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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Raf-1 is a 74-kD serine/threonine kinase located in the cell cytoplasm that is activated by phosphorylation in cells stimulated with a variety of mitogens and growth factors, including hematopoietic growth factors. Using c-raf antisense oligonucleotides to block Raf-1 expression, we have established that Raf-1 is required for hematopoietic growth factor-induced proliferation of murine cell lines stimulated by growth factors whose receptors are members of several different structural classes: (a) the hematopoietin receptor family, including interleukin (IL)-2, IL-3, IL-4, granulocyte colony-stimulating factor, granulocyte/macrophage colony-stimulating factor (GM-CSF), and erythropoietin; (b) the tyrosine kinase receptor class, including Steel factor and CSF-1; and (c) IL-6, leukemia inhibitory factor, and oncostatin M, whose receptors include the gp130 receptor subunit. Although results of previous experiments had suggested that IL-4 does not phosphorylate or activate the Raf-1 kinase, c-raf antisense oligonucleotides inhibited IL-4-induced proliferation of both myeloid and T cell lines, and IL-4 activated Raf-1 kinase activity in an IL-4-dependent myeloid cell line. In colony assays, c-raf antisense oligonucleotides completely inhibited colony formation of unseparated normal murine bone marrow cells stimulated with either IL-3 or CSF-1 and partially inhibited cells stimulated with GM-CSF. In addition, c-raf antisense oligonucleotides completely inhibited both IL-3- and GM-CSF-induced colony formation of CD34+ purified human progenitors stimulated with these same growth factors. Thus, Raf-1 is required for growth factor-induced proliferation of leukemic murine progenitor cell lines and normal murine and human bone marrow-derived progenitor cells regardless of the growth factor used to stimulate cell growth.
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PMID:Raf-1 protein is required for growth factor-induced proliferation of hematopoietic cells. 753 43

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) (10 ng/mL) prolonged human neutrophil survival in culture by at least 36 hours. The addition of H-series compounds at concentrations that are considered to inhibit both protein kinase C (PKC) and cyclic adenylate monophosphate (cAMP)-dependent protein kinase (PKA) counteracted the effect of rhG-CSF. Concomitantly, the inhibition of nucleosomal DNA fragmentation by rhG-CSF was canceled. At lower concentrations, presumably capable of inhibiting only PKA, however, the compounds exhibited marginal effects on rhG-CSF-mediated increase of cell survival. These PKC inhibitors did not influence the priming effect of rhG-CSF significantly, as determined by O2- production stimulated by N-formyl-L-methionyl-L-leucyl phenylalanine (fMLP). Our results suggest that PKC plays an important role in the mechanism by which rhG-CSF promotes neutrophil survival, in striking contrast with the priming effect elicited by rhG-CSF.
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PMID:Role of protein kinase C in neutrophil survival enhanced by granulocyte colony-stimulating factor. 769 71

The proliferation of normal hematopoietic cells is strictly factor dependent, while leukemic cell lines and primary leukemic cells are frequently factor independent. Although autocrine growth stimulation of human leukemias is occasionally observed in vitro, it is possible that mutations of signal-transduction or cell-cycle control genes may also be important in the development of factor independence. We have previously shown that the proto-oncogene Raf-1, a 70-kd serine/threonine protein kinase, is rapidly phosphorylated and activated by hematopoietic growth factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and Steel factor and is likely to be an important intermediate in mitogenic signal transduction pathways in hematopoietic cells. In an effort to better understand the possible role of abnormal signal transduction in the development of factor independence, we compared the state of phosphorylation and associated kinase activity of Raf-1 between a series of factor-dependent human and murine-myeloid normal cells or cell lines and a series of factor-independent myeloid cell lines. In factor-dependent myeloid cells (normal neutrophils; monocytes; and the cell lines MO7, 32Dc13, and FDC-P1), Raf-1 phosphorylation and associated kinase activity was strictly regulated by the supply of growth factor. In contrast, each of eight factor-independent leukemic cell lines examined, HL-60, KG-1, K562, U937, JOSK-S, JOSK-M, JOSK-K, and JOSK-I, expressed hyperphosphorylated Raf-1 with increased Raf-1 associated kinase activity in the absence of growth factor addition. To further explore the relationship of Raf-1 to factor-independent growth, factor-independent sublines were derived from two factor-dependent cell lines, MO7 and FDC-P1, by culture in CSF-deprived medium. Also, several factor-independent sublines were derived by transfection of a cDNA encoding p210BCR/ABL into three different cell lines: MO7, 32Dc13, and FDC-P1. In each case, the new sublines expressed constitutively hyperphosphorylated and activated Raf-1. The correlation of hyperphosphorylation of Raf-1 with factor independence was also observed with primary acute myeloblastic leukemia cells. The rate of "spontaneous" proliferation of primary acute myeloblastic leukemia (AML) cells in vitro correlated with the extent of Raf-1 phosphorylation. These results suggest that the evolution of myeloid leukemic cells to factor independence is associated with phosphorylation and activation of Raf-1, implicating Raf-1 and signal transduction pathways which activate RAf-1 in this process.
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PMID:Factor independence of human myeloid leukemia cell lines is associated with increased phosphorylation of the proto-oncogene Raf-1. 792 78

Expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) by metastatic Lewis lung carcinoma cells (LLC-LN7) was previously shown to contribute to the maintenance of phenotypic characteristics associated with an increased capacity to metastasize. In the present study, pre-incubation of LLC-LN7 cells with neutralizing anti-GM-CSF antibodies diminished the capacity of the tumor cells to form experimental metastases after i.v. inoculation, while pre-incubation with recombinant GM-CSF (rGM-CSF) increased formation of metastases. In the presence of rGM-CSF, the LLC-LN7 cells exhibited an increased capacity to migrate, invade through a reconstituted basement membrane, and adhere to lung tissue. Studies to identify the signal transduction pathway through which GM-CSF enhanced the in vitro metastatic properties of the LLC-LN7 tumor cells implicated protein kinase A (PKA). Signaling through PKA was suggested by the demonstration that the stimulation of tumor-cell motility by GM-CSF was blocked in the presence of the adenylate cyclase inhibitor nicotinic acid, or the PKA inhibitors A3 or KT5720. In addition, the role of PKA as a signaling mechanism for GM-CSF was assessed by using REV-LN7 cells, which are LLC-LN7 cells that have been stably transfected with an expression vector encoding a mutant PKA RI alpha subunit and which, in turn, express a cAMP-resistant PKA. Adherence and invasion by the PKA-defective REV-LN7 cells were not stimulated by rGM-CSF, contrasting with the stimulation observed for wild-type LLC-LN7 cells. These data suggest that rGM-CSF can further enhance the in vitro metastatic characteristics of LLC-LN7 tumor cells and that this is dependent on signal transduction through PKA.
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PMID:Granulocyte-macrophage colony-stimulating factor stimulates the metastatic properties of Lewis lung carcinoma cells through a protein kinase A signal-transduction pathway. 843 41

The receptor for human granulocyte-macrophage (GM)-CSF (GMR) is a heterodimer, consisting of an alpha-chain (GMR alpha) and a beta-chain (GMR beta). While GMR alpha is capable of binding GM-CSF, GMR beta is necessary for signal transduction. Phosphorylation of one or more tyrosine residues in GMR beta is an early event in signaling. We have recently demonstrated that tyrosine 750 (Y750) in GMR beta is a site of GM-CSF-induced phosphorylation and this site may contribute to the maintenance of cellular viability in response to GM-CSF. To investigate possible contributions made by additional GMR beta cytoplasmic tyrosine residues to receptor function, we mutated other selected tyrosine residues to phenylalanine and tested for any defects in signaling. in the present study, we show that Y577 is required for phosphorylation of Shc and an Shc-associated p140 in response to GM-CSF. Y577 is also required for association of Shc with GRB2. Y577 does not appear to be necessary for GM-CSF-induced proliferation and survival. GMR beta with a mutated Y577 is able to transduce signals leading to the activation of the Raf-1 pathway and the Jak-Stat pathway. Interestingly, mutation of Y750 reduced detectable GM-CSF-induced tyrosine phosphorylation of GMR beta, suggesting that the reduction of Shc phosphorylation associated with that mutant might be actually due to a failure to phosphorylate Y577. These data indicate that the phosphorylation of Shc in response to GM-CSF is not required for proliferation or viability signaling in these cells.
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PMID:Tyrosine phosphorylation of Shc is not required for proliferation or viability signaling by granulocyte-macrophage colony-stimulating factor in hematopoietic cell lines. 875 99

The cAMP analogue 8-bromo-cAMP (8BrcAMP) inhibits granulocyte-colony-stimulating factor (G-CSF)-stimulated DNA synthesis in myeloid NFS-60 cells. We examined the effect of 8BrcAMP addition on the G-CSF-stimulated extracellular signal-related protein kinase 1 (Erk-1), p21ras and Raf-1 activation. The Erk-1 activity was not down-regulated by the increase in intracellular cAMP levels, whereas p21ras and Raf-1 activities were, suggesting that Erk-1 activity might not be dependent on upstream p21ras and/or Raf-1 activity in this system. To explore this possibility further, we sought to determine whether there were downstream substrates of Raf-1 that were distinguishable from those of Erk-1 by using two-dimensional SDS/PAGE analysis of the protein phosphorylation patterns of NFS-60 cell cytosolic extracts treated with exogenous Raf-1 or Erk-1 in the presence of [gamma-32P]ATP. The two phosphorylation patterns were found to have many differences. To gain further insights into the possible relevance of these phosphorylation patterns and as an approach to exploring in more detail the inhibitory effect of 8BrcAMP, two-dimensional SDS/PAGE analysis was performed on the cytosolic extracts of 32P-labelled NFS-60 cells treated with G-CSF, in the absence or presence of 8BrcAMP. It was found that the phosphorylated proteins whose appearance was specific to the action of exogenous Raf-1 were sensitive to the action of 8BrcAMP in vivo, whereas those whose appearance was specific to the action of exogenous Erk-1 alone, or common to the actions of Raf-1 and Erk-1, were 8BrcAMP-insensitive. The results are consistent with a Raf-1-independent pathway for Erk-1 activation in G-CSF treated myeloid cells, and a number of potential downstream substrates of these kinases have been identified for further characterization.
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PMID:cAMP suppresses p21ras and Raf-1 responses but not the Erk-1 response to granulocyte-colony-stimulating factor: possible Raf-1-independent activation of Erk-1. 907 46

Activation of the respiratory burst imposes acute metabolic demands on phagocytic cells. These are met by mobilizing internal energy stores and by increasing the utilization of exogenous energy, including glucose in the circulation. To determine whether the increased glucose uptake that is known to be associated with the respiratory burst involves the regulation of glucose transporter molecules, the intrinsic transport properties of glucose transporters on the macrophage cell line RAW 264.7 were determined after activation with PMA, N-formyl-methionine-leucine-phenylalanine (fMLP) and the cytokines granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3). Treatment with PMA resulted in a 2-fold increase in respiratory burst activity within 10 min; this was associated with a 30-50% increase in 2-deoxyglucose uptake and a 4-fold increase in transporter affinity for glucose. Similarly, fMLP, GM-CSF and IL-3 treatments stimulated 2-deoxyglucose uptake that was associated with a 3-4-fold increase in transporter affinity for glucose. To determine whether the changes observed in 2-deoxyglucose uptake in response to PMA, fMLP and growth factors were influenced by phosphorylation of the sugar, 3-O-methylglucose, which is not phosphorylated, was used. Increased 3-O-methylglucose uptake and increased transporter affinity for glucose were also observed after PMA, fMLP and GM-CSF treatments. Whereas both fMLP and GM-CSF stimulated superoxide production, IL-3 failed to activate respiratory burst activity. The protein kinase inhibitors genistein and staurosporine inhibited the increase in 2-deoxyglucose uptake observed with fMLP and GM-CSF, and partly reversed the affinity increase towards that of untreated control cells. In contrast, the phosphatidylinositol 3-kinase inhibitor wortmannin had little effect on 2-deoxyglucose uptake in response to these activators. Western blotting with subtype-specific antisera showed that Glut-3 was the predominant transporter on RAW 264.7 cells. These studies demonstrate that acute regulation of glucose transporters occurs in response to activators that promote respiratory burst activity, and show that this regulation involves both tyrosine kinases and protein kinase C activity.
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PMID:Acute regulation of glucose transport in a monocyte-macrophage cell line: Glut-3 affinity for glucose is enhanced during the respiratory burst. 935 3

Exposure of neutrophils to inflammatory stimuli such as the chemoattractant FMLP leads to activation of responses including cell motility, the oxidative burst, and secretion of proteolytic enzymes. A signaling cascade involving sequential activation of Raf-1, mitogen-activated protein kinase (MEK), and extracellular signal regulated kinase (ERK) is also rapidly activated after agonist exposure. The temporal relationship between these events suggests that the kinases may be involved in triggering the effector functions, but direct evidence of a causal relationship is lacking. To assess the role of the MEK/ERK pathway in the activation of neutrophil responses, we studied the effects of PD098059, a potent and selective inhibitor of MEK. Preincubation of human neutrophils with 50 microM PD098059 almost completely (>90%) inhibited the FMLP-induced activation of MEK-1 and MEK-2, the isoforms expressed by neutrophils. This dose of PD098059 virtually abrogated chemoattractant-induced tyrosine phosphorylation and activation of ERK-1 and ERK-2, implying that MEKs are the predominant upstream activators of these mitogen-activated protein kinases. Pretreatment of neutrophils with the MEK antagonist inhibited the oxidative burst substantially and phagocytosis only moderately. In addition, PD098059 antagonized the delay of apoptosis induced by exposure to granulocyte-macrophage CSF. However, the effects of PD098059 were selective, as it failed to inhibit other responses, including chemoattractant-induced exocytosis of primary and secondary granules, polymerization of F-actin, chemotaxis, or activation of phospholipase A2. We conclude that MEK and ERK contribute to the activation of the oxidative burst and phagocytosis, and participate in cytokine regulation of apoptosis.
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PMID:Importance of MEK in neutrophil microbicidal responsiveness. 955 1

The phosphatidylinositol 3-kinase (PI3K)-signaling pathway has emerged as an important component of cytokine-mediated survival of hemopoietic cells. Recently, the protein kinase PKB/akt (referred to here as PKB) has been identified as a downstream target of PI3K necessary for survival. PKB has also been implicated in the phosphorylation of Bad, potentially linking the survival effects of cytokines with the Bcl-2 family. We have shown that granulocyte/macrophage colony-stimulating factor (GM-CSF) maintains survival in the absence of PI3K activity, and we now show that when PKB activation is also completely blocked, GM-CSF is still able to stimulate phosphorylation of Bad. Interleukin 3 (IL-3), on the other hand, requires PI3K for survival, and blocking PI3K partially inhibited Bad phosphorylation. IL-4, unique among the cytokines in that it lacks the ability to activate the p21ras-mitogen-activated protein kinase (MAPK) cascade, was found to activate PKB and promote cell survival, but it did not stimulate Bad phosphorylation. Finally, although our data suggest that the MAPK pathway is not required for inhibition of apoptosis, we provide evidence that phosphorylation of Bad may be occurring via a MAPK/ERK kinase (MEK)-dependent pathway. Together, these results demonstrate that although PI3K may contribute to phosphorylation of Bad in some instances, there is at least one other PI3K-independent pathway involved, possibly via activation of MEK. Our data also suggest that although phosphorylation of Bad may be one means by which cytokines can inhibit apoptosis, it may be neither sufficient nor necessary for the survival effect.
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PMID:Dissociation of cytokine-induced phosphorylation of Bad and activation of PKB/akt: involvement of MEK upstream of Bad phosphorylation. 963 68

Cytokines are important regulators of hematopoiesis. They exert their actions by binding to specific receptors on the cell surface. Interleukin-5 (IL-5) is a critical cytokine that regulates the growth, activation, and survival of eosinophils. Because eosinophils play a seminal role in the pathogenesis of asthma and allergic diseases, an understanding of the signal transduction mechanism of IL-5 is of paramount importance. The IL-5 receptor is a heterodimer of alpha- and beta-subunits. The alpha-subunit is specific, whereas the beta-subunit is common to IL-3, IL-5, and granulocyte/macrophage colony-stimulating factor (GM-CSF) receptors and is crucial for signal transduction. It has been shown that there are two major signaling pathways of IL-5 in eosinophils. IL-5 activates Lyn, Syk, and JAK2 and propagates signals through the Ras-MAPK and JAK-STAT pathways. Studies suggest that Lyn, Syk, and JAK2 tyrosine kinases and SHP-2 tyrosine phosphatase are important for eosinophil survival. In contrast to their survival-promoting activity, Lyn and JAK2 appear to have no role in eosinophil degranulation or expression of surface adhesion molecules. Raf-1 kinase, on the other hand, is critical for eosinophil degranulation and adhesion molecule expression. Btk is involved in IL-5 stimulation of B cell function. However, it does not appear to be important for eosinophil function. Thus a clear segregation of signaling molecules based on their functional importance is emerging. This review describes the signal transduction mechanism of the IL-3/GM-CSF/IL-5 receptor system and compares and contrasts IL-5 signaling between eosinophils and B cells.
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PMID:The mechanism of IL-5 signal transduction. 973 Sep 44


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