Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Tyrosine phosphorylation and activation of the transcription factor Stat5 occur in response to stimuli like granulocyte-macrophage colony-stimulating factor, interleukin-3, or erythropoietin that stimulate both proliferation and differentiation of hematopoietic cells. It is unclear whether Stat5 is part of a proliferative response or part of the events leading to cellular differentiation. Here we report that agents promoting differentiation but not proliferation of hematopoietic cells, like phorbol ester or both types of interferons (IFNs), activate Stat5 in promonocytic U937 cells. Both IFN types caused tyrosine phosphorylation and DNA binding of predominantly one Stat5 isoform (Stat5a) despite expression of both Stat5a and Stat5b proteins. Monocytic differentiation of U937 cells led to a strong decrease in IFN-gamma-mediated activation of Stat5 but not of Stat1. Transactivation of Stat5-target genes occurred in response to IFN-gamma, which activates both Stat5 and Stat1, but not in response to granulocyte-macrophage colony-stimulating factor, which activates only Stat5. Tyrosine phosphorylation of Stat5 is not generally part of the IFN response. IFN-gamma did not cause Stat5 activation in HeLa cells, despite the expression of both Stat5 isoforms at similar levels. By contrast, IFN-alpha caused tyrosine phosphorylation and DNA binding of exclusively the b isoform of Stat5, and activated Stat5b formed a DNA binding activity previously found in HeLa cells and designated IFN-alpha activation factor 2. Taken together, our results demonstrate that ligand binding of IFN receptors leads to an isoform-specific activation of Stat5 in a restricted number of cell lineages. Moreover, they suggest that Stat5 might be part of the differentiation response of myeloid cells.
Mol Cell Biol 1996 Dec
PMID:Activation of different Stat5 isoforms contributes to cell-type-restricted signaling in response to interferons. 894 49

SHP-1 is an SH2-containing cytoplasmic tyrosine phosphatase that is widely distributed in cells of the hematopoietic system. SHP-1 plays an important role in the signal transduction of many cytokine receptors, including the receptor for erythropoietin, by associating via its SH2 domains to the receptors and dephosphorylating key substrates. Recent studies have suggested that SHP-1 regulates the function of Jak family tyrosine kinases, as shown by its constitutive association with the Tyk2 kinase and the hyperphosphorylation of Jak kinases in the motheaten cells that lack functional SHP-1. We have examined the interactions of SHP-1 with two tyrosine kinases activated during engagement of the erythropoietin receptor, the Janus family kinase Jak-2 and the c-fps/fes kinase. Immunoblotting studies with extracts from mouse hematopoietic cells demonstrated that Jak2, but not c-fes, was present in anti-SHP-1 immunoprecipitates, suggesting that SHP-1 selectively associates with Jak2 in vivo. Consistent with this, when SHP-1 was coexpressed with these kinases in Cos-7 cells, it associated with and dephosphorylated Jak2 but not c-fes. Transient cotransfection of truncated forms of SHP-1 with Jak2 demonstrated that the SHP-1-Jak2 interaction is direct and is mediated by a novel binding activity present in the N terminus of SHP-1, independently of SH2 domain-phosphotyrosine interaction. Such SHP-1-Jak2 interaction resulted in induction of the enzymatic activity of the phosphatase in in vitro protein tyrosine phosphatase assays. Interestingly, association of the SH2n domain of SHP-1 with the tyrosine phosphorylated erythropoietin receptor modestly potentiated but was not essential for SHP-1-mediated dephosphorylation of Jak2 and had no effect on c-fes phosphorylation. These data indicate that the main mechanism for regulation of Jak2 phosphorylation by SHP-1 involves a direct, SH2-independent interaction with Jak2 and suggest the existence of similar mechanisms for other members of the Jak family of kinases. They also suggest that such interactions may provide one of the mechanisms that control SHP-1 substrate specificity.
Mol Cell Biol 1996 Dec
PMID:Direct association with and dephosphorylation of Jak2 kinase by the SH2-domain-containing protein tyrosine phosphatase SHP-1. 894 54

The erythropoietin (EPO) gene is one of the best examples of a mammalian gene controlled by oxygen tension. The DNA elements responsible for hypoxia-induced transcription consist of a short region of the proximal promoter and a <50-bp 3' enhancer. The elements act cooperatively to increase the transcriptional initiation rate approximately 100-fold in response to low oxygen tension in Hep3B cells. Two distinct types of transactivating proteins have been demonstrated to bind the response elements in the human EPO enhancer in vitro: one shows hypoxia-inducible DNA binding activity, while the other activity binds DNA under normoxic and hypoxic conditions. We have investigated the DNA-protein interactions on the human EPO enhancer in living tissue culture cells that produce EPO in a regulated fashion (Hep3B) and in cells that do not express EPO under any conditions tested (HeLa). We have identified in vivo DNA-protein interactions on the control elements in the human EPO enhancer by ligation-mediated PCR technology. We show that the putative protein binding sites in the EPO enhancer are occupied in vivo under conditions of normoxia, hypoxia, and cobalt exposure in EPO-producing cells. These sites are not occupied in cells that do not produce EPO. We also provide evidence for a conformational change in the topography of the EPO enhancer in response to hypoxia and cobalt exposure.
Mol Cell Biol 1997 Feb
PMID:In vivo analysis of DNA-protein interactions on the human erythropoietin enhancer. 900 Dec 39

Interactions between erythropoietin (Epo) and its receptor (EpoR) are critical for the normal proliferation and differentiation of erythroid progenitor cells. EpoR is expressed in low numbers during early stages of erythroid maturation, while higher levels are expressed during later stages, suggesting that the expression of the EpoR gene is tightly regulated throughout erythropoiesis. We used the TF-1 erythroleukemia cell line to analyze the effects of various cytokines and reagents on the regulation of human EpoR gene expression. Human EpoR gene expression was significantly upregulated by IL-1 alpha and the protein inhibitor cycloheximide, but significantly downregulated by the calcium ionophore ionomycin and the phorbol ester PMA. These effects on EpoR gene expression were not due to changes in EpoR mRNA stability, suggesting that these agents directly affected EpoR gene transcription. The selective in vitro modification of EpoR expression by these agents highlights the complexity of human EpoR gene expression, and provides clues to its in vivo regulation.
Blood Cells Mol Dis 1996
PMID:Regulation of expression of the human erythropoietin receptor gene. 907 72

The Janus protein tyrosine kinases (Jaks) play critical roles in transducing growth and differentiation signals emanating from ligand-activated cytokine receptor complexes. The activation of the Jaks is hypothesized to occur as a consequence of auto- or transphosphorylation on tyrosine residues associated with ligand-induced aggregation of the receptor chains and the associated Jaks. In many kinases, regulation of catalytic activity by phosphorylation occurs on residues within the activation loop of the kinase domain. Within the Jak2 kinase domain, there is a region that has considerable sequence homology to the regulatory region of the insulin receptor and contains two tyrosines, Y1007 and Y1008, that are potential regulatory sites. In the studies presented here, we demonstrate that among a variety of sites, Y1007 and Y1008 are sites of trans- or autophosphorylation in vivo and in in vitro kinase reactions. Mutation of Y1007, or both Y1007 and Y1008, to phenylalanine essentially eliminated kinase activity, whereas mutation of Y1008 to phenylalanine had no detectable effect on kinase activity. The mutants were also examined for the ability to reconstitute erythropoietin signaling in gamma2 cells, which lack Jak2. Consistent with the kinase activity, mutation of Y1007 to phenylalanine eliminated the ability to restore signaling. Moreover, phosphorylation of a kinase-inactive mutant (K882E) was not detected, indicating that Jak2 activation during receptor aggregation is dependent on Jak2 and not another receptor-associated kinase. The results demonstrate the critical role of phosphorylation of Y1007 in Jak2 regulation and function.
Mol Cell Biol 1997 May
PMID:Activation of Jak2 catalytic activity requires phosphorylation of Y1007 in the kinase activation loop. 911 18

The growth hormone regulated serine protease inhibitor (SPI) 2.1 and 2.2 gene promoters have been shown to contain a response element similar to the gamma-interferon activated sequence (GAS) family of signal transducer and activator of transcription (STAT) response elements. We have investigated the STAT and cytokine specificity of the SPI 2.1 STAT responsive element using a luciferase (LUC) reporter construct and a cDNA complementation strategy in the COS 7 cell line. Growth hormone was found to stimulate SPI-LUC reporter gene expression via activation of STAT 5, but not STATs 1 or 3, which indicates that the SPI 2.1 STAT responsive element is STAT 5 specific. In addition to the growth hormone receptor, the receptors for prolactin and erythropoietin enhanced gene transcription via the SPI 2.1 STAT responsive element, which indicates that this element is, on the other hand, not cytokine specific. Activation of STAT 5 was also observed after growth hormone treatment of cells transfected with cDNA expression plasmids for several different truncated growth hormone receptor mutants, although this activation was less efficient than with the wild type receptor. Point mutation of individual tyrosines in the growth hormone receptor intracellular domain to phenylalanines had no significant effect on signal transduction via STAT 5. These data, taken together with results from experiments using the phosphatase inhibitor sodium orthovanadate, suggest that STAT 5 may not have an absolute requirement for specific phosphorylated receptor tyrosine docking sites. That receptor tyrosine residues in a variety of amino acid contexts, or phosphorylated Janus kinase (JAK) 2 alone, can facilitate STAT 5 activation could explain the observed lack of cytokine specificity in STAT 5 activation.
Mol Cell Endocrinol 1997 Jun 20
PMID:Specificity of transcription enhancement via the STAT responsive element in the serine protease inhibitor 2.1 promoter. 922 23

The nonlinear pharmacokinetics (1000, 5000, and 10000 IU/kg) and tissue distribution (5000 IU/kg) of erythropoietin (EPO) after intravenous administration of recombinant human EPO (rhuEPO) to rabbits, extent of absolute bioavailability (F) of EPO after subcutaneous administration (5000 IU/kg) to rabbits, and pharmacokinetics of EPO after intravenous administration to 3/4 nephrectomized rats (1000 IU/kg) were investigated. After intravenous administration of rhuEPO, 1000 IU/kg to rabbits, the terminal half-life, t1/2 (296, 368, and 378 min) and mean residence time (255, 318, and 326 min) decreased significantly, however, the total body clearance, CL (0.233, 0.165, and 0.169 ml/min/kg) and nonrenal clearance, CLNR (0.196, 0.141, and 0.120 ml/min/kg) increased significantly when compared with those of 5000 and 10000 IU/kg. The above dose-dependent pharmacokinetic parameters of EPO could be due to saturable metabolism of EPO in rabbits. The affinity of EPO to rabbit tissues studied was very low as reflected to less-than-unity values of tissue to plasma ratios except in the bile. This was supported by a considerably low value of volume of distribution of EPO at steady state (Vss) after intravenous administration of rhuEPO, 1000-10000 IU/kg, to rabbits (0.0524-0.0591 l/kg). After subcutaneous administration of rhuEPO, 5000 IU/kg, to rabbits, the plasma concentration of EPO was reached its peak at 600-720 min and declined slowly with a mean t1/2 of 1040 min. The F value after subcutaneous administration to rabbits was 43.1%. After intravenous administration of rhuEPO, 1000 IU/kg, to control and 3/4 nephrectomized rats, the CL, CLNR, and Vss were not significantly different, however, the MRT and CLR were significantly different between two groups of rats.
Res Commun Mol Pathol Pharmacol 1997 May
PMID:Pharmacokinetics of recombinant human erythropoietin in rabbits and 3/4 nephrectomized rats. 922 57

Fetal nucleated red cells which pass into the maternal circulation during pregnancy are a potential cell source for non-invasive prenatal genetic diagnosis. To sort these rare cells with a high degree of specificity, we focussed our attention on the erythropoietin receptor, a strictly erythroid-specific antigen. We first labelled these receptors with biotin-(sialyl)-erythropoietin, then isolated the erythroid cells by magnetic beads conjugated with streptavidin in a MiniMACS (magnetic cell separator). The effectiveness of this strategy for the enrichment of fetal cells was evaluated by assessing its accuracy for gender prediction in 18 male-bearing pregnancies. Polymerase chain reaction (PCR) results on maternal blood samples sorted for Epo-r and CD71 antigens displayed similar sensitivity (55% Epo-r, 61% CD71) in detecting Y-specific sequences while immunocytochemical studies on four maternal blood samples, sorted after increasing the binding time of the ligand to Epo-r (8 h), showed a substantial improvement in fetal cell recovery and purity. We conclude that sorting by Epo-r/biotin-(sialyl)-erythropoietin provides effective enrichment of fetal nucleated red cells allowing the possibility of direct prenatal cytogenetic analysis by multiprobe fluorescent in-situ hybridization (FISH).
Mol Hum Reprod 1997 May
PMID:Isolation of fetal erythroid cells from maternal blood based on expression of erythropoietin receptors. 923 31

Hematopoietic growth factors (HGFs) act on the hematopoietic cells via binding to specific cell surface receptors. Many HGF receptors have certain common structural features and have therefore been grouped in the superfamily of hematopoietin or cytokine receptors, also referred to as the class I receptor superfamily [1, 2]. Activation of these receptors by their cognate growth factors is mediated through the formation of dimeric or oligomeric complexes of receptor structures. Some HGF receptors are composed of heteromeric complexes, comprising two or three different receptor chains. For instance, this is the case for receptors of interleukins 2, 3, and 5 and granulocyte-macrophage colony-stimulating factor [3]. Other receptor structures, for example, those of granulocyte colony-stimulating factor and erythropoietin, form homodimeric complexes upon growth factor binding [2, 4]. This brief overview begins with an introduction of the major principles of HGF receptor signaling: this is followed by a discussion of the consequences of HGF receptor signaling defects for the development of disorders of the hematopoietic system and the presentation of clinical examples of such diseases.
J Mol Med (Berl) 1997 Jul
PMID:Molecular understanding of hematopoietin/cytokine receptor signaling defects in hematopoietic disorders. 925 10

We observed striking differences between the tumorigenic colony-forming cells present in the spleens of mice late after infection with the anemia-inducing strain of Friend leukemia virus (strain FV-A) and those present after infection with the polycythemia-inducing strain (strain FV-P). Cells within primary colonies derived from FV-A- and FV-P-transformed cells (CFU-FV-A and CFU-FV-P, respectively) contained hemoglobin and spectrin, indicating that the CFU-FV-A and CFU-FV-P were transformed erythroid progenitor cells. The proportion of cells containing hemoglobin was relatively high (> 25%) in newly isolated cell lines derived from CFU-FV-P colonies, whereas cell lines derived from CFU-FV-A colonies had only low levels (0 to 2%) of hemoglobin-containing cells. A high proportion of the cell lines derived from CFU-FV-A colonies responded to pure erythropoietin and accumulated spectrin and hemoglobin, whereas the cell lines derived from CFU-FV-P colonies did not. A cytogenetic analysis indicated that primary CFU-FV-P colony cells were diploid, whereas chromosomal aberrations were observed in the immediate progeny of CFU-FV-A. The presence of unique chromosomal markers in the majority of the cells within individual colonies derived from CFU-FV-A suggested that these colonies originated from single cells. Finally, leukemic progenitor cells transformed by strain FV-A appeared to have an extensive capacity to self-renew (i.e., form secondary colonies in methylcellulose), whereas a significant proportion of the corresponding cells transformed by strain FV-P did not. In addition, the self-renewal capacity of both CFU-FV-A and CFU-FV-P increased as the disease progressed. From these observations, we propose a model for the multistage nature of Friend disease; this model involves clonal evolution and expansion from a differentiating population with limited proliferative capacity to a population with a high capacity for self-renewal and proliferation.
Mol Cell Biol 1981 Aug
PMID:Clonal analysis of the late stages of erythroleukemia induced by two distinct strains of Friend leukemia virus. 927 85


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