Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
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) 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

Thrombopoietin (TPO) plays a central role in megakaryopoiesis and platelet production. It is a ligand for c-mpl, which is a member of the hematopoietic receptor superfamily. We have recently identified several human c-mpl binding peptides which are distinct from TPO, from phage random peptide libraries. PK1M is one of these peptides and is an agonist of c-mpl which is TPO receptor. We show here that PK1M induces the tyrosine phosphorylation of the Janus kinase 2 (JAK2) and the activation of the signal transducer and activation of transcription 5 (STAT5) in TPO-dependent cells like TPO.
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PMID:Signal transduction by the peptide which mimics the activity of thrombopoietin. 962 75

The pathogenesis of the increased number of megakaryocytes and thrombocytosis in essential thrombocythemia (ET) is still unknown. We examined the expression of c-mpl, a receptor of thrombopoietin (TPO), and its signaling molecules in a patient with ET. An 8-year-old girl showed a high platelet count and an increased number of bone marrow megakaryocytes. Neither chromosomal abnormalities nor myelofibrosis was observed. Following the diagnosis of ET, aspirin therapy was begun for the patient, with only modest improvement of symptoms. Her platelet count ranged from 1,200,000/microL to 2,200,000/microL for more than 2 years. In the analyses, the serum TPO level in the patient was 420 attomoles/mL (normal, 760 +/- 320). The level of c-mpl expression in bone marrow mononuclear cells was higher in the patient than in healthy children, while there was no difference in the level of c-mpl expression in CD34+ cells, indicating an expanded pool of megakaryocytic lineage cells. The level of Janus kinase 2 (Jak2) expression was lower in the patient than in a healthy child. These findings indicate that the signal pathway mediated by c-Mpl after binding to TPO may be impaired in ET. Further analysis is needed to clarify the mechanism underlying the development of thrombocytosis in ET patients.
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PMID:Analysis of thrombopoietin and c-mpl expression in a child with essential thrombocythemia. 965 38

During the past 4 years, significant progress has been made in elucidating the earliest events following binding of ligands to members of the cytokine receptor superfamily. This is a rapidly growing family of receptors that currently includes receptors for growth hormone (GH); prolactin; erythropoeitin; granulocyte colony-stimulating factor; granulocyte macrophage colony-stimulating factor; interleukin(IL)s 2-7, 9-13, 15; interferon (IFN)-alpha, beta, and gamma; thrombopoietin; leptin; oncostatin M; leukemia inhibitory factor (LIF); ciliary neurotrophic factor; and cardiotropin-1. Despite their diverse physiological effects in the body, ligands that bind to members of this family share multiple signaling pathways. An early and most likely initiating event for all of them is the activation of one or more members of the Janus (or JAK) family of tyrosine kinases. The activated JAK kinases, which form a complex with the cytokine receptor subunits, phosphorylate themselves as well as the receptor. These phosphorylated tyrosines form binding sites for various signaling molecules that are themselves thought to be phosphorylated by JAK kinases, including 1) signal transducers and activators of transcription (Stats), which regulate transcription; 2) She proteins that recruit Grb2-SOS complexes, thereby initiating the Ras-MAP kinase pathway; and 3) insulin receptor substrate (IRS) proteins that are thought to regulate metabolic events in the cell. Additional other signaling molecules have been implicated in signaling by some cytokines, including protein kinase C, SH2-B beta, and intracellular Ca. This review uses the GH receptor as a model system for studying cytokine signaling and summarizes some of the data used to establish JAK2 as a GH receptor-associated tyrosine kinase and to identify signaling molecules that lie downstream of JAK2. Since these pathways are shared by multiple cytokines, this review also discusses factors that might contribute to specificity of response to different cytokines.
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PMID:Signaling via JAK tyrosine kinases: growth hormone receptor as a model system. 976 3

Thrombopoietin (TPO), the primary physiological regulator of platelet production, was initially thought to be a lineage-specific factor acting predominantly on megakaryocytopoiesis. Detailed studies establish that this cytokine mediates biological effects on a broad spectrum of hematopoietic progenitor cells, including stem cells. TPO is a hormone constitutively produced mainly by the liver and kidney. Plasma TPO levels are regulated by the platelet and megakaryocyte mass through Mpl receptor binding, internalization and degradation. The Mpl receptor is a member of the hematopoietin receptor superfamily lacking intrinsic kinase activity. Upon ligand-induced Mpl homodimerization, the major signaling events for proliferation are mediated through the JAK2/STAT5 pathway, while differentiation might occur through a prolonged activation of the MAPK pathway. Preclinical and clinical studies demonstrate the potential use of TPO in a variety of contexts, but it is too early to evaluate its benefit in reducing platelet transfusion.
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PMID:Thrombopoietin and its receptor. 983 Nov 70

The factor-independent Dami/HEL and Meg-01 and factor-dependent Mo7e leukemic cell lines were used as models to investigate JAK/STAT signal transduction pathways in leukemic cell proliferation. Although Dami/HEL and Meg-01 cell proliferation in vitro was independent of and unresponsive to exogenous cytokines including granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), IL-6, thrombopoietin (TPO), and tumor necrosis factor-alpha (TNF-alpha), the growth of Mo7e cells was dependent on hematopoietic growth factors. When these cell lines were cultured in medium without cytokines, a constitutively activated STAT-like DNA-binding factor was detected in nuclear extracts from both Dami/HEL and Meg-01 cells. However, the STAT-like factor was not detectable in untreated Mo7e cells, but was activated transiently in Mo7e cells in response to cytokine treatments. The constitutively activated and cytokine-induced STAT-like DNA-binding factor in these three cell lines was identified as STAT5 by oligonucleotide competition gel mobility assays and by specific anti-STAT antibody gel supershift assays. Constitutive activation of JAK2 also was detected in the factor-independent cell lines, but not in Mo7e cells without cytokine exposure. Meg-01 cells express a p185 BCR/ABL oncogene, which may be responsible for the constitutive activation of STAT5. Dami/HEL cells do not express the BCR/ABL oncogene, but increased constitutive phosphorylation of Raf-1 oncoprotein was detected. In cytokine bioassays using growth factor-dependent Mo7e and TF-1 cells as targets, conditioned media from Dami/HEL and Meg-01 cells did not show stimulatory effects on cell proliferation. Our results indicate that the constitutive activation of JAK2/STAT5 correlates with the factor-independent growth of Dami/HEL and Meg-01 cells. The constitutive activation of JAK2/STAT5 in Dami/HEL cells is triggered by a mechanism other than autocrine cytokines or the BCR/ABL oncoprotein.
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PMID:Constitutive activation of the JAK2/STAT5 signal transduction pathway correlates with growth factor independence of megakaryocytic leukemic cell lines. 1009 Sep 48

The Janus family of tyrosine kinases (JAKs) plays a critical role in signal transduction by members of the cytokine receptor superfamily. In response to ligand-receptor interaction, these nonreceptor tyrosine kinases are rapidly phosphorylated and activated, triggering tyrosine phosphorylation and activation of downstream signaling intermediates. Upon binding to its receptor, the product of the proto-oncogene c-mpl, thrombopoietin (TPO) activates both JAK2 and TYK2 in multiple cell lines as well as megakaryocytes and platelets. To study whether one or both of these kinases are essential for TPO signal transduction, we engineered a parental human sarcoma cell line (2C4) as well as sarcoma cell lines that are deficient in JAK2 expression (gamma2A) or TYK2 expression (U1A) to express the wild-type Mpl receptor. The ability of TPO to induce tyrosine phosphorylation of Mpl and multiple intracellular substrates in each cell line was then examined. Our results demonstrate that JAK2-deficient cells (gamma2A-Mpl) are unable to initiate TPO-mediated signaling. In contrast, cells that are TYK2-deficient (U1A-Mpl) are able to induce tyrosine phosphorylation of Mpl, JAK2, STAT3, and Shc as efficiently as parental cells (2C4-Mpl). These data indicate that JAK2 is an essential component of Mpl signaling and that, in the absence of JAK2, TYK2 is incapable of initiating TPO-induced tyrosine phosphorylation.
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PMID:Thrombopoietin signal transduction requires functional JAK2, not TYK2. 1022 14

Cell numbers limit the widespread clinical use of cord blood (CB) for gene therapy and marrow replacement in adults; a simple and effective method for ex vivo expansion of CB primitive progenitor cells (PPC) is required. Recently, the combination of thrombopoietin (TPO) and Flk-2/Flt-3 ligand (FL-2) was reported to support slow proliferation of CB-PPC in stroma-free liquid culture. We established a novel culture system in which the murine stromal cell line HESS-5 dramatically supports the rapid expansion of cryopreserved CB-PPC in synergy with TPO/FL-2. Furthermore, while HESS-5 cells directly adhered to human progenitors during culture, the cultured human cells could easily be harvested without contamination by HESS-5 cells. Within 7 days of culture, a 100-fold increase in CD34bright/CD38dim cells was obtained in serum-containing culture. When HESS-5 cells were physically separated from human progenitor cells in the presence of TPO/FL-2, synergy was blocked, suggesting that HESS-5 cells support proliferation of PPC by direct cell-to-cell interaction. The hematopoietic-supportive effects of this xenogeneic coculture system were then assessed in a very short-term (5 days) serum-free culture. Expansion was further enhanced by addition of stem cell factor (SCF) or interleukin-3 (IL-3). As a result, a 50- to 100-fold increase in CD34bright/CD38dim cells was noted. Colony-forming units in culture (CFU-C) and mixed colonies (CFU-GEMM) were enhanced by 10- to 30-fold and 10- to 20-fold, respectively. Moreover, generation of long-term-culture-initiating cells (LTC-IC) from CD34bright/CD38dim cells was amplified by 25-fold. The severe-combined immunodeficient (SCID) mouse-repopulating cell (SRC) assay confirmed extensive ability of the expanded cells to reconstitute long-term hematopoiesis. These results indicate that this xenogeneic coculture system, in combination with human cytokines, can rapidly generate PPC from cryopreserved CB.
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PMID:Rapid ex vivo expansion of human umbilical cord hematopoietic progenitors using a novel culture system. 1034 Apr 7

Thrombopoietin (TPO), the critical regulator of platelet production, acts by binding to its cell surface receptor, c-Mpl. Numerous studies have shown that TPO binding leads to JAK2 kinase activation and Tyr phosphorylation of c-Mpl and several intracellular signaling intermediates, events vital for the biological activity of the hormone. In contrast, virtually nothing is known of the role of Ser or Thr phosphorylation of c-Mpl. By using phosphoamino acid analysis we found that Ser residues of c-Mpl were constitutively phosphorylated in receptor-bearing cells, levels that were increased following exposure of cells to TPO. To identify which residues were modified, and to determine the functional consequences of their phosphorylation, we generated a series of Ser to Ala mutations of a truncated c-Mpl receptor (T69) capable of supporting TPO-induced cell growth. Of the eight Ser within T69 we found that at least four are phosphorylated in TPO-stimulated cells. The mutation of each of these residues alone had minimal effects on TPO-induced proliferation, but substitution of all of the phosphoserine residues with Ala reduced the capacity of the receptor to support cell growth by over 50%. Additionally, the Ser at cytoplasmic position 18 is not detectably phosphorylated. However, the mutation of Ser-18 to Ala nearly abrogates TPO-induced proliferation and co-precipitation of JAK2 with Mpl. This study provides the first systematic analysis of the role of Ser residues in c-Mpl signaling.
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PMID:A structure-function analysis of serine/threonine phosphorylation of the thrombopoietin receptor, c-Mpl. 1091 61

Interferon (IFN)-alpha has proven useful for treating several clinical conditions, including chronic viral hepatitis and chronic myeloproliferative and lymphoproliferative disorders. In addition to its well-known antiviral effects, the cytokine exerts antiproliferative effects on many cell types, helping to explain its therapeutic usefulness in these latter conditions. However, this same property accounts for several undesirable effects, including thrombocytopenia, which can interfere with the successful clinical application of IFN-alpha. Unfortunately, the mechanisms responsible for the myelosuppressive effects of the cytokine are incompletely understood. The effects of IFN-alpha on megakaryocyte (MK) development were studied. Using several marrow cell purification techniques and quantitative culture methods, it was found that IFN-alpha directly inhibits thrombopoietin (TPO)-induced MK growth. Previous studies indicated that Janus kinase (JAK) and its substrates mediate the effects of TPO on cellular proliferation and survival. It was found that IFN-alpha directly suppresses TPO-induced phosphorylation of the JAK2 substrates c-Mpl and STAT 5 in a TPO-dependent hematopoietic cell line and of Mpl and STAT3 in primary murine MK. Moreover, IFN-alpha induces SOCS-1 production in these cells, which has been shown to inhibit TPO-induced cell growth. Because SOCS protein expression is induced by many cytokines and has been reported to extinguish signaling from several hematopoietic cytokine receptors, these results identify a molecular mechanism responsible for cytokine receptor cross-talk.
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PMID:Interferon-alpha directly represses megakaryopoiesis by inhibiting thrombopoietin-induced signaling through induction of SOCS-1. 1097 53


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