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)

The growth-promoting effects of endothelin-1 (ET-1) were examined in adult heart cells. The activity of mitogen-activated protein kinases (MAPKs) was measured in cytosolic extracts of isolated adult feline cardiac myocytes incubated with and without ET-1. Kinase activity was assessed by phosphorylation of the exogenous substrate, myelin basic protein. ET-1 stimulated the activity of MAPK up to 4-fold, with peak activation occurring between five and ten minutes after addition of ET-1. Polyclonal antisera raised against a 14-amino acid sequence of the erk-2 gene product, a MAPK isoform, identified two major bands in cytosolic extracts of the cardiac myocytes. Partial purification of kinase activities using Mono Q anion-exchange chromatography demonstrated two major peaks of myelin basic protein kinase activity. Subsequent immunoblots of the eluted fractions demonstrated that the immunoreactive bands observed in the cytosolic extracts eluted in those fractions possessing kinase activity. Overnight pretreatment of the cardiac myocytes with 100 ng/ml pertussis toxin inhibited the ET-1 stimulated increase in MAPK activity by 50 - 70%, but did not alter stimulation by 100 nM phorbol 12-myristate 13-acetate (PMA). These data suggest that stimulation of MAPK by ET-1 may be mediated by more than one pathway. MAPK has been shown to be activated in the intracellular transmission of growth factor signals. Indicative of a growth effect in this adult heart cell model, myocytes exposed to increasing concentrations of ET-1 demonstrated a dose dependent increase in [3H]-phenylalanine incorporation into cellular protein. This response was blocked by staurosporine and partially inhibited by pretreatment with pertussis toxin, again suggesting the possible involvement of multiple early signals. These data from isolated adult cardiac myocytes further support the hypothesis that ET-1 has a role in the regulation of cardiac growth.
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PMID:Effects of endothelin on mitogen-activated protein kinase activity and protein synthesis in isolated adult feline cardiac myocytes. 863 15

We recently showed that EGF and anisomycin activate two kinases, p45 and p55, whose distinguishing feature is that their detection in in-gel kinase assays is enhanced by copolymerised poly-Glu/Tyr or poly-Glu/Phe (Cano E, Hazzalin CA and Mahadevan LC, Mol. Cell. Biol., 20:117-121). Their activation characteristics and sizes are strikingly similar to those of JNK/SAPKs, which are also strongly activated by anisomycin. However, we show here that p45 and p55 are not JNK/SAPKs but murine forms of MAPKAP kinase-2 because: (i) Detection of immunoprecipitated JNK/SAPKs is completely dependent on the presence of c-Jun as substrate in the in-gel kinase assays, whereas detection of p45 and p55 is not. (ii) Detection of p45 and p55 activity is enhanced by the presence of poly-Glu/Tyr or poly-Glu/Phe, whereas JNK/SAPKs are not detectable under these conditions. (iii) Although the sizes of the murine JNK/SAPKs and MAPKAP K-2 are similar, human JNK/SAPKs migrate at 45 and 55 kDa whereas human MAPKAP K-2 migrates at 50 kDa; the poly-Glu/Tyr-enhanced activity in human cells migrates at 50 KDa. (iv) Purified rabbit muscle MAPKAP K-2 is detectable as two bands of activity on in-gel kinase assays and their detection is enhanced by poly-Glu/Tyr. (v) Finally, the anisomycin-activated poly-Glu/Tyr-enhanced p45 and p55 kinases can be immunoprecipitated from murine cells using an anti-MAPKAP K-2 antibody. Thus, EGF- and anisomycin-activated p45 and p55 are not JNK/SAPKs but MAPKAP K-2, implying that both these agents activate the p38/RK MAP kinase cascade.
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PMID:Identification of anisomycin-activated kinases p45 and p55 in murine cells as MAPKAP kinase-2. 863 2

We have previously shown that extracellular ATP, like norepinephrine (NE) and many other hypertrophy-inducing agents, increases expression of the immediate-early genes c-fos and junB in cultured neonatal cardiac myocytes but that the intracellular signaling pathways activated by ATP and responsible for these changes differ from those stimulated by NE. Furthermore, whereas NE increases incorporation of [14C]phenylalanine (14C-Phe) and cell size in neonatal cardiomyocytes, ATP does not. Since ATP is coreleased with NE from sympathetic nerve endings in the heart, we investigated whether ATP could modulate cardiac hypertrophy induced by adrenergic agonists, such as NE. We report in the present study that extracellular ATP inhibited the increase in incorporation of 14C-Phe into cellular protein and the increase in cell size in neonatal rat cardiac myocytes that was induced by NE, phenylephrine (PE), basic fibroblast growth factor, or endothelin-1. This inhibition was dose dependent, occurred predominantly through P2 purinergic receptors, and was observed even when cells were treated with ATP for as little as 1 hour before the addition of the hypertrophy-inducing agent. ATP also selectively affected changes in gene expression associated with hypertrophy. It prevented PE-stimulated increases in atrial natriuretic factor and myosin light chain-2 mRNA levels, while appearing to augment basal and PE-stimulated skeletal alpha-actin mRNA levels. ATP alone increased sarcoplasmic reticulum Ca2+-ATPase mRNA levels but had no effect when added with PE. ATP did not significantly affect the level of the constitutively expressed mRNA for GAPDH. Neither the PE-stimulated increase in immediate-early gene expression nor the initial induction of mitogen-activated protein kinase activity by PE was inhibited by ATP. These results demonstrate that extracellular ATP can inhibit hypertrophic growth of neonatal cardiac myocytes and differentially alter the changes in gene expression that accompany hypertrophy.
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PMID:Extracellular ATP inhibits adrenergic agonist-induced hypertrophy of neonatal cardiac myocytes. 863 9

We employed neutrophils and enucleate neutrophil cytoplasts to study the activation of the mitogen-activated protein kinases (MAPKs) p44erk1 and p42erk2 in neutrophils by inflammatory agonists that engage G protein-linked receptors. Formyl-methionyl-leucyl-phenylalanine (FMLP) rapidly and transiently activated MAPK in neutrophils and cytoplasts, consistent with a role in signaling for neutrophil functions. FMLP stimulated p2lras activation in neutrophils and Raf-1 translocation from cytosol to plasma membrane in cytoplasts, with kinetics consistent with events upstream of MAPK activation. Insulin, a protein tyrosine kinase receptor (PTKR) agonist, stimulated neutrophil MAPK activation, demonstrating an intact system of PTKR signaling in these post-mitotic cells. FMLP- and insulin-stimulated MAPK activation in cytoplasts were inhibited by Bt2cAMP, consistent with signaling through Raf-1 and suggesting a mechanism for cAMP inhibition of neutrophil function. However, Bt2cAMP had no effect on FMLP-stimulated MAPK activation in neutrophils. The extent of MAPK activation by various chemoattractants correlated with their capacity to stimulate neutrophil and cytoplast homotypic aggregation. Consistent with its effects on MAPK, Bt2cAMP inhibited FMLP-stimulated aggregation in cytoplasts but not neutrophils. Insulin had no independent effect but primed neutrophils for aggregation in response to FMLP. Our studies support a p2lras-, Raf-1-dependent pathway for MAPK activation in neutrophils and suggest that neutrophil adhesion may be regulated, in part, by MAPK.
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PMID:Mitogen-activated protein kinase in neutrophils and enucleate neutrophil cytoplasts: evidence for regulation of cell-cell adhesion. 866 46

The activation of the serine/threonine kinase, Raf-1, serves to connect upstream protein tyrosine kinases to downstream signaling events. We previously reported that FcgammaRI stimulation of interferon gamma-differentiated U937 cells (termed U937IF cells) induces a mobility shift in Erk2. Herein, we report that cross-linking of FcgammaRI receptor in U937IF cells induces a marked tyrosine phosphorylation of Raf-1 (10-fold increase). Tyrosine phosphorylation of Raf-1 is induced by FcgammaRI activation and not by PMA (1 microg/ml), N-formyl-Met-Leu-Phe (1 microM), calcium ionophore (1 microM), thrombin (0.05 unit/ml), FcgammaRII, or FcgammaRIII stimulation. The kinetics of Raf-1 tyrosine phosphorylation is rapid, reaching peak levels 1-2 min after FcgammaRI activation, and the tyrosine phosphorylation of Raf-1 precedes the activation of the respiratory burst. FcgammaRI cross-linking induces the tyrosine phosphorylation of Shc; tyrosine-phosphorylated Shc binds to Grb2 forming a Shc-Grb2 complex. The data provide evidence that the FcgammaRI receptor signals via the upstream activation of nonreceptor protein tyrosine kinases, which leads to the subsequent activation of Ras family GTPases and serine/threonine kinases, Raf-1 and mitogen-activated protein kinase.
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PMID:A role for Shc, Grb2, and Raf-1 in FcgammaRI signal relay. 866 46

The chemotactic peptide f-Met-Leu-Phe (fMLP) stimulates leukocyte functions through binding and activation of a specific G-protein-coupled formyl peptide receptor (FPR). Recent studies have shown that stimulation of neutrophils with fMLP induces the activation of two members of the mitogen-activated protein kinase (MAP kinase) family, ERK1 and ERK2, through mechanisms that are not completely understood but may involve the phosphorylation of the adapter protein SHC by the Src-related kinase Lyn. In this study, transfected fibroblasts expressing the rabbit FPR were used to investigate further the role of Lyn and SHC phosphorylation in fMLP-stimulated MAP kinase activation. Stimulation of transfected cells with fMLP resulted in the time- and dose-dependent increase in tyrosine phosphorylation and activation of ERK1 and ERK2 and the activation of MEK, the MAP kinase/ERK kinase. The activation of both ERKs and MEK was inhibited by preincubation of the cells with pertussis toxin, indicating that activation was dependent upon a Gi/Go-like protein that couples to the receptor. Our data also show that, unlike neutrophils, FPR-transfected fibroblasts do not express the Src-related kinase Lyn. In the absence of Lyn, fMLP stimulation did not result in an increased tyrosine phosphorylation of the adapter protein SHC, whereas it was still able to induce MAP kinase activation. These data suggest that Lyn and SHC are not the only upstream signals for activation of the MAP kinase/ERK pathway by fMLP and demonstrate the potential application of the FPR-transfected cells for the delineation of additional signaling mechanisms stimulated by fMLP.
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PMID:Activation of the mitogen-activated protein kinase pathway by fMet-leu-Phe in the absence of Lyn and tyrosine phosphorylation of SHC in transfected cells. 866 60

The insulin receptor substrate-1 (IRS-1) is the major intracellular substrate of insulin and insulin-like growth factor-I (IGF-I) receptor tyrosine kinase activity, and this protein has been found to be overexpressed in human hepatocellular carcinomas. IRS-1 contains several src homology 2 (SH2) binding motifs that interact following tyrosyl phosphorylation with SH2-containing proteins, and this interaction may be essential for transmitting the growth signal from the cell surface to the nucleus. We have previously reported that overexpression of IRS-1 may induce neoplastic transformation of NIH 3T3 cells. This study examines the role of two SH2-containing molecules, namely the Grb2 adapter and Syp tyrosine phosphatase proteins as important components of the cellular transforming activity of IRS-1. Mutations of tyrosine 897 in the YVNI motif (Y897F) and of tyrosine 1180 in the YIDL motif (Y1180F) reduced the intracellular interaction of IRS-1 with Grb2 and Syp proteins, respectively. Furthermore, a single mutation at either Phe-897 or Phe-1180 substantially but not completely reduced IGF-I-dependent transforming activity of IRS-1, whereas creation of a double mutation of both tyrosine residues (Y897F/Y1180F) strikingly attenuated the transforming activity of IRS-1. Stable expression of the IRS-1 mutant constructs in NIH 3T3 cells was associated with a lower level of activation of the mitogen-activated protein kinase kinase (MAPKK)/MAPK cascade following IGF-I stimulation compared with cells stably transfected with the "wild-type" IRS-1 gene. These results suggest that IRS-1-induced cellular transformation requires an interaction with both Grb2 and Syp signal transduction molecules since neither interaction alone appears to be required, and this event subsequently leads to activation of the MAPKK/MAPK cascade.
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PMID:Neoplastic transformation induced by insulin receptor substrate-1 overexpression requires an interaction with both Grb2 and Syp signaling molecules. 866 27

Exposure of neutrophils to a variety of agonists including chemoattractant peptides and cytokines induces degranulation and activation of the oxidative burst which are required for bacterial killing. The signaling pathways regulating these important functions are incompletely characterized. Mitogen-activated protein (MAP) kinases, which include the extracellular signal-regulated kinases (ERKs), are activated rapidly in neutrophils, suggesting that they may regulate cell activation. We found that neutrophils express two isoforms of MAP/ERK kinase (MEK), mixed-function kinases that are responsible for phosphorylation and activation of ERK. Like MEK-1, MEK-2 was found to reside in the cytosol both before and after stimulation. Studies were undertaken to define the relative abundance and functional contribution of MEK-1 and MEK-2 in neutrophils and to characterize the signaling pathways leading to their activation. Although the abundance of the two isoforms was similar, the activity of MEK-2 was at least 3-fold greater than that of MEK-1. A rise in cytosolic [Ca2+] was insufficient for MEK stimulation, and blunting the [Ca2+] change with intracellular chelators failed to prevent receptor-mediated activation of either isoform, implying that cytosolic Ca2+ transients are not necessary. In contrast, both MEK-1 and MEK-2 were activated by exposure of cells to protein kinase C (PKC) agonists. Conversely, PKC antagonists inhibited the chemotactic stimulation of both isoforms, suggesting that PKC was required for their activation. Despite these similarities, clear differences were also found in the pathways leading to activation of the MEK isoforms. In particular, MEK-2 was considerably more sensitive than MEK-1 to the phosphatidylinositol 3-kinase inhibitor wortmannin. Phosphorylation and activation of ERK-1 and ERK-2 were also reduced by this inhibitor. In summary, MEK-2 is stimulated in formyl-methionyl-leucyl-phenylalanine-treated neutrophils, where it appears to be functionally the predominant isoform. The time course and inhibitor sensitivity of MEK-2 activation parallel those of several components of the microbicidal response, suggesting a signaling role of the MEK-ERK pathway.
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PMID:Chemotactic peptide-induced activation of MEK-2, the predominant isoform in human neutrophils. Inhibition by wortmannin. 870 63

The features of three distinct protein phosphorylation cascades in mammalian cells are becoming clear. These signalling pathways link receptor-mediated events at the cell surface or intracellular perturbations such as DNA damage to changes in cytoskeletal structure, vesicle transport and altered transcription factor activity. The best known pathway, the Ras-->Raf-->MEK-->ERK cascade [where ERK is extracellular-signal-regulated kinase and MEK is mitogen-activated protein (MAP) kinase/ERK kinase], is typically stimulated strongly by mitogens and growth factors. The other two pathways, stimulated primarily by assorted cytokines, hormones and various forms of stress, predominantly utilize p21 proteins of the Rho family (Rho, Rac and CDC42), although Ras can also participate. Diagnostic of each pathway is the MAP kinase component, which is phosphorylated by a unique dual-specificity kinase on both tyrosine and threonine in one of three motifs (Thr-Glu-Tyr, Thr-Phe-Tyr or Thr-Gly-Tyr), depending upon the pathway. In addition to activating one or more protein phosphorylation cascades, the initiating stimulus may also mobilize a variety of other signalling molecules (e.g. protein kinase C isoforms, phospholipid kinases, G-protein alpha and beta gamma subunits, phospholipases, intracellular Ca2+). These various signals impact to a greater or lesser extent on multiple downstream effectors. Important concepts are that signal transmission often entails the targeted relocation of specific proteins in the cell, and the reversible formation of protein complexes by means of regulated protein phosphorylation. The signalling circuits may be completed by the phosphorylation of upstream effectors by downstream kinases, resulting in a modulation of the signal. Signalling is terminated and the components returned to the ground state largely by dephosphorylation. There is an indeterminant amount of cross-talk among the pathways, and many of the proteins in the pathways belong to families of closely related proteins. The potential for more than one signal to be conveyed down a pathway simultaneously (multiplex signalling) is discussed. The net effect of a given stimulus on the cell is the result of a complex intracellular integration of the intensity and duration of activation of the individual pathways. The specific outcome depends on the particular signalling molecules expressed by the target cells and on the dynamic balance among the pathways.
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PMID:Signal-transducing protein phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signalling. 883 13

Insulin receptor substrate-1 (IRS-1) is rapidly phosphorylated on multiple tyrosine residues in response to insulin and binds several Src homology 2 domain-containing proteins, thereby initiating downstream signaling. To assess the tyrosine phosphorylation sites that mediate relevant downstream signaling and biological effects, we created site-directed mutants of IRS-1 and overexpressed them in the Xenopus laevis oocyte. In oocytes overexpressing IRS-1 or IRS-1-895F (Tyr-895 replaced with phenylalanine), insulin activated phosphatidylinositol (PI) 3-kinase, p70 S6 kinase, and mitogen-activated protein kinase and induced oocyte maturation. In contrast, in oocytes overexpressing IRS-1-4F (Tyr-460, Tyr-608, Tyr-939, and Tyr-987 of IRS-1 replaced with phenylalanine), insulin did not activate PI 3-kinase, p70 S6 kinase, and mitogen-activated protein kinase and failed to induce oocyte maturation. These observations indicate that in X. laevis oocytes overexpressing IRS-1, the association of PI 3-kinase rather than Grb2 (growth factor-bound protein 2) with IRS-1 plays a major role in insulin-induced oocyte maturation. Activation of PI 3-kinase may lie upstream of mitogen-activated protein kinase activation and p70 S6 kinase activation in response to insulin.
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PMID:Mutant of insulin receptor substrate-1 incapable of activating phosphatidylinositol 3-kinase did not mediate insulin-stimulated maturation of Xenopus laevis oocytes. 891 May 2


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