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)

Stimulation of hemopoietic cells with IL-3, IL-4, IL-5, granulocyte-macrophage-CSF and Steel factor-(SLF) induced tyrosine phosphorylation of a number of protein substrates. Two of these proteins, designated p42 and p44, were tyrosine phosphorylated rapidly in response to treatment with IL-3, IL-5, granulocyte-macrophage-CSF and SLF, but not IL-4. We demonstrate that these common substrates are members of the mitogen-activated protein kinase (MAP kinase) family of protein serine/threonine kinases. Ion-exchange chromatography yielded a peak of MAP kinase activity eluting at 0.3 to 0.32 M NaCl. Immunoblotting of column fractions with antiphosphotyrosine antibodies showed coelution of the peak of MAP kinase enzyme activity with the p42 and p44 tyrosine phosphorylated species, and with two proteins of 42 and 44 kDa which were immunoreactive with anti-MAP kinase antibodies. Moreover, a characteristic shift in mobility of the p42 and p44 species was observed after factor treatment. Time-course analyses and subsequent ion-exchange chromatography demonstrated SLF activation of MAP kinase activity was maximal after 2 min of factor treatment and decreased to basal levels after 30 min stimulation. By contrast, activation of MAP kinase after IL-5 treatment was not as rapid. Maximal activity was observed 15 min after stimulation and remained elevated for up to 60 min after IL-5 addition. Investigation of the role of protein kinase C in the mechanism of activation by these growth factors demonstrated that specific inhibition of protein kinase C led to a reduction, but not ablation, of the SLF and IL-3 induced stimulation of MAP kinase activity. The use of synthetic peptide substrates confirmed SLF and IL-5 activate isoforms of MAP kinases. These results demonstrate that members of the MAP kinase family are involved in common signal transduction events elicited by IL-3, IL-5, granulocyte-macrophage-CSF and Steel factor, but not those involving IL-4.
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PMID:Multiple hemopoietic growth factors stimulate activation of mitogen-activated protein kinase family members. 138 May 36

Apoptosis of haemopoietic cells can be inhibited by various cytokines, but the specific signalling pathways involved are not well defined. Interleukin (IL)-4 has unique actions since it is unable to activate p21ras or mitogen-activated protein kinase, but can activate PtdIns 3-OH-kinase (PtdIns 3-kinase), the latter effect being shared with most other cytokines. In many cases, IL-4 is able to maintain cell viability by inhibiting apoptosis, but is unable to stimulate continuous proliferation of cells. This led us to investigate the role of PtdIns 3-kinase in inhibition of apoptosis. Two potent inhibitors of PtdIns 3-kinase, wortmannin and LY294002, rapidly induced apoptosis in cells incubated in the presence of IL-4, at concentrations consistent with their ability to inhibit PtdIns 3-kinase activity in whole cells. Interestingly, the same effect was seen in cells in the presence of IL-3 and Steel factor, while cells incubated in the presence of granulocyte-macrophage colony stimulating factor, and to a lesser extent, IL-5 could bypass the effect of wortmannin or LY294002. Therefore, this study suggests that PtdIns 3-kinase activity provides an important, although not a unique signal, required to inhibit apoptosis in haemopoietic cells.
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PMID:Role of phosphatidylinositol 3-OH-kinase activity in the inhibition of apoptosis in haemopoietic cells: phosphatidylinositol 3-OH-kinase inhibitors reveal a difference in signalling between interleukin-3 and granulocyte-macrophage colony stimulating factor. 749 6

The activation of eosinophils by IL-5 plays a crucial role in the pathogenesis of allergic and parasitic disorders. IL-5 has recently been shown to activate Lyn and Jak2 tyrosine kinases, MAP kinases, and STAT1 nuclear factor. We have previously reported that TGF-beta blocks the IL-5-induced activation of eosinophils. In this study, we investigated the effect of TGF-beta on the IL-5-induced signaling molecules in eosinophils. Purified eosinophils from mildly allergic patients were preincubated with TGF-beta and then stimulated with IL-5. The cell lysates were then immunoprecipitated and blotted with antiphosphotyrosine Abs. The activity of the kinases was further studied in the immune-complex kinase assay. We found that TGF-beta inhibited the tyrosine phosphorylation of multiple proteins in eosinophils. The identity of some of the proteins was established by immunoprecipitation. We found that TGF-beta inhibited tyrosine phosphorylation of Lyn, Jak2, and a 44-kDa MAP kinase. In further experiments, it blocked the activation of the above kinases as determined by immune-complex kinase assay. TGF-beta also inhibited phosphorylation of the STAT1 (p91) nuclear protein in eosinophils. We believe that the inhibition of Lyn, Jak2, MAP kinase, and the STAT1 nuclear protein may underlie the inhibitory activity of TGF-beta on eosinophils.
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PMID:Mechanism of inhibition of eosinophil activation by transforming growth factor-beta. Inhibition of Lyn, MAP, Jak2 kinases and STAT1 nuclear factor. 759 7

We have shown that the interaction of interleukin (IL)-5 with the receptor activates Lyn tyrosine kinase within 1 min and Jak2 tyrosine kinase within 1-3 min. IL-5 also stimulates GTP binding to p21ras. The signal is subsequently propagated through the activation of Raf-1, MEK, and MAP kinases as shown by their increased autophosphorylation in vitro and phosphorylation in situ. Jak2 kinase has been shown to phosphorylate STAT nuclear proteins. The activation of STAT nuclear factors was studied by electrophoretic mobility shift assay using a gamma activation site (GAS) probe. We found that IL-5 induces two GAS-binding proteins in eosinophils, one of which is STAT1. We conclude that IL-5 induced signals are propagated through two distinct pathways: (1) Lyn-->Ras-->Raf-1-->MEK-->MAP kinase and (2) Jak2-->STAT1.
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PMID:The interleukin-5/receptor interaction activates Lyn and Jak2 tyrosine kinases and propagates signals via the Ras-Raf-1-MAP kinase and the Jak-STAT pathways in eosinophils. 761 38

The high affinity receptor for GM-CSF consists of a unique alpha subunit and a beta subunit that is shared with receptors for IL-3 and IL-5. Activation of GM-CSF receptor (GMR) triggers two distinct cytoplasmic signalling pathways, JAK2 and Ras, and is sufficient to maintain proliferation of growth factor-dependent cell lines. Shc proteins are phosphorylated upon activation of GMR and may be involved in the transmission of GM-CSF signals to Ras. To define the role of Shc proteins in cells stimulated with GM-CSF, we investigated both the network of interactions that involve Shc after GM-CSF stimulation and the effects of overexpressing Shc proteins on the proliferative response to GM-CSF. Two cytoplasmic complexes, Grb2/Sos and Grb2/p140 bind through the Grb2 SH2 domain to phosphorylated Shc, and are thereby recruited to the beta subunit. Both complexes are stable, even in the absence of ligand, and depend on the direct association of p140 and Sos respectively with the SH3 domains of Grb2. p140 is an uncharacterized protein constitutively phosphorylated on tyrosine and, in its Grb2-bound form, expressed only in hematopoietic cells, the oligomeric complex formed by phosphorylated beta subunit-phosphorylated Shc-Grb2-SoS-p140 is also induced by IL-3 and L-5 stimulation of growth-factor dependent cell lines. Overexpression of wild-type Shc proteins in growth factor-dependent cells increases both MAP kinase activation and proliferation in response to GM-CSF. These effects require the association of Shc with Grb2. Taken together these results indicate that phosphorylation of Shc proteins is a crucial step in the transmission of GM-CSF proliferative stimuli, since it creates a high affinity binding site for the Grb2/SoS complex, whose function is to activate Ras and, for the Grb2/p140 complex, whose function remains unknown.
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PMID:Overexpression of Shc proteins potentiates the proliferative response to the granulocyte-macrophage colony-stimulating factor and recruitment of Grb2/SoS and Grb2/p140 complexes to the beta receptor subunit. 789 32

The high-affinity receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF) consists of a unique alpha chain and a beta c subunit that is shared with the receptors for interleukin-3 (IL-3) and IL-5. Two regions of the beta c chain have been defined; these include a membrane-proximal region of the cytoplasmic domain that is required for mitogenesis and a membrane-distal region that is required for activation of Ras, Raf-1, mitogen-activated protein kinase, and S6 kinase. Recent studies have implicated the cytoplasmic protein tyrosine kinase JAK2 in signalling through a number of the cytokine receptors, including the IL-3 and erythropoietin receptors. In the studies described here, we demonstrate that GM-CSF stimulation of cells induces the tyrosine phosphorylation of JAK2 and activates its in vitro kinase activity. Mutational analysis of the beta c chain demonstrates that only the membrane-proximal 62 amino acids of the cytosolic domain are required for JAK2 activation. Thus, JAK2 activation is correlated with induction of mitogenesis but does not, alone, activate the Ras pathway. Carboxyl truncations of the alpha chain, which inactivate the receptor for mitogenesis, are unable to mediate GM-CSF-induced JAK2 activation. Using baculovirus-expressed proteins, we further demonstrate that JAK2 physically associates with the beta c chain but not with the alpha chain. Together, the results further support the hypothesis that the JAK family of kinase are critical to coupling cytokine binding to tyrosine phosphorylation and ultimately mitogenesis.
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PMID:JAK2 associates with the beta c chain of the receptor for granulocyte-macrophage colony-stimulating factor, and its activation requires the membrane-proximal region. 800 42

The high-affinity receptors for granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 3 (IL-3) and IL-5 consist of two subunits, alpha and beta. The alpha subunits are specific to each cytokine and the same beta subunit (beta c) is shared by these three receptors. Although none of these receptor subunits has intrinsic kinase activity, these cytokines induce protein tyrosine phosphorylation, activation of Ras, Raf-1 and MAP kinase, and transcriptional activation of nuclear proto-oncogenes such as c-myc, c-fos and c-jun. In this paper, we describe a detailed analysis of the signaling potential of the beta c subunit by using a series of cytoplasmic deletion mutants. The human beta c consists of 881 amino acid residues. A C-terminal deletion mutant of beta c at amino acid 763 (beta 763) induced phosphorylation of Shc and activation of Ras, Raf-1, MAP kinase and p70 S6 kinase, whereas a deletion at amino acid 626 (beta 626) induced none of these effects. The beta 763 mutant, as well as the full-length beta c, induced transcription of c-myc, c-fos and c-jun. Deletions at amino acid 517 (beta 517) and 626 (beta 626) induced c-myc and pim-1, but no induction of c-fos and c-jun was observed. GM-CSF increased phosphatidylinositol 3 kinase (PI3-K) activity in anti-phosphotyrosine immunoprecipitates from cells expressing beta 763 as well as beta c, whereas it was only marginally increased from cells expressing beta 517 or beta 626. Thus, there are at least two distinct regions within the cytoplasmic domain of beta c that are responsible for different signals, i.e. a membrane proximal region of approximately 60 amino acid residues upstream of Glu517 is essential for induction of c-myc and pim-1, and a distal region of approximately 140 amino acid residues (between Leu626 and Ser763) is required for activation of Ras, Raf-1, MAP kinase and p70 S6 kinase, as well as induction of c-fos and c-jun.
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PMID:Signal transduction by the high-affinity GM-CSF receptor: two distinct cytoplasmic regions of the common beta subunit responsible for different signaling. 822 33

IL-5 is a member of the hemopoietic cytokine family and has profound effects on the differentiation, survival, migration, and effector function of human eosinophils. Increased tyrosine phosphorylation has been observed as an early event in IL-5 signal transduction in eosinophils; most notably, proteins of 45 and 135 kDa became tyrosine phosphorylated following IL-5 treatment. Some of these phosphotyrosine-containing proteins may represent intermediates in IL-5 signal transduction pathways. This study demonstrates that Jak-2, a tyrosine kinase, is increasingly tyrosine phosphorylated after IL-5 treatment of human eosinophils. Furthermore, we found proteins of 42, 44, and 45 kDa immunoreactive with anti-mitogen-activated protein (MAP) kinase Abs that are expressed in human eosinophils. One of these, the protein of approximately 45 kDa (p45), was tyrosine phosphorylated following treatment of eosinophils with IL-5 and PMA, as seen by anti-phosphotyrosine immunoprecipitation and immunoblotting with anti-MAP kinase Abs. In addition, anti-phosphotyrosine immunoprecipitates of IL-5-treated eosinophils contained enhanced phosphotransferase activity toward a myelin basic protein (MBP) peptide substrate when compared with control-treated eosinophils. In contrast to cytokine-stimulated MAP kinase activation in other cells, there is no evidence of tyrosine phosphorylation or enzymatic activation of p42 MAP kinase in eosinophils after IL-5 treatment. These data suggest that Jak-2 kinase and an activated isoform of MAP kinase, p45, are detected following incubation with IL-5, and may mediate some of this cytokine's effects on eosinophils in a manner unique to the activation pathways previously described for other cells.
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PMID:IL-5 activates a 45-kilodalton mitogen-activated protein (MAP) kinase and Jak-2 tyrosine kinase in human eosinophils. 854 24

Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates the growth and function of several myeloid cell types at different stages of maturation. The effects of GM-CSF are mediated through a high affinity receptor that is composed of two chains: a unique, ligand-specific alpha chain and a beta common chain (beta c) that is also a component of the receptors for interleukin 3 (IL-3) and IL-5. Beta c plays an essential role in the transduction of extra cellular signals to the nucleus through its recruitment of secondary messengers. Several downstream signaling events induced by GM-CSF stimulation have been described, including activation of tyrosine kinases and tyrosine phosphorylation of cellular proteins (including beta c) and activation of the Ras/mitogen-activated protein kinase and the JAK/STAT pathways. A region within the beta c cytoplasmic tail (amino acids 517-763) has been reported to be necessary for tyrosine phosphorylation of the adapter protein, Shc, and for the subsequent GM-CSF-induced activation of Ras. In this paper, we describe a physical association between the tyrosine phosphorylated GM-CSF receptor (GMR)-beta c chain and Shc in vivo. Using a series of cytoplasmic truncation mutants of beta c and various mutant Shc proteins, we demonstrate that the N-terminal phosphotyrosine-binding (PTB) domain of Shc binds to a short region of beta c (amino acids 549-656) that contains Tyr577. Addition of a specific phosphopeptide encoding amino acids surrounding this tyrosine inhibited the interaction between beta c and shc. Moreover, mutation of a key residue within the phosphotyrosine binding pocket of the Shc-PTB domain abrogated its association with beta c. These observations provide an explanation for the previously described requirement for Tyr577 of beta c for GM-CSF-induced tyrosine phosphorylation of Shc and have implications for Ras activation through the GM-CSF, IL-3, and IL-5 receptors.
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PMID:Evidence for a physical association between the Shc-PTB domain and the beta c chain of the granulocyte-macrophage colony-stimulating factor receptor. 864 4

Interleukin (IL) 5 specifically induces the differentiation of eosinophils which are central to the pathogenesis of allergies and asthma. Structurally, IL-5 is a unique member of the short-chain helical bundle subfamily of cytokines. In contrast to other subfamily members which fold unimolecularly into a single helical bundle, IL-5 forms a pair of helical bundles by the interdigitation of two identical monomers covalently linked by a pair of intermolecular disulfide bonds. Although a native IL-5 monomer lacks bioactivity, we recently reported the engineering of an insertional mutant of IL-5 (designated mono5) which folds unimolecularly into a single helical bundle and has biological activity similar to that of native IL-5. Here we demonstrate no differences in signal transduction pathways utilized by mono5 and IL-5, as determined by western blot analysis of early tyrosine phosphorylation events, Jak2 activation, and mitogen-activated protein kinase activation. However, binding studies utilizing conformationally dependent neutralizing anti-IL-5 monoclonal antibodies localized a tertiary structural perturbation near the insert of mono5. This perturbation enabled localization of a limited region of the tertiary structure of IL-5 that engages the IL-5 receptor alpha-chain. Fluorescent labeling studies further revealed that the cysteines of mono5 contained free sulfhydryl groups, thereby demonstrating that the role of the disulfide bonds of IL-5 is the structural maintenance of other functional domains. The retention of conformation epitopes by mono5, but not IL-5, under reducing conditions and the equivalent thermostability of mono5 and IL-5 despite the absence of a disulfide bond in mono5 indicated that the conformation assumed by mono5 is very stable. In addition to providing the structural framework for designing novel IL-5 agonists and antagonists, the knowledge gained from the development of mono5 will enable other helical bundle proteins to be redesigned with therapeutic potential.
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PMID:Engineering of a functional interleukin-5 monomer: a paradigm for redesigning helical bundle cytokines with therapeutic potential in allergy and asthma. 889 59


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