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:2.7.10.2 (focal adhesion kinase)
44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Erythropoietin (EPO) regulates the proliferation and differentiation of erythroid cells through interaction with its receptor (EPOR). Although EPOR is a member of the cytokine receptor superfamily and lacks a kinase domain, EPO induces tyrosine phosphorylation, which is correlated with gene transcription and mitogenesis. Here we demonstrate that EPO induces tyrosine phosphorylation of JAK2 kinase and activates its in vitro autophosphorylation. Using EPOR mutants, phosphorylation and activation of kinase activity correlate with the induction of mitogenesis. Furthermore, JAK2 physically associates with a membrane-proximal region of the EPOR cytoplasmic domain that is required for biological activity. The results support the hypothesis that JAK2 is the kinase that couples EPO binding to tyrosine phosphorylation and mitogenesis.
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PMID:JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin. 834 51

Erythropoietin (EPO) exerts its activities by the induction of multiple signalling pathways through interaction with the erythropoietin receptor (EPOR). Previous studies have suggested that the Ras/MAP kinase as well as the JAK/STAT signalling cascades play significant roles in the induction of EPO-responsive genes. Here we show that, in HCD-57 erythroleukemic cells, both pathways are activated by EPO in a dose-dependent manner with similar sensitivities and kinetics. The activation of signalling molecules is closely related to the proliferative status of the cells. Using an antisense strategy, we were able to show that the downregulation of the JAK2 protein level in HCD-57 cells results in a distinct reduction of the ability to induce not only STAT5 DNA-binding, but also MAP kinase activity. Our results thus provide evidence for a significant contribution of the cytosolic tyrosine kinase JAK2 to the EPO-induced activation of the Ras/MAP kinase cascade.
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PMID:Requirement for JAK2 in erythropoietin-induced signalling pathways. 906 35

The BCR/ABL oncogene encodes an activated tyrosine kinase that causes human chronic myelogenous leukemia. The mechanism of transformation, however, is complex and not well understood. One of the important contributions of BCR to transformation is believed to be dimerization or oligomerization of ABL, thereby activating ABL tyrosine kinase activity. We reasoned that if ABL was dimerized through other mechanisms, activation of the tyrosine kinase activity should also result, and the activated kinase may also be transforming. Erythropoietin is known to activate its receptor by causing dimerization, and therefore a synthetic oncogene was created by linking the extracytoplasmic and transmembrane domains of the EPO receptor with c-ABL. This chimeric receptor was stably expressed in Ba/F3 cells and, in the absence of EPO, had no detectable biological effect on the cells. EPO, however, induced a rapid, dose-dependent activation of ABL tyrosine kinase activity and phosphorylation of several cellular proteins. The major target proteins have been identified, and are very similar to the known substrates of BCR/ABL, including Shc, CBL, CRKL, and several proteins in the cytoskeleton. EPO treatment also resulted in biological effects that were remarkably similar to those of BCR/ABL, including improved viability, altered integrin function, and a weak mitogenic signal. The biological effects were in part dose-dependent, in that low EPO concentrations enhanced viability but did not cause proliferation. At high EPO doses, kinase activation was maximal, and a mitogenic effect was also revealed. In nude mice, Ba/F3 cells expressing this chimeric receptor did not cause detectable disease without administration of pharmacologic doses of EPO. If EPO was given intraperitoneally 5 days a week, however, a dose-dependent lethal leukemia resulted. This ligand-regulatable oncogene mimics some of the biological effects of BCR/ABL, and analysis of ABL mutants in this system will be useful to dissect the signaling pathways that cause CML.
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PMID:A chimeric receptor/oncogene that can be regulated by a ligand in vitro and in vivo. 931 68

Erythropoietin (EPO) is the major hormone regulating the proliferation of erythroid precursors and their differentiation into erythrocytes. Ligand binding to the erythropoietin receptor (EPO-R), a member of the cytokine receptor family, triggers Tyr phosphorylation of the surface form of the receptor, presumably mediated by the Janus kinase (JAK) 2. To study whether non-surface EPO-R can be phosphorylated, Ba/F3 cells stably transfected with EPO-R were treated with pervanadate (PV), which is widely used as a potent tool to inhibit cellular protein Tyr phosphatases, thus resulting in enhanced Tyr phosphorylation of cellular proteins. PV treatment caused the EPO-R to undergo Tyr phosphorylation in a time-dependent and dose-dependent manner. PV-mediated Tyr phosphorylation of EPO-R occurred at several intracellular sites including the endoplasmic reticulum (ER), because both endoglycosidase H (endo H)-resistant EPO-R and the ER-retained EPO-R mutant (DeltaWS1 EPO-R) were Tyr phosphorylated in response to PV. Moreover, in metabolic labelling experiments, endo H-sensitive EPO-R was also phosphorylated. The phosphorylated fraction accounted for only 30-50% of the newly synthesized EPO-R, the fraction that normally exits from the ER. Tyr phosphorylation could not be detected on proteolytic fragments of the EPO-R, suggesting that this is a highly regulated process. Unlike the wild-type (wt) EPO-R, which was phosphorylated both on EPO binding and after inhibition of Tyr phosphatases by PV treatment, an EPO-R mutant (W282R EPO-R) that does not activate JAK2 was phosphorylated after PV treatment but not by EPO binding. Both EPO-R and JAK2 were phosphorylated with similar kinetics by PV treatment, suggesting that JAK2, as well as protein Tyr kinases different from JAK2, might mediate PV-dependent EPO-R phosphorylation. Furthermore the Tyr-phosphorylated ER-retained EPO-R mutant DeltaWS1 co-immunoprecipitated with JAK2 kinase, indicating that the EPO-R might interact with JAK2 while in the ER.
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PMID:Phosphorylation of erythropoietin receptors in the endoplasmic reticulum by pervanadate-mediated inhibition of tyrosine phosphatases. 935 6

Erythropoietin (EPO) and thrombopoietin (c-MPL ligand; TPO) are structurally similar cytokines and support respectively, the proliferation and differentiation for erythroid and megakaryocytic lineages, as well as more primitive progenitors. We studied the effect of these cytokines on the induction of adhesion of human growth-factor-dependent hematopoietic cells to immobilized fibronectin, which is a main component of the extracellular matrix in the bone marrow. MO7ER cells that are genetically engineered to express human EPO receptor and MO7e cells that express endogenous c-MPL were used. Stimulation with either TPO or EPO induced rapid increases in adhesion of M07ER cells to fibronectin without apparent change of expression of integrins. Experiments with inhibitory monoclonal antibodies (mAbs) demonstrated that CD41, which has been reported to be involved in TPO-induced adhesion of megakaryocytic cells, is not responsible for this enhanced adhesion. Anti-beta 1 integrin mAb inhibited adhesion completely, while inhibition by anti-alpha 4 integrin mAb and anti-alpha 5 integrin mAb was partial. Combination of anti-alpha 4 mAb plus anti-alpha 5 mAb completely abolished adhesion, as did anti-beta 1 mAb, suggesting that the adhesion is mediated by both alpha 4 beta 1 and alpha 5 beta 1 integrins. Experiments using inhibitors suggested that ligand binding followed by activation of intracellular tyrosine kinases along with PI3-kinase activation is required. After stimulation of M07ER cells with either TPO or EPO, fibronectin-attached cells, but not cells in suspension, showed tyrosine phosphorylation of focal adhesion kinase, which plays a central role in integrin-mediated signaling. These data suggest that TPO and EPO might be involved in homing/migration to the bone marrow microenvironment by hematopoietic cells that express corresponding receptors.
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PMID:Thrombopoietin and erythropoietin activate inside-out signaling of integrin and enhance adhesion to immobilized fibronectin in human growth-factor-dependent hematopoietic cells. 943 77

Erythropoietin (Epo) is believed to transduce a signal by bringing two Epo receptors into close proximity, enabling cross-phosphorylation. We compared monomeric Epos with homodimers in which two Epo monomers are linked by polyglycine. Monomeric Epo mutant R103A is unable to support Epo-dependent cell growth or trigger Janus kinase 2 and STAT5 activation, even at concentrations greater than 7,000 times that sufficient for wild-type Epo activity. In contrast, R103A homodimer induces proliferation and transduces signal at concentrations similar to that of wild-type Epo monomer and homodimer. These experiments show that two discrete domains on Epo are required for receptor binding and activation. Our results also suggest that the EpoR can be dimerized by different forms and sizes of molecules, as long as two recognition motifs are provided in the same molecule. Design of other dimeric molecules may enhance our understanding of cytokine specificity and signal transduction.
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PMID:Homodimerization restores biological activity to an inactive erythropoietin mutant. 955 5

Erythropoietin and other cytokine receptors are thought to be activated through hormone-induced dimerization and autophosphorylation of JAK kinases associated with the receptor intracellular domains. An in vivo protein fragment complementation assay was used to obtain evidence for an alternative mechanism in which unliganded erythropoietin receptor dimers exist in a conformation that prevents activation of JAK2 but then undergo a ligand-induced conformation change that allows JAK2 to be activated. These results are consistent with crystallographic evidence of distinct dimeric configurations for unliganded and ligand-bound forms of the erythropoietin receptor.
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PMID:Erythropoietin receptor activation by a ligand-induced conformation change. 997 93

Erythropoietin (EPO) and its cell surface receptor (EPOR) play a central role in proliferation, differentiation, and survival of erythroid progenitors. Signals induced by EPO have been studied extensively by using erythroid as well as nonerythroid cell lines, and various controversial results have been reported as to the role of signaling molecules in erythroid differentiation. Here we describe a novel approach to analyze the EPO signaling by using primary mouse fetal liver hematopoietic cells to avoid possible artifacts due to established cell lines. Our strategy is based on high-titer retrovirus vectors with a bicistronic expression system consisting of an internal ribosome entry site (IRES) and green fluorescent protein (GFP). By placing the cDNA for a signaling molecule in front of IRES-GFP, virus-infected cells can be viably sorted by fluorescence-activated cell sorter, and the effect of expression of the signaling molecule can be assessed. By using this system, expression of cell-survival genes such as Bcl-2 and Bcl-XL was found to enhance erythroid colony formation from colony-forming unit-erythroid (CFU-E) in response to EPO. However, their expression was not sufficient for erythroid colony formation from CFU-E alone, indicating that EPO induces signals for erythroid differentiation. To examine the role of EPOR tyrosine residues in erythroid differentiation, we introduced a chimeric EGFR-EPOR receptor, which has the extracellular domain of the EGF receptor and the intracellular domain of the EPOR, as well as a mutant EGFR-EPOR in which all the cytoplasmic tyrosine residues are replaced with phenylalanine, and found that tyrosine residues of EPOR are essential for erythroid colony formation from CFU-E. We further analyzed the function of the downstream signaling molecules by expressing modified signaling molecules and found that both JAK2/STAT5 and Ras, two major signaling pathways activated by EPOR, are involved in full erythroid differentiation.
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PMID:Role of cytokine signaling molecules in erythroid differentiation of mouse fetal liver hematopoietic cells: functional analysis of signaling molecules by retrovirus-mediated expression. 1002 85

Erythropoietin (EPO) and its receptor (EPOR) are required for the development of mature erythrocytes. After binding of ligand, the EPOR activates a variety of signaling pathways that ultimately control cellular proliferation, survival, and specific gene expression. Although erythroid progenitors appear to be the principal EPO-responsive cell type in vivo due to the restricted expression of the EPOR, many growth factor-dependent cell lines expressing the EPOR can respond to EPO by activating many or all of these pathways. In the present study, we have identified a cellular context (the interleukin-2 [IL-2]-dependent HT-2 line) in which the EPO stimulation of the EPOR fails to support cellular proliferation, STAT-5 induction, or MAPK activation, despite efficient phosphorylation of the EPOR and JAK2 and inhibition of apoptosis after withdrawal of IL-2. Interestingly, when we fused HT-2 cells expressing the EPOR with Ba/F3 cells in a complementation assay, the resulting hybridomas proliferated and potently activated STAT-5 and MAPK in response to EPO. These data indicate that an unidentified cellular factor is needed to mediate signaling by the EPOR. Moreover, Ba/F3 cells apparently express this factor(s) and somatic fusions can, therefore, confer EPO-responsiveness to HT-2 cells that lack this factor.
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PMID:Genetic evidence for an additional factor required for erythropoietin-induced signal transduction. 1038

ERYTHROPOIETIN (EPO): Erythropoietin (EPO) is a hormone that promotes the proliferation and differentiation of erythroid progenitor cells and regulates the number of erythrocytes in peripheral blood. EPO is produced mainly by the kidneys, and transcription of the EPO gene is promoted by a reduction in the oxygen concentration in the blood. The existence of EPO was suggested near the end of the 19th century by the discovery that hypoxia increases the production of red blood cells. EPO was identified as a serum factor in the 1950s, and in 1970 Miyake and coworkers succeeded in purifying it by using the urine of patients with aplastic anemia as a starting material. The human EPO gene was cloned in 1985 using a partial amino acid sequence from this purified EPO, and it is well known that recombinant EPO is currently used as a drug to treat anemia associated with chronic renal failure and other illnesses. ACTION OF EPO: When human bone marrow cells are cultured in a semisolid medium containing EPO, they form small erythroblast colonies in five to seven days, and by day 10 large erythroblast colonies appear that resemble fireworks ("burst" colonies). The original cells in the former colonies are called colony forming units-erythroid (CFU-E) or late-stage erythroblast progenitor cells and in the latter colonies they are called burst forming units-erythroid (BFU-E) or early-stage erythroblast progenitor cells. As shown in Figure 1, red blood cells are produced through differentiation from stem cells to BFU-E, CFU-E, and erythroblasts. Although EPO acts on both BFU-E and CFU-E cells, CFU-E cells show greater sensitivity to EPO, and other factors such as stem cell factor (SCF), interleukin (IL)-3, IL-4, and granulocyte macrophage colony-stimulating factor (GM-CSF) must be present together with EPO for BFU-E cell proliferation. In erythroblasts beyond the CFU-E stage, sensitivity to EPO decreases as the cells mature. THE EPO RECEPTOR AND THE CYTOKINE RECEPTOR FAMILY: The EPO receptor gene was cloned by D'Andrea and coworkers in 1989 from murine erythroleukemia cells [1]. It became clear that the EPO receptor belongs to the cytokine receptor family that comprises receptors for the various interleukins, GM-CSF, granulocyte colony-stimulating factor (G-CSF), growth hormone and prolactin. The special characteristic of this family of receptors is that they are switched on (i.e., the receptor is activated) and transduce signals to the interior of the cell by the formation of homo- or hetero-oligomers (dimers or trimers). Moreover, hetero-oligomers of these receptors share a common receptor subunit. As shown in Figure 2, the IL-3, IL-5 and GM-CSF receptors have a common &bgr; subunit, and their ligand specificity is determined by the &agr; subunit. In the same manner, the IL-6, LIF and oncostatin M (OSM) receptors all share gp130, which is the &bgr; subunit of the IL-6 receptor. The IL-2, IL-4 and IL-7 receptors all share the &ggr; subunit of the IL-2 receptor. All the above receptors are activated by the formation of hetero-oligomers, but the G-CSF receptor, EPO receptor, and growth hormone receptor are activated by the formation of homodimers of the same types of molecules [2]. We can see that groups of cytokines such as the interleukins that affect a relatively wide range of cells and have redundant biological activity create this redundancy through the common use of a single receptor subunit. On the other hand, EPO and G-CSF act with high specificity on a relatively limited range of cells, so it was probably unnecessary for their receptors to share one of the subunits. EPO RECEPTOR AND JAK2 KINASE: The signal for cellular proliferation and differentiation into erythroblasts is thought to originate at the EPO receptor. The cytoplasmic domain of the EPO receptor can be divided into two major regions. Roughly half of the cytoplasmic domain, the part lying nearest the plasma membrane, is required for generating the signals for proliferation and differentiation such as the induction of globin synthesis [3, 4]. The remaining half is not required for this signaling, and, conversely, it acts to dampen the signals. It is known that a tyrosine kinase called JAK2 associates with the region near the plasma membrane, undergoes autophosphorylation, and phosphorylates the EPO receptor, and a transcription factor called a STAT [5]. It is thought that JAK2 plays an important role in promoting cellular proliferation. The STAT is activated by the phosphorylation, and it then translocates to the nucleus, recognizes a specific base sequence in the promoter region of its target gene, and initiates transcription. At present, we know that the STAT whose activation is mediated by the EPO receptor is STAT5, and the target genes are CIS [6], which has an SH2 domain (a molecular structure that recognizes a phosphorylated tyrosine) and OSM [7], which is a pleiotropic cytokine. However, activation of STAT5 and activation of the target genes are not unique to the EPO receptor, and they also occur with the IL-2 and IL-3 receptors. Moreover, the JAK2 substrate that is directly linked to cellular proliferation is still unknown. At present, studies are under way to determine the transcription factors specific to EPO and their target genes, as well as the substrates of JAK2. RECEPTOR PHOSPHORYLATION AND CESSATION OF THE SIGNAL: On the other hand, tyrosine phosphorylation of the receptor is necessary at the cytoplasmic tail region far from the plasma membrane, and the signal transduction pathway that originates with this phosphorylated tyrosine and is mediated by proteins with SH2 domains becomes activated. First, a GTP/GDP exchange factor called SOS, which is mediated by Shc and Grb2, migrates to the plasma membrane and converts a ras protein to its GTP form. The activated ras protein then activates the Raf-MAP kinase kinase-MAP kinase cascade, and ultimately initiates the transcription of oncogenes such as c-fos and c-jun. An enzyme called PI3 kinase binds to the tyrosine phosphorylation site of the receptor and a second messenger is born. It is known that this pathway is a requirement for DNA synthesis in certain types of fibroblasts. However, these signal transduction pathways are not unique to the EPO receptor, and they are also activated by most growth factor receptors, so they are not necessarily required for EPO-induced proliferation. Conversely, the tyrosine phosphatase SH-PTP1 (also called HCP) that has an SH2 domain and is specific to blood cells associates with the tyrosine phosphorylation site of the receptor and promotes the dephosphorylation of JAK2. In other words, the role of SH-PTP1 is to stop generation of the signal [8]. Therefore, in mutations lacking this cytoplasmic tail region of the receptor far from the plasma membrane, the receptors do not undergo tyrosine phosphorylation, JAK2 activation continues for a longer period of time, and thus the signal is generated more efficiently. In fact, in one patient with a mild case of familial erythrocytosis a mutation was discovered in which the C-terminus of the EPO receptor was missing 70 amino acids [9]. This was a dominant genetic trait, and the patient's erythroblasts showed an increased sensitivity to EPO. In this family the impairment was not severe enough to be called an illness, and in fact it is said that this patient was proficient enough athletically to compete for a gold medal at the Olympics. More specifically, the reason that athletes undergo training at high altitudes is to boost EPO production because of the lower oxygen partial pressure, and this brings about the desired effect of sustained athletic capability due to a resultant increase in red blood cells. However, the same effect has occurred naturally in this athlete thanks to accelerated receptor capability.
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PMID:Physician Education: The Erythropoietin Receptor and Signal Transduction. 1038 12


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