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)

Receptors that couple to the heterotrimeric G proteins, Gi or Gq, can stimulate phosphoinositide (PI) hydrolysis and mitogen-activated protein kinase (MAPK) activation. PI hydrolysis produces inositol 1,4,5-trisphosphate and diacylglycerol, leading to activation of protein kinase C (PKC), which can stimulate increased MAPK activity. However, the relationship between PI hydrolysis and MAPK activation in Gi and Gq signaling has not been clearly defined and is the subject of this study. The effects of several signaling inhibitors are assessed including expression of a peptide derived from the carboxyl terminus of the beta adrenergic receptor kinase 1 (beta ARKct), which specifically blocks signaling mediated by the beta gamma subunits of G proteins (G beta gamma), expression of dominant negative mutants of p21ras (RasN17) and p74raf-1 (N delta Raf), protein-tyrosine kinase (PTK) inhibitors and cellular depletion of PKC. The Gi-coupled alpha 2A adrenergic receptor (AR) stimulates MAPK activation which is blocked by expression of beta ARKct, RasN17, or N delta Raf, or by PTK inhibitors, but unaffected by cellular depletion of PKC. In contrast, MAPK activation stimulated by the Gq-coupled alpha 1B AR or M1 muscarinic cholinergic receptor is unaffected by expression of beta ARKct or RasN17 expression or by PTK inhibitors, but is blocked by expression of N delta Raf or by PKC depletion. These data demonstrate that Gi- and Gq-coupled receptors stimulate MAPK activation via distinct signaling pathways. G beta gamma is responsible for mediating Gi-coupled receptor-stimulated MAPK activation through a mechanism utilizing p21ras and p74raf independent of PKC. In contrast, G alpha mediates Gq-coupled receptor-stimulated MAPK activation using a p21ras-independent mechanism employing PKC and p74raf. To define the role of G beta gamma in Gi-coupled receptor-mediated PI hydrolysis and MAPK activation, direct stimulation with G beta gamma was used. Expression of G beta gamma resulted in MAPK activation that was sensitive to inhibition by expression of beta ARKct, RasN17, or N delta Raf or by PTK inhibitors, but insensitive to PKC depletion. By comparison, G beta gamma-mediated PI hydrolysis was not affected by beta ARKct, RasN17, or N delta Raf expression or by PTK inhibitors. Together, these results demonstrate that G beta gamma mediates MAPK activation and PI hydrolysis via independent signaling pathways.
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PMID:Distinct pathways of Gi- and Gq-mediated mitogen-activated protein kinase activation. 761 10

The platelet-activating factor (PAF) receptor couples with multiple signaling pathways such as activation of phospholipase C, phospholipase A2, and mitogen-activated protein kinase and the inhibition of adenylate cyclase. The PAF-induced signals are attenuated by repetitive or long standing applications of the agonist (homologous desensitization). To investigate mechanisms underlying the agonist-induced desensitization, we constructed mutant forms of the cloned guinea pig PAF receptor and stably expressed them in Chinese hamster ovary cells. The cells expressing the wild type receptor transiently activated phospholipase C in response to PAF. Intracellular inositol 1,4,5-trisphosphate level and intracellular Ca2+ concentration reached the maximal levels within 20 s and returned to the basal levels in several minutes, even in the continuous presence of the ligand. In contrast, a truncated PAF receptor lacking the carboxyl-terminal cytoplasmic tail induced sustained elevations of inositol 1,4,5-trisphosphate and intracellular Ca2+ concentrations. Similar findings were noted in another mutant, in which the Ser/Thr residues in the carboxyl-terminal tail were substituted with Ala. Both mutant PAF receptors more potently activated the other signals (mitogen-activated protein kinase kinase, arachidonate release, and inhibition of adenylate cyclase) than did the wild type receptor. Thus, while the carboxyl-terminal cytoplasmic tail of the PAF receptor is not required for the forward activation of multiple signals, it does have a critical role for signal attenuation induced by the agonist through phosphate accepters. We also noted that the synthetic peptide of the PAF receptor carboxyl-terminal tail was strongly phosphorylated by the recombinant beta-adrenergic receptor kinase 1, suggesting that it or its relatives might be involved in PAF receptor phosphorylation and homologous desensitization.
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PMID:Role of cytoplasmic tail phosphorylation sites of platelet-activating factor receptor in agonist-induced desensitization. 807 75

Assembly of terminal complement complexes (TCC) C5b-7, C5b-8, and C5b-9 on target cells during acute and chronic inflammation induces hydrolysis of plasma membrane phospholipids and heterotrimeric G protein activation. TCC also stimulate a variety of cellular activities, which include cytokine synthesis, proto-oncogene activation, and mitotic signaling. Now we report that sublytic TCC induced Ras, Raf-1, and extracellular signal-regulated kinase (ERK) 1 activation in JY25 B cell line. When cells were exposed to C5b-9, GTP-bound Ras in anti-C5b-9 immunoprecipitates was increased 3.2-fold at 2 min, while GTP-bound Ras in anti-Ras immunoprecipitates was increased 2-fold at 10 min. Both C5b-9 and C5b-7, but not C5b6, increased Raf-1 kinase activity maximum 3.3-fold at 2 min and 2.8-fold at 5 min, respectively. ERK1 activity was 2-fold increased by C5b-9 at 2 min and by C5b-7 at 10 min, over the C5b6 level. The role of mitogen-activated protein kinase (MAPK) pathway on TCC-inducible mitotic signaling was evaluated by assessing DNA synthesis and activator protein 1 (AP-1) DNA-binding activity. The MAPK/ERK-specific inhibitor PD 098,059 abolished the C5b-9-induced DNA synthesis. Involvement of G protein in the activation of MAPK pathway by TCC was indicated by inhibition of Raf-1 and ERK1 kinase activity, as well as the DNA synthesis by pretreatment of cells with pertussis toxin. Overexpression of beta-adrenergic receptor kinase 1 carboxyl-terminal peptide in JY25 cells also inhibited Raf-1 and ERK1 activity, indicating a direct involvement of G betagamma subunits in the signal transduction generated through activation of MAPK pathway by TCC assembly in the plasma membrane.
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PMID:Activation of Ras and mitogen-activated protein kinase pathway by terminal complement complexes is G protein dependent. 912 5

In this study, prostaglandin (PG) F2alpha was found to activate mitogen-activated protein (MAP) kinase and MAP kinase kinase (MEK) in cultured rat puerperal uterine myometrial cells. PGF2alpha stimulation also led to an increase in phosphorylation of raf-1, son of sevenless (SOS), and Shc. Furthermore, we examined the mechanism by which PGF2alpha induced MAP kinase phosphorylation. Both pertussis toxin (10 ng/ml), which inactivates Gi/Go proteins, and expression of a peptide derived from the carboxyl terminus of the beta-adrenergic receptor kinase 1 (betaARK1), which specifically blocks signaling mediated by the betagamma subunits of G proteins, blocked the PGF2alpha-induced activation of MAP kinase. Ritodrine (1 microM), which is known to relax uterine muscle contraction, attenuated PGF2alpha-induced tyrosine phosphorylation of MAP kinase. Moreover, to examine the role of MAP kinase pathway in uterine contraction, an inhibitor of MEK activity, PD098059, was used. Although MEK inhibitor had no effect on PGF2alpha-induced calcium mobilization, this inhibitor partially inhibited PGF2alpha-induced uterine contraction. These results provide evidence that PGF2alpha stimulates the MAP kinase signaling pathway in cultured rat puerperal uterine myometrial cells through Gbetagamma protein, suggesting that this new pathway may play an important role in the biological action of PGF2alpha on these cells.
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PMID:Role of mitogen-activated protein kinase pathway in prostaglandin F2alpha-induced rat puerperal uterine contraction. 923 56

Angiotensin II (Ang II) induces hypertrophy of cardiac myocytes and hyperplasia of cardiac fibroblasts. To determine the molecular mechanism by which Ang II displayed different effects on cardiac myocytes and fibroblasts, we examined signal transduction pathways leading to activation of extracellular signal-regulated kinases (ERKs). Ang II-induced ERK activation was abolished by pretreatment with pertussis toxin and by overexpression of the Gbetagamma subunit-binding domain of the beta-adrenergic receptor kinase 1 in cardiac fibroblasts but not in cardiac myocytes. Inhibition of protein kinase C strongly inhibited activation of ERKs by Ang II in cardiac myocytes, whereas inhibitors of tyrosine kinases but not of protein kinase C abolished Ang II-induced ERK activation in cardiac fibroblasts. Overexpression of C-terminal Src kinase (Csk), which inactivates Src family tyrosine kinases, suppressed the activation of transfected ERK in cardiac fibroblasts. Ang II rapidly induced phosphorylation of Shc and association of Shc with Grb2. Cotransfection of the dominant-negative mutant of Ras or Raf-1 kinase abolished Ang II-induced ERK activation in cardiac fibroblasts. Overexpression of Csk or the dominant-negative mutant of Ras had no effects on Ang II-induced ERK activation in cardiac myocytes. These findings suggest that Ang II-evoked signal transduction pathways differ among cell types. In cardiac fibroblasts, Ang II activates ERKs through a pathway including the Gbetagamma subunit of Gi protein, tyrosine kinases including Src family tyrosine kinases, Shc, Grb2, Ras, and Raf-1 kinase, whereas Gq and protein kinase C are important in cardiac myocytes.
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PMID:Cell type-specific angiotensin II-evoked signal transduction pathways: critical roles of Gbetagamma subunit, Src family, and Ras in cardiac fibroblasts. 948 62

Terminal differentiation of skeletal muscle cells in culture is inhibited by a number of different growth factors whose subsequent intracellular signaling events are poorly understood. In this study, we have investigated the role of heterotrimeric G proteins in mediating fibroblast growth factor (FGF)-dependent signals that regulate myogenic differentiation. Pertussis toxin, which ADP-ribosylates and inactivates susceptible G proteins, promotes terminal differentiation in the presence of FGF-2, suggesting that Galpha or Gbeta gamma subunits or both are involved in transducing the FGF-dependent signal(s) that inhibits myogenesis. We found that Gbetagamma subunits are likely to be involved since the expression of the C terminus of beta-adrenergic receptor kinase 1, a Gbetagamma subunit-sequestering agent, promotes differentiation in the presence of FGF-2, and expression of the free Gbeta gamma dimer can replace FGF-2, rescuing cells from pertussis toxin-induced differentiation. Addition of pertussis toxin also blocked FGF-2-mediated activation of mitogen-activated protein kinases (MAPKs). Ectopic expression of dominant active mutants in the Ras/MAPK pathway rescued cells from pertussis toxin-induced terminal differentiation, suggesting that the Gbeta gamma subunits act upstream of the Ras/MAPK pathway. It is unlikely that the pertussis toxin-sensitive pathway is activated by other, as yet unidentified FGF receptors since PDGF (platelet-derived growth factor)-stimulated MM14 cells expressing a chimeric receptor containing the FGF receptor-1 intracellular domain and the PDGF receptor extracellular domain were sensitive to pertussis toxin. Our data suggest that FGF-mediated signals involved in repression of myogenic differentiation are transduced by a pertussis toxin-sensitive G-protein-coupled mechanism. This signaling pathway requires the action of Gbeta gamma subunits and activation of MAPKs to repress skeletal muscle differentiation.
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PMID:Regulation of myogenesis by fibroblast growth factors requires beta-gamma subunits of pertussis toxin-sensitive G proteins. 974 95

We investigated the role of arrestins in the trafficking of human alpha2-adrenergic receptors (alpha2-ARs) and the effect of receptor trafficking on p42/p44 MAP kinase activation. alpha2-ARs expressed in COS-1 cells demonstrated a modest level of agonist-mediated internalization, with alpha2c > alpha2b > alpha2a. However, upon coexpression of arrestin-2 (beta-arrestin-1) or arrestin-3 (beta-arrestin-2), internalization of the alpha2b AR was dramatically enhanced and redistribution of receptors to clathrin coated vesicles and endosomes was observed. Internalization of the alpha2c AR was selectively promoted by coexpression of arrestin-3, while alpha2a AR internalization was only slightly stimulated by coexpression of either arrestin. Coexpression of GRK2 had no effect on the internalization of any alpha2-AR subtype, either in the presence or absence of arrestins. Internalization of the alpha2b and alpha2c ARs was inhibited by coexpression of dominant negative dynamin-K44A. However, alpha2-AR-mediated activation of either endogenous or cotransfected p42/p44 mitogen-activated protein (MAP) kinase was not affected by either dynamin-K44A or arrestin-3. Moreover, activation of p42/p44 MAP kinase by endogenous epidermal growth factor, lysophosphatidic acid, and beta2-adrenergic receptors was also unaltered by dynamin-K44A. In summary, our data suggest that internalization of the alpha2b, alpha2c, and to a lesser extent alpha2a ARs, is both arrestin- and dynamin-dependent. However, endocytosis does not appear to be required for alpha2-adrenergic, epidermal growth factor, lysophosphatidic acid, or beta2-adrenergic receptor-mediated p42/p44 MAP kinase activation in COS-1 cells.
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PMID:Role of arrestins in endocytosis and signaling of alpha2-adrenergic receptor subtypes. 1019 13

Agonist-promoted internalization of some G protein-coupled receptors has been shown to mediate receptor desensitization, resensitization, and down-regulation. In this study, we investigated whether opioids induced internalization of the human and rat kappa opioid receptors stably expressed in Chinese hamster ovary cells, the potential mechanisms involved in this process and its possible role in activation of mitogen-activated protein (MAP) kinase. Exposure of the human kappa receptor to the agonists U50,488H, U69,593, ethylketocyclazocine, or tifluadom, but not etorphine, promoted receptor internalization. However, none of these agonists induced significant internalization of the rat kappa opioid receptor. U50, 488H-induced human kappa receptor internalization was time- and concentration-dependent, with 30-40% of the receptors internalized following a 30-min exposure to 1 microM U50,488H. Agonist removal resulted in the receptors gradually returning to the cell surface over a 60-min period. The antagonist naloxone blocked U50, 488H-induced internalization without affecting internalization itself, while pretreatment with pertussis toxin had no effect on U50, 488H-induced internalization. In contrast, incubation with sucrose (0.4-0.8 M) significantly reduced U50,488H-induced internalization of the kappa receptor. While co-expression of the wild type GRK2, beta-arrestin, or dynamin I had no effect on kappa receptor internalization, co-expression of the dominant negative mutants GRK2-K220R, beta-arrestin (319-418), or dynamin I-K44A significantly inhibited receptor internalization. Whether receptor internalization is critical for MAP kinase activation was next investigated. Co-expression of dominant negative mutants of beta-arrestin or dynamin I, which greatly reduced U50,488H-induced internalization, did not affect MAP kinase activation by the agonist. In addition, etorphine, which did not promote human kappa receptor internalization, was able to fully activate MAP kinase. Moreover, U50,488H or etorphine stimulation of the rat kappa receptor, which did not undergo internalization, also effectively activated MAP kinase. Thus, U50,488H-induced internalization of the human kappa opioid receptor in Chinese hamster ovary cells occurs via a GRK-, beta-arrestin-, and dynamin I-dependent process that likely involves clathrin-coated pits. In addition, internalization of the kappa receptor is not required for activation of MAP kinase.
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PMID:U50,488H-induced internalization of the human kappa opioid receptor involves a beta-arrestin- and dynamin-dependent mechanism. Kappa receptor internalization is not required for mitogen-activated protein kinase activation. 1020 34

We previously reported that both carbachol and epidermal growth factor (EGF) are potent inducers of the extracellular signal-regulated protein kinases (ERKs) in isolated gastric canine parietal cells and that induction of these kinases leads to acute inhibitory and chronic stimulatory effects on gastric acid secretion. In this study we investigated the molecular mechanisms responsible for these effects. Both carbachol (100 microM) and EGF (10 nM) induced Ras activation. The role of Ras in ERK2 induction was examined by transfecting parietal cells with a vector expressing hemoagglutinin (HA)-tagged ERK2 (HA-ERK2) together with a dominantly expressed mutant (inactive) ras gene. HA-ERK2 activity was quantitated by in-gel kinase assays. Dominant negative Ras reduced carbachol induction of HA-ERK2 activity by 60% and completely inhibited the stimulatory effect of EGF. Since Ras activation requires the assembly of a multiprotein complex, we examined the effect of carbachol and EGF on tyrosyl phosphorylation of Shc and its association with Grb2 and the guanine nucleotide exchange factor Sos. Western blot analysis of anti-Shc immunoprecipitates with an anti-phosphotyrosine antibody demonstrated that both carbachol and EGF induced tyrosyl phosphorylation of a major 52-kDa shc isoform. Grb2 association with Shc was demonstrated by blotting Grb2 immunoprecipitates with an anti-Shc antibody. Probing of anti-Sos immunoprecipitates with an anti-Grb2 antibody revealed that Sos was constitutively bound to Grb2. To examine the functional role of Sos in ERK2 activation, we transfected parietal cells with the HA-ERK2 vector together with a dominantly expressed mutant (inactive) sos gene. Dominant negative Sos did not affect carbachol stimulation of HA-ERK2 but inhibited the stimulatory effect of EGF by 60%. We then investigated the role of betagamma-subunits in carbachol induction of HA-ERK2. Parietal cells were transfected with the HA-ERK2 vector together with a vector expressing the carboxy terminus of the beta-adrenergic receptor kinase 1, known to block signaling mediated by betagamma-subunits. In the presence of this vector, carbachol induction of HA-ERK2 was inhibited by 40%. Together these data suggest that, in the gastric parietal cells, carbachol activates the ERKs through Ras- and betagamma-dependent mechanisms that require guanine nucleotide exchange factors other than Sos.
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PMID:Carbachol activates ERK2 in isolated gastric parietal cells via multiple signaling pathways. 1036 52

GRK2 is a member of the G protein-coupled receptor kinase (GRK) family, which phosphorylates the activated form of a variety of G protein-coupled receptors (GPCR) and plays an important role in GPCR modulation. It has been recently reported that stimulation of the mitogen-activated protein kinase cascade by GPCRs involves tyrosine phosphorylation of docking proteins mediated by members of the Src tyrosine kinase family. In this report, we have investigated the possible role of c-Src in modulating GRK2 function. We demonstrate that c-Src can directly phosphorylate GRK2 on tyrosine residues, as shown by in vitro experiments with purified proteins. The phosphorylation reaction exhibits an apparent K(m) for GRK2 of 12 nM, thus suggesting a physiological relevance in living cells. Consistently, overexpression of the constitutively active c-Src Y527F mutant in COS-7 cells leads to tyrosine phosphorylation of co-expressed GRK2. In addition, GRK2 can be detected in phosphotyrosine immunoprecipitates from HEK-293 cells transiently transfected with this Src mutant. Interestingly, phosphotyrosine immunoblots reveal a rapid and transient increase in GRK2 phosphorylation upon agonist stimulation of beta(2)-adrenergic receptors co-transfected with GRK2 and wild type c-Src in COS-7 cells. This tyrosine phosphorylation is maximal within 5 min of isoproterenol stimulation and reaches values of approximately 5-fold over basal conditions. Furthermore, GRK2 phosphorylation on tyrosine residues promotes an increased kinase activity toward its substrates. Our results suggest that GRK2 phosphorylation by c-Src is inherent to GPCR activation and put forward a new mechanism for the regulation of GPCR signaling.
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PMID:Agonist-dependent phosphorylation of the G protein-coupled receptor kinase 2 (GRK2) by Src tyrosine kinase. 1056 20


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