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:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Retroviral vectors containing human FMS protooncogene cDNA were reconfigured to allow single-step excision and reinsertion of restriction fragments encoding short segments of the extracellular domain of the colony-stimulating factor 1 receptor (CSF-1R). Fragments ligated into M13 bacteriophages were subjected to random chemical mutagenesis on both strands and recloned into the parental vector to create libraries of FMS genes containing mutations restricted to predefined target cassettes. Transfection of retroviral vector libraries into NIH/3T3 cells gave rise to transformed foci from which cellular DNA was amplified by the polymerase chain reaction (PCR), using primers flanking the mutagenized target sequences. Amplified fragments from individual primary transformants were recloned into intact FMS vector plasmids, and those with transforming activity were subjected to nucleotide sequence analysis. Alternatively, retroviruses rescued from transformed cells by superinfection with helper virus were used to generate secondary transformants containing unique copies of proviral DNA, whose sequences were determined after PCR amplification. Novel activating mutations were identified within sequences separating the third and fourth immunoglobulin-like loops, as well as within non-covalently stabilized loop 4 of the CSF-1R extracellular domain. Thus, FMS mutations able to convert human CSF-1R to an active oncoprotein are not restricted to those previously identified at codon 301. This approach should be generally applicable for defining activating mutations in related growth factor receptors, including those for platelet-derived growth factor and Steel factor (KIT ligand), in which ligand-independent oncoprotein variants have not been identified.
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PMID:Random mutagenesis of CSF-1 receptor (FMS) reveals multiple sites for activating mutations within the extracellular domain. 153 31

Cytokines are known to be important regulators of normal hemopoiesis, acting in concert with components of the bone marrow microenvironment. Interactions with this microenvironment are known to regulate the proliferation, differentiation, and homing of hemopoietic progenitor (CD34+) cells. Adhesive interactions with the extracellular matrix retain CD34+ cells in close proximity to cytokines, but may also provide important costimulatory signals. Thus, the functional states of adhesion receptors are critical properties of CD34+ cells, but the physiological mechanisms responsible for regulating functional properties of cell adhesion receptors on primitive hemopoietic cells are still unknown. We confirm that the integrins very late antigen (VLA)-4 and VLA-5 are expressed on the CD34+ cell lines MO7e, TF1, and on normal bone marrow CD34+ progenitor cells, but in a low affinity state, conferring on them a weak adhesive phenotype on fibronectin (Fn). Herein, we show that the cytokines interleukin (IL)-3, granulocyte-macrophage CSF (GM-CSF), and KIT ligand (KL) are physiological activators of VLA-4 and VLA-5 expressed by MO7e, TF1, and normal bone marrow CD34+ progenitor cells. Cytokine-stimulated adhesion on Fn is dose dependent and transient, reaching a maximum between 15 and 30 min and returning to basal levels after 2 h. This cytokine-dependent activation is specific for VLA-4 and VLA-5, since activation of other beta 1 integrins was not observed. The addition of second messenger antagonists staurosporine and W7 abolished all cytokine-stimulated adhesion to Fn. In contrast, genistein inhibited KL-stimulated adhesion, but failed to inhibit GM-CSF- and IL-3-stimulated adhesion. Our data suggest that cytokines GM-CSF and IL-3 specifically stimulate beta 1 integrin function via an "inside-out" mechanism involving protein kinase activity, while KL stimulates integrin activity through a similar, but initially distinct, pathway via the KIT tyrosine-kinase. Thus, in addition to promoting the survival, proliferation, and development of hemopoietic progenitors, cytokines also regulate adhesive interactions between progenitor cells and the bone marrow microenvironment by modifying the functional states of specific integrins. These data are of importance in understanding the fundamental processes of beta 1 integrin activation and cellular response to mitogenic cytokines as well as on the clinical setting where cytokines induce therapeutic mobilization of hematopoietic progenitors.
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PMID:Cytokines increase human hemopoietic cell adhesiveness by activation of very late antigen (VLA)-4 and VLA-5 integrins. 753 95

Mutations in the KIT transmembrane protein-tyrosine kinase receptor affect erythropoiesis, resulting in fewer committed late progenitors (colony-forming unit erythroid, CFU-E) in the fetal liver. As the survival and proliferation of CFU-Es depend absolutely on erythropoietin (EPO), these results suggest that CFU-Es cannot proliferate or mature further unless both the KIT and EPO receptor signalling pathways are functional. How KIT affects proliferation or differentiation of CFU-Es is not clear. Here we show that the KIT ligand SCF (for stem-cell factor) can replace EPO in supporting the growth and survival of HCD57 cells, an EPO-dependent erythroid-progenitor cell line expressing high levels of KIT. SCF supports the proliferation of 32D cells that express KIT only if they also express the EPO receptor. In HCD57 cells, SCF rapidly induces tyrosine phosphorylation of the EPO receptor, and KIT physically associates with the extended box 2 region in the cytoplasmic domain of the EPO receptor. Our results indicate that KIT may activate the EPO receptor by tyrosine phosphorylation to induce further proliferation and maturation of CFU-Es.
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PMID:Interaction of the erythropoietin and stem-cell-factor receptors. 754 88

The effects of a novel cytokine FLK2/FLT3 ligand (FL) on human fetal bone marrow-derived CD34+CD19+ pro-B cells were analyzed in a stromal-cell-independent, serum-deprived culture system. FL, like interleukin-3 (IL-3), synergized with IL-7 in promoting pro-B cell growth, and differentiation of these cells into CD34-CD19+clgM+slgM- pre-B cells, whereas a small proportion of these cells even differentiate into more mature slgM+ B cells. In contrast, KIT ligand (KL) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were ineffective in promoting IL-7-dependent pro-B cell growth and differentiation. Maximal levels of pro-B cell expansion, generally resulting in 15- to 30-fold increases in cellularity, were obtained in cultures supplemented with optimal doses of FL + IL-7 + IL-3. The addition of mouse bone marrow stromal cells further enhanced the proliferation and differentiation of pro-B cells obtained in the presence of these three cytokines. Under these conditions, cultures could be maintained for more than 4 weeks, and in general 40- to 50-fold increases in cell numbers were observed by 3 weeks of culture. The percentages of clgM+ and slgM+ B cells increased 1.5- to 3-fold and 2-fold, respectively, suggesting that stromal cells may provide additional costimulatory signals for human B-cell growth and differentiation that are different from IL-7, IL-3, and FL. Collectively, our results indicate that FL, in contrast to KL, strongly promotes long-term expansion and differentiation of human pro-B cells in the presence of IL-7 or in combination of IL-7 and IL-3, which is a novel property of this hematopoietic growth factor.
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PMID:The FLK2/FLT3 ligand synergizes with interleukin-7 in promoting stromal-cell-independent expansion and differentiation of human fetal pro-B cells in vitro. 863 36

The stem cell tyrosine kinase 1 (STK1) protein is the human homologue of the murine FLT3 gene product, a receptor belonging to the FMS/KIT family. FLT3 and KIT with their ligands control the growth and differentiation of early human hemopoietic cells. In the present study, 16 cases of acute myeloid leukemia (AML) were examined by flow cytometry for cell surface expression of FLT3 and KIT receptors. All cases were also tested for their proliferative response to human FLT3 ligand (FL) and KIT ligand (KL) and for colony formation in the presence of single or associated cytokines. Among 16 AML cases tested, 10/16 expressed FLT3 receptor and 12/16 expressed KIT receptor, without any correlation with FAB subtype. FL and KL stimulated the proliferation of leukemic blasts in 11/16 AML cases (including five FLT3 or KIT receptor-negative cases), with an additive effect when added simultaneously. By contrast, some receptor-expressing AMLs did not display significant proliferative responses to their respective ligands. FL and KL as single factors induced or significantly increased the colony formation by clonogenic precursor cells respectively in eight and six of 13 cases tested. In some cases growth factor association significantly enhanced colony growth. Taken together these observations provide evidence that the pattern of FLT3 and KIT receptor expression is extremely variable among the AMLs and that receptor presence is not necessarily combined with proliferative and clonogenic response or vice versa.
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PMID:Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts. 884 93

The effects of FLT3/FLK-2 ligand (FL) and KIT ligand (KL) on in vitro expansion of hematopoietic stem cells were studied using lineage-negative (Lin-)Sca-1-positive (Sca-1+) c-kit-positive (c-kit+) marrow cells from 5-fluorouracil (5-FU)-treated mice. As single agents, neither FL nor KL could effectively support the proliferation of enriched cells in suspension culture. However, in combination with interleukin-11 (IL-11), both FL and KL enhanced the production of nucleated cells and progenitors. The kinetics of stimulation by FL was different from that by KL in that the maximal expansion by FL of the nucleated cell and progenitor pools required a longer incubation than with KL. We then tested the reconstituting abilities of cells cultured for 1, 2, and 3 weeks by transplanting the expanded Ly5.1 cells together with "compromised" marrow cells into lethally irradiated Ly5.2 mice. Cells that had been expanded with either cytokine combination were able to maintain the reconstituting ability of the original cells. Only cells that had been incubated with KL and IL-11 for 21 days had less reconstituting ability than fresh marrow cells. These results indicate that there can be significant expansion of progenitors in vitro without compromising the reconstituting ability of stem cells. Addition of IL-3 to permissive cytokine combinations significantly reduced the ability of cultured cells to reconstitute the hematopoiesis of irradiated hosts. These observations should provide a basis for a rational approach to designing cytokine combinations for in vitro expansion of hematopoietic stem cells.
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PMID:In vitro expansion of hematopoietic progenitors and maintenance of stem cells: comparison between FLT3/FLK-2 ligand and KIT ligand. 905 11

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in the human digestive tract, but their molecular etiology and cellular origin are unknown. Sequencing of c-kit complementary DNA, which encodes a proto-oncogenic receptor tyrosine kinase (KIT), from five GISTs revealed mutations in the region between the transmembrane and tyrosine kinase domains. All of the corresponding mutant KIT proteins were constitutively activated without the KIT ligand, stem cell factor (SCF). Stable transfection of the mutant c-kit complementary DNAs induced malignant transformation of Ba/F3 murine lymphoid cells, suggesting that the mutations contribute to tumor development. GISTs may originate from the interstitial cells of Cajal (ICCs) because the development of ICCs is dependent on the SCF-KIT interaction and because, like GISTs, these cells express both KIT and CD34.
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PMID:Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors. 943 54

In an effort to expand human hematopoietic progenitors and stem cells in vitro, we cultured human CD34(+)c-kitlow bone marrow cells in suspension in the presence of KIT ligand, FLK2/FLT3 ligand, interleukin-6 (IL-6), and erythropoietin with or without IL-3 and tested their engrafting capabilities by injecting them into sheep fetuses. As markers for engraftment, we analyzed CD45(+) cells and karyotypes of the colonies grown in methylcellulose culture. In three separate experiments, day-60 engraftment in the bone marrow was seen with both fresh cells and cells cultured in the presence or absence of IL-3. When fetuses were allowed to be born and analyzed for CD45(+) cells, no long-term engraftment was seen with cultured cells. We then pooled the CD45(+) cells of the fetal samples and transplanted them into secondary recipient fetuses. Day-60 engraftment in the secondary recipients was again noted when transplantation in the primary recipients was initiated with fresh cells. There were 3 cases in which cultured cells showed signs of engraftment in the secondary recipients, but the remaining 24 cases showed no signs of engraftment. These data documented that suspension culture for 2 weeks of enriched adult human bone marrow cells can maintain short-term (2 months) engrafting cells, but may not maintain longer term engrafting cells. This sheep/human xenograft model may serve as an excellent method for the evaluation of the engraftment potential of in vitro-expanded cells.
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PMID:Engraftment of cultured human hematopoietic cells in sheep. 957 5

Beta-1 integrins have essential functions in hemopoietic and immune systems by controlling phenomenons such as cell homing and cell activation. The function alpha 4 beta 1 and alpha 5 beta 1 integrins is regulated by divalent cations and, as demonstrated more recently, by mitogenic cytokines which activate them by "inside-out" mechanisms. Using the adhesive interaction of a cytokine-dependent human hemopoietic cell line to immobilized fibronectin, we have analyzed the requirements in divalent cations Mn2+, Mg2+ and Ca2+ for alpha 4 beta 1 and alpha 5 beta 1 activation by "inside-out" mechanisms triggered by cytokines such as granulocyte-macrophage colony stimulating factor or KIT ligand, or by external conformational constraints with the function-activating anti-beta 1 integrin monoclonal antibody 8A2. The intrinsic difference between these two modes of beta 1 integrin activation was revealed by their different requirements in divalent cations. We found that in the absence of any divalent cations, alpha 4 beta 1 and alpha 5 beta 1 were non-functional even after further stimulation by cytokines or 8A2. However, whilst either Ca2+, Mg2+ or Mn2+ were able to restore adhesive functions of alpha 4 beta 1 and alpha 5 beta 1 when activated by 8A2, only Mg2+ and Mn2+ were able to support activation of alpha 4 beta 1 and alpha 5 beta 1 by cytokines. Furthermore, high concentrations of Ca2+ exceeding 20 mM dramatically inhibited cell adhesion to fibronectin induced by Mn2+ and cytokines but not by 8A2. On the contrary, in the presence of both Ca2+ and Mg2+, Mn2+ had an additive effect on the activation of alpha 4 beta 1 and alpha 5 beta 1 by mitogenic cytokines. The presence of the absence of these divalent cations did not inhibit early tyrosine phosphorylation induced by the binding of KIT ligand to its tyrosine-kinase receptor KIT. Therefore, we propose that in hemopoietic cells, Ca2+, Mg2+ and Mn2+ may modulate in vivo alpha 4 beta 1 and alpha 5 beta 1 regulation by mitogenic cytokines, a phenomenon involved in the regulation of hemopoietic progenitor cell homing within the bone marrow.
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PMID:Dual control by divalent cations and mitogenic cytokines of alpha 4 beta 1 and alpha 5 beta 1 integrin avidity expressed by human hemopoietic cells. 978 83

Alternate splicing of mRNA encoding c-KIT results in isoforms which differ in the presence or absence of four amino acids (GNNK) in the juxtamembrane region of the extracellular domain of the receptor. In this study we show that these isoforms of human c-KIT, expressed at similar levels in NIH3T3 cells, display differential effects on various attributes of transformation. The GNNK- isoform strongly promoted anchorage independent growth (colony formation in semi-solid medium), loss of contact inhibition (focus formation), and led to tumorigenicity in nude mice. In contrast, the GNNK+ isoform elicited colony formation but relatively poor focus formation and no tumorigenicity. Saturation binding analysis indicated that the isoforms do not differ significantly in their affinity for the KIT ligand, Steel Factor (SLF). Negligible ligand-independent receptor phosphorylation was observed in either case but, after ligand stimulation, the GNNK- isoform displayed more rapid and extensive tyrosine autophosphorylation and faster internalization. Both isoforms recruited the p85 subunit of phosphatidylinositol 3-kinase and led to similar phosphorylation of its downstream effector c-Akt, but the GNNK- isoform gave rise to more MAP kinase phosphorylation. Thus the c-KIT isoforms display different signalling characteristics and have different transforming activity in NIH3T3 cells.
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PMID:Isoforms of c-KIT differ in activation of signalling pathways and transformation of NIH3T3 fibroblasts. 1052 34


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