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)

Because of the probable causal relationship between constitutive p210(bcr/abl) protein tyrosine kinase activity and manifestations of chronic-phase chronic myelogenous leukemia (CML; myeloid expansion), a key goal is to identify relevant p210 substrates in primary chronic-phase CML hematopoietic progenitor cells. We describe here the purification and mass spectrometric identification of a 155-kD tyrosine phosphorylated protein associated with src homologous and collagen gene (SHC) from p210(bcr/abl)-expressing hematopoietic cells as SHIP2, a recently reported, unique SH2-domain-containing protein closely related to phosphatidylinositol polyphosphate 5-phosphatase SHIP. In addition to an N-terminal SH2 domain and a central catalytic region, SHIP2 (like SHIP1) possesses both potential PTB(NPXY) and SH3 domain (PXXP) binding motifs. Thus, two unique 5-ptases with striking structural homology are coexpressed in hematopoietic progenitor cells. Stimulation of human hematopoietic growth factor responsive cell lines with stem cell factor (SCF), interleukin-3 (IL-3), and granulocyte-macrophage colony-stimulating factor (GM-CSF) demonstrate the rapid tyrosine phosphorylation of SHIP2 and its resulting association with SHC. This finding suggests that SHIP2, like that reported for SHIP1 previously, is linked to downstream signaling events after activation of hematopoietic growth factor receptors. However, using antibodies specific to these two proteins, we demonstrate that, whereas SHIP1 and SHIP2 selectively hydrolyze PtdIns(3,4,5)P3 in vitro, only SHIP1 hydrolyzes soluble Ins(1,3,4,5)P4. Such an enzymatic difference raises the possibility that SHIP1 and SHIP2 may serve different functions. Preliminary binding studies using lysates from p210(bcr/abl)-expressing cells indicate that both Ptyr SHIP2 and Ptyr SHIP1 bind to the PTB domain of SHC but not to its SH2 domain. Interestingly, SHIP2 was found to selectively bind to the SH3 domain of ABL, whereas SHIP1 selectively binds to the SH3 domain of Src. Furthermore, in contrast to SHIP1, SHIP2 did not bind to either the N-terminal or C-terminal SH3 domains of GRB2. These observations suggest (1) that SHIP1 and SHIP2 may have a different hierarchy of binding SH3 containing proteins and therefore may modulate different signaling pathways and/or localize to different cellular compartments and (2) that they may be substrates for tyrosine phosphorylation by different tyrosine kinases. Because recent evidence has clearly implicated both PI(3,4, 5)P3 and PI(3,4)P2 in growth factor-mediated signaling, our finding that both SHIP1 and SHIP2 are constitutively tyrosine phosphorylated in CML primary hematopoietic progenitor cells may thus have important implications in p210(bcr/abl)-mediated myeloid expansion.
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PMID:A novel SH2-containing phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase (SHIP2) is constitutively tyrosine phosphorylated and associated with src homologous and collagen gene (SHC) in chronic myelogenous leukemia progenitor cells. 1019 51

Previous findings indicate that the protein c-KIT and its ligand, stem cell factor (SCF) play a crucial role in the development of melanocytes from their precursors in the embryonic neural crest cells. Using a monoclonal anti-c-KIT antibody, ACK2, which is an antagonistic blocker of c-KIT function, we and colleagues demonstrated that mouse melanocytes disappeared with the injection of ACK2 during certain periods of embryonic and postnatal life. The precise mechanisms of this disappearance, however, remain unclear. Because melanocytes disappeared without any inflammation in these in vivo studies, we suspect that apoptosis was a main cause of their disappearance. In this study, to clarify the underlying mechanism, we studied whether ACK2 induces apoptosis in c-KIT-positive melanoblasts, which appear in mouse neural crest cells cultured with SCF from 9.5 d old mouse embryos. With an in situ apoptosis detection kit, a significant increase in apoptosis was detected after the removal of SCF, which further increased with the addition of ACK2 during SCF-dependent periods. The occurrence of apoptosis in the cultured cells was also demonstrated by a DNA analysis and electron microscopy. Immunohistochemical double staining confirmed that the apoptotic cells were c-KIT positive, and the electron microscopy showed that these apoptotic cells were melanocyte precursors. It was therefore demonstrated that apoptosis was induced in the SCF-dependent c-KIT-positive melanocytes in vitro when the SCF/c-KIT interaction was obstructed. These findings elucidate the mechanism of the regulation of melanocyte development, and the survival and proliferation of these precursor cells, by SCF/c-KIT interaction.
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PMID:Removal of stem cell factor or addition of monoclonal anti-c-KIT antibody induces apoptosis in murine melanocyte precursors. 1023 74

We found that erythropoietin (EPO) and stem cell factor (SCF) activated protein kinase B (PKB/Akt) in EPO-dependent HCD57 erythroid cells. To better understand signals controlling proliferation and viability, erythroid cells that resist apoptosis in the absence of EPO were subcloned and characterized (HCD57-SREI cells). Constitutive activations of PKB/Akt, STAT5a, and STAT5b were noted in these EPO-independent cells. PI3-kinase activity was an upstream activator of PKB/Akt because the PI3-kinase inhibitor LY294002 blocked both constitutive PKB/Akt and factor-dependent PKB/Akt activity. The LY294002 study showed that proliferation and viability of both HCD57-SREI and HCD57 cells correlated with the activity of PKB/Akt; however, PKB/Akt activity alone did not protect these cells from apoptosis. Treatment of HCD57 cells with SCF also activated PKB/Akt, but did not protect from apoptosis. This result suggested that PKB/PI3-kinase activity is necessary but not sufficient to promote viability and/or proliferation. Constitutive STAT5 activity, activated through an unknown pathway not including JAK2 or EPOR, may act in concert with the constitutive PI3-kinase/PKB/Akt pathway to protect the EPO-independent HCD57-SREI cells from apoptosis and promote limited proliferation.
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PMID:Protein kinase B (c-Akt), phosphatidylinositol 3-kinase, and STAT5 are activated by erythropoietin (EPO) in HCD57 erythroid cells but are constitutively active in an EPO-independent, apoptosis-resistant subclone (HCD57-SREI cells). 1033 82

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

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

In vitro maintenance and proliferation of human hematopoietic stem cells is crucial for many clinical applications. Early hematopoietic cells express low levels of FLT-3 and c-kit receptors, as well as the interleukin-6 (IL-6) receptor signal transducing element, gp130, but do not express IL-6 receptor itself. Therefore, we have attempted to maintain human cord blood or bone marrow CD34(+) cells ex vivo in serum-free cultures containing stem cell factor (SCF) and FLT-3 ligand (FL) alone or together with a new recombinant molecule of soluble IL-6 receptor fused to IL-6 (IL6RIL6 chimera). The effect of IL6RIL6 chimera on the proliferation and differentiation of CD34(+) cells was compared with that of each chimera component added separately. The engraftment potential of in vitro-cultured cells was determined using our recently established functional in vivo assay for primitive human severe combined immunodeficiency (SCID)-repopulating cells (SRC). We report here that IL6RIL6 chimera induced significantly higher levels of progenitors and SRC compared with SCF + FL alone or together with IL-6 and soluble IL-6 receptor. IL6RIL6 chimera prolonged in vitro maintenance of SRC for up to 14 days. Stimulation of CD34(+)CD38(-/low) enriched cells with IL6RIL6 chimera maintained the early CD34(+)CD38(-/low) cell subpopulation, which could be detected in vitro for up to 14 days. Moreover, IL6RIL6 chimera preferentially stimulated the growth of early CD34(+)38(-/low) cells, resulting in significantly higher levels of progenitors compared with more mature CD34(+)38(+) cells. Taken together, these findings demonstrate the importance of IL6RIL6 chimera in stimulating the proliferation of early CD34(+). CD38(-)gp130(+)IL-6R(-) cells in vitro and extended maintenance of progenitors and SRC.
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PMID:The soluble interleukin-6 (IL-6) receptor/IL-6 fusion protein enhances in vitro maintenance and proliferation of human CD34(+)CD38(-/low) cells capable of repopulating severe combined immunodeficiency mice. 1041 83

In humans, studies of the erythroid cell lineage are hampered by difficulties in obtaining sufficient numbers of erythroid progenitors. In fact, these progenitors in bone marrow or peripheral blood are scarce and no specific antibodies are available. We describe a new method which allows proliferation in liquid culture of large numbers of pure normal human erythroid progenitors. CD34+ cells were cultured for 7 d in serum-free conditions with the cytokine mixture interleukin (IL)-3/IL-6/stem cell factor (SCF). This resulted in cell expansion and the appearance of a high proportion of CD36+ cells which were purified on day 7. Methylcellulose clones from these cells were composed of 96.6% late BFU-E and 3.4% CFU-GM. These CD36+ cells could be recultured with the same cytokine mixture plus or minus erythropoietin (Epo) for a further 2-7 d. In both conditions further amplification of CD36+ cells was observed, but Epo induced a more dramatic cell expansion. Glycophorin-positive mature cells appeared only in the presence of Epo, and terminal red cell differentiation was observed after 7 d of secondary culture. Cells obtained from adult CD34+ progenitors mostly contained adult haemoglobin, whereas cord blood-derived cells contained equal proportions of adult and fetal haemoglobin. Activation of STAT5 and tyrosine phosphorylation of the Epo receptor and JAK2 were observed after Epo stimulation of these cells. This new method represents a straightforward alternative to the procedures previously described for the purification of normal erythroid progenitors and is useful in the study of erythropoietic regulation.
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PMID:Purification, amplification and characterization of a population of human erythroid progenitors. 1051 92

We have investigated the minimal time required for efficient transduction of human hematopoietic repopulating cells using a surrogate nonobese diabetic (NOD)/severe combined immunodeficient (SCID) xenoengraftment assay. Cord blood CD34+ cells were transduced to high levels over 24-48 hr in the presence of Flt-3 ligand, stem cell factor, interleukin 3, and interleukin 6. Under these conditions, high levels of NOD/SCID repopulating activity were preserved, but the levels of gene marking in engrafting cell populations measured by expression of a reporter transgene were low. Extension of the transduction period by 24 hr (total culture period, 72 hr) under the same cytokine conditions resulted in high levels of gene marking, but on closer analysis expression was limited predominantly to the myeloid population. Efficient transduction of both lymphoid and myeloid lineages could be achieved only if the transduction protocol was extended by a further 24 hr (total culture period, 96 hr), suggesting that myeloid lineage-committed precursors are capable of repopulation, and that over shorter time periods transduction is largely restricted to this population. This adds to the emerging evidence of heterogeneity within the SRC compartment, and has important implications for the interpretation of this assay in stem cell transplantation and gene transfer studies.
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PMID:A defined window for efficient gene marking of severe combined immunodeficient-repopulating cells using a gibbon ape leukemia virus-pseudotyped retroviral vector. 1064 42

The class I(A) phosphoinositide 3-kinases (PI3Ks) consist of a 110-kDa catalytic domain and a regulatory subunit encoded by the p85alpha, p85beta, or p55gamma genes. We have determined the effects of disrupting the p85alpha gene on the responses of mast cells stimulated by the cross-linking of Kit and FcepsilonRI, receptors that reflect innate and adaptive responses, respectively. The absence of p85alpha gene products partially inhibited Kit ligand/stem cell factor-induced secretory granule exocytosis, proliferation, and phosphorylation of the serine/threonine kinase Akt. In contrast, p85alpha gene products were not required for FcepsilonRI-initiated exocytosis and phosphorylation of Akt. LY294002, which inhibits all classes of PI3Ks, strongly suppressed Kit- and FcepsilonRI-induced responses in p85alpha -/- mast cells, revealing the contribution of another PI3K family member(s). In contrast to B lymphocytes, mast cell proliferation was not dependent on Bruton's tyrosine kinase, a downstream effector of PI3K, revealing a distinct pathway of PI3K-dependent proliferation in mast cells. Our findings represent the first example of receptor-specific usage of different PI3K family members in a single cell type. In addition, because Kit- but not FcepsilonRI-initiated signaling is associated with mast cell proliferation, the results provide evidence that distinct biologic functions signaled by these two receptors may reflect differential usage of PI3Ks.
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PMID:Impaired kit- but not FcepsilonRI-initiated mast cell activation in the absence of phosphoinositide 3-kinase p85alpha gene products. 1068 97

Hematopoietic progenitor cells from Fanconi anemia (FA) group C (FA-C) patients display hypersensitivity to the apoptotic effects of gamma interferon (IFN-gamma) and constitutively express a variety of IFN-dependent genes. Paradoxically, however, STAT1 activation is suppressed in IFN-stimulated FA cells, an abnormality corrected by transduction of normal FANCC cDNA. We therefore sought to define the specific role of FANCC protein in signal transduction through receptors that activate STAT1. Expression and phosphorylation of IFN-gamma receptor alpha chain (IFN-gammaRalpha) and JAK1 and JAK2 tyrosine kinases were equivalent in both normal and FA-C cells. However, in coimmunoprecipitation experiments STAT1 did not dock at the IFN-gammaR of FA-C cells, an abnormality corrected by transduction of the FANCC gene. In addition, glutathione S-transferase fusion genes encoding normal FANCC but not a mutant FANCC bearing an inactivating point mutation (L554P) bound to STAT1 in lysates of IFN-gamma-stimulated B cells and IFN-, granulocyte-macrophage colony-stimulating factor- and stem cell factor-stimulated MO7e cells. Kinetic studies revealed that the initial binding of FANCC was to nonphosphorylated STAT1 but that subsequently the complex moved to the receptor docking site, at which point STAT1 became phosphorylated. The STAT1 phosphorylation defect in FA-C cells was functionally significant in that IFN induction of IFN response factor 1 was suppressed and STAT1-DNA complexes were not detected in nuclear extracts of FA-C cells. We also determined that the IFN-gamma hypersensitivity of FA-C hematopoietic progenitor cells does not derive from STAT1 activation defects because granulocyte-macrophage CFU and erythroid burst-forming units from STAT1(-/-) mice were resistant to IFN-gamma. However, BFU-E responses to SCF and erythropoietin were suppressed in STAT(-/-) mice. Consequently, because the FANCC protein is involved in the activation of STAT1 through receptors for at least three hematopoietic growth and survival factor molecules, we reason that FA-C hematopoietic cells are excessively apoptotic because of an imbalance between survival cues (owing to a failure of STAT1 activation in FA-C cells) and apoptotic and mitogenic inhibitory cues (constitutively activated in FA-C cells in a STAT1-independent fashion).
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PMID:The Fanconi anemia protein FANCC binds to and facilitates the activation of STAT1 by gamma interferon and hematopoietic growth factors. 1084 98


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