated the effect of the AII-mediated increase in cAMP on cell number and [3H]thymidine incorporation in T3CHOA/AT1A cells. AII (1 microM) significantly inhibited cell number (51% at 96 h) and [3H]thymidine incorporation of 68% at 24 h) compared to vehicle controls. These effects were blocked by EXP3174, confirming that these responses were mediated through the AT1 receptor. Forskolin (10 microM) and the cAMP analog dibutyryl-cAMP (1 mM) also inhibited [3H]thymidine incorporation by 55 and 25% respectively. We extended our investigation on the effect of AII-stimulated increases in cAMP, to determine the role for established growth related signaling events, i.e., mitogen-activated protein kinase activity an tyrosine phosphorylation of cellular proteins. AII-stimulated mitogen-activated protein kinase activity and phosphorylation of the 42 and 44 kD forms. These events were unaffected by forskolin stimulated increases in cAMP, thus the AII-stimulated mitogen-activated protein kinase activity was independent of cAMP in these cells. AII also stimulated tyrosine phosphorylation of a number of cellular proteins in T3CHO/AT1A cells, in particular at 127 kD protein. The phosphorylation of the 127 kD protein was transient, reaching a maximum at 1 min, and returning to basal levels within 10 min. The dephosphorylation of this protein was blocked by a selective inhibitor of cAMP dependent protein kinase A, H89-dihydrochloride and preexposure to forskolin prevented the AII-induced transient tyrosine phosphorylation of the 127 kD protein. These data suggest that cAMP, and therefore protein kinase A can contribute to AII-mediated growth inhibition by stimulating the dephosphorylation of substrates that are tyrosine phosphorylated in response to AII.
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PMID:A role for cAMP in angiotensin II mediated inhibition of cell growth in AT1A receptor-transfected CHO-K1 cells. 860 15

To elucidate the signal transduction pathway from external stimuli to nuclear gene expression in mechanical stress-induced cardiac hypertrophy, we examined the time course of activation of protein kinases such as Raf-1 kinase (Raf-1), mitogen-activated protein kinase kinase (MAPKK), MAP kinases (MAPKs) and 90-kDa ribosomal S6 kinase (p90rsk) in neonatal rat cardiomyocytes. Mechanical stretch rapidly activated Raf-1 and its maximal activation was observed at 1-2 min after stretch. The activity of MAPKK was also increased by stretch, with a peak at 5 min after stretch. In addition, MAPKs and p90rsk were maximally activated at 8 min and at 10-30 min after stretch, respectively. Next, the relationship between mechanical stress-induced hypertrophy and the cardiac renin-angiotensin system was investigated. When the stretch-conditioned culture medium was transferred to the culture dish of non-stretched cardiac myocytes, the medium activated MAPK activity slightly but significantly, and the activation was completely blocked by the type 1 angiotensin II receptor antagonist, CV-11974. However, activation of Raf-1 and MAPKs provoked by stretching cardiomyocytes was only partially suppressed by pretreatment with CV-11974. These results suggest that mechanical stress activates the protein kinase cascade of phosphorylation in cardiac myocytes in the order of Raf-1, MAPKK, MAPKs and p90rsk, and that angiotensin II, which is secreted from stretched myocytes, activates a part of these protein kinases.
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PMID:Molecular aspects of mechanical stress-induced cardiac hypertrophy. 897 57

Angiotensin II activates p21ras, and mediates cardiac hypertrophic growth through the type 1 angiotensin II receptor in cardiac myocytes. An inhibitor of 3-hydroxy-3-methyglutaryl-coenzyme A (HMG-CoA) reductase has been shown to block the post-translational farnesylation of p21ras and inhibit protein synthesis in several cell types. Primary cultures of neonatal cardiac myocytes were used to determine whether HMG-CoA reductase inhibitors, lovastatin, simvastatin and pravastatin inhibit the angiotensin II-induced hypertrophic growth. Angiotensin II (10(-6) M) significantly increased protein-DNA ratio, RNA-DNA ratio, ratios of protein synthesis and mitogen-activated protein (MAP) kinase activity. Lipid-soluble HMG-CoA reductase inhibitors, lovastatin (10(-6) M) and simvastatin (10(-6) M) partially and significantly inhibited the angiotensin II-induced increases in these parameters, but a water-soluble HMG-CoA reductase inhibitor, pravastatin (10(-6) M) did not. Mevalonate (10(-4) M) overcame the inhibitory effects of lovastatin and simvastatin on angiotensin II-induced increases in these parameters. A selective protein kinase C inhibitor, calphostin C (10(-6) M) partially and significantly prevented angiotensin II-induced increases in these parameters, and treatment with both lovastatin and calphostin C inhibited completely. Angiotensin II increased p21ras activity and membrane association, and lovastatin inhibited them. These studies demonstrate that a lipid-soluble HMG-CoA reductase inhibitor, lovastatin, may prevent angiotensin II-induced cardiac hypertrophy, at least in part, through p21ras/MAP kinase pathway, which is linked to mevalonate metabolism.
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PMID:Lovastatin prevents angiotensin II-induced cardiac hypertrophy in cultured neonatal rat heart cells. 1044 99

The third cytoplasmic loop of the angiotensin (Ang) II type 1 receptor (AT(1)) is important for receptor coupling to G proteins and activation of downstream events. Therefore, we determined whether specific AT(1) sequences were required for kinase activation and inhibition of apoptosis by transfecting wild-type (AT1Rwt) and mutated AT(1) into 293 cells. Ang II stimulated a 19.4-fold increase in extracellular signal-regulated kinase (ERK1/ERK2) activity in 293 cells transfected with AT1Rwt. However, in 293 cells that expressed a receptor in which amino acids 221 and 222 were deleted (AT1R[Del221/222]), Ang II-mediated ERK1/ERK2 activation was inhibited by >85%. In contrast, c-Jun NH(2)-terminal protein kinase (JNK) activation was similar in AT1Rwt- and AT1R(Del221/222)-transfected cells. Activation of ERK1/ERK2 by AT1Rwt was independent of Ca(2+), whereas the low level of ERK1/ERK2 activation by AT1R(Del221/222) was completely Ca(2+) dependent. Activation of ERK1/ERK2 in AT1Rwt required Ras, whereas AT1R(Del221/222) required Rap1. These results demonstrate the presence of 2 different pathways for ERK1/ERK2 activation by Ang II, which differ in their requirements for Ca(2+) and small G proteins (Ras versus Rap1). Furthermore, Ang II prevented serum deprivation-induced apoptosis in cells transfected with AT1Rwt but not AT1R(Del221/222). AKT was only phosphorylated by Ang II in AT1Rwt-transfected cells. Overexpression of constitutively active AKT significantly reduced serum deprivation-induced apoptosis in cells transfected with AT1R(Del221/222). This study shows for the first time a direct link between kinase activation and inhibition of apoptosis dependent on amino acids 221 and 222 in the third cytoplasmic loop of the AT(1).
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PMID:The third cytoplasmic loop of the angiotensin II type 1 receptor exerts differential effects on extracellular signal-regulated kinase (ERK1/ERK2) and apoptosis via Ras- and Rap1-dependent pathways. 1076 5

Stimulation of aldosterone biosynthesis by angiotensin II (AII) is thought to be mediated via the PLC, IP3 and intracellular calcium signalling pathway. MAPK (p42/p44) is involved in cell proliferation, and is also activated by AII, but its role in the adrenal response to dietary sodium is unclear. To study the relationship between AII receptor (ATR), MAPK and PKC isoforms, PKCalpha and PKCepsilon, mature Wistar rats were maintained on low or high sodium diets for 1 week. In adrenals from animals on a sodium deplete diet, total ligand binding to both ATR subtypes decreased in the zona glomerulosa (ZG). Under these conditions, active MAPK in the ZG decreased paralleling a decrease in active PKCalpha. In the inner zones (IZ), largely reflecting medullary events, low sodium did not affect MAPK activity. However active PKCalpha decreased. In adrenals from sodium-loaded animals, type 2 ATR (AT2R) binding was reduced in the ZG, while type 1 ATR (AT1R) increased in the IZ. Active MAPK increased in ZG, as did active PKCalpha and PKCepsilon. In IZ, ERK, PKCalpha and PKCepsilon were unchanged. These results suggest that in the ZG and IZ, two different modes of MAPK regulation may exist, utilising different PKC isoforms.
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PMID:Regulation of MAPK activity in response to dietary sodium in the rat adrenal gland. 1119 66

An analysis of the functional role of a diacidic motif (Asp236-Asp237) in the third intracellular loop of the AT1A angiotensin II (Ang II) receptor (AT1-R) revealed that substitution of both amino acids with alanine (DD-AA) or asparagine (DD-NN) residues diminished Ang II-induced receptor phosphorylation in COS-7 cells. However, Ang II-stimulated inositol phosphate production, mitogen-activated protein kinase, and AT1 receptor desensitization and internalization were not significantly impaired. Overexpression of dominant negative G protein-coupled receptor kinase 2 (GRK2)K220M decreased agonist-induced receptor phosphorylation by approximately 40%, but did not further reduce the impaired phosphorylation of DD-AA and DD-NN receptors. Inhibition of protein kinase C by bisindolylmaleimide reduced the phosphorylation of both the wild-type and the DD mutant receptors by approximately 30%. The inhibitory effects of GRK2K220M expression and protein kinase C inhibition by bisindolylmaleimide on agonist-induced phosphorylation were additive for the wild-type AT1-R, but not for the DD mutant receptor. Agonist-induced internalization of the wild-type and DD mutant receptors was similar and was unaltered by coexpression of GRK2K220M. These findings demonstrate that an acidic motif at position 236/237 in the third intracellular loop of the AT1-R is required for optimal Ang II-induced phosphorylation of its carboxyl-terminal tail by GRKs. Furthermore, the properties of the DD mutant receptor suggest that not only Ang II-induced signaling, but also receptor desensitization and internalization, are independent of agonist-induced GRK-mediated phosphorylation of the AT1 receptor.
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PMID:Agonist-induced signaling, desensitization, and internalization of a phosphorylation-deficient AT1A angiotensin receptor. 1149 23

The agonist-induced internalization of several G protein-coupled receptors is an obligatory requirement for their activation of MAPKs. Studies on the relationship between endocytosis of the angiotensin II (Ang II) type 1 receptor (AT1-R) and Ang II-induced ERK1/2 activation were performed in clone 9 (C9) rat hepatic cells treated with inhibitors of endocytosis [sucrose, phenylarsine oxide (PAO), and concanavalin A]. Although Ang II-induced endocytosis of the AT1-R was prevented by sucrose and PAO, and was partially inhibited by concanavalin A, there was no impairment of Ang II-induced ERK activation. However, the specific epidermal growth factor receptor (EGF-R) kinase inhibitor, AG1478, abolished Ang II-induced activation of ERK1/2. Sucrose and PAO also inhibited EGFinduced internalization of the EGF-R in C9 cells, and the inability of these agents to impair EGF-induced ERK activation suggested that the latter is also independent of receptor endocytosis. In COS-7 cells transiently expressing the rat AT1A-R, Ang II also caused ERK activation through EGF-R transactivation. Furthermore, a mutant AT1A-R with truncated carboxyl terminus and impaired internalization retained full ability to activate ERK1/2 in response to Ang II stimulation. These findings demonstrate that Ang II-induced ERK1/2 activation in C9 hepatocytes is independent of both AT1-R and EGF-R endocytosis and is mediated by transactivation of the EGF-R.
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PMID:Independence of angiotensin II-induced MAP kinase activation from angiotensin type 1 receptor internalization in clone 9 hepatocytes. 1187 20

Different signal transduction cascades have been implicated in angiotensin II (Ang II)-mediated cell growth, such as the extracellular signal-regulated kinase 1/2 (ERK1/2) and the phosphatidylinositol 3-kinase (PI3K) pathways. To identify the downstream targets of PI3K involved in Ang II-induced proliferation, we used both rat aortic smooth muscle (RASM) cells and a CHO cell line stably expressing the rat AT1A receptor. The ERK1/2 and PI3K pathways are independently activated and implicated in Ang II-mediated DNA synthesis and cell number increase in these 2 cell lines. In addition, a specific inhibitor of Akt inhibited Ang II-induced Akt phosphorylation, DNA synthesis and proliferation in CHO-AT1A or RASM cells. A dominant-negative mutant of Akt was also found to selectively block Ang II-induced proliferation of CHO-AT1A cells. To further elucidate the signaling events leading to Akt activation, we used an AT1 receptor mutant (AT1AD74E), deficient for Gq protein coupling, and the intracellular calcium chelator BAPTA-AM. Although altered Akt and ERK1/2 activation was observed in the CHO-AT1AD74E cell line, blockade of intracellular calcium elevation did not affect phosphorylation of these kinases. These results provide the first evidence of a specific and necessary role of Akt in Ang II-induced proliferation through a Gq protein-dependent calcium-independent pathway.
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PMID:Akt is a major downstream target of PI3-kinase involved in angiotensin II-induced proliferation. 1262 64

Increased cardiovascular mortality is an unresolved problem in patients with chronic renal failure. Cardiac hypertrophy is observed in the majority of patients with chronic renal failure undergoing haemodialysis. However, the mechanisms, including signal transduction pathways, responsible for cardiac hypertrophy in renal failure remain unknown. We examined the subcellular localization of protein kinase C (PKC) isoforms and phosphorylation activities of 3 mitogen-activated protein (MAP) kinase families in hypertrophied hearts of progressive renal injury rat model by subtotal nephrectomy (SNx). We also examined the effects of a novel angiotensin II type-1 receptor antagonist, CS-866, on the PKC translocation, MAP kinase activity and cardiac hypertrophy in SNx rats. The left ventricle/body weight ratios were significantly larger in SNx rats than in sham rats at 1, 2, and 4 weeks after surgery. The translocation of PKCalpha and epsilon isoforms to membranous fraction was observed in SNx rat hearts at 1, 2, and 4 weeks after surgery. Activation of extracellular signal regulated kinase (ERK) 1/2, but not p38 MAP kinase and c-Jun N-terminal kinase (JNK), was observed at 1 and 2 weeks after surgery. Angiotensin II receptor blockade with CS-866 (1 mg kg-1 day-1) prevented cardiac hypertrophy, PKC translocation and ERK1/2 activation in SNx rats without significant changes in blood pressure. These data suggest that PKC and ERK1/2 are activated by an angiotensin II receptor-mediated pathway and might play an important role in the progression of cardiac hypertrophy in renal failure.
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PMID:Protein kinase C and extracellular signal regulated kinase are involved in cardiac hypertrophy of rats with progressive renal injury. 1476 70


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