UNIPROT:P04141 - FACTA Search

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Query: UNIPROT:P04141 (granulocyte-macrophage colony-stimulating factor)
6,790 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phosphotyrosine phosphatases (PTPases) regulate cellular metabolic activation by reversing the effects of tyrosine kinases activated earlier in intracellular signaling pathways. We coupled fluorescence-activated cell sorter analysis using anti-CD45 monoclonal antibody with direct measurements of enzyme activity in resolved subcellular fractions to define mechanisms that potentially regulate the availability and activity of CD45-PTPase on neutrophil plasma membranes. Neutrophils in freshly obtained blood as well as neutrophils freshly isolated from blood were found to possess detectable levels of plasma membrane CD45 as assessed by immunofluorescence. However, plasma membranes from these cells were essentially devoid of PTPase catalytic activity, which was largely confined to the specific granules. Granulocyte-macrophage colony-stimulating factor (GM-CSF) upregulated both the catalytic and antigenic components of CD45-PTPase on the plasma membrane of these cells. Upregulation was associated with a shift in the particulate subcellular PTPase catalytic activity from the specific granule fraction to the plasma membrane fraction. The tyrosine kinase inhibitor genistein abrogated GM-CSF-promoted upregulation of plasma membrane CD45 PTPase but did not prevent the GM-CSF-dependent decrease in specific granule catalytic activity. Anti-CD45 antibody immunoprecipitated PTPase activity from both specific granules of resting cells and plasma membranes of GM-CSF-treated cells. However, antiphosphotyrosine immunoprecipitated only activity that had translocated to the plasma membrane, suggesting a role for CD45 phosphorylation in translocation. Western analysis confirmed the tyrosine phosphorylation of CD45 in plasma membranes of GM-CSF-treated neutrophils. Preincubation of plasma membranes of GM-CSF-stimulated neutrophils with cytosol from resting cells resulted in a time- and temperature-dependent loss in membrane PTPase as a consequence of the effects of a cytosolic inactivator. Cytosol obtained from stimulated neutrophils possessed substantially reduced levels of this PTPase inactivator. We conclude that activity of the catalytic component of membrane PTPase in circulating neutrophils is regulated by a cytosolic inactivator. Upon stimulation, intact CD45 PTPase is incorporated into the plasma membrane by a process that requires tyrosine phosphorylation. As a result of inhibition of the cytosolic inactivator, the translocated PTPase expresses full activity, thereby amplifying the potential regulatory influence of the enzyme on the cells' functional response.
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PMID:Cytosolic inactivation of translocated neutrophil plasma membrane protein tyrosine phosphatase. 854 61

Incubation of human neutrophils with 500 pM granulocyte-macrophage colony-stimulating factor (GM-CSF) results in a rapid and time-dependent increase in the phosphorylation of cytosolic phospholipase A2 (cPLA2), which was reflected in a slower electrophoretic mobility of the enzyme. The GM-CSF-induced phosphorylation of cPLA2 was accompanied by a parallel and time-dependent increase in the enzyme activity. Preincubation of neutrophils with the tyrosine kinase inhibitor genistein caused inhibition of the GM-CSF-stimulated phosphorylation and activity of cPLA2. Immunoprecipitation of the enzyme following incubation of neutrophils with [32P]Pi shows that cPLA2 is phosphorylated by GM-CSF. Potato acid phosphatase caused dephosphorylation of the enzyme, indicating that cPLA2 is indeed phosphorylated by GM-CSF. The subcellular distribution of cPLA2 in GM-CSF-stimulated neutrophils revealed that the enzyme resides almost completely in the cytosolic fraction. Addition of Ca2+ to the lysis buffer before homogenization results in the translocation of the phosphorylated and the dephosphorylated forms of the enzyme to the membranes. Translocation of cPLA2 was also achieved after incubation with 0.1 microM N-formylmethionyl-leucyl-phenyl-alanine (fMLP) after GM-CSF stimulation and when neutrophils were challenged with the Ca2+ ionophore A23187. EDTA and EGTA were unable to solubilize the translocated enzyme from the neutrophil membranes, indicating that cPLA2 is attached to the membranes by strong bonds and not merely due to ionic forces exerted by Ca2+. The inability of GM-CSF to promote arachidonic acid mobilization is probably due to the fact that GM-CSF does not cause an increase in intracellular Ca2+, which is necessary for the translocation of the enzyme to the membranes where its substrate(s) reside.
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PMID:Granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes phosphorylation and an increase in the activity of cytosolic phospholipase A2 in human neutrophils. 857 84

Polyoma middle T (PmT)-transformed endothelial cells may represent a unique murine model for human opportunistic vascular tumors. The present study was designed to evaluate the anti-tumor potential of a panel of 13 cytokines against murine PmT-transformed endothelial cells. Interferon gamma (IFNgamma) and transforming growth factor beta 1 (TGFbeta1) substantially decreased in a dose-dependent manner the proliferation of a panel of 6 PmT-transformed cell lines. IFNalpha and tumor necrosis factor alpha(TNFalpha) had marginal anti-proliferative activity, whereas other molecules (interleukins-1, -2, -4, -6 and -13, IFNbeta, leukemia inhibitory factor, oncostatin M, granulocyte-macrophage colony-stimulating factor) caused no growth inhibition. IFNgamma and TGFbeta1 were therefore selected for further analysis of their mechanism of action and in vivo relevance. IFNgamma and TGFbeta1 reduced the activity of phosphatidylinositol-3-kinase and the production of phosphatidylinositol 3,4-biphosphate, without modifying the tyrosine kinase(s) activity associated with PmT. IFNgamma and TGFbeta1 were also tested for their ability to modify the in vivo growth of the PmT-transformed endothelial cells H5V in syngeneic C57B1/6 mice. Treatment with IFNnu and TGFbeta1 significantly delayed tumor growth and increased survival time. In contrast, treatment with IFNalpha and TNFalpha failed to prolong survival. In nude mice, IFNgamma and TGFbeta1 had a transient effect on tumor growth but no effect on survival, suggesting a contribution of T cells to the in vivo anti-tumor activity of these cytokines.
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PMID:Anti-tumor activity of cytokines against opportunistic vascular tumors in mice. 859 25

Interaction of a tyrosine kinase type receptor and its ligand induces receptor-dimerization or -oligomerization followed by transphosphorylation and activation of its intrinsic kinase, which leads to a series of intracellular signals. We have previously reported that the membrane-bound form of Steel factor (SLF) induces more persistent tyrosine kinase activation and longer life span of c-kit encoded protein (KIT) than its soluble form (Miyazawa et al, Blood 85:641, 1995). In this study, we used YB5.B8 monoclonal antibody (MoAb) that recognizes the extracellular domain of KIT to investigate whether immobilized anti-KIT MoAb can substitute for SLF as a potent activator of KIT by cross-linking receptors and further compared its effect with each SLF isoform in a factor-dependent cell line M07e. YB5.B8 MoAb in a soluble state suppressed SLF-induced M07e cell proliferation in a dose-dependent manner. By contrast, once this antibody was immobilized on the goat-antimouse MoAb (GAM)-coated culture plates, it supported the growth of M07e cells in the absence of any growth factors, whereas culture the cells in GAM alone or YB5.B8 without GAM-coated plates resulted in rapid cell-death within 24 hours. As with the natural ligand SLF, immobilized YB5.B8 MoAb synergized with granulocyte-macrophage colony-stimulating factor (GM-CSF) in inducing cell proliferation compared with either YB5.B8 MoAb or GM-CSF alone. Immunoblotting with antiphosphotyrosine MoAb showed that interaction of M07e cells with immobilized YB5.B8 induced tyrosine phosphorylation of a series of intracellular proteins including KIT (145 kD). In addition, cross-linking studies using a water-soluble cross linking reagent bis-sulfosuccinimidyl-suberate showed that immobilized YB5.B8 MoAb induced dimerization and activation of KIT. However, as with stimulation by the membrane-bound form of SLF, the kinetics of KIT activation with YB5.B8 MoAb was more prolonged compared with the cells treated with recombinant soluble SLF. Flow cytometry showed that, unlike the cells treated with soluble SLF, no downmodulation of cell-surface KIT expression was observed in M07e cells cultured with immobilzed YB5.B8 MoAb. These data suggest that immobilized antibodies against hematopoietic receptors may replace their ligand-stimulators; however, their activities may resemble the membrane-bound form rather than the soluble form of natural ligands.
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PMID:Immobilized anti-KIT monoclonal antibody induces ligand-independent dimerization and activation of Steel factor receptor: biologic similarity with membrane-bound form of Steel factor rather than its soluble form. 863 Mar 83

Granulocyte-macrophage colony-stimulating factor (GM-CSF) provokes a proliferative response and induction of early-response genes such as c-fos in target cells. It also induces rapid tyrosine phosphorylation of cellular proteins, including the beta subunit (betac) of its functional receptor. However, locations and functions of phosphorylated tyrosine residues within the betac are unclear. To elucidate the mechanism of the human GM-CSF receptor signal transduction, mutational analyses were made of the cytoplasmic domain of the beta-c, using murine BA/F3 cells. Deletion of the conserved box 1 motif resulted in loss of tyrosine phosphorylation of the betac, thereby indicating an essential role for this motif in activating the tyrosine kinase which phosphorylates betac. A C-terminal truncated mutant at position 589 activated the c-fos promoter, and this activation was diminished by a substitution at tyrosine 577 (Tyr577). However, the same substitution in the full-length betac did not completely abrogate the c-fos promoter activation, hence, redundant signaling pathways probably exist. When we analyzed signaling molecules functioning downstream of the beta-c we found that Tyr577 is essential for Shc phosphorylation, while tyrosine phosphorylation of PTP1D was mediated through Tyr577 as well as through other site(s). We suggest that GM-CSF stimulates at least two modes of signals leading to Ras activation, an event which ultimately gives rise to promoter activation of c-fos.
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PMID:Granulocyte-macrophage colony-stimulating factor provokes RAS activation and transcription of c-fos through different modes of signaling. 863 92

Granulocyte-macrophage colony-stimulating factor (GM-CSF), supports proliferation, differentiation, and functional activation of hemopoietic cells by its interaction with a heterodimeric receptor. Although GM-CSF receptor is devoid of tyrosine kinase enzymatic activity, GM-CSF-induced peripheral blood polymorphonuclear leukocytes (PMN) functional activation is mediated by the phosphorylation of a large number of intracellular signaling molecules. We have previously shown that JAK2 becomes tyrosine-phosphorylated in response to GM-CSF in PMN. In the present study we demonstrate that also the signal transducers and activators of transcription (STAT) family members STAT1 p91 and STAT3 p92 and the product of the c-fps/fes protooncogene become tyrosine-phosphorylated upon GM-CSF stimulation and physically associated with both GM-CSF receptor beta common subunit and JAK2. Moreover GM-CSF was able to induce JAK2 and p93fes catalytic activity. We also demonstrate that the association of the GM-CSF receptor beta common subunit with JAK2 is ligand-dependent. Finally we demonstrate that GM-CSF induces a DNA-binding complex that contains both p91 and p92. These results identify a new signal transduction pathway activated by GM-CSF and provide a mechanism for rapid activation of gene expression in GM-CSF-stimulated PMN.
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PMID:Granulocyte-macrophage colony-stimulating factor stimulates JAK2 signaling pathway and rapidly activates p93fes, STAT1 p91, and STAT3 p92 in polymorphonuclear leukocytes. 863 62

UT-7 is a human megakaryoblastic leukemia cell line with absolute dependence on interleukin-3, granulocyte-macrophage colony-stimulating factor, or erythropoietin (EPO) for growth and survival. We investigated the effect of thrombopoietin (TPO), the ligand for the receptor encoded by c-mpl proto-oncogene, on the proliferation and differentiation of UT-7 and its sublines. We found that UT-7/GM, which is a subline of UT-7, but neither UT-7 nor UT-7/EPO, can proliferate in response to TPO. The subline, UT-7/TPO, was established from UT-7/GM by culture at lower concentrations of TPO. UT-7/TPO cells had morphologically mature megakaryocytic characteristics such as developed demarcation membrane in the cytoplasm and multinucleated appearance. This was also confirmed by the high expression of platelet factor-4 and glycoprotein IIb at the mRNA levels and by the high level of DNA content. UT-7/TPO can be maintained by TPO alone, with a doubling time of 24 hours in log growth phase. In the absence of TPO, the majority of the cells died within a few days. Thus, UT-7/TPO has an absolute dependence on TPO for growth and survival and has mature megakaryocytic features. The mRNA for c-mpl was detected in UT-7/TPO and, to a lesser degree, in UT-7/GM. The mRNA level of NF- E2 p45, reported to be an erythroid-specific transcription factor, was upregulated in UT-7/TPO, whereas it was down-regulated in the erythroid subline, UT-7/EPO. There were no significant differences in GATA-1 and GATA-2 mRNA levels among UT-7 and its sublines. Not only EPO but also TPO induced the tyrosine phosphorylation of JAK2 tyrosine kinase and STAT5-related protein. These findings indicate that UT-7/TPO would be a useful model with which to analyze the gene regulation of megakaryocytic maturation-associated proteins and to study the specific actions of TPO.
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PMID:Establishment and characterization of the thrombopoietin-dependent megakaryocytic cell line, UT-7/TPO. 863 23

Introduction of v-src or c-src527F, a transforming mutant of the c-src proto-oncogene, into the growth factor-dependent cell line FDCP-1 resulted in growth factor independence. Temperature-shift studies with cells carrying the tsLA29 mutant of v-src demonstrated that growth factor independence was oncogene-dependent; that is, the cells were growth factor-independent at the permissive temperature but became growth factor-dependent at the nonpermissive temperature. Introduction of the c-src proto-oncogene did not result in growth factor independence. The c-src2A,527F mutant, which encodes an activated tyrosine kinase but does not transform fibroblasts due to a mutation in the membrane localization sequence, induced growth factor independence. This suggests that the presence of an activated tyrosine kinase is necessary for this process but that membrane localization is not. Bioassays indicated that conditioned medium from growth factor-independent cells contained a growth factor identified by antibody neutralization studies as granulocyte-macrophage colony-stimulating factor (GM-CSF). Secretion of GM-CSF was confirmed by a quantitative enzyme-linked immunosorbent assay (ELISA) specific for GM-CSF. The presence of GM-CSF mRNA in src-infected FDCP-1 cells was demonstrated by PCR amplification of cDNAs with primers specific for GM-CSF. While GM-CSF mRNA was detected in FDCP/ts29 cells grown at 34 degrees C, it was not observed in cells infected with the tsLA29 mutant grown at the nonpermissive temperature of 39 degrees C. Transfection of v-src-infected FDCP-1 cells with a GM-CSF promoter reporter plasmid revealed src-dependent expression of luciferase; that is, while expression was observed at the permissive temperature, no expression was detected in FDCP/ts29 clone 6 cells grown at the nonpermissive temperature. No expression of the GM-CSF promoter reporter plasmid was observed in uninfected FDCP-1 cells.
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PMID:Induction of growth factor-independence and GM-CSF secretion by the v-src oncogene in murine myeloid cells does not require membrane association of pp60v-src. 864 39

Fc-gamma receptor III (Fc gamma RIII, CD16) type A is expressed on natural killer cells, on a small subset of peripheral blood monocytes and on mature macrophages. Along with differentiation into macrophages, monocytes will express Fc gamma RIII when cultured with transforming growth factor-beta (TGF-beta). In view of the involvement of granulocyte-macrophage colony-stimulating factor (GM-CSF) in myeloid cell differentiation, we investigated the effect of this cytokine on Fc gamma RIII expression in cultures of peripheral blood monocytes. GM-CSF antagonized TGF-beta-induced expression of Fc gamma RIII on monocytes in vitro in a dose-dependent way. The effect of GM-CSF persisted in cultures until at least day 7. The suppression was at the mRNA level, as shown by Northern analyses with a CD16 specific probe, and the signalling pathway involved tyrosine kinase activity. Interferon-gamma and interleukin-2 had no effect on the induced expression of Fc gamma RIII by TGF-beta, while interleukin-4, similar to GM-CSF, antagonized this induction. Our findings suggest that regulatory cytokine networks can drive monocytes into different effector functions and differentiation pathways.
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PMID:Granulocyte-macrophage colony-stimulating factor antagonizes the transforming growth factor-beta-induced expression of Fc gamma RIII (CD16) on human monocytes. 866 30

The stimulating effect of nerve growth factor (NGF) on phagocytosis, parasite killing, and interleukin-1beta (IL-1beta) production of murine peritoneal macrophages was assessed. In the presence of various doses of NGF, macrophages showed the increased phagocytosis of both nonspecific hydrophilic microspheres and sheep red blood cells (SRBC) opsonized with anti-SRBC antibodies (Ab) or complement in a dose-dependent manner. NGF also enhanced killing of Leishmania donovani promastigotes by macrophages, and its ability was comparable with that of an optimal dose of recombinant granulocyte-macrophage colony-stimulating factor or recombinant interferon-gamma. The addition of NGF to peritoneal macrophages and monocyte-macrophage J774A.1 cells led to a significant release of IL-1beta in a dose-dependent manner and expression of IL-1beta mRNA. Because pretreatment of peritoneal macrophages and J774A.1 cells with K-252a, a tyrosine kinase inhibitor, completely suppressed these NGF-mediated stimulating effects and p140trk phosphorylation and because flow cytometric analysis with specific Ab against two distinct NGF receptors showed the expression of p140trk, unlike p75LNGFR, on the surface of macrophages, the stimulating activity of NGF to murine macrophages may be mediated through p140trk. Thus, NGF may act as an activator for murine macrophages in the process of inflammatory and immune actions.
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PMID:Functional properties of murine macrophages promoted by nerve growth factor. 897 55

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